CN114478759A

CN114478759A - Anti-human hemoglobin antibody, application thereof and diagnostic kit

Info

Publication number: CN114478759A
Application number: CN202011259381.7A
Authority: CN
Inventors: 崔鹏; 何志强; 孟媛; 钟冬梅; 姜瑢瑢; 何雯雯; 范凌云
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2020-11-12
Filing date: 2020-11-12
Publication date: 2022-05-13

Abstract

The invention discloses an anti-human hemoglobin antibody and application thereof and a diagnostic kit, and relates to the technical field of antibodies. The anti-human hemoglobin antibody or functional fragment thereof disclosed by the invention has a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody has better binding specificity and affinity to human hemoglobin, can be used for detecting the human hemoglobin in a sample such as a stool sample, and provides more antibody choices for the detection of the human hemoglobin and the diagnosis of related diseases.

Description

Anti-human hemoglobin antibody, application thereof and diagnostic kit

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-human hemoglobin antibody and application thereof and a diagnostic kit.

Background

Hemoglobin (Hb), also called Fecal Occult Blood (FOB), in stool is currently clinically recognized as one of the tumor markers for rectal and colon cancers. Occult blood means bleeding with digestive tract bleeding amount below 5ml/d, no visible blood color, and no erythrocyte destruction under microscope. The fecal occult blood test is a routine diagnosis test of gastrointestinal bleeding, has great significance for diagnosing gastrointestinal diseases, and is also an important screening means for digestive tract tumors.

Clinical studies prove that the trace hemorrhage with cancer and tumor in feces is the only abnormal phenomenon that can be found out in early intestinal cancer, so that occult blood experiments become the hot spot of continuous and intensive research of people. In the past, chemical methods are used for detecting fecal occult blood, and in recent years, a colloidal gold immunochromatography method is developed, and compared with the traditional chemical methods, the immunoassay method for detecting hemoglobin monoclonal antibodies has the advantages that the sensitivity and specificity are greatly improved, the immunoassay method is not influenced by diet or related medicines, and the immunoassay method plays an important role in diagnosis and treatment of early intestinal cancer.

The fecal occult blood test is the main diagnostic indicator of gastrointestinal bleeding and tumors. At present, a screening test method commonly used in fecal occult blood tests is a simple chemical examination method designed by utilizing heme in hemoglobin (Hb), which is a main component of blood, and having the effect similar to peroxidase. The methods have many oxidation color developing agents, such as benzidine, o-toluidine, o-tolidine, guaiazulene, reduced phenolphthalein, aminopyridine, colorless malachite green, tetramethylbenzidine, diphenylamine, and dimethylbenzidine. The method is simple and easy to implement, but has low specificity and more interference factors. Animal blood, meat, liver, chlorophyll-rich food, iron, vitamin C and Chinese medicine can all cause false positive.

Compared with the method, the colloidal gold immunochromatography based on the immunological principle has the obvious advantages of fast diagnosis, better specificity without being interfered by substances such as animal blood and the like, simple operation and no need of special equipment, and the main raw material is the specific monoclonal antibody aiming at the FOB. In techniques for detecting antigens or antibodies based on immunological principles, the HOOK effect occurs due to inappropriate ratios of antigen to antibody. When the HOOK effect occurs, the ratio of antigen to antibody may be adjusted as required by those skilled in the art.

However, the existing monoclonal antibodies aiming at human hemoglobin are few, and the sensitivity, the affinity and the specificity are all defective.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an anti-human hemoglobin antibody, application thereof and a detection kit. The antibody has better binding specificity and affinity to human hemoglobin, can be used for detecting the human hemoglobin in samples such as stool samples, and provides more antibody choices for the detection of the human hemoglobin and the diagnosis or auxiliary diagnosis of related diseases taking the human hemoglobin as a marker.

The invention is realized by the following steps:

in one aspect, the present invention provides an antibody against human hemoglobin or a functional fragment thereof, the antibody or functional fragment thereof having the following complementarity determining regions:

CDR-VH 1: G-X1-S-X2-T-S-X3-Y-S-W-H; wherein: x1 is F or Y; x2 is L, V or I; x3 is N or D;

CDR-VH 2: Y-X1-H-Y-S-G-S-X2-X3-Y-N-P-S-X4-K-S; wherein: x1 is I or L; x2 is S or T; x3 is S or T; x4 is L or I;

CDR-VH 3: A-X1-T-Y-Y-X2-F-Y-A; wherein: x1 is Q, R or K; x2 is A or G;

CDR-VL 1: A-S-X1-S-X2-D-S-Y-G-N-S-F-X3-H; wherein: x1 is Q or E; x2 is I, V or L; x3 is I, V or L;

CDR-VL 2: X1-A-S-N-X2-A-S; wherein: x1 is I, V or L; x2 is I, V or L;

CDR-VL 3: Q-Q-N-X1-E-X2-P-Y; wherein: x1 is Q, N or H; x2 is D or N.

The antibody or the functional fragment thereof with the complementarity determining region structure can be specifically combined with human hemoglobin, has higher affinity to the human hemoglobin, is used for detecting the human hemoglobin, and has better sensitivity and specificity. The antibody can be used for diagnosing or assisting in diagnosing related tumors with human hemoglobin as a marker, such as detecting FOB and assisting in diagnosing intestinal cancer. The invention provides more antibody choices for the detection of human hemoglobin and the diagnosis or auxiliary diagnosis of related diseases using the human hemoglobin as a marker.

In alternative embodiments, in CDR-VH1, X1 is Y; in CDR-VH2, X1 is I; in CDR-VH3, X2 is G; in CDR-VL1, X1 is E; in CDR-VL3, X2 is D.

In each CDR, when CDR-VH1, X1 is Y; in CDR-VH2, X1 is I; in CDR-VH3, X2 is G; in CDR-VL1, X1 is E; in CDR-VL3, when X2 is D, the affinity of the antibody for human hemoglobin is further improved.

In some embodiments, X2 is L in CDR-VH 1.

In some embodiments, X2 is V in CDR-VH 1.

In some embodiments, X2 is I in CDR-VH 1.

In some embodiments, in CDR-VH1, X3 is N.

In some embodiments, in CDR-VH1, X3 is D.

In some embodiments, X2 is S in CDR-VH 2.

In some embodiments, X2 is T in CDR-VH 2.

In some embodiments, X3 is S in CDR-VH 2.

In some embodiments, X3 is T in CDR-VH 2.

In some embodiments, X4 is L in CDR-VH 2.

In some embodiments, X4 is I in CDR-VH 2.

In some embodiments, in CDR-VH3, X1 is Q.

In some embodiments, X1 is R in CDR-VH 3.

In some embodiments, in CDR-VH3, X1 is K.

In some embodiments, X2 is I in CDR-VL 1.

In some embodiments, X2 is V in CDR-VL 1.

In some embodiments, X2 is L in CDR-VL 1.

In some embodiments, X3 is I in CDR-VL 1.

In some embodiments, X3 is V in CDR-VL 1.

In some embodiments, X3 is L in CDR-VL 1.

In some embodiments, X1 is I in CDR-VL 2.

In some embodiments, X1 is V in CDR-VL 2.

In some embodiments, X1 is L in CDR-VL 2.

In some embodiments, X2 is I in CDR-VL 2.

In some embodiments, X2 is V in CDR-VL 2.

In some embodiments, X2 is L in CDR-VL 2.

In some embodiments, X1 is Q in CDR-VL 3.

In some embodiments, X1 is N in CDR-VL 3.

In some embodiments, X1 is H in CDR-VL 3.

In alternative embodiments, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 1-51:

in alternative embodiments, the antibody or functional fragment thereofWith human hemoglobin by K_D≤1×10^-9Affinity binding in mol/L.

In an alternative embodiment, K_D≤1×10^-9mol/L、K_D≤9×10^-10mol/L、K_D≤8×10^-10mol/L、K_D≤7×10^-10mol/L、K_D≤6×10^-10mol/L、K_D≤5×10^-10mol/L、K_D≤4×10^-10mol/L、K_D≤3×10^- ¹⁰mol/L、K_D≤2×10^-10mol/L、K_D≤1×10^-10mol/L、K_D≤9×10^-11mol/L、K_D≤8×10^-11mol/L、K_D≤7×10^-11mol/L、K_D≤6×10^-11mol/L、K_D≤5×10^-11mol/L、K_D≤4×10^-11mol/L、K_D≤3×10^- ¹¹mol/L、K_D≤2×10^-11mol/L or K_D≤1×10^-11mol/L。

In an alternative embodiment, K_D≤9×10^-10mol/L。

In an alternative embodiment, K_D≤8×10^-11mol/L。

In an alternative embodiment, 1.14 × 10^-11mol/L≤K_D≤7.97×10^-11mol/L。

K_DThe detection of (2) is carried out with reference to the method in the examples of the present invention.

In alternative embodiments, in CDR-VH1, X1 is F; in CDR-VH2, X1 is L; in CDR-VH3, X2 is A; in CDR-VL1, X1 is Q; in CDR-VL3, X2 is N.

In alternative embodiments, each complementarity determining region of the antibody, or functional fragment thereof, is selected from any one of the following combinations of mutations 52-58:

in alternative embodiments, the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L in sequence as set forth in SEQ ID NOS: 1-4, and/or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H in sequence as set forth in SEQ ID NOS: 5-8.

In general, the variable regions of the heavy chain (VH) and light chain (VL) can be obtained by linking the CDRs and FRs numbered below in a combined arrangement as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4.

It is noted that in other embodiments, each framework region amino acid sequence of an antibody or functional fragment thereof provided herein can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the corresponding framework region described above (SEQ ID NO:1, 2, 3, 4, 5, 6, 7, or 8).

In alternative embodiments, the antibody further comprises a constant region.

In alternative embodiments, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

In alternative embodiments, the species of the constant region is derived from sheep, goat, cow, horse, cow, pig, rat, dog, cat, rabbit, camel, donkey, mouse, deer, mink, duck, goose, turkey, chicken, or human.

In alternative embodiments, the constant region is derived from a mouse.

In alternative embodiments, the light chain constant region sequence of the constant region is set forth in SEQ ID NO. 9 and the heavy chain constant region sequence of the constant region is set forth in SEQ ID NO. 10.

In alternative embodiments, the functional fragment is selected from any one of F (ab ') 2, Fab, scFv, Fab', and Fv of the antibody.

Functional fragments of the above antibodies typically have the same binding specificity as the antibody from which they are derived. It will be readily understood by those skilled in the art from the disclosure of the present invention that functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction cleavage of disulfide bonds. Based on the disclosure of the structure of the intact antibody, the above-described functional fragments are readily available to those skilled in the art.

Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques also known to those skilled in the art or synthesized by, for example, automated peptide synthesizers, such as those sold by Applied BioSystems and the like.

In another aspect, the present invention provides the use of the anti-human hemoglobin antibody or functional fragment thereof according to any one of the above methods for preparing a reagent or a kit for diagnosis or auxiliary diagnosis of tumor, wherein the marker of tumor comprises human hemoglobin.

The antibody provided by the invention can be used for detecting human hemoglobin, so that the antibody can be used for diagnosis or auxiliary diagnosis of related tumors using the human hemoglobin as a marker.

In an alternative embodiment, the tumor is an intestinal cancer.

In an alternative embodiment, the intestinal cancer is selected from colorectal cancer and colon cancer.

Fecal Occult Blood (FOB) is considered as one of tumor markers of rectal cancer and colon cancer, but based on the common knowledge of the skilled person and the specific binding capacity and affinity of the antibody of the invention to human hemoglobin, the antibody of the invention can be used for detecting any tumor with human hemoglobin as a marker. Therefore, the antibody provided by the invention is also used for other tumors related to one of the human hemoglobin markers, and belongs to the protection scope of the invention.

In an alternative embodiment, the test sample in the reagent or kit is human stool.

In another aspect, the present invention provides a reagent or a kit for diagnosis or auxiliary diagnosis of a tumor associated with human hemoglobin as a marker, comprising the anti-human hemoglobin antibody or functional fragment thereof as described in any one of the above.

In another aspect, the present invention provides a reagent or a kit for detecting human hemoglobin, which comprises the antibody against human hemoglobin or a functional fragment thereof as described in any one of the above.

In an alternative embodiment, the antibody or functional fragment thereof in the above-described reagent or kit is labeled with a detectable label.

Detectable labels are substances having properties, such as luminescence, color development, radioactivity, etc., which can be observed directly by the naked eye or detected by an instrument, by which qualitative or quantitative detection of the respective target substance can be achieved.

In alternative embodiments, the detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze the development of a substrate, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

In the actual use process, one skilled in the art can select a suitable marker according to the detection condition or actual requirement, and whatever marker is used belongs to the protection scope of the present invention.

In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (e.g., including, but not limited to, Fluorescein Isothiocyanate (FITC) hydroxyphoton (FAM), tetrachlorofluorescein (TET), etc. or analogs thereof), rhodamine-based dyes and derivatives thereof (e.g., including, but not limited to, red Rhodamine (RBITC), Tetramethylrhodamine (TAMRA), rhodamine b (tritc), etc. or analogs thereof), Cy-series dyes and derivatives thereof (e.g., including, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or analogs thereof), Alexa-series dyes and derivatives thereof (e.g., including, but not limited to, Alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or analogs thereof), and protein-based dyes and derivatives thereof (e.g., including, but not limited to, Phycoerythrin (PE), Phycocyanin (PC), phycocyanin, pyrenochrome, and derivatives thereof, Allophycocyanin (APC), polymethacrylic flavin-chlorophyll protein (precP), etc.).

In alternative embodiments, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme.

In alternative embodiments, the radioisotope includes, but is not limited to²¹²Bi、¹³¹I、¹¹¹In、⁹⁰Y、¹⁸⁶Re、²¹¹At、¹²⁵I、¹⁸⁸Re、¹⁵³Sm、²¹³Bi、³²P、⁹⁴mTc、⁹⁹mTc、²⁰³Pb、⁶⁷Ga、⁶⁸Ga、⁴³Sc、⁴⁷Sc、¹¹⁰mIn、⁹⁷Ru、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁸Cu、⁸⁶Y、⁸⁸Y、¹²¹Sn、¹⁶¹Tb、¹⁶⁶Ho、¹⁰⁵Rh、¹⁷⁷Lu、¹⁷²Lu and¹⁸F。

in alternative embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridyl ruthenium and its derivatives, acridinium ester and its derivatives, dioxane and its derivatives, lotrine and its derivatives, and peroxyoxalate and its derivatives.

In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

In another aspect, the present invention provides a nucleic acid molecule encoding the above antibody or functional fragment thereof.

In another aspect, the present invention provides a vector comprising the nucleic acid molecule described above.

In another aspect, the present invention provides a recombinant cell comprising the vector described above.

In another aspect, the present invention provides a method of preparing an antibody or functional fragment thereof, comprising: culturing a recombinant cell capable of recombinantly expressing the anti-human hemoglobin antibody or functional fragment thereof according to any one of the above, and isolating and purifying the antibody or functional fragment thereof from the culture product.

Based on the disclosure of the amino acid sequence of the antibody or its functional fragment, it is easy for those skilled in the art to think that the antibody or its functional fragment can be prepared by genetic engineering techniques or other techniques (chemical synthesis, hybridoma cells), for example, the antibody or its functional fragment can be obtained by separation and purification from the culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment against human hemoglobin as described above, and therefore, it is within the scope of the present invention to prepare the antibody or its functional fragment by any technique.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the result of reducing SDS-PAGE of the anti-human hemoglobin antibody of example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit dosages herein, some are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. m.j. goal, 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), Handbook of Experimental Immunology (Handbook of Experimental Immunology) (ed. D.M.Weir and C.C.Black well), Gene Transfer Vectors for Mammalian Cells (ed. J.M.Miller and M.P.Calos) (ed. J.M.and M.P.Calos) (ed. 1987), Methods in Current Generation (Current Protocols in Molecular Biology) (ed. F.M.Ausubel.et al, 1987), PCR, Polymerase Chain Reaction (ed. PCR: The Polymerase Chain Reaction) (ed. Mullis et al, 1994), and Methods in Current Immunology (ed. J.1991).

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Restriction enzyme, Prime Star DNA polymerase, was purchased from Takara in this example. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART^TMRACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kit purchased fromTiangen corporation. Primer synthesis and gene sequencing were performed by Invitrogen corporation.

1 construction of recombinant plasmid

(1) Antibody Gene preparation

mRNA is extracted from a hybridoma cell strain (7G2) secreting an anti-human hemoglobin antibody, a DNA product is obtained by an RT-PCR method, the product is added with A by rTaq DNA polymerase for reaction and then inserted into a pMD-18T vector, the product is transformed into DH5 alpha competent cells, after colonies grow out, the Heavy Chain and Light Chain genes are respectively taken for cloning 4 clones, and the clones are sent to a gene sequencing company for sequencing.

(2) Sequence analysis of antibody variable region genes

Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 336bp, belongs to VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 357bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.

(3) Construction of recombinant antibody expression plasmid

pcDNA^TM3.4

vector is a constructed recombinant antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, BamHI, EcoRI and the like are introduced into the expression vector and are named as pcDNA3.4A expression vector, and the vector is called 3.4A expression vector for short in the following; according to the sequencing result of the antibody variable region gene in the pMD-18T, VL and VH gene specific primers of the antibody are designed, two ends of the primers are respectively provided with HindIII and EcoRI restriction sites and protective bases, and a Light Chain gene fragment of 0.74KB and a Heavy Chain gene fragment of 1.4KB are amplified by a PCR amplification method.

The gene fragments of the Heavy Chain and the Light Chain are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, the Heavy Chain gene and the Light Chain gene are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.

2 Stable cell line selection

(1) Transient transfection of recombinant antibody expression plasmid into CHO cells and determination of expression plasmid activity

Plasmid was diluted with ultrapure water to 40. mu.g/100. mu.L and CHO cells were conditioned at 1.43X 10⁷cells/ml are put into a centrifuge tube, 100 mu L of plasmid and 700 mu L of cells are mixed, transferred into an electric rotating cup, electrically rotated, sampled and counted on days 3, 5 and 7, and sampled and detected on day 7.

Coating liquid (main component NaHCO)₃) Diluting human hemoglobin antigen to 3 μ g/ml, 100 μ L per well, and standing overnight at 4 deg.C; the next day, washing liquid (main component Na)₂HPO₄NaCl) for 2 times, patting dry; adding blocking solution (20% BSA + 80% PBS), and drying at 37 deg.C for 1 hr in each well; adding diluted cell supernatant at 100 μ L/well, 37 deg.C for 30 min; washing with washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100 mu L per well at 37 ℃ for 30 min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 muL/hole, containing citric acid, sodium acetate, acetanilide and carbamide peroxide), and adding a developing solution B (50 muL/hole, containing citric acid, EDTA-2 Na, TMB and concentrated HCl) for 10 min; adding stop solution (50 μ L/well, EDTA-2 Na + concentrated H)₂SO₄) (ii) a OD readings were taken at 450nm (reference 630nm) on the microplate reader. The results show that the reaction OD after the cell supernatant is diluted 1000 times is still larger than 1.0, and the reaction OD of the wells without the cell supernatant is smaller than 0.1, which indicates that the antibody generated after the plasmid is transiently transformed has activity on the human hemoglobin antigen.

(2) Linearization of recombinant antibody expression plasmids

The following reagents were prepared: buffer 50 mu L, DNA 100 mu g/tube, Puv I enzyme 10 mu L, sterile water to 500 mu L, 37 ℃ water bath enzyme digestion overnight; sequentially extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1 and then chloroform (water phase); precipitating with 0.1 volume (water phase) of 3M sodium acetate and 2 volumes of ethanol on ice, rinsing with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water after ethanol is completely volatilized, and finally measuring concentration.

(3) Stable transfection of recombinant antibody expression plasmid, pressurized screening of stable cell lines

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10⁷cells/ml are put into a centrifuge tube, 100 mu L of plasmid is mixed with 700 mu L of cells, and the mixture is transferred into an electric rotating cup and is electrically rotated, and the next day is counted; 25umol/L MSX 96-well pressure culture for about 25 days.

Observing the marked clone holes with cells under a microscope, and recording the confluence degree; taking culture supernatant, and sending the culture supernatant to a sample for detection; selecting cell strains with high antibody concentration and relative concentration, transferring the cell strains into 24 holes, and transferring the cell strains into 6 holes after 3 days; after 3 days, the seeds were kept and cultured in batches, and the cell density was adjusted to 0.5X 10⁶cells/ml, 2.2ml, cell density 0.3X 10⁶cells/ml, 2ml for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample sending detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter to transfer TPP for seed preservation and passage.

3 recombinant antibody production

(1) Cell expanding culture

After the cells are recovered, the cells are cultured in a shaking flask with the specification of 125ml, the inoculation volume is 30ml, the culture medium is 100% Dynamis culture medium, and the cells are placed in a shaking table with the rotation speed of 120r/min, the temperature of 37 ℃ and the carbon dioxide of 8%. Culturing for 72h, inoculating and expanding culture at an inoculation density of 50 ten thousand cells/ml, wherein the expanding culture volume is calculated according to production requirements, and the culture medium is 100% Dynamis culture medium. Then the culture is expanded every 72 h. When the cell amount meets the production requirement, the production is carried out by strictly controlling the inoculation density to be about 50 ten thousand cells/ml.

(2) Shake flask production and purification

Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8 percent. Feeding in a flowing mode: daily feeding was started when the culture was carried out for 72h in a shake flask, 3% of the initial culture volume was fed daily to HyCloneTM Cell BoostTM Feed 7a, and one thousandth of the initial culture volume was fed daily to Feed 7b, up to day 12 (day 12 feeding). Glucose was supplemented with 3g/L on the sixth day. Samples were collected on day 13. Affinity purification was performed using a proteinA affinity column. Mu.g of the purified antibody was subjected to reducing SDS-PAGE, and 4. mu.g of an external control antibody was used as a control, and the electrophoretogram showed two bands, 1 of which Mr was 50KD (heavy chain, SEQ ID NO:14) and the other Mr was 28KD (light chain, SEQ ID NO:13), as shown in FIG. 1 below, after the reducing SDS-PAGE.

Example 2

Detection of antibody Performance

(1) Example 1 Activity assay of antibodies and mutants thereof

The antibody (WT) of example 1 was analyzed and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:12, where the amino acid sequence of each complementarity determining region is as follows:

CDR-VH1：G-F(X1)-S-L(X2)-T-S-D(X3)-Y-S-W-H；

CDR-VH2：Y-L(X1)-H-Y-S-G-S-S(X2)-S(X3)-Y-N-P-S-I(X4)-K-S；

CDR-VH3：A-K(X1)-T-Y-Y-A(X2)-F-Y-A；

the variable region amino acid sequence of the light chain is shown as SEQ ID NO. 11, wherein the amino acid sequences of the complementarity determining regions of the light chain are as follows:

CDR-VL1：A-S-Q(X1)-S-I(X2)-D-S-Y-G-N-S-F-L(X3)-H；

CDR-VL2：I(X1)-A-S-N-V(X2)-A-S；

CDR-VL3：Q-Q-N-N(X1)-E-N(X2)-P-Y。

based on the anti-human hemoglobin antibody (WT) of example 1, mutation was performed in the complementarity determining regions at sites involved in the activity of the antibody by a number of analyses, wherein X1, X2, X3, and X4 were all mutation sites. See table 1 below.

TABLE 1 mutant sites associated with antibody Activity

Antibody binding activity assay in table 1:

coating liquid (main component NaHCO)₃) Diluting human hemoglobin to 3 μ g/ml, 100 μ l per well, overnight at 4 ℃; the next day, washing liquid (main component Na)₂HPO₄NaCl) for 2 times, patting dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1 hr, and adding blocking solution (120 μ l per well); adding diluted purified antibody 100 μ l/well at 37 deg.C for 30 min; washing with washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100 mu l per well at 37 ℃ for 30 min; washing with washing solution for 5 times, and drying; adding color development liquid A (50 μ L/well containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide) and adding color development liquid B (50 μ L/well containing 1.05g/L citric acid, 0.186g/L LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10 min; stop solution (50. mu.l/well, containing 0.75 g/EDTA-2 Na and 10.2ml/L concentrated H) was added₂SO₄) 50 μ l/well; OD readings were taken at 450nm (reference 630nm) on the microplate reader. The results are shown in Table 2 below.

TABLE 2 Activity data of WT antibodies and mutants thereof

As can be seen from the data in table 2, WT, mutation 1 to mutation 4 had higher activity on human hemoglobin compared to mutation 5 and mutation 6, with the activity of mutation 1 being the best.

(2) Affinity detection of antibodies and mutants thereof

(a) Based on mutation 1, other sites were mutated, and the sequence of each mutation is shown in table 3 below.

TABLE 3 mutation sites related to antibody affinity

Affinity assay

Using AMC sensor, the purified antibody is diluted to 10 mug/ml by PBST, and the human hemoglobin is diluted by PBST in gradient;

the operation flow is as follows: buffer 1(PBST, main component Na)₂HPO₄+ NaCl + TW-20), immobilized antibody in antibody solution for 300s, incubation in buffer 2(PBST) for 180s, binding in antigen solution for 420s, dissociation in buffer 2 for 1200s, sensor regeneration with 10mM pH 1.69GLY solution and buffer 3, and data output. K_DRepresents the equilibrium dissociation constant, i.e., affinity; kon denotes the binding rate; kdis denotes the off-rate. The results are shown in Table 4 below.

Table 4 affinity assay data

As can be seen from the data in Table 4, the affinity of the mutant 1 and the mutants in the series is higher, which indicates that the antibodies obtained by mutation in the way of the mutation in Table 3 have higher affinity on the basis of the mutant 1.

(b) Based on WT, mutation is carried out on other sites, and the affinity of each mutant is detected, the sequence of each mutation is shown in Table 5, and the corresponding affinity data is shown in Table 6.

TABLE 5 mutations with WT as backbone

TABLE 6 affinity assay results for WT antibodies and their mutants

	K_D(M)	kon(1/Ms)	kdis(1/s)
				WT	7.32E-10	9.18E+04	6.72E-05
WT 1	6.32E-10	8.13E+04	5.14E-05
				WT 2	9.01E-10	6.78E+04	6.11E-05
WT 3	5.95E-10	8.75E+04	5.21E-05
				WT 4	9.28E-10	7.12E+04	6.61E-05
WT 5	1.07E-09	5.07E+04	5.44E-05
				WT 6	7.57E-10	6.96E+04	5.27E-05

As can be seen from the data in Table 6, the affinity of WT and its series of mutants is also better, which indicates that the antibodies obtained by mutation in the way of the mutation in Table 5 have better affinity on the basis of WT.

(3) Evaluation of stability against naked antibody

Placing the antibody in a temperature range of 4 ℃ (refrigerator), -80 ℃ (refrigerator) and 37 ℃ (thermostat) for 21 days, taking samples in 7 days, 14 days and 21 days for state observation, and performing activity detection on the samples in 21 days, wherein the result shows that under three examination conditions, no obvious protein state change is seen in 21 days of placing the antibody, and the activity does not show a descending trend along with the rise of the examination temperature, which indicates that the antibody is stable. The following table 7 shows the OD results of the enzyme immunity activity assay for 21 days of the mutation 1 antibody test.

TABLE 7

Antibody concentration (ng/ml)	1000	125	0
				4℃Day 21 samples	1.684	1.080	0.033
21 days samples at-80 deg.C	1.665	1.017	0.042
				21 day samples at 37 deg.C	1.626	0.999	0.078

(4) Evaluation of Performance

The mutant antibody and WT were used as coating antibodies, respectively, and were used in combination with a labeled antibody (from Fenpeng Bio Inc.), and the performance differences of the antibodies in the above examples were compared on a colloidal gold platform using a double antibody sandwich method, so that the mutant antibody could achieve a better performance level than WT.

1 marking

(1) Preparing colloidal gold: heating a chloroauric acid solution to boiling by adopting a traditional sodium citrate reduction method, quickly adding a trisodium citrate solution in a certain proportion, uniformly stirring, stopping heating when the color of the solution becomes wine red and does not change any more, and cooling to room temperature to obtain a colloidal gold solution with the concentration of four parts per million;

(2) marking: adding 0.2M K into the colloidal gold solution₂CO₃Adjusting the pH value of the solution to 7.0-10.0;

(3) centrifuging: adding labeled antibody (obtained from Fipeng organism) into the pH-adjusted colloidal gold solution, mixing, adding blocking agent, stopping labeling, centrifuging at 4 deg.C for 7min at 10000rpm, and removing supernatant; (4) redissolving: resuspending to 100uL, and performing ultrasonic treatment for 2-3 times; (5) paving gold: the resuspended concentrated gold was diluted and spread on a glass cellulose membrane and then lyophilized in a lyophilizer (1-2h) or dried overnight in a 37 ℃ dry room.

2. Coating quilt

(1) Assembling the nitrocellulose membrane and the colloidal gold PVC base plate for later use; (2) the antibodies in tables 3 and 5 were diluted to 1.0-2.0mg/mL, and the line was drawn uniformly on the NC membrane using a gold-spraying membrane drawing instrument, and then dried in a 37 ℃ incubator for at least 45 min. Assembling the cutting strips, and detecting the sample adding.

3. Application of antibody in colloidal gold detection

The assembled test strip is used for detecting whether the detected sample contains the FOB protein, so that the detection effect of the antibody obtained in the previous embodiment on the FOB protein is determined. Whether the test material contains FOB is detected by a double antibody sandwich method. During detection, the FOB protein is firstly combined with the FOB antibody marked by the colloidal gold to form a FOB protein-colloidal gold mark-FOB antibody compound, the FOB protein-colloidal gold mark-FOB antibody compound swims forwards along the nitrocellulose membrane due to capillary action, and when the FOB protein-colloidal gold mark-FOB antibody compound reaches a detection line, the FOB protein-colloidal gold mark-FOB antibody compound is combined with the FOB antibody obtained in the embodiment to form a FOB antibody-FOB protein-colloidal gold mark-FOB antibody compound, so that the FOB antibody-FOB protein-colloidal gold mark-FOB antibody compound is enriched on the detection line, and a red precipitation line is formed. The FOB protein-colloidal gold labeled-FOB antibody compound which is not combined with the FOB antibody on the detection line passes through the detection line, is captured by the goat anti-mouse IgG antibody, and is enriched on the quality control line to form a red precipitation line. And judging as a positive result when the detection line and the quality control line have the red precipitation line at the same time. If the sample does not contain FOB protein, when the FOB antibody labeled by the colloidal gold which is not combined with the FOB protein reaches the detection line, a FOB antibody-FOB protein-colloidal gold labeled-FOB antibody compound cannot be formed, and the FOB antibody compound labeled by the colloidal gold which is not combined with the FOB protein passes through the detection line and is only enriched on the quality control line to form a red precipitation line, and the result is judged to be negative.

The results are shown in Table 8 below.

TABLE 8

Remarking: the gold mark color development is formed by adding a number C, and the smaller the number behind the number C is, the stronger the color development is, and the higher the activity is; higher numbers after C indicate weaker color development and lower activity; the sign with a "+" after the number is slightly stronger than the non-coloration by 0.5-1C, and the sign with a "-" after the number is slightly lower than the non-coloration by 0.5-1C. B means no color development or weak color development.

The results show that the antibody provided by the embodiment of the invention has obvious activity when used for immunoassay by a gold-labeled platform sandwich method, and can detect the antigen under a high-concentration sample.

The detection rates of 100 positive samples and 200 negative samples were measured according to the above method, and the data are shown in Table 9.

TABLE 9

As can be seen from Table 9, the antibodies provided in the examples of the present invention were able to detect the results accurately.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Dongguan City of Pengzhi Biotech Co., Ltd

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Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn

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Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

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Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala

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Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr

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435 440

Claims

1. An antibody or functional fragment thereof against human hemoglobin, said antibody or functional fragment thereof having the following complementarity determining regions:

CDR-VH 3: A-X1-T-Y-Y-X2-F-Y-A; wherein: x1 is Q, R or K; x2 is A or G;

CDR-VL 2: X1-A-S-N-X2-A-S; wherein: x1 is I, V or L; x2 is I, V or L;

CDR-VL 3: Q-Q-N-X1-E-X2-P-Y; wherein: x1 is Q, N or H; x2 is D or N.

2. The anti-human hemoglobin antibody or functional fragment thereof according to claim 1,

in CDR-VH1, X1 is Y;

in CDR-VH2, X1 is I;

in CDR-VH3, X2 is G;

in CDR-VL1, X1 is E;

in CDR-VL3, X2 is D;

preferably, in CDR-VH1, X2 is L;

preferably, in CDR-VH1, X2 is V;

preferably, in CDR-VH1, X2 is I;

preferably, in CDR-VH1, X3 is N;

preferably, in CDR-VH1, X3 is D;

preferably, in CDR-VH2, X2 is S;

preferably, in CDR-VH2, X2 is T;

preferably, in CDR-VH2, X3 is S;

preferably, in CDR-VH2, X3 is T;

preferably, in CDR-VH2, X4 is L;

preferably, in CDR-VH2, X4 is I;

preferably, in CDR-VH3, X1 is Q;

preferably, in CDR-VH3, X1 is R;

preferably, in CDR-VH3, X1 is K;

preferably, in CDR-VL1, X2 is I;

preferably, in CDR-VL1, X2 is V;

preferably, in CDR-VL1, X2 is L;

preferably, in CDR-VL1, X3 is I;

preferably, in CDR-VL1, X3 is V;

preferably, in CDR-VL1, X3 is L;

preferably, in CDR-VL2, X1 is I;

preferably, in CDR-VL2, X1 is V;

preferably, in CDR-VL2, X1 is L;

preferably, in CDR-VL2, X2 is I;

preferably, in CDR-VL2, X2 is V;

preferably, in CDR-VL2, X2 is L;

preferably, in CDR-VL3, X1 is Q;

preferably, in CDR-VL3, X1 is N;

preferably, in CDR-VL3, X1 is H;

preferably, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 1-51:

3. the anti-human hemoglobin antibody or functional fragment of claim 2, wherein the antibody or functional fragment thereof binds human hemoglobin with K_D≤1×10^-9Affinity binding of mol/L; preferably, K_D≤8×10^- ¹¹mol/L。

4. The anti-human hemoglobin antibody or functional fragment thereof according to claim 1,

in CDR-VH1, X1 is F;

in CDR-VH2, X1 is L;

in CDR-VH3, X2 is A;

in CDR-VL1, X1 is Q;

in CDR-VL3, X2 is N;

preferably, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 52-58:

5. the anti-human hemoglobin antibody or functional fragment thereof of any one of claims 1-4, wherein the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L in sequence as shown in SEQ ID Nos. 1-4, and/or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H in sequence as shown in SEQ ID Nos. 5-8;

preferably, the antibody further comprises a constant region;

preferably, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD;

preferably, the species of the constant region is derived from sheep, goat, cow, horse, cow, pig, rat, dog, cat, rabbit, camel, donkey, mouse, deer, mink, duck, goose, turkey, chicken, or human;

preferably, the constant region is derived from a mouse;

preferably, the light chain constant region sequence of the constant region is shown as SEQ ID NO. 9, and the heavy chain constant region sequence of the constant region is shown as SEQ ID NO. 10;

preferably, the functional fragment is selected from any one of F (ab ') 2, Fab, scFv, Fab' and Fv of the antibody.

6. Use of an anti-human hemoglobin antibody or functional fragment thereof according to any one of claims 1-5 for the preparation of a reagent or kit for the diagnosis or the aided diagnosis of a tumor, wherein the marker of the tumor comprises human hemoglobin;

preferably, the tumor is an intestinal cancer, preferably, the intestinal cancer is selected from the group consisting of rectal cancer and colon cancer;

preferably, the sample detected in the reagent or the kit is human feces.

7. A reagent or kit for the diagnosis or aided diagnosis of a tumor associated with human hemoglobin as a marker, comprising the antibody or functional fragment thereof according to any one of claims 1 to 5.

8. A reagent or a kit for detecting human hemoglobin, comprising the anti-human hemoglobin antibody or functional fragment thereof according to any one of claims 1 to 5.

9. The reagent or kit according to claim 7 or 8, wherein the antibody or functional fragment thereof is labeled with a detectable label;

preferably, the detectable label is selected from the group consisting of a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label;

preferably, the fluorescent dye is selected from fluorescein dyes and derivatives thereof, rhodamine dyes and derivatives thereof, Cy dyes and derivatives thereof, Alexa dyes and derivatives thereof, and protein dyes and derivatives thereof;

preferably, the enzyme catalyzing the color development of the substrate is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme;

preferably, the radioisotope is selected from the group consisting of²¹²Bi、¹³¹I、¹¹¹In、⁹⁰Y、¹⁸⁶Re、²¹¹At、¹²⁵I、¹⁸⁸Re、¹⁵³Sm、²¹³Bi、³²P、⁹⁴mTc、⁹⁹mTc、²⁰³Pb、⁶⁷Ga、⁶⁸Ga、⁴³Sc、⁴⁷Sc、¹¹⁰mIn、⁹⁷Ru、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁸Cu、⁸⁶Y、⁸⁸Y、¹²¹Sn、¹⁶¹Tb、¹⁶⁶Ho、¹⁰⁵Rh、¹⁷⁷Lu、¹⁷²Lu and¹⁸F；

preferably, the chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, lowhine and its derivatives, and peroxyoxalate and its derivatives;

preferably, the nanoparticle-based label is selected from the group consisting of nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles;

preferably, the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latexes;

preferably, the colloidal metal is selected from the group consisting of colloidal gold, colloidal silver and colloidal selenium.

10. A method of producing an antibody or functional fragment thereof, comprising: culturing a recombinant cell capable of recombinantly expressing the anti-human hemoglobin antibody or functional fragment thereof according to any one of claims 1 to 5, and isolating and purifying the antibody or functional fragment thereof from the culture product.