WO2024061058A1 - 一种抗生物素抗体或其功能性片段及其应用 - Google Patents

一种抗生物素抗体或其功能性片段及其应用 Download PDF

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WO2024061058A1
WO2024061058A1 PCT/CN2023/118384 CN2023118384W WO2024061058A1 WO 2024061058 A1 WO2024061058 A1 WO 2024061058A1 CN 2023118384 W CN2023118384 W CN 2023118384W WO 2024061058 A1 WO2024061058 A1 WO 2024061058A1
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antibody
functional fragment
cdr
biotin
derivatives
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PCT/CN2023/118384
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English (en)
French (fr)
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张顺
汤定斌
易维京
杜雪花
周胜男
徐小霞
温雅雯
徐娅
李强
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重庆艾生斯生物工程有限公司
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Publication of WO2024061058A1 publication Critical patent/WO2024061058A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • 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

Definitions

  • the present invention relates to the field of antibody technology, and in particular to an anti-biotin antibody or functional fragment thereof and its application.
  • Biotin is vitamin B7, also known as vitamin H, coenzyme R, etc. It is a water-soluble vitamin that regulates the rise of glycogen, the circulation of fatty acids and the metabolism of certain amino acids, and maintains the normal functions of the human body. . Biotin is abundant in the food people consume and can also be taken in through medicines. As a medicine, biotin can treat hereditary metabolic diseases such as biotin-responsive basal ganglionopathy and biotinidase deficiency. With supplementation As the dosage and concentration of biotin continue to increase, biotin in circulating blood will interfere with and affect immune detection.
  • Streptavidin-biotin is the most commonly used signal amplification system in immunoassays. Streptavidin is a carbohydrate protein commonly found in egg whites and consists of four identical subunits. Each subunit contains a biotin-binding site, so a normal avidin can theoretically bind four biotins. Streptavidin has a very strong affinity with biotin, and its dissociation constant is approximately 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 urea solutions with concentrations as high as 8M, it can maintain structural integrity and maintain affinity for biotin.
  • biotin in the human extracorporeal circulation As people's biotin intake increases, the concentration of biotin in the human extracorporeal circulation will increase. If the "streptavidin-biotin" system is used to detect samples, the high concentration of free biotin in the sample will Interfere with the ability of streptavidin to capture analytes, causing false increases or decreases in test results, affecting clinical judgment.
  • the simplest method to directly resist biotin interference is to increase the amount of avidin, such as increasing the concentration of avidin magnetic beads, to increase the biotin loading capacity of the reaction system.
  • avidin such as increasing the concentration of avidin magnetic beads
  • this approach usually increases the reagent concentration. costs, and the improvements are limited. Therefore, there is an urgent need for a low-cost, simple and efficient method to improve biotin interference.
  • the present invention provides an anti-biotin antibody or functional fragment thereof and its application. use. It solves the problem of biotin interference in the immune detection process existing in the existing technology.
  • the present invention provides an anti-biotin antibody or a functional fragment thereof, which has the following complementarity determining region:
  • CDR-VH1 T-X1-Y-X2-N, where 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, where 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 functional fragment thereof provided by the present invention has the above-mentioned complementary determining region structure, which can specifically bind to free biotin and has good affinity for biotin.
  • the antibody or functional fragment thereof is used to detect organisms. element, with better specificity and sensitivity.
  • the invention reduces interference in the immunodetection of the biotin-streptavidin system and provides more possibilities for accurate measurement of various analytes.
  • the antibody or functional fragment thereof 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, where 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 inventor of the present invention found that when the above-mentioned mutation site in each complementarity-determining region is the above-mentioned amino acid residue, the antibody shows better affinity for free biotin.
  • the antibody or functional fragment thereof has any combination of the following complementarity determining regions:
  • the antibody or functional fragment thereof includes a light chain backbone region represented by the following sequence:
  • FR1-L as shown in SEQ ID NO:1
  • FR2-L as shown in SEQ ID NO:2
  • SEQ ID NO:3 FR3-L
  • FR4-L as shown in SEQ ID NO:4;
  • the heavy chain backbone 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- as shown in SEQ ID NO:8 H;
  • the antibody also contains a constant region
  • the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD;
  • the species source of the constant region is cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock, or human being;
  • the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.
  • the heavy chain variable region (VH) and the light chain variable region (VL) can be obtained by connecting the following numbered CDRs and FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • each framework region of the antibody or functional fragment thereof provided by the invention can be the same as the above corresponding framework region (SEQ ID NO: 1, 2, 3, 4, 5, 6 , 7 or 8) 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.
  • Functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they are derived.
  • the functional fragments of the above-mentioned antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or methods of cleaving disulfide bonds through chemical reduction.
  • enzymatic digestion including pepsin or papain
  • cleaving disulfide bonds through chemical reduction.
  • the present invention discloses the structure of the complete antibody, those skilled in the art can easily obtain the above-mentioned functional fragments.
  • the present invention provides a detection reagent or a kit, which comprises the above-mentioned anti-biotin antibody or a functional fragment thereof.
  • the sample detected by the detection reagent or the kit is selected from body fluids such as whole blood, serum, plasma, urine or saliva.
  • the antibody or functional fragment thereof in the reagent or kit is labeled with a detectable label
  • the detectable markers include but are not limited to fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents and nanoparticle markers;
  • the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives, rhodamine dyes and their derivatives. substances, Cy series dyes and their derivatives, Alexa series dyes and their derivatives and protein dyes and their derivatives;
  • the fluorescein dyes and their derivatives include, but are not limited to, fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs ;
  • the rhodamine dyes and their derivatives include but are not limited to red rhodamine (RBITC), tetramethyl rhodamine (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.
  • 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
  • protein dyes and their derivatives include but are not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydinophycoxanthin-chlorophyll protein (preCP), etc.
  • the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and 6-phosphate. glucose deoxygenase;
  • 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;
  • the chemiluminescent reagents include but are not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridyl and its derivatives, acridinium ester and its derivatives, dioxygen Cycloethane and its derivatives, lopranine and its derivatives and peroxyoxalate and its derivatives;
  • the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles;
  • the colloids include but are not limited to colloidal metals, disperse dyes, dye-labeled microspheres and latex;
  • the colloidal metal includes but is not limited to colloidal gold, colloidal silver and colloidal selenium.
  • a third aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof.
  • the present invention provides a vector containing the above nucleic acid molecule.
  • the present invention provides a recombinant cell comprising the above-mentioned vector.
  • a sixth aspect of the present invention provides a method for preparing the antibody or functional fragment thereof, which includes: culturing the above-mentioned recombinant cells, and isolating and purifying the antibody or functional fragment thereof from the culture product.
  • a seventh aspect of the present invention provides the use of the anti-biotin antibody or functional fragment thereof for anti-biotin interference in immunoassays.
  • Anti-interference in the present invention refers to the ability to remove analytes when measuring analytes using the biotin-streptavidin system.
  • Biotin other than biotin in the biotin-streptavidin system such as free biotin, other biotin produced by metabolism, etc.
  • the present invention discloses the amino acid sequence of the antibody or its functional fragment
  • those skilled in the art can easily imagine using genetic engineering technology or other technologies (chemical synthesis, hybridoma cells) to prepare the antibody or its functional fragment, for example It is easy for those skilled in the art to separate and purify the antibody or functional fragment thereof from the culture product of the recombinant cell capable of recombinantly expressing the antibody or functional fragment thereof as described in any one of the above. Based on this, No matter what technology is used to prepare the antibody of the present invention or its functional fragment, it all falls within the protection scope of the present invention.
  • the present invention has the following beneficial effects:
  • the anti-biological antibody or its functional fragment of the present invention contains specific heavy chain CDRs and light chain CDRs, can specifically recognize and bind to free biotin in the sample, has high sensitivity and specificity, and can be used as a biotin interference in the sample.
  • the detergent, in the biotin-streptavidin system can accurately and efficiently detect various analytes without causing false increases or decreases in test results, even if the sample is in a high-concentration biotin environment. , the analyte can still be stably detected through the biotin-streptavidin system, achieving accurate clinical diagnosis.
  • the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer.
  • Dissolve compound 2 (1g, 3.87mmol) in pyridine (20mL); add 4,4'-bismethoxytrityl chloride (2g, 6mmol); then add DMAP (50mg, 0.38mmol); fully After mixing and dissolving, transfer the above mixed solution to a 100 mL stainless steel jar lined with PTFE; stir the above reaction solution at 75°C for 18 hours; after cooling to room temperature, pour the reaction solution into 200 mL of deionized water, and use 200 mL of deionized water. Extract twice with ethyl acetate.
  • PBS phosphate buffer solution
  • Freund's complete adjuvant and the complete antigen Biotin-KLH protein with a concentration of 2 mg/ml were mixed and emulsified in equal volumes.
  • This embodiment takes serum amyloid A (SAA) detection as an example.
  • SAA serum amyloid A
  • the antibody screening scheme is the key to successfully developing the antibodies of the present invention.
  • 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.
  • the antigen is coated on an enzyme-labeled plate, which is made of polystyrene.
  • the binding with the antigen relies on adsorption or hydrophobic interaction and does not affect the active groups of the antigen itself.
  • Hybridoma cell sequencing Take the hybridoma cells that are positive in the ELISA test, wash them with DPBS and directly lyse them with lysis buffer, and then use reverse transcription reaction to obtain cDNA.
  • the designed rabbit anti-specific light and heavy chain primers, TAQ enzyme and the obtained cDNA samples were used to conduct in vitro amplification experiments to obtain rabbit anti-light and heavy chain PCR products. Then the gel recovery and T-vector construction experiments were carried out through the kit, and then the sequencing company sent it for testing.
  • Antibody fermentation Take Expi 293 cells in the logarithmic phase of growth, count them with trypan blue, record the viability rate, and inoculate them at a certain proportion into disposable shake flasks and incubate them in an incubator. After 24 hours, take the required number of cells and transfer them to a new fermentation bottle. , mix the calculated ratio of transfection reagent and plasmid solution 1:1, let it stand for a short time, then slowly pour it into a new shake flask and place it in an incubator for incubation. After 18-22 hours, add a certain proportion of feed material. Collect the fermentation supernatant after 5-6 days. Antibody WT was obtained through ProG column affinity chromatography purification.
  • 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, 100ul/well, and incubated at 37°C for 30 minutes; secondary antibody (goat antibody Rabbit-HRP) incubation: wash the plate, dilute the goat anti-rabbit-HRP (1:5000), add 100ul/well, and incubate at 37°C for 30 minutes; use a chromogenic solution to develop color for 3 minutes, stop, and use a microplate reader to read at OD450.
  • 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.
  • 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.
  • mutation 1 has the best activity, so mutation 1 is used as the backbone sequence to screen mutation sites with better anti-interference activity. Some results are shown in Table 3.
  • 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.
  • goat anti-rabbit-HRP binds to the antibody, under the action of the substrate , will produce a strong light signal, and 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 binds to the antibody After binding, it is still in a free state, so it will be washed away after washing the plate. Therefore, with the addition of free biotin, the luminescence signal will decrease.
  • Binding ability 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, measure the intensity and difference of the absorbance value of each mutation OD450 As shown in Table 6.
  • mutant 1 As can be seen from Table 6, the overall change of mutant 1 is not very large, indicating that the above-mentioned mutated antibody still has good free biotin binding ability.
  • AMH Anti-Müllerian Hormone
  • AMH detection kit The amount of R1 biotin antibody is 50ul; the amount of R2 enzyme-labeled antibody is 50ul; the amount of avidin magnetic beads is 30ul; the sample volume is 20ul. The results are shown in Table 7.
  • Purified mutation 1 can bind to free biotin and significantly improve the decrease in signal value caused by free biotin. And as the concentration of purified mutation 1 increases, its ability to resist the interference of free biotin is also significantly enhanced. Purified mutation 1 of 0.5 mg/ml can basically resist the interference of 2400ng/ml free biotin.

Abstract

一种抗生物素抗体或其功能性片段及其应用，所述抗体或其功能性片段具有如下互补决定区：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

一种抗生物素抗体或其功能性片段及其应用
本发明要求2022年09月20日向中国国家知识产权局提交的专利申请号为202211141046.6，发明名称为“一种抗生物素抗体或其功能性片段及其应用”的在先申请的优先权。该件在先申请的全文通过引用的方式结合于本发明中。
技术领域
本发明涉及抗体技术领域，尤其涉及一种抗生物素抗体或其功能性片段及其应用。
背景技术
生物素即维生素B7，又称为维生素H、辅酶R等，是一种水溶性维生素，其对于糖元的异升，脂肪酸的循环以及某些氨基酸的新陈代谢具有调控作用，维持着人体的正常功能。生物素在人们摄取的食物中具有丰富的来源，也能够通过药品摄入，生物素作为药品，可以治疗生物素反应性基底神经节病和生物素酶缺乏症等遗传性代谢病，随着补充生物素用量和浓度的不断增加，循环血液中的生物素会对免疫检测产生干扰和影响。
“链霉亲和素-生物素”是免疫检测中最常用的信号放大系统。链霉亲和素是蛋清中常见的糖类蛋白，由四个相同的亚基组成。每一个亚基都包含一个生物素结合位点，因此一个理论上正常的亲和素能够结合四个生物素。链霉亲和素与生物素具有非常强烈的亲和力，其解离常数大约是1.3*10-15M，是已知自然界中最强的非共价相互作用之一。链霉亲和素的蛋白结构非常稳定，即使在浓度高达8M的尿素溶液中，也能够维持结构的完整性，保持对生物素的亲和力，并且在结合生物素后，“链霉亲和素-生物素”结构的稳定性进一步增强，此外，“链霉亲和素-生物素”的结合与抗体-抗原的结合类似，有极高的特异性，能够在复杂的溶液环境中相互结合。
随着人们生物素摄入的增加，会导致人体外循环中生物素浓度的上升，若使用“链霉亲和素-生物素”系统进行样本的检测，那么样本中高浓度的游离生物素就会干扰链霉亲和素捕获分析物的能力，从而造成检测结果假性升高或者降低，影响临床判断。目前最简单直接抗生物素干扰的方法是提高亲和素的加入量，如加大亲和素磁珠的浓度，以提高反应体系对生物素的载量，但是这种做法通常会增加试剂的成本，而且改善程度有限。所以亟需一种能够低成本、简单的高效改善生物素干扰的方法。
发明内容
针对现有技术中所存在的不足，本发明提供了一种抗生物素抗体或其功能性片段及其应 用。其解决了现有技术中存在的免疫检测过程中生物素干扰的问题。
本发明一方面，提供一种抗生物素抗体或其功能性片段，所述抗体或其功能性片段具有如下互补决定区：
CDR-VH1：T-X1-Y-X2-N，其中X1是Y、A或N；X2是I或M；
CDR-VH2：M-X3-W-A-T-G-X4-T-Y-Y-A-X5-W-A-K-G，其中X3是I，X4是N、G或R，X5是S或N；
CDR-VH3：T-X6-G-T-Y-D-K-X7-H-F，其中X6是A、S或T，X7是S或N；
CDR-VL1：Q-X8-S-Q-S-X9-Y-X10-N-N-Y-L-A，其中X8是A或S，X9是Y、V或L，X10是K、N或A；
CDR-VL2：X11-A-X12-T-L-A-S，其中X11是D、G或S，X12是A或S；
CDR-VL3：X13-G-G-Y-D-C-X14-S-A-D-C-X15-A，其中X13是Q或L，X14是R、G、S、Q或L，X15是A或S。
本发明提供的抗生物素的抗体或其功能性片段具有上述互补决定区结构，其能够特异性结合游离的生物素，对生物素具有较好的亲和力，用该抗体或其功能性片段检测生物素，具有较好的特异性和灵敏度。本发明为生物素-链霉亲和素体系的免疫检测降低了干扰，为各种分析物的精确测量提供了更多的可能。
在可选的实施方式中，所述抗体或其功能性片段具有如下互补决定区：
CDR-VH1：T-X1-Y-X2-N，其中X1是Y、A或N；X2是M；
CDR-VH2：M-X3-W-A-T-G-X4-T-Y-Y-A-X5-W-A-K-G，其中X3是I，X4是N、G或R，X5是S或N；
CDR-VH3：T-X6-G-T-Y-D-K-X7-H-F，其中X6是A、S或T，X7是N；
CDR-VL1：Q-X8-S-Q-S-X9-Y-X10-N-N-Y-L-A，其中X8是A，X9是Y、V或L，X10是K、N或A；
CDR-VL2：X11-A-X12-T-L-A-S，其中X11是D、G或S，X12是S；
CDR-VL3：X13-G-G-Y-D-C-X14-S-A-D-C-X15-A，其中X13是L，X14是R、G、S、Q或L，X15是A或S。
本发明的发明人发现，在各互补决定区中的上述突变位点为上述氨基酸残基时，该抗体对游离的生物素表现出更好的亲和力。
在可选的实施方式中，所述抗体或其功能性片段具有如下互补决定区中的任一种组合：

在可选的实施方式中，所述抗体或其功能性片段包括如下序列所示的轻链骨架区：
如SEQ ID NO:1所示的FR1-L；如SEQ ID NO:2所示的FR2-L；如SEQ ID NO:3所示的 FR3-L；如SEQ ID NO:4所示的FR4-L；和/或
如下序列所示的重链骨架区：
如SEQ ID NO:5所示的FR1-H；如SEQ ID NO:6所示的FR2-H；如SEQ ID NO:7所示的FR3-H；如SEQ ID NO:8所示的FR4-H；
优选的，所述抗体还包含恒定区；
优选的，所述恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中的任意一者的恒定区；
优选的，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人；
优选的，所述功能性片段选自所述抗体的VHH、F(ab')2、Fab’、Fab、Fv和scFv中的任意一种。
通常情况下，重链可变区(VH)和轻链的可变区(VL)可由以下编号的CDR与FR按如下组合排列连接获得：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
需要说明的是，在其他的实施例中，本发明提供的抗体或其功能性片段的各骨架区氨基酸序列可以与上述对应骨架区(SEQ ID NO:1、2、3、4、5、6、7或8)可以具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同源性。
上述抗体的功能性片段通常具有与其来源抗体相同的结合特异性。本领域技术人员根据本发明记载的内容容易理解到，上述抗体的功能性片段可以通过比如酶消化的方法(包括胃蛋白酶或木瓜蛋白酶)和/或通过化学还原分裂二硫键的方法获得。在本发明公开了完整抗体的结构基础上，本领域技术人员容易获得上述的功能性片段。
上述抗体的功能性片段还可以通过也是本领域技术人员所知的重组遗传学技术或通过例如自动肽合成仪合成获得。
本发明第二方面，提供一种检测试剂或试剂盒，其包括上述抗生物素抗体或其功能性片段，优选的，所述检测试剂或试剂盒检测的样本选自全血、血清、血浆、尿液或唾液等体液。
在可选的实施方式中，所述试剂或试剂盒中的所述抗体或其功能性片段标记有可被检测的标记物；
优选的，所述可被检测的标记物包括但不限于荧光染料、催化底物显色的酶、放射性同位素、化学发光试剂和纳米颗粒类标记物；
优选的，所述荧光染料包括但不限于荧光素类染料及其衍生物、罗丹明类染料及其衍生 物、Cy系列染料及其衍生物、Alexa系列染料及其衍生物和蛋白类染料及其衍生物；
在可选的实施方式中，所述荧光素类染料及其衍生物包括但不限于异硫氰酸荧光素(FITC)羟基光素(FAM)、四氯光素(TET)等或其类似物；所述罗丹明类染料及其衍生物包括但不限于红色罗丹明(RBITC)、四甲基罗丹明(TAMRA)、罗丹明B(TRITC)等或其类似物；所述Cy系列染料及其衍生物包括但不限于Cy2、Cy3、Cy3B、Cy3.5、Cy5、Cy5.5、Cy3等或其类似物；所述Alexa系列染料及其衍生物包括但不限于AlexaFluor350、405、430、488、532、546、555、568、594、610、33、647、680、700、750等或其类似物；所述蛋白类染料及其衍生物包括但不限于藻红蛋白(PE)、藻蓝蛋白(PC)、别藻蓝蛋白(APC)、多甲藻黄素-叶绿素蛋白(preCP)等。
优选的，所述催化底物显色的酶包括但不限于辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶；
优选的，所述放射性同位素包括但不限于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和18F；
优选的，所述化学发光试剂包括但不限于鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物；
优选的，所述纳米颗粒类标记物包括但不限于纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒；
优选的，所述胶体包括但不限于胶体金属、分散型染料、染料标记的微球和乳胶；
优选的，所述胶体金属包括但不限于胶体金、胶体银和胶体硒。
本发明第三方面，提供一种核酸分子，所述核酸分子可编码上述抗体或其功能性片段。
本发明第四方面，提供一种载体，所述载体含有上述核酸分子。
本发明第五方面，提供一种重组细胞，含有上述的载体。
本发明第六方面，提供一种制备所述的抗体或其功能性片段的方法，其包括：培养上述重组细胞，从培养产物中分离纯化得到所述抗体或其功能性片段。
本发明第七方面，提供所述的抗生物素抗体或其功能性片段在免疫检测中用于抗生物素干扰的用途。
本发明所述抗干扰是指利用生物素-链酶亲和素体系对分析物进行测量时，能够清除除了 生物素-链酶亲和素体系中生物素以外的其他生物素，例如游离生物素、代谢产生的其他生物素等。
在本发明公开了抗体或其功能性片段的氨基酸序列的基础上，本领域技术人员容易想到采用基因工程技术或其他技术(化学合成、杂交瘤细胞)制备得到该抗体或其功能性片段，例如从能够重组表达如上任一项所述的抗体或其功能性片段的重组细胞的培养产物中分离纯化得到该抗体或其功能性片段，这对本领域技术人员来说是容易实现的，基于此，无论采用何种技术制备本发明的抗体或其功能性片段，其均属于本发明的保护范围。
相比于现有技术，本发明具有如下有益效果：
本发明的抗生物抗体或其功能片段，包含特定的重链CDR和轻链CDR，能够特异性的识别并结合样本中游离的生物素，具有高灵敏度和特异性，可以作为样本中生物素干扰的清洁剂，在生物素-链霉亲和素体系中，准确高效的实现对各种分析物的检测，不会造成检测结果假性升高或者降低，使样本即使处于高浓度生物素的环境中，依然能够通过生物素-链霉亲和素体系稳定的检测出分析物，实现临床诊断的准确判断。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。
除非另有定义，否则本文使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。本文描述的那些方法和材料类似或等同的任何方法和材料都可用于本文的制剂或单位剂量的实践或测试。除非另有说明，否则本文采用或考虑的技术是标准方法。材料、方法和实例仅是说明性而非限制性的。
实施例1抗体的制备
1、全抗原的制备
(1)半抗原的制备
a、化合物2的合成
将D-生物素(2.44g,10mmol)溶解在无水甲醇中(75mL)；然后加入浓硫酸(1mL)；氮气保护下将上述反应溶液回流(70℃)7小时；将反应液冷却至室温后于45℃减压真空浓缩；将所得残余物用甲基叔丁基醚(75mL)打浆后过滤，少量甲基叔丁基醚清洗滤饼，所得固体于45℃减压真空干燥，得到约2g白色粉末。MS Found：[M+H]+＝259.34
b、化合物3的合成
将化合物2(1g,3.87mmol)溶解在吡啶(20mL)中；加入4,4'-双甲氧基三苯甲基氯(2g,6mmol)；然后再加入DMAP(50mg,0.38mmol)；充分混合溶解后，将上述混合液转移至100mL带四氟内衬的不锈钢闷罐中；将上述反应溶液在75℃下搅拌18小时；冷却至室温后将反应液倒入200mL去离子水中，用200mL乙酸乙酯萃取2次，合并有机相后，将有机相用200mL 1N盐酸洗一次，100mL饱和碳酸氢钠洗一次，100mL饱和食盐水洗一次后用无水硫酸钠干燥，于45℃减压真空浓缩；所得残余物用柱层析纯化，得到约1.68g白色固体。MS Found：[M+H]+＝561.71
c、化合物4的合成
将化合物3(1g,1.78mmol)溶于THF(20mL)中；将上述溶液用氮气保护后用冰水浴降温冷却至0℃；将氢化钠(60％,100mg,2.67mmol)一次性加入到上述反应混合液中；将上述反 应混合液在0℃下搅拌30分钟；将乙酰氯(210mg,2.67mmol)滴加到上述反应混合液中，控制内温小于20摄氏度；滴加完毕后不撤冷浴，在0-20℃搅拌3小时；将反应液倒入200mL去离子水中，用200mL乙酸乙酯萃取2次，合并有机相，用100mL饱和食盐水洗一次后用无水硫酸钠干燥，于45℃减压真空浓缩；将残余物于45℃减压真空干燥，得到约1.1g黄色固体。MS Found：[M+H]+＝603.75
d、化合物5的合成
将化合物4(1g,1.66mmol)溶于甲醇(20mL)中；用冰水浴降温冷却至0℃；加入氯化氢的甲醇饱和溶液(20mL)；不撤冷浴，自然升温至室温；搅拌15小时后，将反应液于45℃减压真空浓缩；所得残余物用柱层析纯化，得到约0.34g白色固体。MS Found：[M+H]+＝301.37
e、化合物6的合成
将化合物5(0.34g,1.13mmol)溶于THF(20mL)中；将上述溶液用氮气保护后用冰水浴降温冷却至0℃；将氢化钠(60％,68mg,1.7mmol)一次性加入到上述反应混合液中；将上述反应混合液在0℃下搅拌30分钟；将Fomc-丙氨酸酰氯(0.56g,1.7mmol)溶于超干THF(10mL)中后，滴加到上述反应混合液中，控制内温小于20摄氏度；滴加完毕后不撤冷浴，在0-20℃搅拌3小时；将反应液倒入200mL去离子水中，用200mL乙酸乙酯萃取2次，合并有机相，用100mL饱和食盐水洗一次后用无水硫酸钠干燥，于45℃减压真空浓缩；将残余物用柱层析纯化，得到约0.5g黄色固体。MS Found：[M+H]+＝594.75
f、化合物7的合成
将化合物6(0.5g,0.84mmol)溶于甲醇(10mL)中；将上述溶液用氮气保护后用冰水浴降温冷却至0℃；将哌啶(0.7g,8.4mmol)一次性加入到上述反应混合液中；将上述反应混合液在0-20℃下搅拌3小时；将反应液于45℃减压真空浓缩；将残余物于45℃减压真空干燥；将所得黄色油状残余物溶于甲醇(10mL)中；加入一水合氢氧化锂(0.1g,2.5mmol)，将反应混合物在60℃下搅拌1.5小时后冷却至室温；将上述混合物于45℃减压真空浓缩；将残余物用Pre-HPLC纯化，得到约167mg白色固体。MS Found：[M+H]+＝358.43
g、化合物8的合成
将化合物7(167mg,0.46mmol)溶于DMF(5mL)中；氮气保护下加入双琥珀酰亚胺辛二酸酯(172mg,0.46mmol)，并在氮气保护下将反应液在室温搅拌1-2小时；将反应液直接用Pre-HPLC纯化，得到约98mg白色固体。MS Found：[M+H]+＝611.68
(2)全抗原的制备
全抗原-BSA的制备：称取1mg化合物8，溶于500ul DMSO当中；取3mg KLH，加1mL20mM磷酸盐缓冲溶液(PBS，pH＝7.4)稀释，再逐滴将半抗原Ⅰ液缓慢加入到KLH中，室温反应过夜；然后用20mM PBS溶液透析，除去未反应的小分子半抗原，得到完全抗原Biotin-KLH，并通过紫外吸收扫描方法进行鉴定。
2、家兔免疫基抗体检测
将弗氏完全佐剂与浓度为2mg/ml的完全抗原Biotin-KLH蛋白按等体积混合并乳化，挑选4只2月龄1.5公斤左右的健康新西兰大白兔，采用背部皮下五点注射的免疫方式对兔子进行初免，免疫剂量为800ug/只。在免疫的第一个月每周对兔子进行免疫，第二个月开始，每月对兔子进行免疫。初免用弗式完全佐剂，其余免疫用弗式不完全佐剂，加强免疫的免疫原用量为初免用量的一半。36天后开始采取兔耳静脉采血，离心收集上清利用ELISA检测血清效价。对兔血清效价进行监测，待兔血清效价合格，取脾脏分离淋巴细胞用于细胞融合。
3、杂交瘤细胞融合
取免疫后兔子的耳动脉血，ELISA测定免疫效价；将效价达标的兔子处死，取脾脏，研磨，裂红，制备B细胞。将兔B细胞与骨髓瘤细胞SP2/0按1：1混合，然后进行电融合，得到兔鼠杂交瘤细胞。随后将杂交瘤细胞加入1.2％甲基纤维素半固体培养基中，再添加HAT、抗支原体药物、饲养层细胞充分混匀，铺板。置培养箱培养3-4天后补加DMEM完全培养基，连续培养13天后，取细胞上清进行ELISA检测(初筛、复查)。
4、抗体筛选
本实施例以血清淀粉样蛋白A(SAA)检测为例
抗体筛选方案是成功开发本发明抗体的关键。在抗体筛选阶段，在使用生物素-亲和素体系检测SAA的体系中，加入游离生物素干扰体系信号，筛选能恢复体系信号值的杂交瘤克隆。
具体实验方案：1ug/ml SAA抗原4℃包被8小时；洗板后，使用100ul标记生物素的SAA抗体(50ng/ml，使用1％BSA+0.1％PBST配置以达到封闭效果)孵育30min；加入50ul的50ng/ml的游离生物素，同时加入50ul细胞株的上清液(对照加入等体积的空白培养基)以及50ul亲和素-HRP(1：2000倍稀释)，37℃孵育30min；洗板，使用显色液显色3min，终止，使用酶标仪在OD450下读数。
对照组无游离生物素干扰，故信号值保持较高水平(1.7282)，使用50ng/ml游离生物素干扰生物素-亲和素体系后，信号值明显降低(0.3833)，细胞株上清抗体可结合游离生物素，发挥抗干扰作用，从而保证标记生物素的SAA抗体与亲和素结合不受到游离亲和素的干扰，明显提高信号值，据此筛选出抗干扰能力最好的细胞株用于后续实验。
所述抗原包被在酶标板上，所述酶标板为聚苯乙烯，与抗原的结合依靠吸附或疏水作用等，不会影响抗原本身的活性基团。
5、抗体制备
杂交瘤细胞序列测定。取ELISA检测筛选为阳性的杂交瘤细胞，经DPBS清洗后利用裂解液直接裂解细胞，然后利用反转录反应获取cDNA。利用设计的兔抗特异性轻重链引物、TAQ酶以及获取的cDNA样本进行体外扩增实验，获得兔抗轻重链PCR产物。随后通过试剂盒进行胶回收与T载体构建实验，随后测序公司送测。
抗体发酵。取生长对数期内Expi 293细胞，经台盼蓝计数、记活率后按一定比例接种至一次性摇瓶内放于恒温箱孵育，24h后取所需细胞数的细胞转移至新发酵瓶中，将计算好比例的转染试剂和质粒溶液1：1混合均匀再静置少量时间后缓慢倒入新摇瓶中放于恒温箱孵育，18-22h后再加入一定比例的补料，于5-6天后收集发酵上清。ProG柱亲和层析纯化获得抗体WT。
实施例2抗体性能的验证
1、ELISA亲和力测试
(1)亲和力检测(抗体与全抗原亲和力)实验步骤
全抗原包被(1ug/ml)4℃孵育8小时；一抗孵育：洗板后，纯化后抗体(1ug/ml)三倍梯度稀释，100ul/孔，37℃孵育30min；二抗(羊抗兔-HRP)孵育：洗板，羊抗兔-HRP(1：5000)稀释后，100ul/孔，37℃孵育30min；使用显色液显色3min，终止，使用酶标仪在OD450下读数。
分析实施例1步骤5的抗体(WT)序列，其重链可变区如SEQ ID NO:9所示，其中，重链可变区上的各互补决定区的氨基酸序列如下：
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。
其轻链可变区如SEQ ID NO:10所示，其中，轻链可变区上的各互补决定区的氨基酸序列如下：
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。
在实施例1的抗生物素抗体(WT)基础上，在互补决定区中对于抗体活性有关的位点进行突变，其中，X1-X15均为突变位点。见下表1。并对抗生物素抗体的WT和突变体进行亲和力检测，结果如表2所示。
表1与抗体活性有关的突变位点
表2抗体活性分析数据

从表2可以看出，突变1的活性效果最佳，因而以突变1作为骨架序列，筛选抗干扰活性较好的突变位点，部分结果如表3所示。
表3与抗体亲和力有关的突变位点

对突变1的突变体进行亲和力检测，抗原浓度为1ug/ml，测量每个突变OD450的吸光值，结果如表4所示。
表4突变亲和力测试
由表4可知，突变1的突变体的亲和力与突变体1相似。
(2)ELISA竞争法检测对游离生物素结合能力
竞争法(抗体与游离生物素结合能力)实验步骤：全抗原包被(1ug/ml)4℃孵育8小时；游离生物素1ug/mL，3倍梯度稀释，60ul/孔，抗体稀释至500ng/mL，加抗体40ul/孔，37℃孵育30min；二抗(羊抗兔-HRP)孵育后洗板，羊抗兔-HRP(1：5000)稀释后，100ul/孔，37℃孵育30min；使用显色液显色3min，终止，使用酶标仪在OD450下读数。突变体1与游离生物素结合能力结果如表5所示。
表5游离生物素结合能力

由表5可知：随游离生物素使用量升高，ELISA信号值明显降低，从添加0ug/ml-1ug/ml，WT的信号改变为1.0743，突变1的信号改变为1.0821，突变1相对于WT，对游离生物素具有更强的结合能力。
因为，抗体能与全抗原和游离生物素结合，当没有游离生物素时，抗体只能与全抗原结合，被固定在板上，当羊抗兔-HRP与抗体结合，在底物作用下时，会产生强烈的光信号，而当溶液中存在游离生物素时，抗体会与游离生物素结合，而羊抗兔-HRP会与抗体结合，在底物下显色，因为游离生物素与抗体结合以后依旧是游离的状态，所以洗板后，会被清洗掉，所以随着游离生物素的添加，发光信号会降低。
突变体1的突变体与游离生物素结合能力：突变位点如表3所示，游离生物素的浓度为0ug/ml和1ug/ml时，测量每个突变OD450的吸光值强弱，差值如表6所示。
表6突变游离生物素结合能力
由表6可以看出，突变体1的突变体整体变化并不是很大，说明上述突变的抗体依然具有很好的游离生物素结合力。
实施例3化学发光法抗干扰能力测试
选择抗缪勒氏管激素(AMH)检测试剂盒(化学发光法)，在样本中加入不同浓度的游离生物素作为干扰，在试剂R1组分中加入不同浓度的抗体(突变1)，验证不同浓度抗体消除游离生物素干扰的效果。
具体实验方案：使用中元汇吉AMH检测试剂盒，在样本中加入不同浓度的游离Biotin，在R1组分中加入不同浓度纯化后抗体，使用中元汇吉EXI1800全自动化学发光免疫分析仪测量样本数值。
AMH检测试剂盒：R1生物素抗体使用量50ul；R2酶标抗体使用量50ul；亲和素磁珠使用量30ul；样本加样量20ul。结果如表7所示。
表7抗干扰能力测试
结果表明，不同剂量的游离生物素会对AMH检测信号值造成干扰，干扰程度随游离生物素浓度升高而增强，纯化突变1可结合游离生物素，明显改善游离生物素造成的信号值降低，且随纯化突变1使用浓度的升高，其抗游离生物素干扰能力也明显增强，0.5mg/ml的纯化突变1基本可抵抗2400ng/ml游离生物素的干扰。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (10)

  1. 一种抗生物素抗体或其功能性片段，其特征在于：所述抗体或其功能性片段具有如下互补决定区：
    CDR-VH1：T-X1-Y-X2-N，其中X1是Y、A或N；X2是I或M；
    CDR-VH2：M-X3-W-A-T-G-X4-T-Y-Y-A-X5-W-A-K-G，其中X3是I，X4是N、G或R，X5是S或N；
    CDR-VH3：T-X6-G-T-Y-D-K-X7-H-F，其中X6是A、S或T，X7是S或N；
    CDR-VL1：Q-X8-S-Q-S-X9-Y-X10-N-N-Y-L-A，其中X8是A或S，X9是Y、V或L，X10是K、N或A；
    CDR-VL2：X11-A-X12-T-L-A-S，其中X11是D、G或S，X12是A或S；
    CDR-VL3：X13-G-G-Y-D-C-X14-S-A-D-C-X15-A，其中X13是Q或L，X14是R、G或S，X15是A或S。
    优选地，所述抗体或其功能性片段具有如下互补决定区：
    CDR-VH1：T-X1-Y-X2-N，其中X1是Y、A或N；X2是M；
    CDR-VH2：M-X3-W-A-T-G-X4-T-Y-Y-A-X5-W-A-K-G，其中X3是I，X4是N、G或R，X5是S或N；
    CDR-VH3：T-X6-G-T-Y-D-K-X7-H-F，其中X6是A、S或T，X7是N；
    CDR-VL1：Q-X8-S-Q-S-X9-Y-X10-N-N-Y-L-A，其中X8是A，X9是Y、V或L，X10是K、N或A；
    CDR-VL2：X11-A-X12-T-L-A-S，其中X11是D、G或S，X12是S；
    CDR-VL3：X13-G-G-Y-D-C-X14-S-A-D-C-X15-A，其中X13是L，X14是R、G、S、Q或L，X15是A或S。
  2. 根据权利要求1所述的一种抗生物素抗体或其功能性片段，其特征在于：所述抗体或其功能性片段具有如下互补决定区中的任一种组合：

  3. 根据权利要求1所述的一种抗生物素抗体或其功能性片段，其特征在于：所述抗体或其功能性片段包括如下序列所示的轻链骨架区：
    如SEQ ID NO:1所示的FR1-L；如SEQ ID NO:2所示的FR2-L；如SEQ ID NO:3所示的FR3-L；如SEQ ID NO:4所示的FR4-L；和/或
    如下序列所示的重链骨架区：
    如SEQ ID NO:5所示的FR1-H；如SEQ ID NO:6所示的FR2-H；如SEQ ID NO:7所示的 FR3-H；如SEQ ID NO:8所示的FR4-H；
    优选的，所述抗体还包含恒定区；
    优选的，所述恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中的任意一者的恒定区；
    优选的，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人；
    优选的，所述功能性片段选自所述抗体的VHH、F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
  4. 一种检测试剂或试剂盒，其包括权利要求1-3任一项所述的抗生物素抗体或其功能性片段，优选的，所述检测试剂或试剂盒检测的样本选自全血、血清、血浆、尿液或唾液的体液。
  5. 根据权利要求4所述的试剂或试剂盒，其特征在于，所述试剂或试剂盒中的所述抗体或其功能性片段标记有可被检测的标记物；
    优选的，所述可被检测的标记物包括但不限于荧光染料、催化底物显色的酶、放射性同位素、化学发光试剂和纳米颗粒类标记物；
    优选的，所述荧光染料包括但不限于荧光素类染料及其衍生物、罗丹明类染料及其衍生物、Cy系列染料及其衍生物、Alexa系列染料及其衍生物和蛋白类染料及其衍生物；
    优选的，所述催化底物显色的酶包括但不限于辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶；
    优选的，所述放射性同位素包括但不限于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和18F；
    优选的，所述化学发光试剂包括但不限于鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物；
    优选的，所述纳米颗粒类标记物包括但不限于纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒；
    优选的，所述胶体包括但不限于胶体金属、分散型染料、染料标记的微球和乳胶；
    优选的，所述胶体金属包括但不限于胶体金、胶体银和胶体硒。
  6. 一种核酸分子，其特征在于，所述核酸分子可编码权利要求1-3任一项所述的抗体或其功能性片段。
  7. 一种载体，其特征在于，所述载体含有权利要求6所述的核酸分子。
  8. 一种重组细胞，其特征在于，含有权利要求7所述的载体。
  9. 一种制备如权利要求1-3所述的抗体或其功能性片段的方法，其包括：培养权利要求8所述的重组细胞，从培养产物中分离纯化得到所述抗体或其功能性片段。
  10. 权利要求1-3所述的抗生物素抗体或其功能性片段在免疫检测中用于抗生物素干扰的用途。
PCT/CN2023/118384 2022-09-20 2023-09-12 一种抗生物素抗体或其功能性片段及其应用 WO2024061058A1 (zh)

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