CN115586335B

CN115586335B - Polymerase-antibody combination for rapid immunohistochemistry in melanoma surgery

Info

Publication number: CN115586335B
Application number: CN202211375416.2A
Authority: CN
Inventors: 伍进; 赵松庆; 王健夫; 陈硕
Original assignee: Guizhou Meixinda Medical Technology Co ltd
Current assignee: Guizhou Meixinda Medical Technology Co ltd
Priority date: 2021-11-04
Filing date: 2022-11-04
Publication date: 2023-10-13
Anticipated expiration: 2042-11-04
Also published as: CN115586335A; CN115840047A; CN115840047B

Abstract

The application provides a polymerase-antibody combination for rapid immunohistochemistry in melanoma surgery. The present application relates to polymerase/antibody conjugates and combinations thereof for rapid immunohistochemistry in oncology, kits comprising said polymerase/antibody conjugates or combinations thereof, and their use in the diagnosis of cancer, such as melanoma.

Description

Polymerase-antibody combination for rapid immunohistochemistry in melanoma surgery

Technical Field

The present application relates generally to rapid identification and genotyping of tumors, and more particularly to polymerase/antibody conjugates and combinations thereof, kits comprising the same, methods and uses thereof for rapid identification and genotyping of tumors such as brain tumors, lymphomas, melanomas and metastatic cancers in surgery, and for determining tumor tissue surgical resection margin negativity.

Background

Rapid and accurate intraoperative identification of the importance of a tumor for optimal brain tumor resection

The number of primary brain tumors occurring annually worldwide is about 250,000. Brain tumors were graded from low (I) to high (IV) malignancy according to their histiocyte morphology and growth rate. Surgical removal of brain tumors is the first choice of treatment and is generally the only treatment for low brain tumors. Excision of brain tumors can reduce intracranial pressure to relieve symptoms and reduce the volume of tumor mass that needs to be treated by subsequent radiation or chemotherapy. There is generally a need for maximum effort to clear invasive brain tumors during surgery, otherwise residual invasive cancer cells continue to grow resulting in tumor recurrence, thereby reducing overall survival of the patient. However, the process is not limited to the above-mentioned process, After infiltration of the peripheral fragile functional normal brain tissue by the tumor, complete removal of the brain tumor becomes almost impossible ^[1] . Currently, preoperative imaging localization and intraoperative precise identification of tumors are necessary options to promote optimal tumor resection ^[2] . Most non-invasive brain-image scans will only accurately locate brain tumor masses at the millimeter level. There remains a need in the surgical procedure for differential cancer diagnosis at the tissue cell level through tissue morphology to enable the surgeon to make an important decision as to whether to resect or not. This is especially true for malignant brain cancers. Hematoxylin and eosin tissue and cell staining on intraoperative frozen sections with century history is still necessary today and is the main method of intraoperative brain tumor differential diagnosis and marginal determination.

Intrinsic quality defect of hematoxylin and eosin staining of intraoperative frozen sections

Methods for identifying tumors on frozen sections using hematoxylin and eosin stained slides can only rely on histocytomorphology visual inspection by a pathologist under a microscope. Thus, hematoxylin and eosin staining quality of frozen section tissue has been the primary limiting factor in determining the accuracy of intra-operative frozen section cancer diagnosis. In essence, the frozen section hematoxylin and eosin staining technique has two inherent problems that are difficult to overcome. The first problem is a defect caused by freezing. The soft nature of brain tissue and the high moisture content during tissue freezing introduce ice crystals into the tissue, resulting in the formation of freezing artifacts throughout the tissue, thereby distorting the cell morphology. For example, frozen tissue often creates irregularities in the nuclear profile of oligodendrogliomas, making it look similar to astrocytomas and thus difficult to distinguish. A second inherent problem with cryo-section hematoxylin and eosin staining is the inability to resolve histological structures based on molecular level differences due to their non-specific chemical staining properties. These inherent defects present a significant challenge and burden to the clinical pathologist who makes a diagnosis based on the tissue morphology of frozen sections, which can only rely on the micro-histological morphology to identify and differentiate cancer tissue in a short period of time. For example, in frozen section of Caesalpinia sappan In terms of sperm and eosin staining, it is very difficult to distinguish meningiomas, peripheral schwannomas and spindle cell proliferation. Distinguishing reactive gliosis from low glioma is one of the most difficult differential diagnostic challenges in surgical neuropathology ^[3] . There is a clear need in brain surgery to distinguish metastatic cancers, meningiomas, medulloblastomas, melanomas, gliomas with IDH 1R 132H mutations, astrocytomas, secondary glioblastomas, etc., and a single chemical staining of frozen sections by hematoxylin and eosin is not adequate for rapid identification of these brain tumor tissue types.

Attempts to improve diagnosis in brain tumor surgery

1) Coherent raman scattering microscopy can amplify spontaneous raman signals 10,000 times, and can perform real-time histological imaging without tissue processing, slicing and staining ^[4] . This brain tissue imaging technique demonstrated some agreement with the results of hematoxylin and eosin staining of frozen sections ^[4] . However, this technique has limited specificity and is not universally applicable.

2) Rapid evaporative ionization mass spectrometry is an emerging technique that can be used to characterize human tissue in vivo in near real time by analyzing aerosols released during electrosurgical dissection. The instrument is still very expensive. Whether the instrument performance can exceed the hematoxylin and eosin staining of frozen sections is yet to be observed ^[5] 。

3) Confocal microscopy for real-time histopathological imaging of brain tumors is another in vivo imaging technique that relies on tumor-specific fluorescent contrast agents. Its role in intraoperative interpretation remains to be assessed ^[6] 。

4) Rapid immunohistochemical staining of sentinel lymph node Immunohistochemistry (IHC) was accomplished in 20 minutes using a polyHRP-labeled anti-cytokeratin secondary antibody ^[7] . Dako (california) has proposed a rapid immunohistochemical staining kit EnVision that can stain frozen sections at 37 ℃ in about 20 minutes. The two-step method of staining with labeled secondary antibody is increased by one than that of direct methodThe step dyeing procedure requires more time. However, according to feedback information applied by the Mohs clinic, the actual process takes about 45 minutes.

5) For low cell infiltration gliomas, the staining of intraoperative frozen sections with hematoxylin and eosin is difficult to discriminate due to staining non-specificity and low chromatic aberration. Rapid and sensitive genotyping methods have been developed for detecting somatic cell mononucleotide isocitrate dehydrogenase 1 (IDH 1), real-time PCR for IDH1 mutation, and mass spectrometry for 2-hydroxyglutarate, a metabolite of mutant IDH. The method showed the results within 60 minutes.

While the technological advances described above have facilitated assessment in brain oncology from different angles, they have been compared against cryosectional hematoxylin and eosin staining procedures to show limited improvement in certain cases of hematoxylin and eosin staining difficulties.

Studies and applications of polymerase antibody conjugates in the prior art

In order to obtain direct coupling of the polymerase to the antibody and thus greater amplification of the developed signal, numerous previous inventions have described numerous specific methods for preparing and coupling the polymerase to the antibody. For example, patent CN1300942a describes various preparation methods, one of which uses focusing is immunoassays in solution, and the other uses focusing is immunohistochemical staining on conventional paraffin-embedded tissue sections, but staining incubation times of more than 60 minutes are merely too slow to be used for intra-operative detection. European patent EP0175560A2 describes a method for the preparation of antibodies coupled to a variety of enzymes to form a polymerase/antibody for use in quantitative determination of binding of the antibody to an antigen. However, the determination of the immunological binding reaction described in this patent is carried out in solution without hindrance. Immunohistochemical staining of biological tissues is quite different from the immune response in solution, antibodies and antibody conjugates face complex and irregular steric hindrance of tissues, and many antibodies can react easily with antigen binding in solution but cannot react with antigen binding in tissues, so that the immune response in solution cannot be simply and directly generalized to the staining of biological tissues for immunohistochemistry, wherein the technical crossing is not obvious. CN 1945333a discloses a reagent for rapid immunohistochemical detection of breast cancer lymph node metastasis and a detection method using the reagent, which is characterized in that a plurality of antibodies which can distinguish metastatic cancer cells from lymph node resident cells are directly marked into a reagent, and the metastatic cancer cells in the lymph node are specifically colored through one-step reaction. The technology is characterized in that the increase of the signal amplification gain of the polymerase is realized by the mixed coupling of the polymerase and a plurality of breast cancer metastasis positive antibodies to increase the antigen density correspondingly positioned in tissues, but the method still needs 15-20 minutes to realize the intraoperative diagnosis of breast cancer lymph node metastasis.

Thus, there remains a need for a more accurate and reliable method for more rapid identification for sample detection from brain tumor tissue. (1) In order to significantly improve the accuracy of diagnosis in brain oncology, staining signals must be specifically enhanced on histological features at a specific molecular level; (2) All these tests must be completed in less than 10-15 minutes to meet the time constraints of the intraoperative test; (3) It is necessary to have a plurality of reagents with molecular specific staining to form an antibody combination, and based on the results of staining of these antibody combinations, multiple tumor types can be more accurately identified.

In view of the above, the present invention satisfies the above-described needs by optimizing the distribution of the molecular weight of direct coupling of a polymerase with an antibody and random diversity to achieve high sensitivity and high speed staining of a single antigen, and forming a necessary diagnostic combination in tumor surgery with a plurality of direct conjugates of a polymerase with an antibody related to brain tumor, lymphoma, melanoma, metastatic cancer, and achieving immunohistochemical differential diagnosis in brain tumor surgery which has been difficult in the past.

Disclosure of Invention

The invention adopts the direct coupling of the polymerase with the antibody, and then forms a matched antibody combination for rapidly identifying the tumor by using a plurality of polymerase/antibody conjugates of specific antibodies, and implements rapid and accurate immunohistochemical staining on frozen tissue sections in tumor operation.

In one aspect, the invention provides a polymerase/antibody conjugate formed by a polymerase and an antibody. In the polymerase/antibody conjugates of the invention, each polymerase antibody conjugate may comprise: (i) One or more polymerases, (ii) an antibody that recognizes an analyte of interest.

In some embodiments according to any of the embodiments above, the polymerase/antibody conjugate comprises a plurality of polymerases. In some embodiments, the polymerase/antibody conjugate comprises a plurality of enzyme molecules, each polymerase/antibody conjugate comprising a different number of enzyme molecules. In some embodiments, the polymerase/antibody conjugate comprises a plurality of polymerases, wherein the number of enzyme molecules of each polymerase in the plurality of polymerases can be the same or different. In some embodiments, the polymerase/antibody conjugate comprises a plurality of polymerases, wherein the combined structure of the plurality of polymerases can differ.

In some embodiments according to any of the embodiments described above, the polymerase/antibody conjugates of the invention may comprise a plurality of the polymerase and the antibody, wherein each polymerase comprises a different number of enzyme molecules, and the molecular weight of the conjugate forms a multi-dispersed distribution characterized by a molecular weight of about 400kDa to about 2,000kDa per polymerase/antibody conjugate. The polydisperse distribution refers to a number of polymerase/antibody conjugates that is not zero between each partition within the molecular weight range as defined above. For example, between 400 and 600kDa, the number is about 2-8%, such as 4%, 5%, 6%, 7% or 8%, preferably 4% -6% of the number of the whole molecular weight range; between 600-800kDa at about 5-10%, e.g. 6%, 7%, 8%, 9% or 10%, preferably 7% -9%; between 800 and 1000kDa 6-13%, e.g. 7%, 8%, 9%, 10%, 11% or 12%, preferably 9% -11%; between 1000 and 1200kDa 7-14%, e.g. 8%, 9%, 10%, 11%, 12%, 13% or 14%, preferably 9% -12%; between 1200-1400kDa 9-16%, e.g. 9%, 10%, 11%, 12%, 13%, 14%, 15% or 16%, preferably 11% -13%; between 1400 and 1600kDa, 10-16%, e.g. 10%, 11%, 12%, 13%, 14%, 15% or 16%, preferably 12% -14%; between 1600 and 2000kDa, 12-18%, e.g. 12%, 13%, 14%, 15%, 16%, 17% or 18%, preferably 13% -15%. Further, in more specific examples, for example, in preferred exemplary polymerase/antibody conjugates (e.g., as used in the examples), the molecular weight is between 400 and 600kDa and the number is about 4% -6% of the number of the entire molecular weight range; between about 7% -9% of 600-800 kDa; between about 9% -11% of 800-1000 kDa; between 1000-1200kDa and about 10% -12%; between about 11% -13% of 1200-1400 kDa; between about 12% -14% between 1400-1600 kDa; between 1600-2000kDa approximately 13% -15%.

More specifically, the molecular weight of the polymerase/antibody conjugates of the invention may be 300kDa to about 10,000kDa, preferably 400kDa to about 10,000kDa, 400kDa to about 5,000kDa, 500kDa to about 5,000kDa or 750kDa to about 5,000kDa, with different molecular weights in each interval within this range, and the molecular weights of the conjugates forming a polydisperse distribution as described above.

In one embodiment, the three-dimensional structure of the polymerase/antibody conjugate exhibits a random distribution within each molecular weight interval, e.g., a polymerase/antibody conjugate comprising randomly different three-dimensional structures within the 400kDa to about 8,00kDa interval; polymerase/antibody conjugates comprising additional types of randomly distinct three-dimensional structures in the 800kDa to about 1,200kDa interval; polymerase/antibody conjugates comprising other types of randomly distinct three-dimensional structures in the 1,200kda to about 1,600kda interval; other types of polymerase/antibody conjugates comprising randomly different three-dimensional structures in the 1,600kDa to about 2,000kDa interval, wherein the three-dimensional structures comprised by the polymerase/antibody conjugates in the four intervals may be different.

In some embodiments according to any of the above embodiments, the polymerase/antibody conjugates of the invention may comprise a plurality of polymerases, each polymerase/antibody conjugate having a total number of polymerase molecules that is different from the total number of polymerase molecules, e.g., may comprise 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240, and/or 250 enzyme molecules, or one or more of the above numbers.

In some embodiments according to any of the embodiments above, the distribution of molecular weights of the polymerase/antibody conjugates of the invention is non-uniform across the molecular weight intervals and the composition of its three-dimensional structure is random. For example, the three-dimensional structure of the polymerase/antibody conjugates of the invention is structured randomly and varies from one molecular weight interval to another. In some embodiments according to any of the embodiments above, the polymerase/antibody conjugates of the present invention comprise a plurality of enzyme molecules or polymerases, wherein the plurality of enzyme molecules or polymerases may comprise the same or different enzyme types. For example, all enzyme molecules or polymerases in the polymerase/antibody conjugates of the invention can be horseradish peroxidase.

In some embodiments according to any of the embodiments described above, the enzyme molecules of the polymerase used in the polymerase of the polymerase/antibody conjugates of the present invention may include horseradish peroxidase (HRP), beta-D-galactosidase, alkaline phosphatase, superoxide dismutase, luciferase, lactate dehydrogenase, galactose oxidase, or the like.

In some embodiments, the polymerase/antibody conjugates of the invention may be formed by covalent attachment of a polymerase to an antibody.

In some embodiments according to any of the embodiments above, the polymerase/antibody conjugates of the invention may comprise a primary antibody (i.e., a primary antibody). In some embodiments, the primary antibody may comprise a full length antibody, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or an antibody fusion protein, or an antigen binding fragment thereof. In some embodiments according to any of the embodiments above, the primary antibody may comprise an antibody or antigen binding fragment thereof that targets the antigen of interest of the invention and its associated disease, such as cancer. In some embodiments according to any of the embodiments above, the primary antibody may be selected from antibodies or antigen binding fragments thereof that target the antigen of interest of the invention and diseases related thereto, such as cancer. In further embodiments, the cancer comprises or is selected from brain tumors, such as brain glioma, meningioma, medulloblastoma, ependymoma, schwannoma, intracranial neuroepithelial tumors, chordoma; epithelioid sarcoma; lymphomas; small cell carcinoma; synovial tumor; neuroendocrine cell tumors, including olfactory cell tumors, medulloblastoma, neuroblastoma, pituitary adenoma; melanoma; lymphomas; metastatic tumors; oligodendroglioma, IDH1 mutant tumor, astrocytoma, schwannoma, choroid plexus tumor, papillary tumor, spindle tumor, atypical deformity/tumor-like. In still further embodiments according to any of the above embodiments, the primary antibody or antigen binding fragment may comprise one or more, e.g., any two, three, four, five, six, seven or eight of anti-Pan-CK, anti-GFAP, anti-EMA, anti-synapsin (syntenysin), anti-CD 45, anti-CD 34, anti-Mart-1, and anti-Sox-10 antibodies or antigen binding fragments thereof. In still further embodiments according to any of the above embodiments, the primary antibody or antigen binding fragment may comprise one or more, e.g., any two, three, four, five, six, seven or eight of anti-Pan-CK, anti-GFAP, anti-EMA, anti-synapsin (syntenin), anti-CD 45, anti-Mart-1, and anti-Sox-10 antibodies or antigen binding fragments thereof.

In some embodiments according to any of the embodiments described above, the polymerase molecules used in the polymerase/antibody conjugates of the invention may be covalently linked via a crosslinking reagent. In some embodiments, the enzyme molecules of the polymerase may be directly covalently linked. In some embodiments, the enzyme molecules of the polymerase are covalently linked in a linear fashion. In some embodiments, the enzyme molecules of the polymerase are covalently linked in a branched fashion. In some embodiments, the enzyme molecules of the polymerase are covalently linked in a mixed linear and branched fashion.

In some embodiments according to any of the embodiments described above, the polymerase used in the polymerase/antibody conjugates of the invention may have a molecular weight of about 300kDa to about 5 megadaltons (MDa). In some embodiments, the polymerase has a molecular weight of at least about 500 kDa. In some embodiments, the polymerase has a molecular weight of less than or about 5 MDa. In some embodiments, the polymerase has a molecular weight of at least about 750 kDa. In some embodiments, the polymerase has a molecular weight of at least about 1, 2, 3, or 4 MDa.

In some embodiments according to any of the embodiments above, the polymerase is first formed before being coupled to the antibody.

In another aspect, the invention provides a combination of polymerase/antibody conjugates comprising a plurality of the polymerase/antibody conjugates described above, e.g., two, three, four, five, six, seven or eight different polymerase/antibody conjugates. Preferably, the combination of the polymerase/antibody conjugates of the present invention comprises or consists of a polymerase/antibody conjugate against Pan-CK, anti-GFAP, anti-EMA and/or anti-CD 34, anti-synaptophysin (synthapsin), anti-CD 45, and anti-Mart-1 and/or anti-Sox-10 antibodies, or antigen binding fragments thereof. More preferably, the combination of the polymerase/antibody conjugates of the present invention comprises anti-Mart-1 and/or anti-Sox-10 antibodies, and at least one (e.g., two, three, four, five and six) of the following antibodies: anti-Pan-CK, anti-GFAP, anti-EMA, anti-CD 34, anti-synaptophysin, and anti-CD 45 antibodies or antigen binding fragments thereof. In the combination of the polymerase/antibody conjugates of the invention, each polymerase/antibody conjugate may comprise: (i) One or more polymerases, (ii) an antibody that recognizes an analyte of interest.

In some embodiments according to any of the embodiments described above, each polymerase/antibody conjugate in the combination of the polymerase/antibody conjugates of the invention may comprise a plurality of enzyme molecules or a plurality of polymerases, wherein the plurality of enzyme molecules or the plurality of polymerases may comprise the same or different enzyme types, e.g., all of the enzyme molecules or the polymerases in the polymerase/antibody conjugates of the invention may be horseradish peroxidase.

In some embodiments according to any of the embodiments above, in the combination of the polymerase/antibody conjugates of the invention, the enzyme molecule of the polymerase may include horseradish peroxidase, beta-D-galactosidase, alkaline phosphatase, superoxide dismutase, luciferase, lactate dehydrogenase, galactose oxidase, and the like.

In some embodiments according to any of the embodiments above, each of the polymerase/antibody conjugates in the combination of the invention comprises a primary antibody and a polymerase, wherein the primary antibody may comprise a full length antibody, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a human antibody or antibody fusion protein, or an antigen binding fragment.

In some embodiments according to any of the embodiments above, each polymerase/antibody conjugate in the combination of the polymerase/antibody conjugates of the invention comprises a primary antibody and a polymerase, wherein the primary antibody in each polymerase/antibody conjugate comprises an antibody to a different antigen.

In some embodiments according to any of the embodiments above, each polymerase/antibody conjugate in the combination of the polymerase/antibody conjugates of the invention comprises a primary antibody, which may comprise or be selected from antibodies or antigen binding fragments that target different antigens of interest and their associated diseases such as cancer, and a polymerase. In a further embodiment, the cancer comprises or is selected from: brain tumors, such as brain glioma, oligodendroglioma, IDH1 mutant tumor, astrocytoma, schwannoma, choroid plexus tumor, papillary tumor, spindle tumor, atypical deformity/tumor-like, meningioma, medulloblastoma, ependymoma, schwannoma, intracranial neuroepithelial tumor, chordoma; epithelioid sarcoma; lymphomas; small cell carcinoma; synovial tumor; neuroendocrine cell tumors, including olfactory cell tumors, medulloblastoma, neuroblastoma, pituitary adenoma; melanoma; lymphomas; metastatic tumors, and the like.

In still further embodiments according to any of the above embodiments in combination, the primary antibody or antigen-binding fragment thereof may comprise or be selected from two or more, such as three, four, five, six, seven or eight antibodies or antigen-binding fragments thereof: anti-Pan-CK, anti-GFAP, anti-EMA, anti-synaptophysin, anti-CD 34, anti-CD 45, anti-Mart-1 and anti-Sox-10 antibodies or antigen binding fragments.

In some embodiments according to any of the above embodiments of the combination, in the combination of the polymerase/antibody conjugates of the invention, each polymerase/antibody conjugate comprises a primary antibody and a polymerase, wherein the primary antibody may be an antibody directed against a different antigen. Preferably, for example, the combination of the polymerase/antibody conjugates of the present invention may comprise an anti-Mart-1 and/or anti-Sox-10 antibody and at least one (e.g., two, three, four, five and six) antibody selected from the group consisting of: anti-Pan-CK, anti-GFAP, anti-EMA, anti-synaptophysin, anti-CD 34, anti-CD 45.

In some embodiments according to any of the embodiments above, in the combination of the polymerase/antibody conjugates of the invention, each polymerase/antibody conjugate comprises a primary antibody and a polymerase, wherein each polymerase/antibody conjugate comprises a primary antibody or antigen binding fragment thereof directed against a different antigen. Preferably, the primary antibody is selected from two or more, e.g., any three, four, five, six, seven or eight of anti-Pan-CK, anti-GFAP, anti-EMA, anti-synaptophysin, anti-CD 34, anti-CD 45, anti-Mart-1 and anti-Sox-10 antibodies or antigen binding fragments thereof.

In some embodiments according to any of the embodiments described above, the polymerase molecules used in the combination of the polymerase/antibody conjugates of the invention may be covalently linked via a crosslinking reagent. In some embodiments, the enzyme molecules of the polymerase may be directly covalently linked. In some embodiments, the enzyme molecules of the polymerase are covalently linked in a linear fashion. In some embodiments, the enzyme molecules of the polymerase are covalently linked in a branched fashion. In some embodiments, the enzyme molecules of the polymerase are covalently linked in a mixed linear and branched fashion.

In some embodiments according to any of the embodiments above, the population of polymerases comprising multiple polymerases in the combination of the polymerase/antibody conjugates of the invention comprises a size distribution of the polymerase characterized by a number of enzyme molecules per polymerase. In some embodiments, the population of polymerases comprising a plurality of polymerases comprises a polydisperse distribution of the polymerases characterized by a structure of the polymerase.

In a further embodiment according to any of the above embodiments, the size distribution of the population molecular weight of the polymerase comprising a plurality of the polymerases in the combination of the polymerase/antibody conjugates of the invention is non-uniform and random. In some embodiments, the population of polymerases comprising a plurality of polymerases exhibits a random polydispersity profile with different molecular weights within each interval ranging from 300kDa to about 5000 kDa.

In some embodiments according to any of the embodiments described above, the polymerase used in the combination of the polymerase/antibody conjugates of the invention may have a molecular weight of about 300kDa to about 5 megadaltons (MDa). In some embodiments, the polymerase has a molecular weight of at least about 500 kDa. In some embodiments, the polymerase has a molecular weight of less than or about 5 MDa. In some embodiments, the polymerase has a molecular weight of at least about 750 kDa. In some embodiments, the polymerase has a molecular weight of at least about 1, 2, 3, or 4 MDa.

In some embodiments, the polymerase is first formed before being coupled to the antibody.

In another aspect of the invention, the invention provides a kit comprising a polymerase/antibody conjugate or a combination of polymerase/antibody conjugates of the invention as described above, the kit comprising a polymerase/antibody conjugate or a combination of polymerase/antibody conjugates as described above, wherein each of the polymerase/antibody conjugates in the polymerase/antibody conjugate or the combination of polymerase/antibody conjugates as described above may comprise a plurality of primary antibodies, preferably the plurality of primary antibodies may be antibodies against different antigens.

In further embodiments according to any of the above embodiments, the plurality of primary antibodies may comprise two or more of the following, such as three, four, five, six, seven or eight antibodies or antigen binding fragments: anti-Pan-CK, anti-GFAP, anti-EMA, anti-synaptophysin, anti-CD 34, anti-CD 45, anti-Mart-1 and anti-Sox-10 antibodies or antigen binding fragments thereof.

In a further embodiment according to any of the above embodiments, the plurality of primary antibodies may be selected from two or more of the following, such as three, four, five, six, seven or eight antibodies or antigen binding fragments: anti-Pan-CK, anti-GFAP, anti-EMA, anti-synaptophysin, anti-CD 34, anti-CD 45, anti-Mart-1 and anti-Sox-10 antibodies or antigen binding fragments.

The kits of the invention comprise a polymerase/antibody conjugate as described above, or a combination thereof, and instructions for use, wherein each of the plurality of polymerase/antibody conjugates may comprise: (i) One or more polymerases, (ii) an antibody that recognizes an analyte of interest. For example, the kit may comprise a polymerase/antibody conjugate of two or more, such as three, four, five, six, seven or eight antibodies or antigen binding fragments: anti-Pan-CK, anti-GFAP, anti-EMA, anti-synaptophysin, anti-CD 34, anti-CD 45, anti-Mart-1 and anti-Sox-10 antibodies or antigen binding fragments thereof.

In some embodiments, the kits of the invention further comprise a substrate for a polymerase.

In a further aspect, the invention provides a method for detecting an analyte of interest in a tissue sample, comprising:

contacting a tissue sample comprising an analyte of interest with a polymerase/antibody conjugate comprising a plurality of enzyme molecules and an antibody that recognizes the analyte of interest to form a complex comprising the analyte of interest and the polymerase/antibody conjugate; substantially removing the polymerase/antibody conjugate that does not form the complex; and contacting the tissue with substrates of the plurality of enzyme molecules, thereby detecting the target analyte. In some embodiments, the tissue is frozen tissue. In some embodiments the tissue sample is a continuous paraffin or a continuous frozen tissue section.

In some embodiments according to any of the above embodiments of the invention, the method further comprises a closing step, followed by the following steps: contacting a tissue comprising an analyte of interest with a polymerase/antibody conjugate comprising a plurality of enzyme molecules and an antibody that recognizes the analyte of interest to form a complex comprising the analyte of interest and the polymerase/antibody conjugate; wherein the sealing step comprises contacting the tissue with a sealing agent. In some embodiments, the tissue is frozen tissue. In some embodiments, the blocking agent comprises skim milk, BSA, casein, or animal serum.

In a further aspect, the invention provides the use of a polymerase/antibody conjugate or combination of polymerase/antibody conjugates as described above for the preparation of a reagent or combination of reagents or kit for detecting an analyte of interest in a tissue.

In a further aspect of the invention, the invention provides the use of a polymerase/antibody conjugate or combination of polymerase/antibody conjugates as described above for the preparation of a reagent or combination of reagents or kit for identifying the type or grade of cancer (e.g. of an intraoperative sample) or determining the negativity of surgical resection margin of tumour tissue.

In a further aspect of the invention, the invention provides the use of a polymerase/antibody conjugate or combination of polymerase/antibody conjugates as described above for the preparation of a reagent or combination of reagents or kit for the identification or diagnosis of melanoma (e.g. of an intraoperative sample).

In a further aspect of the invention, the invention provides the use of a polymerase/antibody conjugate or combination of polymerase/antibody conjugates as described above for the preparation of a reagent or combination of reagents or kit for the identification or diagnosis of metastatic cancer (e.g. of an intraoperative sample).

In a further aspect of the invention, the present invention provides the use of a polymerase/antibody conjugate or combination of polymerase/antibody conjugates as described above for the preparation of a reagent or combination of reagents or kit for the identification and/or diagnosis of brain tumors (e.g. of an intraoperative sample), such as gliomas, meningiomas, medulloblastomas, lymphomas and the like.

Drawings

The features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will become more readily apparent to those skilled in the art by reference to the following detailed description of illustrative embodiments and the accompanying drawings, in which:

FIG. 1 shows the results of 10 minute quick staining (20X) of frozen prostate cancer sections using the preferred exemplary peroxidase/Pan-CK antibody conjugates of the invention. Prostate cancer epithelial cells were positive (as indicated by the arrow).

FIG. 2 shows the results of 10 minute flash staining (20X) of brain glioma frozen sections using the preferred exemplary peroxidase/GFAP antibody conjugates of the present invention. The thin areas of glioma stained positively (as indicated by the arrows).

FIG. 3 shows the results of 10 minute quick staining (20X) of frozen kidney tissue sections using the preferred exemplary peroxidase/EMA antibody conjugates of the invention. Kidney tissue epithelial cells stained positively (as indicated by the arrow).

FIG. 4 shows the results of 10 minute quick staining (20X) of liver neuroendocrine tumor tissue frozen sections using a preferred exemplary polyperoxidase/synapsin antibody conjugate of the present invention. Neuroendocrine tumor tissue synaptoproteins are stained sexually (as indicated by the arrows).

FIG. 5 shows representative results (20X) of 10 minute quick staining of tonsil frozen sections using a preferred exemplary peroxidase/CD 45 antibody conjugate of the invention. Tonsil lymphocytes stained positively for CD45 (as indicated by the arrow).

FIG. 6 shows representative results (20X) of 10 minute quick staining of melanoma frozen sections using the preferred exemplary peroxidase/Mart 1 antibody conjugates of the invention. Melanoma cells stained positive for Mart-1 (as indicated by the arrow).

FIG. 7 shows representative results (20X) of rapid staining of melanoma frozen sections using preferred exemplary peroxidase/Sox 10 antibody conjugates of the invention. Melanoma cells stained positive for Sox10 (as indicated by the arrow)

Fig. 8A to 8H show the results of rapid staining of low grade glioma frozen serial sections using the combination of the exemplary peroxidase/antibody conjugates of the invention (10X). 8A, the dyeing result of the polyperoxidase/Pan-CK antibody conjugate shows that the low-grade glioma is negative; 8B, a peroxidase/anti-GFAP antibody conjugate, and the low-grade glioma was positive; 8C, dyeing and staining the polyperoxidase/synapsin antibody conjugate, wherein the low-grade glioma is weak positive; 8D, dyeing the polyperoxidase/EMA antibody conjugate, wherein the low-grade glioma is negative; 8E, dyeing and staining the polyperoxidase/CD 45 antibody conjugate, wherein the low-grade glioma is negative; 8F, dyeing and staining the polyperoxidase/Mart 1 antibody conjugate, wherein the low-grade glioma is negative; 8G, dyeing and staining the polyperoxidase/Sox 10 antibody conjugate, wherein the low-grade glioma is negative; 8H.H & E staining control.

Fig. 9A to 9H show the results of rapid staining of high grade glioma frozen serial sections using the combination of the exemplary peroxidase/antibody conjugates of the invention (10X). 9A, dyeing and staining the polyperoxidase/Pan-CK antibody conjugate, wherein the high-grade glioma is negative; 9B, the dyeing result of the peroxidase/GFAP antibody conjugate shows that the high-grade glioma is positive; 9C, dyeing the polyperoxidase/EMA antibody conjugate to obtain a negative high-grade glioma; 9D, dyeing the polyperoxidase/synapsin antibody conjugate, wherein the high-grade glioma is negative; 9E, dyeing the polyperoxidase/CD 45 antibody conjugate to obtain a negative high-grade glioma; 9F, dyeing and staining the polyperoxidase/Mart 1 antibody conjugate, wherein the high-grade glioma is negative; 9G, dyeing and staining the polyperoxidase/Sox 10 antibody conjugate, wherein the high-grade glioma is negative; 9H.H & E staining control.

FIGS. 10A through 10H show the results of rapid staining of metastatic cancer frozen serial sections using the combination of exemplary peroxidase/antibody conjugates of the invention. 10A. Dyeing the polyperoxidase/Pan-CK antibody conjugate, wherein the metastatic cancer cells are positive; 10B. Dyeing the polyperoxidase/EMA antibody conjugate, wherein the metastatic cancer is weak positive; 10C, the result of dyeing the polyperoxidase/GFAP antibody conjugate shows that the metastatic cancer is negative; 10D, staining the polyperoxidase/synapsin antibody conjugate, wherein the metastatic cancer is negative; 10E. Dyeing the polyperoxidase/CD 45 antibody conjugate, wherein the metastatic cancer is negative; 10F, dyeing the polyperoxidase/Mart 1 antibody conjugate, wherein the metastatic cancer is negative; 10G. Dyeing the polyperoxidase/Sox 10 antibody conjugate, and making the metastatic cancer negative; 10h.h & e staining control.

FIGS. 11A through 11H show the results of rapid staining (10X) of frozen serial sections of malignant melanoma using a preferred exemplary combination of the peroxidase/antibody conjugates of the invention. 11A. Dyeing the polyperoxidase/Pan-CK antibody conjugate, wherein melanoma cells are negative; 11B. Dyeing the polyperoxidase/EMA antibody conjugate, wherein melanoma is negative; 11C, the result of dyeing the polyperoxidase/GFAP antibody conjugate shows that melanoma is negative; 11D, staining the polyperoxidase/synapsin antibody conjugate, and making melanoma negative; 11E. dyeing the polyperoxidase/CD 45 antibody conjugate, wherein melanoma is negative; 11F. Dyeing the polyperoxidase/Mart 1 antibody conjugate, positive for melanoma (cytoplasm); 11G. Dyeing the polyperoxidase/Sox 10 antibody conjugate, positive for melanoma (nucleus); 11h.h & e staining control.

Fig. 12A to 12H show the results of rapid staining (20X) of malignant melanoma paraffin serial sections using a preferred exemplary combination of the peroxidase/antibody conjugates of the invention. 12A. Dyeing the polyperoxidase/Pan-CK antibody conjugate, wherein melanoma cells are negative; 12B. Dyeing the polyperoxidase/EMA antibody conjugate, wherein melanoma is negative; 12C, the result of dyeing the polyperoxidase/GFAP antibody conjugate shows that melanoma is negative; 12D, staining the polyperoxidase/synapsin antibody conjugate, and making melanoma negative; 12E. dyeing the polyperoxidase/CD 45 antibody conjugate, wherein melanoma is negative; 12F. Dyeing the polyperoxidase/Mart 1 antibody conjugate, positive for melanoma (cytoplasm); 12G. Polyperoxidase/Sox 10 antibody conjugate staining (nucleus), melanoma positive; FIG. 12H staining of the peroxidase/CD 34 antibody conjugate, melanoma cells were negative.

Detailed Description

To address the non-specific and low color-difference problems inherent in ice-section hematoxylin and eosin staining based intraoperative oncological diagnosis, the present invention employs a rapid, sensitive and specific polymerase/antibody conjugate or combination thereof to shorten the time to immunohistochemical staining on fresh/frozen tissue. The polymer HRP-specific antibody conjugate which is specially designed and optimized can be directly combined with specific antigen, the labeled secondary antibody in a two-step method is not needed, and various tumor cells with different colors can be displayed on fresh tissues or frozen sections in less than 10 minutes at room temperature. This novel 10-15 minute rapid immunohistochemical staining is suitable for intraoperative cancer identification.

Immunohistochemical staining of paraffin-embedded tissue sections using a polymerase-labeled secondary antibody plus a set of antibody combinations directed against tumors such as brain tumors to achieve differential diagnosis is the current standard pathological diagnostic procedure. Since the staining process using this technique takes several hours to complete, it cannot be directly used for differential diagnosis of tumors in frozen tissues during surgery. The inventor establishes a set of combinations of the polymerase/antibody conjugates comprising a plurality of specific antibodies against different antigens by using the technology of the polymerase/antibody conjugates of the invention, and the combinations can quickly immunohistochemical staining of tissue sections such as frozen sections within 10-15 minutes, thereby realizing quick identification and diagnosis of tumors in surgery and guiding the surgery immediately and accurately.

The invention adopts covalent linkage of polymerase-antibody to form conjugate. In one aspect, the invention provides polymerase/antibody conjugates, wherein each polymerase/antibody conjugate comprises: (i) One or more polymerases, (ii) an antibody that recognizes an analyte of interest. In some embodiments, each polymerase has multiple enzyme molecules.

In some embodiments, a polymerase used in a polymerase/antibody conjugate of the invention may comprise a plurality of enzyme molecules, e.g., comprising at least about 6 enzyme molecules, such as at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 150, at least 160, at least 180, at least 200, at least 220, at least 240, about 250 enzyme molecules, or one or more of the foregoing. In some embodiments, the polymerase comprises less than about 250, 200, 180, 160, 150, 140, 120, 100, 80, 75, 60, 50, 40, 25, 20, 15, or 10 enzyme molecules, or one or more of the foregoing numbers of enzyme molecules. In some embodiments, the polymerase/antibody conjugates of the invention comprise between about 6 and about 80, between about 10 and about 80, between about 20 and about 80, between about 30 and about 80, between about 6 and about 100, between about 10 and about 100, between about 20 and about 100, between about 30 and about 100, between about 40 and about 100, between about 6 and about 200, between about 10 and about 200, between about 20 and about 200, between about 30 and about 200, between about 50 and about 200, between about 100 and about 200, between about 6 and about 250, between about 10 and about 250, between about 20 and about 250, between about 30 and about 250, between about 50 and about 250, between about 100 and about 250, or one or more of the foregoing numbers of enzyme molecules per polymerase/antibody conjugate.

In some embodiments, the polymerase/antibody conjugates of the invention comprise a plurality of polymerases, wherein each polymerase comprises about the same number of enzyme molecules. In some embodiments, the polymerase/antibody conjugates of the invention comprise a plurality of polymerases, wherein each polymerase comprises a different number of enzyme molecules. In some embodiments, the polymerase/antibody conjugates of the invention comprise a plurality of polymerases, wherein the plurality of polymerases exhibits a distribution of the number of enzyme molecules per polymerase. In some embodiments, the polymerase/antibody conjugate comprises a plurality of polymerases, wherein the plurality of polymerases have random differences in shape.

In some embodiments, the polymerase/antibody conjugates of the invention may comprise a plurality of the polymerase and the antibody, wherein each polymerase/antibody conjugate comprises a different number of enzyme molecules and antibodies, the molecular weight size of the conjugate forming a polydisperse distribution. The polydisperse distribution means that the number of polymerase/antibody conjugates between the various partitions within the molecular weight range as defined above is not zero and the polydisperse distribution is characterized by a molecular weight of about 400kDa to about 2,000kDa per polymerase/antibody conjugate. For example, between 400 and 600kDa, the number is about 2-8%, such as 4%, 5%, 6%, 7% or 8%, preferably 4% -6% of the number of the entire molecular weight range; between 600-800kDa at about 5-10%, e.g. 6%, 7%, 8%, 9% or 10%, preferably 7% -9%; between 800 and 1000kDa 6-13%, e.g. 7%, 8%, 9%, 10%, 11% or 12%, preferably 9% -11%; between 1000 and 1200kDa 7-14%, e.g. 8%, 9%, 10%, 11%, 12%, 13% or 14%, preferably 9% -12%; between 1200-1400kDa 9-16%, e.g. 9%, 10%, 11%, 12%, 13%, 14%, 15% or 16%, preferably 11% -13%; between 1400 and 1600kDa, 10-16%, e.g. 10%, 11%, 12%, 13%, 14%, 15% or 16%, preferably 12% -14%; between 1600 and 2000kDa, 12-18%, e.g. 12%, 13%, 14%, 15%, 16%, 17% or 18%, preferably 13% -15%. In particular, the molecular weight of the polymerase/antibody conjugates of the invention may be from 300kDa to about 10,000kDa, preferably from 400kDa to about 10,000kDa, from 400kDa to about 5,000kDa, from 500kDa to about 5,000kDa or from 750kDa to about 5,000kDa, with different molecular weights in each interval of the range and the molecular weights of the conjugates forming a polydisperse distribution as described above.

Immunohistochemistry is very different from antigen-antibody binding in liquids due to the complexity of the environment in which the antigens of biological tissues are located: the formation of a spatially diverse and random three-dimensional structure blocks a wide variety and excessive polymerase/antibody conjugates. In order for the polymerase/antibody conjugate to quickly and efficiently access and bind to the antigen, the present invention adopts the following scheme: 1) Limiting oversized polymerase/antibody conjugates; 2) Selecting multiple molecular weight polymerase/antibody conjugates; 3) Three-dimensional structure of diverse or random polymerase/antibody conjugates. Through repeated experiments, the invention adopts the polymerase/antibody conjugate with the molecular weight of the conjugate being in the range of about 400kDa to about 2,000kDa, and the molecular weight of the polymerase/antibody conjugate shows random multi-dispersity distribution, namely the polymerase/antibody conjugate with different molecular weights and different three-dimensional structures in each interval in the range of 400kDa to about 2,000kDa, thereby realizing the rapid approaching and further combining of various polymerase-antibody conjugates and corresponding antigens in frozen tissues. For example, between 400 and 600kDa, the number is about 2-8%, such as 4%, 5%, 6%, 7% or 8%, preferably 4% -6% of the number of the entire molecular weight range; between 600-800kDa at about 5-10%, e.g. 6%, 7%, 8%, 9% or 10%, preferably 7% -9%; between 800 and 1000kDa 6-13%, e.g. 7%, 8%, 9%, 10%, 11% or 12%, preferably 9% -11%; between 1000 and 1200kDa 7-14%, e.g. 8%, 9%, 10%, 11%, 12%, 13% or 14%, preferably 9% -12%; between 1200-1400kDa 9-16%, e.g. 9%, 10%, 11%, 12%, 13%, 14%, 15% or 16%, preferably 11% -13%; between 1400 and 1600kDa, 10-16%, e.g. 10%, 11%, 12%, 13%, 14%, 15% or 16%, preferably 12% -14%; between 1600 and 2000kDa, 12-18%, e.g. 12%, 13%, 14%, 15%, 16%, 17% or 18%, preferably 13% -15%. And for example, a polymerase/antibody conjugate comprising randomly distinct three-dimensional structures in the 400kDa to about 8,00kDa range; polymerase/antibody conjugates comprising additional types of randomly distinct three-dimensional structures in the 800kDa to about 1,200kDa interval; polymerase/antibody conjugates comprising other types of randomly distinct three-dimensional structures in the 1,200kda to about 1,600kda interval; other types of polymerase/antibody conjugates comprising randomly different three-dimensional structures in the 1,600kDa to about 2,000kDa interval, wherein the three-dimensional structures comprised by the polymerase/antibody conjugates in the four intervals may be different.

The implementation of the combined distribution is achieved by a combination of several variables; 1) Each polymerase/antibody conjugate consists of at least one polymerase; 2) The number of enzymes contained in each polymerase is one to several hundred; 3) The three-dimensional structure of the polymerase is diverse and random; 4) The polymerase binds to multiple sites of the antibody; 5) The manner in which each of the different polymerases binds to a different site of an antibody is random. These multiple variables form end products in the antibody/polymer coupling reaction that are multi-dispersed in the distribution of the molecular weight of the polymerase/antibody conjugate, while being diverse and random in their three-dimensional structure. The polymerase/antibody conjugate with the molecular weight distribution characteristics can be quickly combined with antigens in heterogeneous fresh or frozen slice tissues, a complex of the antigens and the polymerase/antibody conjugate is formed only by 5 minutes or less of incubation time, the polymerase/antibody conjugate which does not form the complex is further removed by washing, and the tissue-bound polymerase is contacted with a substrate of the enzyme, so that target analytes can be quickly detected.

In some embodiments, the polymerase/antibody conjugates of the invention may contain a plurality of polymerases, each of which contains a different number of enzyme molecules, e.g., may contain 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240, and/or 250 enzyme molecules, or one or more of the foregoing numbers.

In some embodiments according to any of the embodiments above, the distribution of molecular weights of the polymerase/antibody conjugates of the invention is non-uniform across each molecular weight interval and its three-dimensional structure is diverse and random.

In some embodiments according to any of the above embodiments, the polymerase/antibody conjugate of the invention may have a molecular weight between about 400kDa and about 500kDa, 600kDa, 700kDa, 800kDa, 900kDa, 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa or l0000kDa, and/or a molecular weight between about 500kDa and about 600kDa, 700kDa, 800kDa, 900kDa, 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa or l0000kDa, and/or a molecular weight between about 600kDa and about 700kDa, 800kDa, 900kDa, 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa or l0000kDa, and/or having a molecular weight between about 700kDa and about 800kDa, 900kDa, 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa or l0000kDa, and/or having a molecular weight between about 800kDa and about 900kDa, 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa or l0000kDa, and/or having a molecular weight between about 900kDa and about 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa or l0000kDa, and/or having a molecular weight between about 1000kDa and about 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa or l0000 kDa.

In some embodiments according to any of the embodiments above, the population of polymerases comprising a plurality of polymerases comprises a distribution of sizes of the polymerases characterized by a number of enzyme molecules per polymerase. In some embodiments, the population of polymerases comprising a plurality of polymerases comprises a random distribution of shapes of the polymerases characterized by a structure of the polymerase. In a further embodiment, the distribution of molecular weights of a population of polymerases comprising a plurality of polymerases is non-uniform and random. In some embodiments according to any of the embodiments above, the population of polymerases comprising a plurality of the polymerases exhibits a random polydispersity distribution, having different molecular weights within each interval ranging from 300kDa to about 5000kDa, e.g., between 400-600kDa in an amount of about 2-8%, e.g., 4%, 5%, 6%, 7% or 8%, preferably 4% -6% of the number of the entire molecular weight range; between 600-800kDa at about 5-10%, e.g. 6%, 7%, 8%, 9% or 10%, preferably 7% -9%; between 800 and 1000kDa 6-13%, e.g. 7%, 8%, 9%, 10%, 11% or 12%, preferably 9% -11%; between 1000 and 1200kDa 7-14%, e.g. 8%, 9%, 10%, 11%, 12%, 13% or 14%, preferably 9% -12%; between 1200-1400kDa 9-16%, e.g. 9%, 10%, 11%, 12%, 13%, 14%, 15% or 16%, preferably 11% -13%; between 1400 and 1600kDa, 10-16%, e.g. 10%, 11%, 12%, 13%, 14%, 15% or 16%, preferably 12% -14%; between 1600 and 2000kDa, 12-18%, e.g. 12%, 13%, 14%, 15%, 16%, 17% or 18%, preferably 13% -15%. More specifically, the molecular weight of the polymerase/antibody conjugates of the invention may be 300kDa to about 10,000kDa, preferably 400kDa to about 10,000kDa, 400kDa to about 5,000kDa, 500kDa to about 5,000kDa or 750kDa to about 5,000kDa, with different molecular weights in each interval within this range, and the molecular weights of the conjugates forming a polydisperse distribution as described above.

In some embodiments according to any of the above embodiments, the polymerase/antibody conjugates of the invention comprise a plurality of enzyme molecules or polymerases, wherein the plurality of enzyme molecules or polymerases may comprise the same or different enzyme types, e.g., all of the enzyme molecules or polymerases in the polymerase/antibody conjugates of the invention may comprise horseradish peroxidase.

In some embodiments according to any of the above embodiments, the polymerase/antibody conjugates of the present invention comprise a polymerase and an antibody, and the enzyme molecule of the polymerase may comprise horseradish peroxidase (HRP), beta-D-galactosidase, alkaline phosphatase, superoxide dismutase, luciferase, lactate dehydrogenase, galactose oxidase, and the like.

In some embodiments according to any of the embodiments described above, the polymerase/antibody conjugates of the invention may be formed by covalent attachment of a polymerase to an antibody.

In some embodiments according to any of the embodiments above, the polymerase/antibody conjugates of the invention may comprise a primary antibody. In some embodiments, the primary antibody may comprise a full length antibody, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a human antibody or antibody fusion protein, or an antigen binding fragment.

Examples of antibody binding fragments include, but are not limited to, fab, F (ab ') 2, fab' fragments, fd fragments, single chain antibody molecules (e.g., scFv), fv fragments, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

In some embodiments according to any of the above embodiments, the antibody comprises IgG, igM, igE, igA, or IgD.

In some embodiments according to any of the embodiments above, the primary antibody may comprise or be selected from antibodies or antigen binding fragments that target the antigen of interest of the invention and diseases associated therewith, such as cancer. In a further embodiment, the cancer comprises: brain tumors such as brain glioma, meningioma, medulloblastoma, ependymoma, schwannoma, intracranial neuroepithelial tumors, chordoma; epithelioid sarcoma; lymphomas; small cell carcinoma; synovial tumor; neuroendocrine cell tumors, including olfactory cell tumors, medulloblastoma, neuroblastoma, pituitary adenoma; melanoma; lymphomas; metastatic tumors, and the like. In still further embodiments according to any of the above embodiments, the primary antibody or antigen binding fragment may comprise or be selected from the group consisting of: anti-Pan-CK, anti-GFAP, anti-EMA, anti-synaptophysin, anti-CD 34, anti-CD 45, anti-S-100, anti-NSE, anti-Vimentin (Vimentin), anti-LCA, anti-AFP, anti-CD 34, anti-Ki-67, anti-P63, anti-NFP, anti-IDH 1, anti-BRAF, anti-Chromogranin (chromagranin), anti-LCA, anti-Mart-1, and anti-Sox-10 antibodies or antigen binding fragments thereof.

In some embodiments according to any of the embodiments above, the polymerase/antibody conjugates of the invention comprise a polymerase molecule and an antibody, wherein the polymerase molecule can be covalently linked via a crosslinking reagent. For example, the enzyme molecules of the polymerase may be covalently linked directly, or covalently linked in a linear fashion, or covalently linked in a branched fashion, or covalently linked in a mixed linear and branched fashion.

In another aspect of the invention, the invention provides a combination of a polymerase/antibody conjugate comprising a plurality of the polymerase/antibody conjugates of the invention as described above. In the combination of the polymerase/antibody conjugates of the present invention, each of the plurality of polymerase/antibody conjugates may comprise: (i) One or more polymerases, (ii) an antibody that recognizes an analyte of interest.

In some embodiments according to any of the above embodiments, each polymerase/antibody conjugate in the combination of the polymerase/antibody conjugates of the invention may comprise a plurality of enzyme molecules, e.g., comprising at least about 6 enzyme molecules, such as at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 150, at least 160, at least 180, at least 200, at least 220, at least 240, at least 250 enzyme molecules, or an enzyme molecule of one or more of the foregoing numbers. In some embodiments, the polymerase comprises less than about 250, 200, 180, 160, 150, 140, 120, 100, 80, 75, 60, 50, 40, 25, 20, 15, or 10 enzyme molecules, or one or more of the foregoing numbers of enzyme molecules. In some embodiments according to any of the above embodiments, the polymerase/antibody conjugate comprises between about 6 and about 80, between about 10 and about 80, between about 20 and about 80, between about 30 and about 80, between about 6 and about 100, between about 10 and about 100, between about 20 and about 100, between about 30 and about 100, between about 40 and about 100, between about 6 and about 200, between about 10 and about 200, between about 20 and about 200, between about 30 and about 200, between about 50 and about 200, between about 100 and about 200, between about 6 and about 250, between about 10 and about 250, between about 20 and about 250, between about 30 and about 250, between about 50 and about 250, between about 100 and about 250, or one or more of the foregoing numbers of enzyme molecules per polymerase/antibody conjugate.

In some embodiments according to any of the above embodiments, each polymerase/antibody conjugate in the combination of the invention comprises a plurality of polymerases and antibodies, wherein each polymerase comprises a different number of enzyme molecules, the molecular weight size of the conjugate forming a multi-dispersed distribution characterized by a molecular weight of each polymerase antibody conjugate of about 400kDa to about 2,000 kDa. In one embodiment, the molecular weight of the polymerase/antibody conjugates in the combination of the polymerase/antibody conjugates of the invention exhibits a random, polydisperse distribution, i.e., the polymerase/antibody conjugates have different molecular weights and different three-dimensional structures within each interval ranging from 400kDa to about 2,000 kDa. The polydisperse distribution refers to a number of polymerase/antibody conjugates that is not zero between each partition within the molecular weight range as defined above. For example, between 400 and 600kDa, the number is about 2-8%, such as 4%, 5%, 6%, 7% or 8%, preferably 4% -6% of the number of the whole molecular weight range; between 600-800kDa at about 5-10%, e.g. 6%, 7%, 8%, 9% or 10%, preferably 7% -9%; between 800 and 1000kDa 6-13%, e.g. 7%, 8%, 9%, 10%, 11% or 12%, preferably 9% -11%; between 1000 and 1200kDa 7-14%, e.g. 8%, 9%, 10%, 11%, 12%, 13% or 14%, preferably 9% -12%; between 1200-1400kDa 9-16%, e.g. 9%, 10%, 11%, 12%, 13%, 14%, 15% or 16%, preferably 11% -13%; between 1400 and 1600kDa, 10-16%, e.g. 10%, 11%, 12%, 13%, 14%, 15% or 16%, preferably 12% -14%; between 1600 and 2000kDa, 12-18%, e.g. 12%, 13%, 14%, 15%, 16%, 17% or 18%, preferably 13% -15%. More specifically, the molecular weight of the polymerase/antibody conjugate in the above combination of the invention may be 300kDa to about 10,000kDa, preferably 400kDa to about 10,000kDa, 400kDa to about 5,000kDa, 500kDa to about 5,000kDa or 750kDa to about 5,000kDa, with different molecular weights in each interval within this range, and the molecular weight of the conjugate forming a multi-dispersed distribution as described above.

In some embodiments according to any of the embodiments above, each polymerase/antibody conjugate of the combination of the invention may contain a heterogeneous molecular weight of the polymerase, each polymerase may contain a different number of enzyme molecules, such as may contain 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240 and/or 250 enzyme molecules, or one or more of the above numbers.

In some embodiments according to any of the embodiments above, the distribution of the molecular weight of each polymerase/antibody conjugate in the combination of the polymerase/antibody conjugates of the invention may be non-uniform and random.

In some embodiments according to any of the above embodiments, each of the combinations of the polymerase/antibody conjugates of the invention may have a molecular weight between about 400kDa and about 500kDa, 600kDa, 700kDa, 800kDa, 900kDa, 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa or l0000kDa, and/or a molecular weight between about 500kDa and about 600kDa, 700kDa, 800kDa, 900kDa, 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa or l0000kDa, and/or a molecular weight between about 600kDa and about 700kDa, 800kDa, 900kDa, 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000, 8000, 9000kDa or l0000kDa, and/or having a molecular weight between about 700kDa and about 800kDa, 900kDa, 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa or l0000kDa, and/or having a molecular weight between about 800kDa and about 900kDa, 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa or l0000kDa, and/or having a molecular weight between about 900kDa and about 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa or l0000kDa, and/or having a molecular weight between about 1000kDa and about 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa or l0000 kDa.

In some embodiments according to any of the embodiments described above, each polymerase/antibody conjugate in the combination of the present invention may comprise a plurality of enzyme molecules or a plurality of polymerases, wherein the plurality of enzyme molecules or the plurality of polymerases may comprise the same enzyme type, e.g., all enzyme molecules or polymerases in the present polymerase/antibody conjugate are horseradish peroxidase.

In some embodiments according to any of the embodiments described above, each polymerase/antibody conjugate in the combination of the polymerase/antibody conjugates of the invention may comprise a plurality of enzyme molecules or a plurality of polymerases, wherein the plurality of enzyme molecules or the plurality of polymerases may comprise different enzyme types, e.g., some of the enzyme molecules or the polymerases in the polymerase/antibody conjugates of the invention are horseradish peroxidase and others are alkaline phosphatase; or wherein the plurality of enzyme molecules or plurality of polymerases can comprise the same enzyme type, e.g., the enzyme molecule or polymerase in the polymerase/antibody conjugate of the invention is horseradish peroxidase.

In some embodiments according to any of the embodiments above, in the combination of the polymerase/antibody conjugates of the present invention, the polymerase may comprise or be selected from horseradish peroxidase, beta-D-galactosidase, alkaline phosphatase, superoxide dismutase, luciferase, lactate dehydrogenase, galactose oxidase, and the like.

In some embodiments according to any of the embodiments above, each polymerase/antibody conjugate in the combination of the polymerase/antibody conjugates of the invention comprises a primary antibody and a polymerase, wherein the primary antibody comprised by each polymerase/antibody conjugate may be an antibody directed against a different antigen. For example, the combination of the polymerase/antibody conjugates of the invention comprises or is selected from two or more of, e.g., three, four, five, six, seven or eight antibodies or antigen binding fragments: anti-Pan-CK, anti-GFAP, anti-EMA, anti-synaptophysin, anti-CD 34, anti-CD 45, anti-Mart-1 and anti-Sox-10 antibodies or antigen binding fragments thereof.

In some embodiments according to any of the embodiments above, each polymerase/antibody conjugate in the combination of the polymerase/antibody conjugates of the invention comprises a primary antibody, which may comprise or be selected from an antibody or antigen binding fragment thereof that targets the antigen of interest of the invention and its associated disease, such as cancer. In further embodiments, the cancer comprises a brain tumor such as brain glioma, oligodendroglioma, IDH1 mutant tumor, astrocytoma, schwannoma, choroid plexus neuroma, papillary tumor, spindle tumor, atypical deformity/tumor-like, meningioma, ependymoma, schwannoma, intracranial neuroepithelial tumor, chordoma; epithelioid sarcoma; lymphomas; small cell carcinoma; synovial tumor; neuroendocrine cell tumors, including olfactory cell tumors, medulloblastoma, neuroblastoma, pituitary adenoma; melanoma; lymphomas; metastatic tumors, and the like. In still further embodiments, the cancer is selected from brain tumors such as brain glioma, oligodendroglioma, IDH1 mutant tumor, astrocytoma, schwannoma, choroid plexus neuroma, papillary tumor, spindle tumor, atypical deformity/tumor-like, meningioma, ependymoma, schwannoma, intracranial neuroepithelial tumor, chordoma; epithelioid sarcoma; lymphomas; small cell carcinoma; synovial tumor; neuroendocrine cell tumors, including olfactory cell tumors, medulloblastoma, neuroblastoma, pituitary adenoma; melanoma; lymphomas; metastatic tumors, and the like. In still further embodiments according to any of the above embodiments, the primary antibody or antigen-binding fragment may comprise or be selected from two or more, such as three, four, five, six, seven or eight antibody or antigen-binding fragments: anti-Pan-CK, anti-GFAP, anti-EMA, anti-synaptophysin, anti-CD 34, anti-CD 45, anti-Mart-1 and anti-Sox-10 antibodies or antigen binding fragments thereof.

In some embodiments according to any of the embodiments above, in the combination of the polymerase/antibody conjugates of the invention, each polymerase/antibody conjugate comprises a primary antibody and a polymerase, wherein the primary antibody may be an antibody directed against a different antigen. For example, a combination of the polymerase/antibody conjugates of the invention may comprise or be selected from two or more antibodies or antigen binding fragments of: anti-Pan-CK, anti-GFAP, anti-EMA, anti-synaptophysin, anti-CD 45, anti-S-100, anti-NSE, anti-vimentin, anti-LCA, anti-AFP, anti-CD 34, anti-Ki-67, anti-P63, anti-NFP, anti-IDH 1, anti-BRAF, anti-Chromogranin, anti-LCA, anti-Mart-1, and anti-Sox-10 antibodies or antigen binding fragments; preferably, six, seven or eight antibodies are included or selected from anti-Pan-CK, anti-GFAP, anti-EMA, anti-synaptophysin (syntenin), anti-CD 34, anti-CD 45, anti-Mart-1 and anti-Sox-10 antibodies.

In some embodiments according to any of the embodiments described above, preferably, for example, the combination of the polymerase/antibody conjugates of the present invention may comprise a polymerase/Mart-1 antibody conjugate and/or a polymerase/Sox-10 antibody conjugate and at least one (e.g., two, three, four, five, and six) polymerase/antibody conjugate selected from the group consisting of: polymerase/Pan-CK antibody conjugate, polymerase/GFAP antibody conjugate, polymerase/EMA antibody conjugate, polymerase/synapsin (Synatophysin) antibody conjugate, polymerase/CD 34 antibody conjugate, polymerase/CD 45 antibody conjugate. For example, the combination of the polymerase/antibody conjugates of the present invention may include or consist of a polymerase/Mart-1 antibody conjugate or a polymerase/Sox-10 antibody conjugate, a polymerase/GFAP antibody conjugate, a polymerase/Pan-CK antibody conjugate, and a polymerase/synapsin antibody conjugate. Preferably, the combination of the polymerase/antibody conjugates of the present invention may comprise or consist of a polymerase/Mart-1 antibody conjugate or a polymerase/Sox-10 antibody conjugate, a polymerase/GFAP antibody conjugate, a polymerase/Pan-CK antibody conjugate, a polymerase/synapsin antibody conjugate, a polymerase/CD 45 antibody conjugate, and optionally a polymerase/EMA antibody conjugate, a polymerase/CD 34 antibody conjugate.

In some embodiments according to any of the embodiments above, in the combination of the polymerase/antibody conjugates of the invention, each polymerase/antibody conjugate comprises a primary antibody and a polymerase, wherein the primary antibody or antigen binding fragment thereof contained by each polymerase/antibody conjugate is different. Preferably, the primary antibody comprises or is selected from one or more of anti-Pan-CK, anti-GFAP, anti-EMA, anti-synaptophysin, anti-CD 34, anti-CD 45, anti-Mart-1 and anti-Sox-10 antibodies or antigen binding fragments, e.g., three, four, five, six, seven or eight of the above antibodies.

In some embodiments according to any of the embodiments above, the population of polymerases comprising multiple polymerases in the combination of the polymerase/antibody conjugates of the invention comprises a size distribution of the polymerase characterized by a number of enzyme molecules per polymerase. In some embodiments, the population of polymerases comprising a plurality of polymerases comprises a shape distribution of the polymerases characterized by a structure of the polymerase. The molecular weight size of the polymerase/antibody conjugate forms a multi-dispersed distribution, which means that the number of polymerase/antibody conjugates between each partition within the molecular weight range as defined above is not zero. For example, between 400 and 600kDa, the number is about 2-8%, such as 4%, 5%, 6%, 7% or 8%, preferably 4% -6% of the number of the entire molecular weight range; between 600-800kDa at about 5-10%, e.g. 6%, 7%, 8%, 9% or 10%, preferably 7% -9%; between 800 and 1000kDa 6-13%, e.g. 7%, 8%, 9%, 10%, 11% or 12%, preferably 9% -11%; between 1000 and 1200kDa 7-14%, e.g. 8%, 9%, 10%, 11%, 12%, 13% or 14%, preferably 9% -12%; between 1200-1400kDa 9-16%, e.g. 9%, 10%, 11%, 12%, 13%, 14%, 15% or 16%, preferably 11% -13%; between 1400 and 1600kDa, 10-16%, e.g. 10%, 11%, 12%, 13%, 14%, 15% or 16%, preferably 12% -14%; between 1600 and 2000kDa, 12-18%, e.g. 12%, 13%, 14%, 15%, 16%, 17% or 18%, preferably 13% -15%. Preferably, the polymerase/antibody conjugates in the exemplary combination of the polymerase/antibody conjugates of the invention (e.g., as used in the examples) have a molecular weight between 400 and 600kDa in an amount of about 4% to 6% of the total number of molecular weight ranges; between about 7% -9% of 600-800 kDa; between about 9% -11% of 800-1000 kDa; between 1000-1200kDa and about 10% -12%; between about 11% -13% of 1200-1400 kDa; between about 12% -14% between 1400-1600 kDa; between 1600-2000kDa approximately 13% -15%. More specifically, the molecular weight of the polymerase/antibody conjugate in the above combination of the invention may be 300kDa to about 10,000kDa, preferably 400kDa to about 10,000kDa, 400kDa to about 5,000kDa, 500kDa to about 5,000kDa or 750kDa to about 5,000kDa, with different molecular weights in each interval within this range, and the molecular weight of the conjugate forming a multi-dispersed distribution as described above.

In some embodiments according to any of the embodiments above, the size distribution of the population molecular weight of the polymerase comprising a plurality of the polymerases in the combination of the polymerase/antibody conjugates of the invention is non-uniform and random. In some embodiments according to any of the above embodiments, the population of polymerases comprising a plurality of polymerases exhibits a random polydispersity profile with different molecular weights within each interval ranging from 300kDa to about 5000 kDa. In some embodiments, the invention provides combinations of 6, 7 or 8 polyperoxidase/antibody conjugates formed by the polyperoxidase with 6, 7 or 8 antibodies respectively associated with tumor such as brain tumor, lymphoma, melanoma, metastatic carcinoma differentiation. The combination of the polymerase/antibody conjugates can be applied to the field related to tumors such as brain tumors and melanoma, and can be particularly used for the differential diagnosis of tumor brain tumors and melanoma in surgery and the determination of the cutting margin due to the characteristic of rapid staining of fresh and frozen tissues. In further embodiments, the intraoperative brain tumor combinations of the invention include a plurality (e.g., 4, 5, 6, 7, or 8) of peroxidase/antibody conjugates: a peroxidase/Pan-CK antibody conjugate, a peroxidase/GFAP antibody conjugate, a peroxidase/EMA antibody conjugate, a peroxidase/synapsin antibody conjugate, a peroxidase/CD 45 antibody conjugate, a peroxidase/CD 34 antibody conjugate, a peroxidase/Mart-1 antibody conjugate, and/or a peroxidase/Sox-10 antibody conjugate. In still further embodiments, an exemplary intraoperative brain tumor combination of the invention consists of at least 6, 7, or 8 polyperoxidases/antibodies: a peroxidase/Pan-CK antibody conjugate, a peroxidase/GFAP antibody conjugate, a peroxidase/EMA antibody conjugate, a peroxidase/synapsin antibody conjugate, a peroxidase/CD 45 antibody conjugate, a peroxidase/CD 34 antibody conjugate, a peroxidase/Mart-1 antibody conjugate, and/or a peroxidase/Sox-10 antibody conjugate. The combination is designed for determining tumor grade and identifying and diagnosing tumors such as metastatic cancer, melanoma, glioma, meningioma, medulloblastoma and the like in an operation.

A multimeric peroxidase-Pan-CKAntibody conjugate: squamous epithelium, glandular epithelium and transitional cells from benign or malignant sources (e.g., small cell carcinoma, chordoma, synovial and epithelioid sarcoma) are stained. In recent years, it has been used for differential diagnosis of intracranial neuroepithelial tumors and metastatic cancers. In metastatic cancer cells, it stains the cytoplasm in a positive diffusion mode with high diffusivity and relatively poor specificity.

A multimeric peroxidase-GFAPAntibody conjugate: staining was positive for normal, reactive and neoplastic astrocytes, ependymal cells and oligodendrocytes; ganglion cells, neurons, metastatic cancers of epithelial origin, fibroblasts and their origin are negative for staining. The antibody was used to diagnose brain glioma and showed positive diffuse cytoplasmic staining. In metastatic cancers, lymphomas, tumors of medullary and other embryonic origin, it shows focal staining negatives.

A multimeric peroxidase-EMAAntibody conjugate: EMA is found in various glandular epithelium, such as breast, endocrine and acrocrine glands and pancreas, while being gastrointestinalLittle or no EMA is expressed in the skin, cervical intimal epithelium and prostate. EMA was positive in meningiomas, ependymomas (90% of membrane response), schwannomas and chordoma.

A multimeric peroxidase/synapsin antibody conjugate: is mainly used for recognizing neuroendocrine cell tumors, including olfactory cell tumors, medulloblastoma, neuroblastoma, pituitary adenoma and small cell carcinoma. It stains negatively on gliomas, and positively stains strongly in normal neurons and medulloblastomas. Used in combination with GFAP, pan-CK, they can be used for differential diagnosis of medulloblastoma, metastatic carcinoma, glioma with primitive neuronal components, and are particularly useful for the identification of medulloblastoma and metastatic carcinoma.

A multimeric peroxidase-CD45Antibody conjugate: for identifying lymphomas. It is negative staining in gliomas and metastatic cancers.

A multimeric peroxidase-Mart-1 or Sox-10Antibody conjugate: for identifying melanoma.

In a further embodiment according to any of the embodiments above, the use of a combination of a portion of the peroxidase/antibody conjugates as described above may also result in a pathological diagnosis applicable in brain oncology as shown in the examples. For example, a peroxidase/Pan-CK antibody conjugate, a peroxidase/GFAP antibody conjugate and a peroxidase/CD 45 antibody conjugate are selected, and the kit is applied to the rapid diagnosis and identification of glioma, lymphoma, glioma or metastatic tumor in neurosurgery operation [8]. Table 1 summarizes the specific applications of the exemplary combination of the peroxidase/antibody conjugates used in the examples of the present invention in oncology.

Table 1 identification of combinations of exemplary peroxidase/antibody conjugates of the invention for use in different tumor surgery

+: positive; -: negative of

In another aspect of the invention, the invention provides a kit comprising a polymerase/antibody conjugate or a combination of polymerase/antibody conjugates of the invention as described above, said kit comprising a plurality of polymerase/antibody conjugates as described above or a combination of polymerase/antibody conjugates as described above, wherein each of said plurality of polymerase/antibody conjugates or combinations of polymerase/antibody conjugates as described above may comprise a plurality of primary antibodies, preferably said plurality of primary antibodies may be directed against different antigens, such as different tumor antigens.

In a further embodiment according to any of the above embodiments, the plurality of primary antibodies may comprise or be selected from one or more of the following, such as two, three, four, five, six, seven or eight antibodies or antigen binding fragments: anti-Pan-CK, anti-GFAP, anti-EMA, anti-synaptophysin, anti-CD 34, anti-CD 45, anti-Mart-1 and anti-Sox-10 antibodies or antigen binding fragments.

In a further embodiment according to any of the embodiments above, the kit of the invention comprises a) a polymerase/antibody conjugate, and b) instructions for use. In other embodiments, the kit comprises a) a polymerase/antibody conjugate, and b) a substrate for the polymerase. In a further embodiment, the kit of the invention comprises a) a polymerase/antibody conjugate, b) a substrate for the polymerase, and c) instructions for use. In some embodiments, the polymerase is a polymeric-HRP.

In a further embodiment according to any of the embodiments above, the kit comprises a) a combination of a polymerase/antibody conjugate, and b) instructions for use. In some embodiments, the kit comprises a) a combination of a polymerase/antibody conjugate, and b) a substrate for the polymerase. In some embodiments, the kit comprises a) a combination of a polymerase/antibody conjugate, b) a substrate for the polymerase, and c) instructions for use. In some embodiments, the polymerase is a polymeric-HRP.

In a further aspect, the invention provides a method for detecting an analyte of interest in a tissue, comprising:

contacting a tissue comprising an analyte of interest with a polymerase/antibody conjugate comprising a plurality of enzyme molecules and an antibody that recognizes the analyte of interest to form a complex comprising the analyte of interest and the polymerase/antibody conjugate; substantially removing the polymerase/antibody conjugate that does not form the complex; and contacting the tissue with substrates of the plurality of enzyme molecules in the polymerase/antibody conjugate, thereby detecting the analyte of interest. In some embodiments, the tissue is frozen tissue.

In some embodiments, the invention provides methods for detecting an analyte of interest in a tissue, comprising: (a) Contacting a tissue sample comprising an analyte of interest with a polymerase/antibody conjugate at an incubation temperature of between about 15 ℃ and about 45 ℃ for a period of about 1 minute to about 1 hour to form a complex comprising the analyte of interest and at least one polymerase/antibody conjugate, wherein the antibody is capable of specifically binding to the analyte of interest; (b) Removing the polymerase/antibody conjugate that does not form the complex; (c) Contacting the tissue sample with a substrate for the enzyme, thereby detecting the analyte of interest.

In some embodiments, a tissue sample (e.g., a tissue section) comprising an analyte of interest is contacted with a polymerase/antibody conjugate at an incubation temperature between about 15 ℃ and about 45 ℃ for about 3 minutes to about 1 hour to form a complex comprising the analyte of interest and at least one polymerase/antibody conjugate, and the polymerase/antibody conjugate comprises a primary antibody capable of specifically binding to the analyte of interest. In the above embodiments, the tissue sample is a continuous frozen tissue section or a continuous paraffin section.

In a further embodiment according to any of the above embodiments, a tissue sample (e.g., a tissue section) comprising a series of analytes of interest (e.g., analyte a and analyte B) is contacted with a combination of polymerase/antibody conjugates (e.g., a polymerase/antibody conjugate that specifically binds analyte a and a polymerase/antibody conjugate that specifically binds analyte B) at an incubation temperature of about 15 ℃ and about 45 ℃ for about 3 minutes to about 1 hour to form a series of complexes comprising the analytes of interest and at least one polymerase/antibody conjugate, and the polymerase/antibody conjugates comprise primary antibodies capable of specifically binding to their respective analytes of interest. In some embodiments, the tissue sample is a continuous frozen tissue section or a continuous paraffin section.

In some embodiments according to any of the above embodiments, the incubation temperature is between about 15 ℃ and about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 ℃, between about 20 ℃ and about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 ℃, between about 25 ℃ and about 30 ℃, preferably between about 25 ℃ and about 37 ℃.

In some embodiments according to any of the above embodiments, the incubation time is between about 1 minute and about 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, between 5 minutes and about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, between 10 minutes and about 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, between about 15 minutes and about 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, between about 20 minutes and about 25, 30, 35, 40, 45, 50, 55, or 60 minutes, between about 25 minutes and about 30, 35, 40, 45, 50, 55, or 60 minutes, between about 30 minutes and about 35, 40, 45, 50, 55, or 60 minutes.

In more specific embodiments, the incubation time is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes.

After incubation, the tissue sample is typically washed with a wash buffer comprising, for example, PBS, TBS, MOPS, MES, HEPES or bicarbonate buffer, optionally containing a detergent, such as tween 20 (e.g., 0.01-0.2% tween 20). For example, an exemplary wash buffer is 10mM PBS containing 0.05% Tween 20.

The washing steps may be performed 2 to 6 times, preferably 3, 4 or 5 times, each washing step lasting for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes or more.

In some embodiments, the wash temperature is between about 15 ℃ and about 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 ℃, between about 20 ℃ and about 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 ℃, between about 25 ℃ and about 30 ℃, or between about 25 ℃ and about 37 ℃.

In some embodiments, the washing temperature is about 15 ℃, 16 ℃, 17 ℃, 18 ℃, 19 ℃, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, or 45 ℃.

In some embodiments, the washing time is between about 1 minute and about 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes.

In some embodiments, the wash time is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes.

In some embodiments according to any of the above embodiments of the invention, the method further comprises a closing step comprising the steps of: contacting a tissue comprising an analyte of interest with a polymerase/antibody conjugate comprising a plurality of enzyme molecules and an antibody that recognizes the analyte of interest to form a complex comprising the analyte of interest and the polymerase/antibody conjugate; wherein the sealing step comprises contacting the tissue with a sealing agent.

In some embodiments, the method further comprises a blocking step comprising a blocking step prior to incubating the antibody conjugate with the tissue, wherein the blocking step comprises contacting the tissue with a blocking agent.

In some embodiments, the blocking agent comprises skim milk, BSA, casein, or animal serum.

In some embodiments, the blocking agent comprises a buffer, such as TBS or PBS, preferably TBS or PBS containing BSA.

In some embodiments, the blocking agent comprises a buffer system selected from PBS, TBS, MOPS, MES, HEPES and bicarbonate, optionally containing serum albumin, e.g., 1-5% Bovine Serum Albumin (BSA), horse serum albumin, goat serum albumin, rabbit serum albumin, or gelatin, and tween 20, e.g., 0.001-0.05% tween 20.

In some embodiments, the blocking agent comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% skim milk.

In some embodiments, the blocking agent comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% BSA.

In some embodiments, the blocking temperature is between about 15 ℃ and about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 ℃, between about 20 ℃ and about 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 ℃, between about 25 ℃ and about 30 ℃, preferably between about 25 ℃ and about 37 ℃.

In some embodiments, the sealing temperature is about 15 ℃, 16 ℃, 17 ℃, 18 ℃, 19 ℃, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, or 45 ℃.

In some embodiments, the blocking time is between about 3 minutes and about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, between 5 minutes and about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, between 10 minutes and about 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, between about 15 minutes and about 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, between about 20 minutes and about 25, 30, 35, 40, 45, 50, 55, or 60 minutes, between about 25 minutes and about 30, 35, 40, 45, 50, 55, or 60 minutes, or between about 30 minutes and about 35, 40, 45, 50, 55, or 60 minutes.

In some embodiments, the method further comprises a detection step. After the washing step, a detector comprising an enzyme substrate, such as DAB for HRP or fast red for AP, is added to the tissue sample.

In some embodiments, the detection reagent comprises a buffer, such as PBS or TBS buffer, optionally with BSA and/or polyethylene glycol.

In some embodiments according to any of the above embodiments of the invention, the enzymatic reaction is detected using a spectrophotometer. In some embodiments, the enzymatic reaction is detected using a chemical photometer. In some embodiments, the enzymatic reaction is detected using a fluorescence detector. In some embodiments, the enzymatic reaction is detected using a colorimetric (colorimetric) signal detector. In some embodiments, the enzymatic reaction is detected using an optical microscope or a fluorescence microscope.

In some embodiments according to any of the above embodiments of the invention, the tissue is frozen tissue. In some embodiments, the tissue is paraffin-embedded tissue. In some embodiments, the tissue sample is a clinical smear or cultured cells or tissue. Preferably, the tissue (sample) is a continuous frozen tissue section or paraffin section.

In some embodiments, the tissue is a tissue slice greater than about 5 μm thick. In some embodiments, the tissue is a tissue slice of about 5 μm thickness. In some embodiments, the tissue is a tissue slice that is less than about 5 μm thick. In some embodiments, the tissue is a tissue slice having a thickness of about 1.5 μm to about 5.5 μm. In some embodiments, the tissue is a tissue slice having a thickness of about 4.5 μm to about 7.5 μm.

In some embodiments, the method further comprises a fixing step. In some embodiments, the tissue is fixed in a fixative comprising an aldehyde. Preferably, the fixative comprising an aldehyde such as formalin (formaldehyde) and glutaraldehyde, more preferably, the fixative is 1-10% formalin. In another embodiment, the tissue is fixed in an acetone fixative. In another embodiment, the tissue is fixed in a methanol fixative. In another embodiment, the tissue is fixed in 50-90% alcohol fixative.

The invention has the advantages that:

methods of treating primary tumors (e.g., brain tumors) typically involve surgical removal of the tumor. However, since tumor infiltration is adjacent to delicate brain tissue, the bottom-of-penetration clearance of tumors (e.g., brain tumors) is very difficult, in part because the accuracy of evaluating intraoperative tumor diagnosis such as brain tumors by hematoxylin and eosin staining of frozen sectioned tissue is inadequate. The invention adopts covalent linkage of polymerase/antibody to form conjugate, synthesizes the antibodies respectively related to 5-8 tumors (such as brain tumor, lymphoma, melanoma and metastatic cancer) by the polymerase, forms the combination of 5-8 polymerase/antibody conjugates, is applied to the field related to the identification of tumors (such as brain tumor, lymphoma, melanoma and metastatic cancer), particularly can be used for the identification diagnosis of tumors (such as brain tumor, lymphoma, melanoma and metastatic cancer) in operation and the determination of the cutting margin due to the characteristic of rapid staining of fresh and frozen tissues, and improves the accuracy of tumor diagnosis. Application of this technique will significantly improve surgical management, especially for certain invasive and invasive brain cancers.

The specific technical scheme of the application is as follows.

1. A combination of polymerase/antibody conjugates comprising or selected from two, three, four or more of the following polymerase/antibody conjugates: polymerase/Pan-CK antibody conjugates, polymerase/EMA antibody conjugates, polymerase/CD 34 antibody conjugates, polymerase/synapsin antibody conjugates, polymerase/Mart-1 antibody conjugates, polymerase/Sox-10 antibody conjugates, polymerase/GFAP antibody conjugates, and polymerase/CD 45 antibody conjugates,

wherein each polymerase/antibody conjugate comprises:

(i) One or more polymerases;

(ii) At least one antibody that recognizes an analyte of interest, wherein each polymerase has a plurality of enzyme molecules, wherein each polymerase/antibody conjugate comprises a different number of enzymes and antibodies, the molecular weight sizes of which form a multi-dispersed distribution, wherein the polymerase/antibody conjugates have different molecular weights and different three-dimensional structures in each interval ranging from 400kDa to 2,000kDa, wherein the polymerase/antibody conjugates have a number of 2% -8% of the total molecular weight range between 400kDa and 600kDa, a number of 5% -10% between 600kDa and 800kDa, a number of 6% -13% between 800kDa and 1000kDa, a number of 7% -14% between 1000kDa and 1200kDa, a number of 9% -16% between 1200kDa and 1400kDa, a number of 10% -18% between 1400kDa and 1600kDa and 12% -18% between 1600 kDa.

2. The combination of polymerase/antibody conjugates of scheme 1 comprising, or consisting of, the following polymerase antibody conjugates: polymerase/GFAP antibody conjugates, polymerase/Pan-CK antibody conjugates, polymerase/EMA antibody conjugates and/or polymerase/CD 34 antibody conjugates, and polymerase/synapsin antibody conjugates.

3. The combination of polymerase/antibody conjugates of scheme 1 comprising a polymerase/Mart-1 antibody conjugate and/or a polymerase/Sox-10 antibody conjugate, and at least one polymerase antibody conjugate selected from the group consisting of: polymerase/GFAP antibody conjugates, polymerase/Pan-CK antibody conjugates, and polymerase/synapsin antibody conjugates, or consist thereof.

4. The combination of polymerase/antibody conjugates of scheme 1 comprising a polymerase/Mart-1 antibody conjugate and/or a polymerase/Sox-10 antibody conjugate, and at least one polymerase antibody conjugate selected from the group consisting of: polymerase/GFAP antibody conjugates, polymerase/Pan-CK antibody conjugates, polymerase/CD 45 antibody conjugates, polymerase/synapsin antibody conjugates, polymerase/EMA antibody conjugates, and polymerase/CD 34 antibody conjugates.

5. The combination of polymerase/antibody conjugates of scheme 1 comprising a polymerase/EMA antibody conjugate and/or a polymerase/CD 34 antibody conjugate, and at least one polymerase antibody conjugate selected from the group consisting of: polymerase/GFAP antibody conjugate, polymerase/Pan-CK antibody conjugate, polymerase/synapsin antibody conjugate, and polymerase/CD 45 antibody conjugate.

6. The combination of polymerase/antibody conjugates of scheme 1 comprising, or consisting of, the following polymerase antibody conjugates: polymerase/Pan-CK antibody conjugate, polymerase/EMA antibody conjugate or polymerase/CD 34 antibody conjugate, polymerase/synapsin antibody conjugate or polymerase/Mart-1 antibody conjugate or polymerase/Sox-10 antibody conjugate, polymerase/GFAP antibody conjugate, and polymerase/CD 45 antibody conjugate.

7. The combination of polymerase/antibody conjugate according to scheme 1 consisting of a polymerase/Pan-CK antibody conjugate, a polymerase/EMA antibody conjugate, a polymerase/CD 34 antibody conjugate, a polymerase/synapsin antibody conjugate, a polymerase/Mart-1 antibody conjugate, a polymerase/Sox-10 antibody conjugate, a polymerase/GFAP antibody conjugate, and a polymerase/CD 45 antibody conjugate.

8. The combination of a polymerase/antibody conjugate of any of claims 1-7, wherein the polymerase is selected from horseradish peroxidase (HRP), beta-D-galactosidase, alkaline phosphatase, superoxide dismutase, luciferase, lactate dehydrogenase, galactose oxidase.

9. A kit comprising: a combination of the polymerase/antibody conjugates of any of the above schemes 1-8, and instructions for use.

10. The kit according to the above-mentioned scheme 9, further comprising a substrate for a polymerase molecule.

11. Use of a combination of a polymerase/antibody conjugate according to any of the above schemes 1-8 in the preparation of a reagent or reagent combination or kit for identifying and/or diagnosing melanoma in a tissue sample.

12. The use according to claim 11, wherein the tissue sample is a surgical sample.

13. The use according to any of the above aspects 11 or 12, wherein the tissue sample comprises (continuous) paraffin or frozen tissue sections.

14. The use according to any one of the above schemes 11-13, wherein the melanoma is malignant melanoma.

Examples

Hereinafter, the present invention will be described in more detail with reference to specific examples in which preferred exemplary peroxidase antibody conjugates of the present invention are used. The following examples and descriptions are for illustrative purposes only and are not to be construed as limiting the spirit of the invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used in the examples described below are commercially available conventional reagents unless otherwise specified.

EXAMPLE 1 pretreatment of frozen tissue on glass slides

Frozen tissue sections placed on slides were fixed in a fixative solution of 10% formalin or low-temperature acetone for 30 seconds. Then, the slide glass was washed with Phosphate Buffered Saline (PBS) for about 20 seconds; if the tissue sections are enriched in endogenous peroxidases, the slides are treated with a 4% or so hydrogen peroxide blocking solution (4% hydrogen peroxide in water) for 1 to 2 minutes, after which the slides are washed with water and wash buffer, respectively, for about 20 seconds.

EXAMPLE 2 dyeing of the polyperoxidase/Pan-CK antibody conjugate

The peroxidase/Pan-CK antibody conjugate was diluted to an appropriate concentration (1-30 μg/ml) and added to frozen prostate cancer tissue sections pretreated as above, and reacted for 3 to 5 minutes. The sections were washed 3 times with phosphate-buffered saline (PBS) for 20 seconds. A substrate universal DAB staining solution (Vector Lab, SK-4100) was added and reacted for 3 minutes. The slide was washed with water for 10 seconds to 1 minute 3 times. Mu.l of general hematoxylin solution (Sigma-Aldrich) was added for staining for 20 seconds to 1 minute. Wash with water for 15 seconds (or longer) 3 times. Mu.l of a Universal sealant (Vector Lab) coverslip was added. The whole staining was completed within 10 minutes. A micrograph of the tissue staining is shown in fig. 1: the conjugate of the present invention, the polyperoxidase/Pan-CK antibody conjugate, was stained rapidly on frozen prostate cancer sections within 10 minutes, and the prostate cancer cell plasma was stained specifically.

Example 3 dyeing of a polyperoxidase/GFAP antibody conjugate

The peroxidase/GFAP antibody conjugate was diluted to an appropriate concentration (1-30. Mu.g/ml) and added to the brain tumor frozen sections pretreated as above, and reacted for 3 to 5 minutes. The sections were washed 3 times with phosphate-buffered saline (PBS) for 20 seconds. A substrate universal DAB staining solution (Vector Lab, SK-4100) was added and reacted for 3 minutes. The slide was washed with water for 10 seconds to 1 minute 3 times. Mu.l of general hematoxylin solution (Sigma-Aldrich) was added for staining for 20 seconds to 1 minute. Wash with water for 15 seconds (or longer) 3 times. Mu.l of a Universal sealant (Vector Lab) coverslip was added. The whole staining was completed within 10 minutes. Microphotographs of tissue staining thereof are shown in fig. 2: the polyperoxidase/GFAP antibody conjugate of the present invention was stained rapidly within 10 minutes on brain tumor frozen sections and brain astrocytes were specifically stained for their cytoplasm.

Example 4 dyeing of a Polyperoxidase/EMA antibody conjugate

The peroxidase/EMA conjugate was diluted to the appropriate concentration (1-30 μg/ml) and added to frozen kidney tissue sections pretreated as above, and reacted for 3 to 5 minutes. The sections were washed 3 times with phosphate-buffered saline (PBS) for 20 seconds. A substrate universal DAB staining solution (Vector Lab, SK-4100) was added and reacted for 3 minutes. The slide was washed with water for 10 seconds to 1 minute 3 times. Mu.l of general hematoxylin solution (Sigma-Aldrich) was added for staining for 20 seconds to 1 minute. Wash with water for 15 seconds (or longer) 3 times. Mu.l of a Universal sealant (Vector Lab) coverslip was added. The whole staining was completed within 10 minutes. A micrograph of the tissue staining is shown in fig. 3: the polyperoxidase/EMA antibody conjugate of the present invention stained rapidly at 10 minutes of frozen kidney tissue sections and kidney parenchymal cancer cell plasma was stained specifically.

Example 5 dyeing of a polyperoxidase/synapsin antibody conjugate

The peroxidase/synapsin antibody conjugate was diluted to the appropriate concentration (1-30 μg/ml) and added to frozen liver tissue sections pretreated as above, and reacted for 3 to 5 minutes. The sections were washed 3 times with phosphate-buffered saline (PBS) for 20 seconds. A substrate universal DAB staining solution (Vector Lab, SK-4100) was added and reacted for 3 minutes. The slide was washed with water for 10 seconds to 1 minute 3 times. Mu.l of general hematoxylin solution (Sigma-Aldrich) was added for staining for 20 seconds to 1 minute. Wash with water for 15 seconds (or longer) 3 times. Mu.l of a Universal sealant (Vector Lab) coverslip was added. The whole staining was completed within 10 minutes. A micrograph of the tissue staining is shown in fig. 4: the polyperoxidase/synapsin antibody conjugate of the present invention stains rapidly within 10 minutes on frozen sections of liver tissue, neuroendocrine tumor cytoplasm specific staining.

EXAMPLE 6 dyeing of the polyperoxidase/CD 45 antibody conjugate

The peroxidase/CD 45 antibody conjugate was diluted to the appropriate concentration (1-30. Mu.g/ml) and added to frozen tonsil tissue sections pretreated as above, and reacted for 3 to 5 minutes. The sections were washed 3 times with phosphate-buffered saline (PBS) for 20 seconds. A substrate universal DAB staining solution (Vector Lab, SK-4100) was added and reacted for 3 minutes. The slide was washed with water for 10 seconds to 1 minute 3 times. Mu.l of general hematoxylin solution (Sigma-Aldrich) was added for staining for 20 seconds to 1 minute. Wash with water for 15 seconds (or longer) 3 times. Mu.l of a Universal sealant (Vector Lab) coverslip was added. The whole staining was completed within 10 minutes. A micrograph of the tissue staining is shown in fig. 5: the polyperoxidase/CD 45 antibody conjugate of the present invention stained rapidly at 10 minutes for tonsil frozen sections, in which lymphocyte membranes were specifically stained.

EXAMPLE 7 dyeing of the polyperoxidase/Mart-1 antibody conjugate

The peroxidase/Mart 1 antibody conjugate was diluted to the appropriate concentration (1-30. Mu.g/ml) and added to the melanoma frozen tissue sections pretreated as above, and reacted for 3 to 5 minutes. The sections were washed 3 times with phosphate-buffered saline (PBS) for 20 seconds. A substrate universal DAB staining solution (Vector Lab, SK-4100) was added and reacted for 3 minutes. The slide was washed with water for 10 seconds to 1 minute 3 times. Mu.l of general hematoxylin solution (Sigma-Aldrich) was added for staining for 20 seconds to 1 minute. Wash with water for 15 seconds (or longer) 3 times. Mu.l of a Universal sealant (Vector Lab) coverslip was added. The whole dyeing time was completed within 10 minutes. A micrograph of the tissue staining is shown in fig. 6: the polyperoxidase/Mart 1 antibody conjugate of the present invention stained rapidly at 10 minutes in frozen sections of melanoma where the cytoplasm of melanoma was stained specifically.

EXAMPLE 8 dyeing of the polyperoxidase/Sox-10 antibody conjugate

The peroxidase/Sox-10 antibody conjugate was diluted to an appropriate concentration (1-30 μg/ml) and added to melanoma frozen sections pretreated as above, and reacted for 3 to 5 minutes. The sections were washed 3 times with phosphate-buffered saline (PBS) for 20 seconds. A substrate universal DAB staining solution (Vector Lab, SK-4100) was added and reacted for 3 minutes. The slide was washed with water for 10 seconds to 1 minute 3 times. Mu.l of general hematoxylin solution (Sigma-Aldrich) was added for staining for 20 seconds to 1 minute. Wash with water for 15 seconds (or longer) 3 times. Mu.l of a Universal sealant (Vector Lab) coverslip was added. The whole staining was completed within 10 minutes. A micrograph of the tissue staining is shown in fig. 7: the polyperoxidase/Sox 10 antibody conjugates of the present invention are fast staining in frozen sections of melanoma where the nuclei of melanoma are specifically stained.

Example 9 staining of Low grade gliomas in successive frozen sections with the exemplary combination of the Polyperoxidase/antibody conjugates of the invention

The peroxidase/Pan-CK antibody conjugate, the peroxidase/GFAP antibody conjugate, the peroxidase/EMA antibody conjugate, the peroxidase/synapsin antibody conjugate, the peroxidase/CD 45 antibody conjugate, the peroxidase/Mart 1 antibody conjugate, and the peroxidase/Sox-10 antibody conjugate were diluted to appropriate concentrations (1-30 μg/ml), respectively, and added to a series of frozen sections pretreated as above, which were diagnosed as low-grade glioma by classical pathology, and reacted for 3 to 5 minutes, respectively. The sections were washed 3 times with phosphate-buffered saline (PBS) for a total of 20 seconds. The substrate universal DAB staining solution (Vector Lab, SK-4100) was added to each section and reacted for 3 minutes. The slides were washed with water for 10 seconds to 1 minute 3 times, respectively. Mu.l of general hematoxylin solution (Sigma-Aldrich) was added to each section for staining for 20 seconds to 1 minute, followed by washing with water for 15 seconds (or longer) 3 times. Mu.l of a Universal sealant (Vector Lab) coverslip was added to each section. The whole staining was completed within 10 minutes. The microphotographs of tissue staining of a series of low grade glioma frozen sections are shown in fig. 8A-8G: 8A, the dyeing result of the polyperoxidase/anti-Pan-CK antibody conjugate shows that the low-grade glioma is negative; 8B, a peroxidase/anti-GFAP antibody conjugate, and the low-grade glioma was positive; 8C, dyeing and staining the polyperoxidase/anti-synapsin antibody conjugate, wherein the low-grade glioma is weak positive; 8D, dyeing and staining the polyperoxidase/anti-EMA antibody conjugate, wherein the low-grade glioma is negative; 8E, dyeing and staining the polyperoxidase/anti-CD 45 antibody conjugate, wherein the low-grade glioma is negative; 8F, dyeing and staining the polyperoxidase/anti-Mart 1 antibody conjugate, wherein the low-grade glioma is negative; 8G, dyeing and staining the polyperoxidase/anti-Sox 10 antibody conjugate, wherein the low-grade glioma is negative; 8H.H & E staining control. From the above results, the diagnostic results of the combination of the present exemplary peroxidase/antibody conjugates are consistent with those of classical pathology.

Example 10 staining of high grade glioma with exemplary peroxidase/antibody conjugates of the invention in serial frozen sections

The peroxidase/Pan-CK antibody conjugate, the peroxidase/GFAP antibody conjugate, the peroxidase/EMA antibody conjugate, the peroxidase/synapsin antibody conjugate, the peroxidase/CD 45 antibody conjugate, the peroxidase/Mart 1 antibody conjugate, and the peroxidase/Sox-10 antibody conjugate were diluted to appropriate concentrations (1-30 μg/ml), respectively, and added to a series of frozen sections pretreated as above, which were diagnosed as high-grade glioma by classical pathology, and reacted for 3 to 5 minutes, respectively. The sections were washed 3 times with phosphate-buffered saline (PBS) for a total of 20 seconds. The substrate universal DAB staining solution (Vector Lab, SK-4100) was added to each section and reacted for 3 minutes. The slides were washed with water for 10 seconds to 1 minute 3 times, respectively. Mu.l of general hematoxylin solution (Sigma-Aldrich) was added to each section for staining for 20 seconds to 1 minute, followed by washing with water for 15 seconds (or longer) 3 times. Mu.l of a Universal sealant (Vector Lab) coverslip was added to each section. The whole staining was completed within 10 minutes. The microphotographs of tissue staining of a series of high grade glioma frozen sections are shown in fig. 9A-9G: 9A, dyeing and staining the polyperoxidase/Pan-CK antibody conjugate, wherein the high-grade glioma is negative; 9B, the dyeing result of the peroxidase/anti-GFAP antibody conjugate shows that the high-grade glioma is positive; 9C, dyeing the polyperoxidase/EMA antibody conjugate to obtain a negative high-grade glioma; 9D, dyeing the polyperoxidase/synapsin antibody conjugate, wherein the high-grade glioma is negative; 9E, dyeing the polyperoxidase/CD 45 antibody conjugate to obtain a negative high-grade glioma; 9F, dyeing and staining the polyperoxidase/Mart 1 antibody conjugate, wherein the high-grade glioma is negative; 9G, dyeing and staining the polyperoxidase/Sox 10 antibody conjugate, wherein the high-grade glioma is negative; 9H.H & E staining control. From the above results, it can be seen that the diagnostic results of the combination of the exemplary peroxidase/antibody conjugates of the present invention are consistent with the results of classical pathological diagnosis.

Example 11 staining of metastatic cancers in successive frozen sections with exemplary peroxidase/antibody conjugates of the invention

The peroxidase/Pan-CK antibody conjugate, the peroxidase/GFAP antibody conjugate, the peroxidase/EMA antibody conjugate, the peroxidase/synapsin antibody conjugate, the peroxidase/CD 45 antibody conjugate, the peroxidase/Mart 1 antibody conjugate, and the peroxidase/Sox-10 antibody conjugate were diluted to appropriate concentrations (1-30 μg/ml), respectively, and added to a series of frozen sections pretreated as above, which were diagnosed as metastatic cancer by classical pathology, and reacted for 3 to 5 minutes, respectively. The sections were washed 3 times with phosphate-buffered saline (PBS) for a total of 20 seconds. The substrate universal DAB staining solution (Vector Lab, SK-4100) was added to each section and reacted for 3 minutes. The slides were washed with water for 10 seconds to 1 minute 3 times, respectively. Mu.l of general hematoxylin solution (Sigma-Aldrich) was added to each section for staining for 20 seconds to 1 minute, followed by washing with water for 15 seconds (or longer) 3 times. Mu.l of a Universal sealant (Vector Lab) coverslip was added to each section. The whole staining was completed within 10 minutes. A series of microphotographs of tissue staining of frozen sections of metastatic cancer are shown in fig. 10A-10G: 10A. Dyeing the polyperoxidase/Pan-CK antibody conjugate, wherein the metastatic cancer cells are positive; 10B. Dyeing the polyperoxidase/EMA antibody conjugate, wherein the metastatic cancer is weak positive; 10C, the result of dyeing the polyperoxidase/anti-GFAP antibody conjugate shows that the metastatic cancer is negative; 10D, staining the polyperoxidase/synapsin antibody conjugate, wherein the metastatic cancer is negative; 10E. Dyeing the polyperoxidase/CD 45 antibody conjugate, wherein the metastatic cancer is negative; 10F, dyeing the polyperoxidase/Mart 1 antibody conjugate, wherein the metastatic cancer is negative; 10G. Dyeing the polyperoxidase/Sox 10 antibody conjugate, and making the metastatic cancer negative; 10h.h & e staining control. From the above results, the diagnostic results of the combination of the present exemplary peroxidase/antibody conjugates are consistent with those of classical pathology.

Example 12 staining of melanoma in successive frozen sections with exemplary peroxidase/antibody conjugates of the invention

The peroxidase/Pan-CK antibody conjugate, the peroxidase/GFAP antibody conjugate, the peroxidase/EMA antibody conjugate, the peroxidase/synapsin antibody conjugate, the peroxidase/CD 45 antibody conjugate, the peroxidase/Mart 1 antibody conjugate, and the peroxidase/Sox-10 antibody conjugate were diluted to appropriate concentrations (1-30 μg/ml), respectively, and added to a series of frozen sections pretreated as above, which were diagnosed as malignant melanoma by classical pathology, and reacted for 3 to 5 minutes, respectively. The sections were washed 3 times with phosphate-buffered saline (PBS) for a total of 20 seconds. The substrate universal DAB staining solution (Vector Lab, SK-4100) was added to each section and reacted for 3 minutes. The slides were washed with water for 10 seconds to 1 minute 3 times, respectively. Mu.l of general hematoxylin solution (Sigma-Aldrich) was added to each section for staining for 20 seconds to 1 minute, followed by washing with water for 15 seconds (or longer) 3 times. Mu.l of a Universal sealant (Vector Lab) coverslip was added to each section. The whole staining was completed within 10 minutes. The results are shown in fig. 11A to 11H, showing that the polyperoxidase/Mart 1 antibody conjugate and the polyperoxidase/Sox-10 antibody conjugate stained malignant melanoma positive, and that the other polyperoxidase/antibody conjugates stained melanoma negative, indicating that the diagnostic results of the combination of the present exemplary polyperoxidase/antibody conjugate are consistent with the results of the classical pathological diagnosis.

Example 13 staining of melanoma in continuous paraffin sections with exemplary peroxidase/antibody conjugates of the invention

The peroxidase/Pan-CK antibody conjugate, the peroxidase/GFAP antibody conjugate, the peroxidase/EMA antibody conjugate, the peroxidase/synapsin antibody conjugate, the peroxidase/CD 45 antibody conjugate, the peroxidase/Mart 1 antibody conjugate, and the peroxidase/Sox-10 antibody conjugate were diluted to appropriate concentrations (1-30 μg/ml), respectively, and added to a series of paraffin sections diagnosed as malignant melanoma by classical pathology through conventional dewaxing and antigen retrieval, and reacted for 3 to 10 minutes, respectively. The sections were washed 3 times with phosphate-buffered saline (PBS) for a total of 20 seconds. The substrate universal DAB staining solution (Vector Lab, SK-4100) was added to each section and reacted for 3 minutes. The slides were washed with water for 10 seconds to 1 minute 3 times, respectively. Mu.l of general hematoxylin solution (Sigma-Aldrich) was added to each section for staining for 20 seconds to 1 minute, followed by washing with water for 15 seconds (or longer) 3 times. Mu.l of a Universal sealant (Vector Lab) coverslip was added to each section. The whole staining was completed within 10 to 20 minutes. The results are shown in fig. 12A to 12H, showing that each of the other peroxidase/antibody conjugates stained melanoma was negative, except that the peroxidase/Mart 1 antibody conjugate and the peroxidase/Sox-10 antibody conjugate stained malignant melanoma were positive, indicating that the diagnostic results of the combination of the present exemplary peroxidase/antibody conjugate were consistent with the results of the classical pathological diagnosis.

The above antibody combinations are merely exemplary, and in addition, other polymerases can be coupled thereto, and different colors can be stained on the same slice tissue by using different color reaction substrates to perform relevant identification and localization of tumor.

Document index

[1]Timothy J.Brown,Matthew C.Brennan,Michael Li,Association of the Extent of Resection with Survivalin Glioblastoma A Systematic Review and Meta-analysis,JAMA Oncol.2016；2(11):1460-1469

[2]Daniel A Orringer,Alexandra Golbyand Ferenc Jolesz,Neuronavigation in the surgical management of brain tumors:currentandfuture trends,ExpertRevMed Devices.2012Sep；9(5):491–500.

[3]Richard A.Prayson,Mark L.Cohen,Differential Diagnosis in Surgical Neuropathology,Humana Press,May 19,2010.

[4]Todd Hollon,Spencer Lewis,Christian W.Freudiger,X.SunneyXie,and Daniel A.Orringer,Improving the accuracy of brain tumor surgery via Raman-based technology Neurosurg Focus 40(3):E9,2016

[5]Edward R.St Johna,Merja Rossib,Pamela Pruskib,Ara Darzia,Zoltan Takats:Intraoperative tissue identification by mass spectrometric technologies,TrAC Trendsin Analytical Chemistry,Volume85,PartA,2016,On-site and In-vivo Instrumentation and Applications.

[6]Jennifer eschbacher,Nikolayl.Martirosyan,Peternaka Ji,NaDer Sanai,Mark C.Preul,KrisA.Smith,Stephen W.Coons,and Robert F.Spetzler:In vivo intraoperative confocal microscopy forreal-time histopathological imaging of brain tumors.J NeurosurgVolume116,2012

[7]Young Jin Cho:Intraoperative Examination of Sentinel Lymph Nodes by Ultrarapid Immunohistochemistry in Breast Cancer,Jpn J Clin Oncol 2006；36(8)489-493.

[8]Xuebin Zhang,Jiayu Liu,Xiaoling Yan,Rapid intraoperative immunocytochemistry of central nervous system tumors,Int J Clin Exp Pathol.2020；13(1):44-48.

Claims

1. Combinations of polymerase/antibody conjugates comprising a polymerase/Mart-1 antibody conjugate and/or a polymerase/Sox-10 antibody conjugate, a polymerase/Pan-CK antibody conjugate, a polymerase/EMA antibody conjugate and/or a polymerase/CD 34 antibody conjugate, a polymerase/synapsin antibody conjugate and a polymerase/GFAP antibody conjugate, and optionally a polymerase/CD 45 antibody conjugate,

wherein each polymerase/antibody conjugate comprises:

(i) One or more polymerases;

2. The combination of polymerase/antibody conjugates of claim 1, consisting of the following polymerase antibody conjugates: polymerase/Pan-CK antibody conjugate, polymerase/EMA antibody conjugate or polymerase/CD 34 antibody conjugate, polymerase/synapsin antibody conjugate or polymerase/Mart-1 antibody conjugate or polymerase/Sox-10 antibody conjugate, polymerase/GFAP antibody conjugate, and polymerase/CD 45 antibody conjugate.

3. The combination of polymerase/antibody conjugates of claim 1 consisting of a polymerase/Pan-CK antibody conjugate, a polymerase/EMA antibody conjugate, a polymerase/CD 34 antibody conjugate, a polymerase/synapsin antibody conjugate, a polymerase/Mart-1 antibody conjugate, a polymerase/Sox-10 antibody conjugate, a polymerase/GFAP antibody conjugate, and a polymerase/CD 45 antibody conjugate.

4. The combination of a polymerase/antibody conjugate of any of claims 1-3, wherein the polymerase is selected from horseradish peroxidase (HRP), beta-D-galactosidase, alkaline phosphatase, superoxide dismutase, luciferase, lactate dehydrogenase, galactose oxidase.

5. A kit comprising: the combination of the polymerase/antibody conjugate of any of claims 1-4, and instructions for use.

6. The kit of claim 5, further comprising a substrate for a polymerase molecule.

7. Use of a combination of a polymerase/antibody conjugate according to any one of claims 1-6 for the preparation of a reagent or a combination of reagents or a kit for identifying and/or diagnosing melanoma in a tissue sample.

8. The use of claim 7, wherein the tissue sample is a surgical sample.

9. The use of claim 7 or 8, wherein the tissue sample comprises a continuous paraffin or frozen tissue section.

10. The use according to any one of claims 7-9, wherein the melanoma is malignant melanoma.