CN116041506B - Monoclonal antibody pair and detection kit for sugar-deficient transferrin - Google Patents
Monoclonal antibody pair and detection kit for sugar-deficient transferrin Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/79—Transferrins, e.g. lactoferrins, ovotransferrins
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Food Science & Technology (AREA)
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- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
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Abstract
The application discloses a monoclonal antibody pair of sugar-deleted transferrin and a detection kit. Comprising an anti-CDT monoclonal antibody M1 and an anti-CDT monoclonal antibody M2, said anti-CDT monoclonal antibody M1 and anti-CDT monoclonal antibody M2 binding to different epitopes of CDT antigen, respectively, said anti-CDT monoclonal antibody M1 and anti-CDT monoclonal antibody M2 not binding to TRF. The detection kit prepared by the antibody can directly test the natural CDT in a blood or body fluid sample, does not need to separate CDT from other serum proteins and transferrin isomers other than CDT, and does not need to pretreat the sample.
Description
Technical Field
The application relates to the technical field of biological detection, in particular to a monoclonal antibody pair of sugar-deleted transferrin and a detection kit.
Background
Human transferrin (Human Transferrin, TRF) is an iron-binding plasma single chain glycoprotein with a molecular weight of about 80kDa that can be transported through the blood circulation to the bone marrow, liver and spleen where red blood cells are produced. Structurally, it is a polypeptide chain consisting of 679 amino acids, with two iron binding sites and two N-linked oligosaccharide chains linked to Asn432 and Asn630, the two sugar chain sites being capable of binding 2, 3 or 4 sugar chains, respectively, each of which is terminated by a negatively charged sialic acid molecule. Sugar-deficient transferrin (Carbohydrate-deficient Transferrin CDT) is an isoform of transferrin, and is referred to clinically as asialotransferrin, monosialotein and bissialyltransferase subtypes and is referred to as CDT.
Alcohol abuse is the most common cause of elevated CDT levels, the only marker approved by the U.S. food and drug administration for use in identifying heavy alcohol use. In chronic alcohol abusers, ethanol and its by-product acetaldehyde inhibit the enzyme responsible for the addition of carbohydrate side chains and induces the production of sialidases that cleave terminal sialic acid residues from side chains. Thus, in patients who drink large amounts over a long period of time, the number of carbohydrate components per transferrin molecule is reduced, thereby increasing the level of CDT.
There are many other causes of elevated CDT, such as a) elevated levels of CDT in the blood of patients with bile ducts blocked or damaged by disease, for example, cholestatic liver disease, primary biliary cirrhosis, primary sclerosing cholangitis, gall stones or liver/pancreatic cancer patients; b) Transferrin variants, which can be produced by amino acid substitutions, at least 38 genetic transferrin variants resulting from amino acid substitutions have been reported to have three phenotypes: TRFC, TRFB and TRFD, wherein TRFC is the normal form of transferrin in blood, TRFB and TRFD are two variants, TRFD is very rare in caucasians, less than one ten thousandth in prevalence, but has been shown to result in elevated CDT levels, TRFD is more common in africans; c) Congenital glycosylation disorder syndrome (Congenital disorders of glycosylation, CDG), a very rare group of syndromes, a genetic defect of enzymes that add side chains to proteins, with severe mental retardation and other physical abnormalities in affected children, and CDT levels in the range of 50-100%, and in heterozygotes or disease carriers, CDT levels in the range of 10-25%; d) Under certain physiological or pathological conditions, such as pregnancy, rheumatoid arthritis, malignancy, alcoholism, etc., the glycan structure is characteristically altered and CDT levels in the blood are elevated.
In the current method for detecting CDT, CDT is required to be separated from other serum proteins and transferrin isomers other than CDT due to lack of CDT antibodies specifically binding to CDT. The different charges and isoelectric points of the transferrin isomers of CDT and non-CDT can be used to separate CDT and non-CDT isomers by anion exchange isoelectric chromatography, isoelectric focusing (IEF) electrophoresis, or capillary electrophoresis and capillary zone charged swimming. Many commercial CDT test products developed since 1993, including CDTect-RIA, CDT-EIA (Pharmacia & Upjohn in Uppsala, sweden),% CDT-TIA (Axis-Shield in Norway Olympic),% CDTri-TIA (Bio-Rad Laboratories, calif., USA) and Tinaquat-% CDT/transferrin (Roche Diagnostic GmbH, germany) were also required to separate CDT and non-CDT isomers on anion exchange microcolumns prior to immunoassay quantitative detection of CDT, whereas the methods of rapid CDT determination based on immunoaffinity liquid chromatography and electrospray mass spectrometry developed in recent years were expensive in materials and instruments and could not be routinely used.
Disclosure of Invention
The application provides a monoclonal antibody pair of high-specificity and high-affinity sugar-deleted transferrin and a detection kit, which solve the problems that CDT antibodies which are specifically combined with CDT are lack in the existing CDT detection method, and CDT needs to be separated from other serum proteins and transferrin isomers other than CDT before detection.
The application provides a monoclonal antibody pair of sugar-deleted transferrin, comprising an anti-CDT monoclonal antibody M1 and an anti-CDT monoclonal antibody M2, wherein the anti-CDT monoclonal antibody M1 and the anti-CDT monoclonal antibody M2 are respectively combined with different epitopes of CDT antigen, and neither the anti-CDT monoclonal antibody M1 nor the anti-CDT monoclonal antibody M2 is combined with TRF, wherein the anti-CDT monoclonal antibody M1 is produced by a hybridoma cell strain TRF 1 of the anti-human desugared transferrin with the preservation number of CCTCC NO: C2022391, the anti-CDT monoclonal antibody M2 is produced by a hybridoma cell strain TRF 2 of the anti-human desugared transferrin with the preservation number of CCTCC NO: C2022392, and the hybridoma cell strain TRF 1 of the anti-human desugared transferrin and the hybridoma cell strain TRF 2 are preserved for 21 months in 20212 months, and the anti-human desugared transferrin is obtained by the following steps: china center for type culture Collection; the request for preservation is Shenzhen Shangtai bioengineering Co., ltd, and the preservation address is Wuhan in China.
Further, the CDT antigen is a native CDT antigen or a recombinant CDT antigen prepared using protein recombination techniques.
Further, the preparation method of the anti-CDT monoclonal antibody M1 and the anti-CDT monoclonal antibody M2 comprises the following steps:
s1, performing animal immunization by taking the recombinant CDT antigen as an antigen;
s2, extracting cells of the animal in the S1 and fusing the cells with myeloma to generate hybridoma cells;
s3, selecting the hybridoma cells producing the anti-CDT monoclonal antibody M1 and the anti-CDT monoclonal antibody M2 by sandwich ELISA by using the recombinant CDT antigen;
s4, confirming the hybridoma cells by using a natural CDT antigen through a sandwich ELISA method in the step S3;
s5, reversely screening the hybridoma cells by using TRF through a sandwich ELISA method which is the same as that of the step S3, so as to obtain a hybridoma cell strain TRF M1 for producing the anti-CDT monoclonal antibody M1 and a hybridoma cell strain TRF M2 for producing the anti-human desugared transferrin of the anti-CDT monoclonal antibody M2.
The application also provides a high-specificity and high-affinity detection kit of the sugar-deleted transferrin, which comprises a reagent R1, a reagent R2, a calibrator and a quality control product, wherein the reagent R1 comprises the latex microsphere conjugate of the anti-CDT monoclonal antibody M1 and the latex microsphere conjugate of the anti-CDT monoclonal antibody M2.
Further, the calibrator and quality control comprise CDT antigens as described above.
Further, the preparation method of the latex microsphere conjugate of the anti-CDT monoclonal antibody M1 and the latex microsphere conjugate of the anti-CDT monoclonal antibody M2 comprises the following steps:
s1, sensitization: placing the emulsion solution into a centrifuge tube, adding a sensitization buffer solution, adding the anti-CDT monoclonal antibody M1 or the anti-CDT monoclonal antibody M2, uniformly mixing, and placing the mixture at 37 ℃ for reaction for 1 hour;
s2, sealing: adding a sealing buffer solution, performing ultrasonic treatment for 2 minutes, sealing at 37 ℃ for 1 hour, and centrifuging to remove unreacted parts in the supernatant after sealing;
s3, adding 1mL of sensitization buffer solution, and carrying out ultrasonic treatment on the latex, wherein the absorbance of the latex microsphere conjugate of the anti-CDT monoclonal antibody M1 at 700nm is regulated to be 0.8Abs by the sensitization buffer solution.
Further, the sensitization buffer was 20mM MES at pH6.1, the sensitization buffer was 10mM PBS at pH 7.5, and the blocking buffer included 10mM PBS at pH 7.5, 10% bovine serum albumin, and 0.095% w/w sodium azide.
Further, the concentration of the solution after adding the anti-CDT monoclonal antibody M1 or the anti-CDT monoclonal antibody M2 in the step S1 is 0.03-0.09 mg/mL.
The application also provides a hybridoma cell pair which is respectively used for producing the monoclonal antibody pair, wherein the hybridoma cell pair is a hybridoma cell strain TRF M1 of anti-human desugared transferrin with the preservation number of CCTCC NO: C2022391 for producing the anti-CDT monoclonal antibody M1 and a hybridoma cell strain TRF M2 of anti-human desugared transferrin with the preservation number of CCTCC NO: C2022392 for producing the anti-CDT monoclonal antibody M2, the hybridoma cell strain TRF M1 of the anti-human desugared transferrin and the hybridoma cell strain TRF M2 of the anti-human desugared transferrin are both preserved in the year 2022 and the day 12 and 21, and the preservation of human is requested by Takara biological engineering Co.
Compared with the prior art, the application has the beneficial effects that:
(1) The anti-CDT monoclonal antibody M1 and the anti-CDT monoclonal antibody M2 are antibodies capable of recognizing the natural CDT structure, are two monoclonal antibody pairs capable of recognizing different CDT epitopes respectively, and can be used for directly testing the natural CDT in a blood or body fluid sample without separating the CDT from other serum proteins and transferrin isomers other than the CDT and without preprocessing the sample.
(2) The CDT and TRF proteins are only different in that glycosylation sites are deleted, and two CDT monoclonal antibodies capable of recognizing different epitopes cannot be prepared simultaneously by a conventional method due to smaller antigen recognition epitope space, so that a kit for CDT specificity detection can only select a competition method.
(3) Preparing CDT antigen by protein recombination technology, using the CDT antigen as antigen to perform animal immunization, then fusing cells to obtain hybridoma cells producing antibodies, screening the hybridoma cells producing CDT antibodies by sandwich ELISA, confirming with natural CDT and performing reverse screening with TRF to obtain hybridoma cell strain TRF 1 producing anti-CDT monoclonal antibody M1 and hybridoma cell strain TRF M2 producing anti-human desugared transferrin of anti-CDT monoclonal antibody M2. In the prior art, TRF with isoelectric point of 5.4 is incubated with neuraminidase together to artificially prepare CDT with isoelectric point of 5.7, the protein denaturation treatment often leads to incomplete confirmation of protein conformation, the preparation of CDT antigen of the application overcomes the problems, and the specificity of the anti-CDT monoclonal antibody M1 and the anti-CDT monoclonal antibody M2 prepared by the application to natural CDT is ensured through natural CDT confirmation and reverse screening with TRF.
(4) The detection kit is based on a latex enhanced immunoturbidimetry method of transmission or scattering, can improve the accuracy of measurement and expand the measurement range; the matched use of general automatic analysis equipment, such as a full-automatic biochemical analyzer or a specific protein analyzer, can reduce the economic cost.
(5) The CDT detection kit can be used for directly detecting the concentration of CDT in a sample and simultaneously calculating the CDT: TRF ratio, improves the diagnostic specificity of CDT in patients with increased TRF.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments of the present application will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a complete Mass spectrum (extract Mass) of recombinant human serum Transferrin (TRF);
FIG. 2 is a complete Mass spectrum (identity Mass) of recombinant glycosyl deleted transferrin (CDT);
FIG. 3 is a gel electrophoresis of recombinant TRF and recombinant CDT;
FIG. 4 is a graph showing the absorbance detection results of monoclonal antibodies at different concentrations.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application. The present application will be specifically described with reference to the following specific examples.
The embodiment of the application provides a monoclonal antibody pair of sugar-deficient transferrin, which comprises an anti-CDT monoclonal antibody M1 and an anti-CDT monoclonal antibody M2 which can be used in a paired manner, wherein the anti-CDT monoclonal antibody M1 and the anti-CDT monoclonal antibody M2 are respectively combined with different epitopes of CDT antigens, and neither the anti-CDT monoclonal antibody M1 nor the anti-CDT monoclonal antibody M2 is combined with TRF, wherein the anti-CDT monoclonal antibody M1 is produced by a hybridoma cell strain TRF 1 of the anti-human desugar transferrin with a preservation number of CCTCC NO: C2022391, the anti-CDT monoclonal antibody M2 is produced by a hybridoma cell strain TRF 2 of the anti-human desugar transferrin with a preservation number of CCTCC NO: C2022392, and the hybridoma cell strain TRF 1 of the anti-human desugar transferrin is required to be preserved for a biological engineering of a deep-cut in the upper limit of the human being required for a year of 21.
Specifically, the CDT antigen is a native CDT antigen or a recombinant CDT antigen prepared using protein recombination techniques.
Specifically, the preparation method of the anti-CDT monoclonal antibody M1 and the anti-CDT monoclonal antibody M2 comprises the following steps:
s1, performing animal immunization by taking the recombinant CDT antigen as an antigen;
s2, extracting cells of the animal in the S1 and fusing the cells with myeloma to generate hybridoma cells;
s3, selecting the hybridoma cells producing the anti-CDT monoclonal antibody M1 and the anti-CDT monoclonal antibody M2 by sandwich ELISA by using the recombinant CDT antigen;
s4, confirming the hybridoma cells by using a natural CDT antigen through a sandwich ELISA method in the step S3;
s5, reversely screening the hybridoma cells by using TRF through a sandwich ELISA method which is the same as that of the step S3, so as to obtain a hybridoma cell strain TRF M1 for producing the anti-CDT monoclonal antibody M1 and a hybridoma cell strain TRF M2 for producing the anti-human desugared transferrin of the anti-CDT monoclonal antibody M2.
The embodiment of the application also provides a high-specificity and high-affinity detection kit for the sugar-deleted transferrin, which comprises a reagent R1, a reagent R2, a calibrator and a quality control product, wherein the reagent R1 comprises the latex microsphere conjugate of the anti-CDT monoclonal antibody M1 and the latex microsphere conjugate of the anti-CDT monoclonal antibody M2.
Specifically, the calibrator and quality control comprise CDT antigens as described above.
Specifically, the preparation method of the latex microsphere conjugate of the anti-CDT monoclonal antibody M1 and the latex microsphere conjugate of the anti-CDT monoclonal antibody M2 comprises the following steps:
s1, sensitization: placing the emulsion solution into a centrifuge tube, adding a sensitization buffer solution, adding the anti-CDT monoclonal antibody M1 or the anti-CDT monoclonal antibody M2, uniformly mixing, and placing the mixture at 37 ℃ for reaction for 1 hour;
s2, sealing: adding a sealing buffer solution, performing ultrasonic treatment for 2 minutes, sealing at 37 ℃ for 1 hour, and centrifuging to remove unreacted parts in the supernatant after sealing;
s3, adding 1mL of sensitization buffer solution, and carrying out ultrasonic treatment on the latex, wherein the absorbance of the latex microsphere conjugate of the anti-CDT monoclonal antibody M1 at 700nm is regulated to be 0.8Abs by the sensitization buffer solution.
Specifically, the sensitization buffer was 20mM MES at pH6.1, the sensitization buffer was 10mM PBS at pH 7.5, and the blocking buffer included 10mM PBS at pH 7.5, 10% bovine serum albumin, and 0.095% w/w sodium azide.
Specifically, the concentration of the solution after the anti-CDT monoclonal antibody M1 or the anti-CDT monoclonal antibody M2 is added in the step S1 is 0.03-0.09 mg/mL.
The embodiment of the application also provides a hybridoma cell pair, wherein the hybridoma cell pair is respectively used for producing the monoclonal antibody pair, the hybridoma cell pair is a hybridoma cell strain TRF 1 of anti-human desugared transferrin with the preservation number of CCTCC NO: C2022391 for producing the anti-CDT monoclonal antibody M1 and a hybridoma cell strain TRF M2 of anti-human desugared transferrin with the preservation number of CCTCC NO: C2022392 for producing the anti-CDT monoclonal antibody M2, the hybridoma cell strain TRF M1 of the anti-human desugared transferrin and the hybridoma cell strain TRF M2 of the anti-human desugared transferrin are preserved in the year of 2022 and 12 months 21, and the preservation of human is requested to be made by Takara bioengineering limited on Shenzhen.
The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available and, unless otherwise indicated, the techniques not described in detail are carried out according to standard methods well known to those skilled in the art. The cell lines, reagents and vectors mentioned in the present application are commercially available or otherwise publicly available, by way of example only, are not exclusive of the present application, and can be replaced with other suitable tools or biological materials, respectively.
Example 1 preparation, identification and use of immunogens
Preparation of immunogens
The coding sequence for human serum transferrin was assembled from restriction fragments of full-length cDNA clones from a human liver cDNA library. The latest TRF expressed by human transferrin gene is selected, and the sequence of the TRF is SEQ ID NO.1, as follows:
high-level recombinant human serum Transferrin (TRF) was synthesized using molecular biology methods of gene synthesis, and the size and purity of the TRF molecules were judged by electrophoretic analysis (FIG. 1, agreement with TRF isolated from natural serum), with complete glycosylation sites and complete function.
And then using protein recombination technology to replace single amino acid residues involved in anion binding, salt effect, receptor interaction and metal binding regulation with isotopically substituted amino acids, wherein asparagine residues at 432 and 630 are mutated to aspartic acid (figure 2), and deglycosylated-TRF (deglyco-TRF) without glycosylation sites, namely CDT antigen, is obtained.
Identification of immunogens
The structure and isoelectric point of CDT antigen were verified to be consistent with native CDT by isoelectric focusing gel electrophoresis (fig. 3).
The specificity and accuracy of the CDT antigen are detected by ELISA sandwich method, specifically, the anti-CDT monoclonal antibody M1 (anti-deglyco-TRF M1) is coated, the CDT antigen or natural CDT sandwich is used, and the anti-CDT monoclonal antibody M2 (anti-deglyco-TRF M2) is used.
Application of immunogen
The CDT antigen is also used as a main raw material of a matched calibrator and quality control product in the CDT detection kit. A calibrator prepared with CDT antigen or native CDT protein of the present application was mixed with two latex microspheres sensitized with two monoclonal antibodies M1 and M2, respectively, having different epitopes to cause agglutination, and turbidity changes of the agglutination were measured by transmitted or scattered light signals of the instrument.
Example 2 preparation of antibodies
CDT antigen obtained in example 1 was used as antigen, and BALB/c mice of 5 weeks of age were immunized subcutaneously. The extracted spleen cells are fused with mouse myeloma to produce hybridoma cells. Hybridoma cells producing CDT antibodies were selected by sandwich ELISA and confirmed with native CDT. Reverse screening was performed with TRF to confirm the specificity of the anti-CDT monoclonal antibody to native CDT.
Immunization of animals
CDT antigen solution at 0.5mg/mL was mixed with the same volume of adjuvant and emulsified, and 5 week old BALB/c mice were immunized subcutaneously. The immunization was boosted every two weeks for 3 total immunizations. Blood samples were taken prior to each immunization to detect antibody activity in the blood. After 14 days of the last immunization, 100 μl of antigen emulsion was injected into the peritoneal cavity of the mice, and after 3 days the spleens were removed for fusion.
Cell fusion
The mouse spleen cells, myeloma cells (P3X163 Ag8.653) and polyethylene glycol 4000 were mixed in HAT selective medium and fused for 2 minutes, and the mixture was dispensed into 96-well plates and cultured in a carbon dioxide incubator at 37℃to prepare hybridoma cells.
Selection and confirmation of hybridoma cells
The hybridoma cells which can produce antibodies in the hybridoma cells prepared by screening are detected and selected by a sandwich ELISA method, and the specific steps comprise:
a. 10. Mu.g/mL of anti-mouse antibody in PBS was added to 96-well microwell plates, 200. Mu.L per well, and reacted overnight at 4 ℃;
b. washing the microplate once with PBS solution, and blocking with PBS solution containing 0.5% BSA;
c. 100 μl of culture supernatant of hybridoma cells was added to a microplate and reacted at room temperature for 1 hour;
d. washing with PBS washing solution containing 0.05% Tween;
e. CDT antigen or TRF was added and reacted at room temperature for 1 hour and washed as above;
f. 100. Mu.L of an alkaline phosphatase (HRP enzyme) -labeled anti-transferrin polyclonal antibody (obtained by purchase) was added and allowed to react at room temperature for 1 hour;
h. after washing again, adding a substrate to start color development;
i. absorbance at 490nm was measured with a microplate reader.
Hybridoma cells having antibody activity against CDT antigen but not against TRF are selected.
The hybridoma cells of the CDT monoclonal antibody generated by the same sandwich ELISA are confirmed by the natural CDT of human serum, so that the hybridoma cells are obtained, and the monoclonal antibody generated by the hybridoma cells can be specifically combined with the natural CDT in the human serum, so that the target antibody can be screened.
Example 3 screening of paired antibodies
Screening monoclonal antibodies, taking out two monoclonal antibodies from the monoclonal antibodies, taking one monoclonal antibody as a capture antibody and the other monoclonal antibody as a labeled antibody (HRP enzyme label), coating the capture antibody on an antigen plate, adding an antigen (CDT antigen or natural CDT), incubating, washing off unbound antigen, adding the labeled antibody, incubating, washing off unbound labeled antibody, and finally adding a color development liquid for color development. If color development is possible, it is indicated that the labeled antibody specifically binds to the antigen, and the capture antibody and the labeled antibody are a pair of paired antibodies. If color development is not possible, it means that the labeled antibody cannot bind to the antigen and is eluted, and the capture antibody and the labeled antibody are not paired antibodies. The obtained anti-CDT monoclonal antibody M1 and anti-CDT monoclonal antibody M2 are the matched antibodies of natural CDT, wherein the anti-CDT monoclonal antibody M1 is generated by a hybridoma cell strain TRF M1 of the anti-human desugared transferrin with the preservation number of CCTCC NO: C2022391, and the anti-CDT monoclonal antibody M2 is generated by a hybridoma cell strain TRF M2 of the anti-human desugared transferrin with the preservation number of CCTCC NO: C2022392, and has good correlation with the concentration of the natural CDT tested by control.
Example 4 preparation and use of CDT immunoturbidimetry detection kit
Preparation of CDT immunoturbidimetry detection kit
The CDT latex immunoturbidimetry detection kit comprises: r1 reagent, R2 reagent, calibrator and quality control product. The R1 reagent is a buffer solution containing a coagulant, the R2 reagent mainly contains a mixed suspension of an anti-CDT monoclonal antibody M1 latex microsphere conjugate and an anti-CDT monoclonal antibody M2 latex microsphere conjugate, and the calibrator and the quality control product are prepared by dissolving CDT antigens prepared in example 1 with different concentrations in a specific buffer solution.
Preparation of anti-CDT monoclonal antibody M1/M2 latex microsphere conjugate (monoclonal antibody concentration optimization): 100. Mu.L of a 10% solution of latex having a particle size of 0.13 μm was placed in a 1.5mL centrifuge tube, 825. Mu.L of 20mM MES having a pH of 6.1 was added thereto, and a certain amount of 10mg/mL of the anti-CDT M1 monoclonal antibody was further added thereto so that the concentration of the antibody in the solution became 0.02mg/mL, 0.03mg/mL, 0.05mg/mL, 0.06mg/mL, 0.07mg/mL, 0.08mg/mL and 0.09mg/mL, and the mixture was uniformly mixed and allowed to react at 37℃for 1 hour. After sensitization, 100. Mu.L of blocking buffer (10 mM PBS, pH 7.5, 10% bovine serum albumin and 0.095% w/w sodium azide) was added and after 2 minutes of sonication, blocking was performed at 37℃for 1 hour. After the completion of the blocking, centrifugation was performed to remove unreacted portions of the supernatant. 1mL of 10mM PBS pH 7.5 was added and the latex sonicated. The absorbance at 700nm of the latex microsphere conjugate of anti-CDT monoclonal antibody M1 was adjusted to 0.8Abs with 10mM PBS pH 7.5.
anti-CDT monoclonal antibody M2 latex microsphere conjugates were prepared by the same procedure using anti-CDT monoclonal antibody M2 priming.
Use of CDT immunoturbidimetry detection kit
Measurements were made using a Hitachi automatic analyzer Model 7180. After reacting 150 mu L R reagent with 5 mu L of calibrator with each concentration for 0-5 min, adding 50 mu LR2 reagent, recording absorbance A1 30 seconds after adding R2 reagent, reacting for 1-5 min, recording absorbance A2, taking the difference between absorbance A2 and A1 as the reactivity, and taking the calibrator concentration as the abscissa and the reactivity as the ordinate as a calibration curve. And then testing the sample according to the steps, and taking the absorbance of the tested sample into a calibration curve equation to calculate the concentration of the sample. The absorbance was measured at 570nm and the measuring points were 19-34.
The different concentrations of CDT antigen were detected using R2 reagents prepared from different concentrations of anti-CDT M1 monoclonal antibodies prepared in the above procedure, and the results are shown in FIG. 4. As shown in FIG. 4, the sensitivity was insufficient at an antibody concentration of 0.02mg/mL in the reaction solution, but an increase in CDT-dependent absorbance was confirmed at 0.03mg/mL to 0.09mg/mL, indicating that this range is the range of use of the antibody.
The detection principle can be an immunonephelometry or a nephelometry, and the detection instrument can be applicable to, but not limited to, biochemical analysis and specific protein analyzers.
CDT can be calculated by testing the concentration of CDT and the concentration of TRF, respectively, as described above: ratio of TRF.
Example 5 Performance test of CDT immunoturbidimetry detection kit
a. Blank limit
The measurement was repeated 20 times in the above-mentioned procedure using 5ul of physiological saline as a test sample having a CDT concentration of 0, and the results of the test blank are shown in Table 1.
| Number of tests | Test concentration (mg/dL) |
| 1 | 0 |
| 2 | 0.1 |
| 3 | -0.3 |
| 4 | -0.2 |
| 5 | 0.1 |
| 6 | 0 |
| 7 | 0.2 |
| 8 | -0.2 |
| 9 | 0.1 |
| 10 | -0.2 |
| 11 | 0.2 |
| 12 | -0.3 |
| 13 | -0.1 |
| 14 | 0 |
| 15 | 0 |
| 16 | -0.4 |
| 17 | -0.1 |
| 18 | -0.3 |
| 19 | 0.1 |
| 20 | 0 |
| ave | -0.065 |
| SD | 0.1785 |
| ave+2SD | 0.292 |
The ave+2SD value of CDT reagent was 0.292, indicating that the reagent had a good blank.
b. Repeatability of
Serum samples at 4 different CDT concentrations were tested in duplicate 10 times for each sample and the test repeatability results are shown in table 2.
| Number of tests | Sample 1 (mg/dL) | Sample 2 (mg/dL) | Sample 3 (mg/dL) | Sample 4 (mg/dL) |
| 1 | 1.2 | 4.9 | 17.3 | 25.6 |
| 2 | 1.3 | 4.9 | 17.2 | 25.7 |
| 3 | 1.3 | 4.6 | 17.1 | 25.4 |
| 4 | 1.3 | 4.9 | 17.4 | 25.3 |
| 5 | 1.3 | 4.8 | 17.6 | 25.5 |
| 6 | 1.3 | 4.8 | 17.8 | 25.8 |
| 7 | 1.2 | 4.7 | 17.7 | 24.9 |
| 8 | 1.3 | 4.7 | 17.2 | 24.8 |
| 9 | 1.4 | 4.9 | 17.8 | 25.6 |
| 10 | 1.3 | 4.7 | 17.9 | 25.4 |
| ave | 1.29 | 4.79 | 17.5 | 25.4 |
| SD | 0.0568 | 0.1101 | 0.2944 | 0.3266 |
| CV | 4.40％ | 2.30％ | 1.68％ | 1.29％ |
Serum samples with different concentrations are tested, and the variation coefficient of test repeatability is less than 5%, so that the CDT detection kit has good repeatability.
c. Linear range
Dissolving purified natural CDT in negative serum, respectively preparing low-concentration CDT and high-concentration CDT, mixing the high-concentration CDT and the low-concentration CDT into 4 different concentrations, respectively detecting the CDT samples with 6 different concentrations by using a detection kit, testing each concentration for 3 times, and respectively obtaining the average value of detection results. And (3) taking the dilution concentration as an independent variable, and taking the average value of the detection result as a dependent variable to solve a linear regression equation. The results are shown in Table 3.
The linearity of the CDT detection kit is in the range of 1.2-120mg/dL, the correlation coefficient is 0.9998, the absolute deviation is smaller than 1.2mg/dL, and the relative deviation is within +/-5%, which indicates that the linearity is good.
The above description is only of the preferred embodiments of the present application and is not intended to limit the application, but any modifications, equivalents, improvements, etc. within the principles of the present application should be included in the scope of the present application.
Claims (6)
1. A high-specificity, high-affinity, glyco-deleted transferrin paired monoclonal antibody comprising an anti-CDT monoclonal antibody M1 and an anti-CDT monoclonal antibody M2, said anti-CDT monoclonal antibody M1 and anti-CDT monoclonal antibody M2 respectively binding to different epitopes of a CDT antigen, said anti-CDT monoclonal antibody M1 and anti-CDT monoclonal antibody M2 not binding to TRF, wherein said anti-CDT monoclonal antibody M1 is produced by a hybridoma cell line TRF M1 of anti-human desugared transferrin deposited with the accession number cctccc NO: C2022391, said anti-CDT monoclonal antibody M2 is produced by a hybridoma cell line TRF M2 of anti-human desugared transferrin deposited with the accession number cctccc NO: C2022392, said CDT antigen is a natural CDT antigen or a recombinant CDT antigen prepared using a protein recombination technique, and said hybridoma cell line TRF M1 and hybridoma cell line TRF 2 are both obtained using a recombinant protein recombination technique as an immunogen.
2. A kit for detecting high-specificity, high-affinity sugar-deficient transferrin, comprising a reagent R1, a reagent R2, a calibrator and a quality control, wherein the reagent R1 comprises the latex microsphere conjugate of the anti-CDT monoclonal antibody M1 of claim 1 and the latex microsphere conjugate of the anti-CDT monoclonal antibody M2 of claim 1.
3. The test kit of claim 2, wherein the calibrator and quality control comprise the CDT antigen of claim 1.
4. The detection kit as claimed in claim 2, wherein the preparation method of the latex microsphere conjugate of the anti-CDT monoclonal antibody M1 and the anti-CDT monoclonal antibody M2 comprises the following steps:
s1, sensitization: placing the emulsion solution into a centrifuge tube, adding a sensitization buffer solution, adding the anti-CDT monoclonal antibody M1 or the anti-CDT monoclonal antibody M2, uniformly mixing, and placing the mixture at 37 ℃ for reaction for 1 hour;
s2, sealing: adding a sealing buffer solution, performing ultrasonic treatment for 2 minutes, sealing at 37 ℃ for 1 hour, and centrifuging to remove unreacted parts in the supernatant after sealing;
s3, adding 1mL of sensitization buffer solution, and carrying out ultrasonic treatment on the latex, wherein the absorbance of the latex microsphere conjugate of the anti-CDT monoclonal antibody M1 at 700nm is regulated to be 0.8Abs by the sensitization buffer solution.
5. The test kit of claim 4, wherein the priming buffer is 10mM PBS at pH 7.5 and the blocking buffer comprises 10mM PBS at pH 7.5, 10% bovine serum albumin, and 0.095% w/w sodium azide.
6. The test kit according to claim 4, wherein the concentration of the solution after adding the anti-CDT monoclonal antibody M1 or the anti-CDT monoclonal antibody M2 in the step S1 is 0.03-0.09 mg/mL.
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| EP1378521A1 (en) * | 2002-07-05 | 2004-01-07 | Dade Behring Marburg GmbH | Anti-Carbohydrate Deficient Transferrin (CDT) spezific antibody, its production and use |
| CN1732261A (en) * | 2002-12-24 | 2006-02-08 | 日东纺绩株式会社 | Marker proteins for diagnosing liver disease and method of diagnosing liver disease using the same |
| JP2007254428A (en) * | 2006-03-25 | 2007-10-04 | Nissui Pharm Co Ltd | Antibody against natural unmodified cdt, method for producing antibody, hybridoma and immunoassay method |
| CN113866406A (en) * | 2021-10-18 | 2021-12-31 | 深圳上泰生物工程有限公司 | Kit for specifically detecting sugar-deficient transferrin |
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| JP5199067B2 (en) * | 2006-03-31 | 2013-05-15 | 日水製薬株式会社 | Immunoagglutination reagent kit and antigen measurement method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1378521A1 (en) * | 2002-07-05 | 2004-01-07 | Dade Behring Marburg GmbH | Anti-Carbohydrate Deficient Transferrin (CDT) spezific antibody, its production and use |
| CN1732261A (en) * | 2002-12-24 | 2006-02-08 | 日东纺绩株式会社 | Marker proteins for diagnosing liver disease and method of diagnosing liver disease using the same |
| JP2007254428A (en) * | 2006-03-25 | 2007-10-04 | Nissui Pharm Co Ltd | Antibody against natural unmodified cdt, method for producing antibody, hybridoma and immunoassay method |
| CN113866406A (en) * | 2021-10-18 | 2021-12-31 | 深圳上泰生物工程有限公司 | Kit for specifically detecting sugar-deficient transferrin |
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Inventor after: Wu Xiangdong Inventor after: Zhang Ning Inventor after: Chen Xiaoru Inventor before: Wu Xiangdong Inventor before: Zhang Ning Inventor before: Zhou Liang Inventor before: Chen Xiaoru |
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