CN118742574A - An anti-biotin antibody or its functional fragment and its application - Google Patents

Abstract

A biotin antibody or its functional fragment and its application, the antibody or its functional fragment having the following complementary determining regions: CDR-VH1: T-X1-Y-X2-N; CDR‑VH2：M‑X3‑W‑A‑T‑G‑X4‑T‑Y‑Y‑A‑X5‑W‑A‑K‑G；CDR‑VH3：T‑X6‑G‑T‑Y‑D‑K‑X7‑H‑F；CDR‑VL1：Q‑X8‑S‑Q‑S‑X9‑Y‑X10‑N‑N‑Y‑L‑A；CDR‑VL2：X11‑A‑X12‑T‑L‑A‑S；CDR‑VL3：X13‑G‑G‑Y‑D‑C‑X14‑S‑A‑D‑C‑X15‑A。

Description

An anti-biotin antibody or its functional fragment and its application

The present invention claims the priority of a prior application with patent application number 202211141046.6 filed with the State Intellectual Property Office of China on September 20, 2022, and entitled “An anti-biotin antibody or its functional fragment and its application”. The entire text of the prior application is incorporated into the present invention by reference.

Technical Field

The present invention relates to the field of antibody technology, and in particular to an anti-biotin antibody or a functional fragment thereof and application thereof.

Background Art

Biotin, also known as vitamin B7, is a water-soluble vitamin that regulates the rise of glycogen, the circulation of fatty acids, and the metabolism of certain amino acids, maintaining the normal functions of the human body. Biotin is abundant in the food people eat, and can also be taken in through medicines. As a medicine, biotin can treat inherited metabolic diseases such as biotin-responsive basal ganglia and biotinidase deficiency. With the continuous increase in the amount and concentration of supplemental biotin, the biotin in the circulating blood will interfere with and affect immune detection.

"Streptavidin-biotin" is the most commonly used signal amplification system in immunoassays. Streptavidin is a common carbohydrate protein in egg white, composed of four identical subunits. Each subunit contains a biotin binding site, so a theoretically normal avidin can bind to four biotins. Streptavidin has a very strong affinity for biotin, and its dissociation constant is about 1.3* ^10-15 M, which is one of the strongest non-covalent interactions known in nature. The protein structure of streptavidin is very stable. Even in a urea solution with a concentration of up to 8M, it can maintain the integrity of the structure and maintain its affinity for biotin. After binding to biotin, the stability of the "streptavidin-biotin" structure is further enhanced. In addition, the binding of "streptavidin-biotin" is similar to the binding of antibody-antigen, has extremely high specificity, and can bind to each other in a complex solution environment.

As people's biotin intake increases, the biotin concentration in the human extracorporeal circulation will increase. If the "streptavidin-biotin" system is used to test samples, the high concentration of free biotin in the sample will interfere with the ability of streptavidin to capture analytes, causing a false increase or decrease in the test results, affecting clinical judgment. Currently, the simplest and most direct way to resist biotin interference is to increase the amount of avidin added, such as increasing the concentration of avidin magnetic beads to increase the biotin loading capacity of the reaction system, but this approach usually increases the cost of the reagents and the degree of improvement is limited. Therefore, there is an urgent need for a low-cost, simple and efficient method to improve biotin interference.

Summary of the invention

In view of the deficiencies in the prior art, the present invention provides an anti-biotin antibody or a functional fragment thereof and its application. It solves the problem of biotin interference in the immune detection process in the prior art.

In one aspect, the present invention provides an anti-biotin antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof has the following complementary determining regions:

CDR-VH1: T-X1-Y-X2-N, wherein X1 is Y, A or N; X2 is I or M;

CDR-VH2: M-X3-W-A-T-G-X4-T-Y-Y-A-X5-W-A-K-G, wherein X3 is I, X4 is N, G or R, and X5 is S or N;

CDR-VH3: T-X6-G-T-Y-D-K-X7-H-F, where X6 is A, S or T, and X7 is S or N;

CDR-VL1: Q-X8-S-Q-S-X9-Y-X10-N-N-Y-L-A, where X8 is A or S, X9 is Y, V or L, and X10 is K, N or A;

CDR-VL2: X11-A-X12-T-L-A-S, where X11 is D, G or S, and X12 is A or S;

CDR-VL3: X13-G-G-Y-D-C-X14-S-A-D-C-X15-A, where X13 is Q or L, X14 is R, G, S, Q or L, and X15 is A or S.

The anti-biotin antibody or its functional fragment provided by the present invention has the above complementary determining region structure, can specifically bind to free biotin, has good affinity for biotin, and has good specificity and sensitivity when detecting biotin using the antibody or its functional fragment. The present invention reduces interference in immunoassay of the biotin-streptavidin system and provides more possibilities for accurate measurement of various analytes.

In an optional embodiment, the antibody or its functional fragment has the following complementarity determining region:

CDR-VH1: T-X1-Y-X2-N, where X1 is Y, A or N; X2 is M;

CDR-VH2: M-X3-W-A-T-G-X4-T-Y-Y-A-X5-W-A-K-G, wherein X3 is I, X4 is N, G or R, and X5 is S or N;

CDR-VH3: T-X6-G-T-Y-D-K-X7-H-F, where X6 is A, S or T, and X7 is N;

CDR-VL1: Q-X8-S-Q-S-X9-Y-X10-N-N-Y-L-A, where X8 is A, X9 is Y, V or L, and X10 is K, N or A;

CDR-VL2: X11-A-X12-T-L-A-S, where X11 is D, G or S, and X12 is S;

CDR-VL3:X13-G-G-Y-D-C-X14-S-A-D-C-X15-A, where X13 is L, X14 is R, G, S, Q or L, and X15 is A or S.

The inventors of the present invention have found that when the mutation site in each complementary determining region is the amino acid residue, the antibody exhibits better affinity for free biotin.

In an optional embodiment, the antibody or its functional fragment has any combination of the following complementary determining regions:

In an optional embodiment, the antibody or its functional fragment comprises a light chain framework region shown in the following sequence:

FR1-L as shown in SEQ ID NO:1; FR2-L as shown in SEQ ID NO:2; FR3-L as shown in SEQ ID NO:3; FR4-L as shown in SEQ ID NO:4; and/or

The heavy chain framework region is shown in the following sequence:

FR1-H as shown in SEQ ID NO:5; FR2-H as shown in SEQ ID NO:6; FR3-H as shown in SEQ ID NO:7; FR4-H as shown in SEQ ID NO:8;

Preferably, the antibody further comprises a constant region;

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

Preferably, the species of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans;

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

Typically, the heavy chain variable region (VH) and the light chain variable region (VL) can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the antibodies or their functional fragments provided by the present invention may 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 with the above-mentioned corresponding framework regions (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8).

The functional fragments of the above antibodies usually have the same binding specificity as the antibodies from which they are derived. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, a person skilled in the art can easily obtain the above functional fragments.

The functional fragments of the above antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automatic peptide synthesizer.

In a second aspect, the present invention provides a detection reagent or a kit, which comprises the above-mentioned anti-biotin antibody or a functional fragment thereof. Preferably, the sample detected by the detection reagent or the kit is selected from body fluids such as whole blood, serum, plasma, urine or saliva.

In an optional embodiment, the antibody or functional fragment thereof in the reagent or kit is labeled with a detectable marker;

Preferably, the detectable markers include, but are not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent agents, and nanoparticle markers;

Preferably, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, and protein dyes and their derivatives;

In an optional embodiment, the fluorescein dyes and their derivatives include but are not limited to fluorescein isothiocyanate (FITC), hydroxyapatite (FAM), tetrachloroapatite (TET), etc. or their analogs; the rhodamine dyes and their derivatives include but are not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs; the Cy series dyes and their derivatives include but are not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or their analogs; the Alexa series dyes and their derivatives include but are not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs; the protein dyes and their derivatives include but are not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.

Preferably, the enzyme that catalyzes the color development of the substrate includes but is not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and 6-phosphoglucose deoxidase;

Preferably, the radioactive isotopes include but are not limited to 212Bi, 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 and 18F;

Preferably, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives;

Preferably, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles;

Preferably, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex;

Preferably, the colloidal metal includes but is not limited to colloidal gold, colloidal silver and colloidal selenium.

The third aspect of the present invention provides a nucleic acid molecule, which can encode the above-mentioned antibody or a functional fragment thereof.

In a fourth aspect, the present invention provides a vector comprising the above-mentioned nucleic acid molecule.

In a fifth aspect, the present invention provides a recombinant cell comprising the above-mentioned vector.

In a sixth aspect, the present invention provides a method for preparing the antibody or a functional fragment thereof, comprising: culturing the above-mentioned recombinant cells, and isolating and purifying the antibody or a functional fragment thereof from the culture product.

In a seventh aspect, the present invention provides use of the anti-biotin antibody or a functional fragment thereof for preventing biotin interference in immunoassays.

The anti-interference of the present invention refers to the ability to remove other biotins other than biotin in the biotin-streptavidin system, such as free biotin, other biotin produced by metabolism, etc., when measuring the analyte using the biotin-streptavidin system.

Based on the amino acid sequence of the antibody or its functional fragment disclosed in the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, hybridoma cells) to prepare the antibody or its functional fragment, for example, separating and purifying the antibody or its functional fragment from the culture product of a recombinant cell that can recombinantly express the antibody or its functional fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment of the present invention, it belongs to the protection scope of the present invention.

Compared with the prior art, the present invention has the following beneficial effects:

The anti-biological antibody or its functional fragment of the present invention comprises specific heavy chain CDR and light chain CDR, can specifically recognize and bind to free biotin in the sample, has high sensitivity and specificity, can be used as a cleaning agent for biotin interference in the sample, and can accurately and efficiently detect various analytes in the biotin-streptavidin system without causing false increase or decrease in the test results, so that even if the sample is in an environment with high concentration of biotin, the analyte can still be stably detected by the biotin-streptavidin system, thereby achieving accurate judgment of clinical diagnosis.

DETAILED DESCRIPTION

The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available or can be prepared by known methods. If no specific conditions are specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are followed.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Any method and material similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein. Unless otherwise indicated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

Example 1 Preparation of Antibodies

1. Preparation of complete antigen

(1) Preparation of hapten

a. Synthesis of Compound 2

Dissolve D-biotin (2.44 g, 10 mmol) in anhydrous methanol (75 mL); then add concentrated sulfuric acid (1 mL); reflux the reaction solution (70°C) for 7 hours under nitrogen protection; cool the reaction solution to room temperature and concentrate it under reduced pressure at 45°C; slurry the residue with methyl tert-butyl ether (75 mL) and filter it, wash the filter cake with a small amount of methyl tert-butyl ether, and dry the solid under reduced pressure at 45°C to obtain about 2 g of white powder. MS Found: [M+H] ⁺ = 259.34

b. Synthesis of Compound 3

Compound 2 (1 g, 3.87 mmol) was dissolved in pyridine (20 mL); 4,4'-bismethoxytrityl chloride (2 g, 6 mmol) was added; DMAP (50 mg, 0.38 mmol) was then added; after thorough mixing and dissolution, the mixture was transferred to a 100 mL stainless steel jar with a PTFE lining; the reaction solution was stirred at 75°C for 18 hours; after cooling to room temperature, the reaction solution was poured into 200 mL deionized water, extracted twice with 200 mL ethyl acetate, and after the organic phases were combined, the organic phases were washed once with 200 mL 1N hydrochloric acid, once with 100 mL saturated sodium bicarbonate, and once with 100 mL saturated saline, and then dried with anhydrous sodium sulfate, and concentrated under reduced pressure at 45°C; the residue was purified by column chromatography to obtain about 1.68 g of a white solid. MS Found: [M+H]+＝561.71

c. Synthesis of Compound 4

Compound 3 (1 g, 1.78 mmol) was dissolved in THF (20 mL); the solution was protected with nitrogen and then cooled to 0°C in an ice-water bath; sodium hydride (60%, 100 mg, 2.67 mmol) was added to the reaction mixture at once; the reaction mixture was stirred at 0°C for 30 minutes; acetyl chloride (210 mg, 2.67 mmol) was added dropwise to the reaction mixture, and the internal temperature was controlled to be less than 20°C; after the addition was completed, the cold bath was not removed and the mixture was stirred at 0-20°C for 3 hours; the reaction solution was poured into 200 mL of deionized water, extracted twice with 200 mL of ethyl acetate, the organic phases were combined, washed once with 100 mL of saturated brine, dried with anhydrous sodium sulfate, and concentrated under reduced pressure at 45°C; the residue was dried under reduced pressure at 45°C to obtain about 1.1 g of yellow solid. MS Found: [M+H] ⁺ = 603.75

d. Synthesis of Compound 5

Dissolve compound 4 (1 g, 1.66 mmol) in methanol (20 mL); cool to 0°C with an ice-water bath; add a saturated methanol solution of hydrogen chloride (20 mL); do not remove the cooling bath, and naturally warm to room temperature; after stirring for 15 hours, concentrate the reaction solution under reduced pressure at 45°C; purify the residue by column chromatography to obtain about 0.34 g of a white solid. MS Found: [M+H]+＝301.37

e. Synthesis of Compound 6

Compound 5 (0.34 g, 1.13 mmol) was dissolved in THF (20 mL); the solution was protected with nitrogen and then cooled to 0°C in an ice-water bath; sodium hydride (60%, 68 mg, 1.7 mmol) was added to the reaction mixture at once; the reaction mixture was stirred at 0°C for 30 minutes; Fomc-alanine chloride (0.56 g, 1.7 mmol) was dissolved in ultra-dry THF (10 mL) and then added dropwise to the reaction mixture, and the internal temperature was controlled to be less than 20°C; after the addition was completed, the cooling bath was not removed and the mixture was stirred at 0-20°C for 3 hours; the reaction solution was poured into 200 mL of deionized water, extracted twice with 200 mL of ethyl acetate, the organic phases were combined, washed once with 100 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure at 45°C; the residue was purified by column chromatography to obtain about 0.5 g of a yellow solid. MS Found: [M+H] ⁺ = 594.75

f. Synthesis of Compound 7

Compound 6 (0.5 g, 0.84 mmol) was dissolved in methanol (10 mL); the solution was protected with nitrogen and then cooled to 0°C in an ice-water bath; piperidine (0.7 g, 8.4 mmol) was added to the reaction mixture at once; the reaction mixture was stirred at 0-20°C for 3 hours; the reaction solution was concentrated under reduced pressure at 45°C; the residue was dried under reduced pressure at 45°C; the obtained yellow oily residue was dissolved in methanol (10 mL); lithium hydroxide monohydrate (0.1 g, 2.5 mmol) was added, the reaction mixture was stirred at 60°C for 1.5 hours and then cooled to room temperature; the mixture was concentrated under reduced pressure at 45°C; the residue was purified by Pre-HPLC to obtain about 167 mg of white solid. MS Found: [M+H]+＝358.43

g. Synthesis of Compound 8

Compound 7 (167 mg, 0.46 mmol) was dissolved in DMF (5 mL); disuccinimidyl suberate (172 mg, 0.46 mmol) was added under nitrogen protection, and the reaction solution was stirred at room temperature for 1-2 hours under nitrogen protection; the reaction solution was directly purified by Pre-HPLC to obtain about 98 mg of white solid. MS Found: [M+H] ⁺ = 611.68

(2) Preparation of complete antigen

Preparation of complete antigen-BSA: Weigh 1 mg of compound 8 and dissolve it in 500 ul DMSO; take 3 mg of KLH, add 1 mL of 20 mM phosphate buffer solution (PBS, pH = 7.4) to dilute, then slowly add hapten I solution dropwise into KLH and react overnight at room temperature; then dialyze with 20 mM PBS solution to remove unreacted small molecule haptens to obtain complete antigen Biotin-KLH, which was identified by UV absorption scanning method.

2. Rabbit immune-based antibody detection

Freund's complete adjuvant and complete antigen Biotin-KLH protein at a concentration of 2mg/ml were mixed and emulsified in equal volumes. Four healthy New Zealand white rabbits of about 2 months old and 1.5 kg were selected, and the rabbits were immunized by five-point subcutaneous injection on the back. The immunization dose was 800ug/rabbit. The rabbits were immunized weekly in the first month of immunization, and the rabbits were immunized monthly starting from the second month. Freund's complete adjuvant was used for the initial immunization, and Freund's incomplete adjuvant was used for the remaining immunizations. The amount of immunogen used for the booster immunization was half of the amount used for the initial immunization. After 36 days, blood was collected from the rabbit ear vein, and the supernatant was collected by centrifugation and the serum titer was detected by ELISA. The rabbit serum titer was monitored, and when the rabbit serum titer was qualified, the spleen was taken to separate lymphocytes for cell fusion.

3. Hybridoma cell fusion

Blood from the ear artery of the immunized rabbit was taken and the immune titer was determined by ELISA. The rabbits with the titer reaching the standard were killed, the spleen was taken, ground, and the red blood cells were lysed to prepare B cells. Rabbit B cells were mixed with myeloma cells SP2/0 at a ratio of 1:1, and then electrofused to obtain rabbit-mouse hybridoma cells. The hybridoma cells were then added to 1.2% methylcellulose semi-solid culture medium, and HAT, anti-mycoplasma drugs, and feeder layer cells were added to mix thoroughly and plated. After 3-4 days of culture in an incubator, DMEM complete culture medium was added. After 13 days of continuous culture, the cell supernatant was taken for ELISA detection (initial screening, re-examination).

4. Antibody Screening

This example takes serum amyloid A (SAA) detection as an example

The antibody screening scheme is the key to successfully developing the antibodies of the present invention. In the antibody screening stage, free biotin is added to the system using the biotin-avidin system to detect SAA to interfere with the system signal, and hybridoma clones that can restore the system signal value are screened.

Specific experimental plan: 1ug/ml SAA antigen was coated at 4℃ for 8 hours; after washing the plate, 100ul biotin-labeled SAA antibody (50ng/ml, prepared with 1% BSA+0.1% PBST to achieve blocking effect) was used for incubation for 30min; 50ul of 50ng/ml free biotin was added, and 50ul of cell line supernatant (an equal volume of blank culture medium was added as a control) and 50ul avidin-HRP (1:2000 dilution) were added, and incubated at 37℃ for 30min; the plate was washed, and color was developed with color developing solution for 3min, and the plate was terminated and read at OD450 using an enzyme reader.

There was no interference from free biotin in the control group, so the signal value remained at a high level (1.7282). After using 50 ng/ml free biotin to interfere with the biotin-avidin system, the signal value was significantly reduced (0.3833). The antibodies in the supernatant of the cell line can bind to free biotin and play an anti-interference role, thereby ensuring that the binding of the biotin-labeled SAA antibody to avidin is not interfered by free avidin, significantly improving the signal value. Based on this, the cell line with the best anti-interference ability was screened for subsequent experiments.

The antigen is coated on an ELISA plate, which is made of polystyrene. The antigen is combined with the plate by adsorption or hydrophobic effect, and will not affect the active groups of the antigen itself.

5. Antibody Preparation

Hybridoma cell sequence determination. Take the hybridoma cells that are positive in ELISA detection, wash with DPBS, and then directly lyse the cells with lysis buffer, and then use reverse transcription reaction to obtain cDNA. Use the designed rabbit anti-specific light and heavy chain primers, TAQ enzyme and the obtained cDNA samples for in vitro amplification experiments to obtain rabbit anti-light and heavy chain PCR products. Then use the kit to perform gel recovery and T vector construction experiments, and then send them to the sequencing company for testing.

Antibody fermentation. Take Expi 293 cells in the logarithmic growth phase, count with trypan blue, record the viability, and inoculate them into disposable shake flasks in a certain proportion and incubate them in a constant temperature incubator. After 24 hours, take the required number of cells and transfer them to a new fermentation bottle. Mix the calculated transfection reagent and plasmid solution in a 1:1 ratio, let it stand for a short time, then slowly pour it into a new shake flask and incubate it in a constant temperature incubator. After 18-22 hours, add a certain proportion of feed, and collect the fermentation supernatant after 5-6 days. Antibody WT is obtained by ProG column affinity chromatography purification.

Example 2 Verification of antibody performance

1. ELISA affinity test

(1) Affinity test (antibody and full antigen affinity) experimental steps

Full antigen coating (1ug/ml) was incubated at 4°C for 8 hours; primary antibody incubation: after washing the plate, the purified antibody (1ug/ml) was diluted three times in a gradient manner, 100ul/well, and incubated at 37°C for 30min; secondary antibody (goat anti-rabbit-HRP) incubation: after washing the plate, goat anti-rabbit-HRP (1:5000) was diluted, 100ul/well, and incubated at 37°C for 30min; color development was performed with color developing solution for 3min, terminated, and read at OD450 using an enzyme reader.

The antibody (WT) sequence of step 5 of Example 1 was analyzed, and its heavy chain variable region was shown in SEQ ID NO: 9, wherein the amino acid sequences of each complementary determining region on the heavy chain variable region were as follows:

CDR-VH1: T-A(X1)-Y-I(X2)-N;

CDR-VH2: M-I(X3)-W-A-T-G-N(X4)-T-Y-Y-A-S(X5)-W-A-K-G;

CDR-VH3: T-T(X6)-G-T-Y-D-K-S(X7)-H-F.

The light chain variable region is shown in SEQ ID NO: 10, wherein the amino acid sequences of the complementary determining regions on the light chain variable region are as follows:

CDR-VL1: Q-S(X8)-S-Q-S-L(X9)-Y-K(X10)-N-N-Y-L-A;

CDR-VL2: G(X11)-A-A(X12)-T-L-A-S;

CDR-VL3: Q(X13)-G-G-Y-D-C-R(X14)-S-A-D-C-S(X15)-A.

Based on the anti-biotin antibody (WT) of Example 1, mutations were performed on sites related to antibody activity in the complementary determining region, wherein X1-X15 were all mutation sites. See Table 1 below. Affinity tests were performed on the WT and mutants of the anti-biotin antibody, and the results are shown in Table 2.

Table 1 Mutation sites related to antibody activity

Table 2 Antibody activity analysis data

As can be seen from Table 2, mutation 1 has the best activity effect. Therefore, mutation 1 was used as the backbone sequence to screen mutation sites with better anti-interference activity. Some of the results are shown in Table 3.

Table 3 Mutation sites related to antibody affinity

The affinity test was performed on the mutants of mutation 1, the antigen concentration was 1ug/ml, and the absorbance value of OD450 of each mutation was measured. The results are shown in Table 4.

Table 4 Mutation affinity test

As shown in Table 4 , the affinity of the mutant of mutation 1 is similar to that of mutant 1.

(2) ELISA competitive assay to detect free biotin binding capacity

Competition method (antibody binding ability to free biotin) experimental steps: full antigen coating (1ug/ml) incubated at 4℃ for 8 hours; free biotin 1ug/mL, 3-fold gradient dilution, 60ul/well, antibody diluted to 500ng/mL, antibody 40ul/well, incubated at 37℃ for 30min; secondary antibody (goat anti-rabbit-HRP) incubation, wash the plate, goat anti-rabbit-HRP (1:5000) diluted, 100ul/well, incubated at 37℃ for 30min; color development with colorimetric solution for 3min, stop, and read at OD450 using an enzyme reader. The results of mutant 1 binding ability to free biotin are shown in Table 5.

Table 5 Free biotin binding capacity

It can be seen from Table 5 that as the amount of free biotin used increases, the ELISA signal value decreases significantly. From the addition of 0ug/ml-1ug/ml, the signal of WT changes to 1.0743, and the signal of mutant 1 changes to 1.0821. Mutant 1 has a stronger binding ability to free biotin than WT.

Because the antibody can bind to the whole antigen and free biotin. When there is no free biotin, the antibody can only bind to the whole antigen and be fixed on the plate. When the goat anti-rabbit-HRP binds to the antibody, it will produce a strong light signal under the action of the substrate. When there is free biotin in the solution, the antibody will bind to the free biotin, and the goat anti-rabbit-HRP will bind to the antibody and develop color under the substrate. Because the free biotin is still in a free state after binding to the antibody, it will be washed away after washing the plate. Therefore, with the addition of free biotin, the luminescent signal will decrease.

Binding ability of mutants of mutant 1 to free biotin: The mutation sites are shown in Table 3. When the concentration of free biotin is 0ug/ml and 1ug/ml, the absorbance value of each mutant OD450 is measured, and the difference is shown in Table 6.

Table 6 Mutation free biotin binding capacity

As can be seen from Table 6, the overall change of mutant 1 is not very large, indicating that the above mutated antibodies still have good free biotin binding ability.

Example 3 Chemiluminescence Anti-interference Ability Test

The anti-Mullerian hormone (AMH) detection kit (chemiluminescence method) was selected, different concentrations of free biotin were added to the sample as interference, and different concentrations of antibodies (mutation 1) were added to the reagent R1 component to verify the effect of different concentrations of antibodies in eliminating the interference of free biotin.

Specific experimental plan: Use the Zhongyuan Huiji AMH detection kit, add different concentrations of free Biotin to the sample, add different concentrations of purified antibodies to the R1 component, and use the Zhongyuan Huiji EXI1800 fully automatic chemiluminescence immunoassay analyzer to measure the sample values.

AMH detection kit: R1 biotin antibody usage 50ul; R2 enzyme-labeled antibody usage 50ul; avidin magnetic beads usage 30ul; sample loading 20ul. The results are shown in Table 7.

Table 7 Anti-interference ability test

The results showed that different doses of free biotin would interfere with the AMH detection signal value, and the degree of interference increased with the increase of free biotin concentration. Purified mutant 1 can bind to free biotin and significantly improve the decrease in signal value caused by free biotin. Moreover, as the concentration of purified mutant 1 increased, its ability to resist interference from free biotin was also significantly enhanced. 0.5 mg/ml of purified mutant 1 can basically resist the interference of 2400 ng/ml free biotin.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims (10)

1. An anti-biotin antibody or functional fragment thereof, characterized in that: the antibody or functional fragment thereof has the following complementarity determining regions:

   CDR-VH1: T-X1-Y-X2-N, wherein X1 is Y, A or N; x2 is I or M;

   CDR-VH2: M-X3-W-A-T-G-X4-T-Y-Y-A-X5-W-A-K-G, wherein X3 is I, X4 is N, G or R, X5 is S or N;

   CDR-VH3: T-X6-G-T-Y-D-K-X7-H-F, wherein X6 is A, S or T and X7 is S or N;

   CDR-VL1: Q-X8-S-Q-S-X9-Y-X10-N-N-Y-L-A, wherein X8 is A or S, X9 is Y, V or L, and X10 is K, N or A

   CDR-VL2: X11-A-X12-T-L-A-S, wherein X11 is D, G or S and X12 is A or S

   CDR-VL3: X13-G-G-Y-D-C-X14-S-A-D-C-X15-A, wherein X13 is Q or, X14 is R, G or S, and X15 is A or S.

   Preferably, the antibody or functional fragment thereof has the following complementarity determining regions:

   CDR-VH1: T-X1-Y-X2-N, wherein X1 is Y, A or N; x2 is M;

   CDR-VH2: M-X3-W-A-T-G-X4-T-Y-Y-A-X5-W-A-K-G, wherein X3 is I, X4 is N, G or R, X5 is S or N;

   CDR-VH3: T-X6-G-T-Y-D-K-X7-H-F, wherein X6 is A, S or T and X7 is N;

   CDR-VL1: Q-X8-S-Q-S-X9-Y-X10-N-N-Y-L-A, wherein X8 is A, X9 is Y, V or L, and X10 is K, N or A

   CDR-VL2: X11-A-X12-T-L-A-S, wherein X11 is D, G or S and X12 is S

   CDR-VL3: X13-G-G-Y-D-C-X14-S-A-D-C-X15-A, wherein X13 is, X14 is R, G, S, Q or, and X15 is A or S.
2. An anti-biotin antibody or functional fragment thereof according to claim 1, wherein: the antibody or functional fragment thereof has any one of the following combinations of complementarity determining regions:
3. an anti-biotin antibody or functional fragment thereof according to claim 1, wherein: the antibody or functional fragment thereof comprises a light chain framework region as shown in the following sequence:

   FR1-L shown as SEQ ID NO. 1; FR2-L shown in SEQ ID NO. 2; FR3-L shown in SEQ ID NO. 3; FR4-L shown in SEQ ID NO. 4; and/or

   Heavy chain framework regions shown in the following sequences:

   FR1-H shown in SEQ ID NO. 5; FR2-H shown in SEQ ID NO. 6; FR3-H shown in SEQ ID NO. 7; FR4-H shown in SEQ ID NO. 8;

   preferably, the antibody further comprises a constant region;

   preferably, the constant region is selected from the group consisting of the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD;

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

   preferably, the functional fragment is selected from any one of VHH, F (ab ') 2, fab', fab, fv and scFv of the antibody.
4. A detection reagent or kit comprising an anti-biotin antibody or a functional fragment thereof according to any one of claims 1 to 3, preferably wherein the sample detected by the detection reagent or kit is selected from whole blood, serum, plasma, urine or saliva.
5. The reagent or kit according to claim 4, wherein the antibody or functional fragment thereof in the reagent or kit is labeled with a detectable label;

   preferably, the detectable label includes, but is not limited to, fluorescent dyes, enzymes that catalyze the development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels;

   Preferably, the fluorescent dye includes, but is not limited to, fluorescein-based dye and its derivatives, rhodamine-based dye and its derivatives, cy-based dye and its derivatives, alexa-based dye and its derivatives, and protein-based dye and its derivatives;

   Preferably, the enzyme that catalyzes the development of a substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase;

   Preferably, the radioisotope includes, but is not limited to 212Bi、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 and 18F;

   Preferably, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine and its derivatives, and peroxyoxalate and its derivatives;

   preferably, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles;

   preferably, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex;

   preferably, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
6. A nucleic acid molecule encoding the antibody or functional fragment thereof of any one of claims 1-3.
7. A vector comprising the nucleic acid molecule of claim 6.
8. A recombinant cell comprising the vector of claim 7.
9. A method of making the antibody or functional fragment thereof of claims 1-3, comprising: culturing the recombinant cell of claim 8, and isolating and purifying the antibody or functional fragment thereof from the culture product.
10. Use of an anti-biotin antibody or a functional fragment thereof according to claims 1-3 for anti-biotin interference in immunodetection.