CN117384294A

CN117384294A - Mouse anti-goose IgY monoclonal antibody and application

Info

Publication number: CN117384294A
Application number: CN202311708815.0A
Authority: CN
Inventors: 王河川; 石元朔; 马玉岭; 陈新新; 苑晓松; 潘悦; 刘星; 路轲; 王芳; 魏彦辉
Original assignee: Beijing Solarbio Technology Co ltd
Current assignee: Beijing Solarbio Technology Co ltd
Priority date: 2023-12-13
Filing date: 2023-12-13
Publication date: 2024-01-12
Anticipated expiration: 2043-12-13
Also published as: CN117384294B

Abstract

The invention belongs to the technical field of poultry immunology, and provides a mouse anti-goose IgY monoclonal antibody and application thereof. According to the invention, after fusion screening is carried out on immunized mice, two monoclonal antibodies with good specificity and stability are obtained, and an ELISA detection technology based on a double-antibody sandwich method is established, so that the content of IgY in geese can be detected. The method has the advantages of good specificity and high sensitivity by means of an enzyme chromogenic amplification system, and can detect samples with low content of goose IgY.

Description

Mouse anti-goose IgY monoclonal antibody and application

Technical Field

The invention belongs to the technical field of poultry immunology, and particularly relates to a mouse anti-goose IgY monoclonal antibody and application thereof.

Background

Geese are herbivores, the dietary structure is also obviously changed along with the continuous improvement of the living standard of people, the demands of people on poultry, particularly ecological raised poultry, are steadily improved, and the meat goose industry is rapidly developed in recent years. The goose contains various amino acids necessary for human growth and development, the composition of the goose is close to the proportion of amino acids required by human body, the goose is full-value and high-quality protein, the fat content in the goose is low, the quality is good, the content of unsaturated fatty acid is high, and particularly, the linolenic acid content is higher than that of other meats, so that the goose is very beneficial to human health. The melting point of the goose fat is also very low, the texture is soft, and the goose fat is easy to digest and absorb by human bodies.

IgY detection methods are various, and common methods include a single immunodiffusion method, an immunoturbidimetry method and the like, but the methods have the disadvantages of long time consumption, poor accuracy and poor repeatability. The sensitivity of the chemiluminescence method is higher than that of an enzyme-linked immunosorbent assay (ELISA), but the instrument and equipment required to be detected are more expensive than that of an ELISA used for the ELISA, so that the ELISA double-antibody sandwich method has high sensitivity and good repeatability and is widely applied to the defects of the method.

Most ELISA kits which are currently available on the market are ELISA kits developed for IgG of human, rat and mouse, but the development of IgG ELISA kits for animals such as pigs, cattle, horses, sheep, rabbits, chickens and the like of small species is less, and the development of goose IgY ELISA kits is more rare. At present, some goose IgY detection kits sold in domestic markets have difficulty in ensuring detection sensitivity and result accuracy due to mixed fish and dragon.

Disclosure of Invention

The invention aims to provide a mouse anti-goose IgY monoclonal antibody and application thereof.

The invention utilizes the mouse monoclonal antibody technology to carry out fusion screening on Balb/c mice after immunization, obtains two monoclonal antibodies with good specificity and stability, carries out antibody pairing, and develops a double-antibody sandwich ELISA kit. Can be applied to the detection of the content of the IgY in geese, and overcomes various defects of other original methods.

To achieve the object of the present invention, in a first aspect, the present invention provides a mouse anti-goose IgY monoclonal antibody (2H 6), the heavy chain 3 CDR regions of which are respectively: DYYMK, DIIPNTGDTFFNRKFKD and SIVTAYRDY (SEQ ID NOS: 12-14), the 3 CDR regions of the light chain are: KASRDIDQYLA, YTSTLQP and LQYDSLYT (SEQ ID NO: 9-11).

Further, the light chain variable region of the antibody is: an amino acid sequence shown in SEQ ID NO. 3; or polypeptide with the same function obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 3;

the heavy chain variable region of the antibody is: an amino acid sequence shown in SEQ ID NO. 4; or a polypeptide having the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO. 4.

In a second aspect, the invention provides nucleic acid molecules encoding said antibodies.

In a third aspect, the invention provides biological materials comprising the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors or engineering bacteria.

In a fourth aspect, the invention provides the use of said antibodies in the detection of goose IgY (including for non-disease diagnostic purposes).

In a fifth aspect, the invention provides a goose IgY detection reagent or kit prepared from the antibodies.

In a sixth aspect, the invention provides a goose IgY enzyme-linked immunosorbent assay kit, comprising: the antibody and the labeled mouse anti-goose IgY monoclonal antibody (2F 2), and the kit further comprises at least one of a substrate chromogenic solution, a blocking solution, a stopping solution and the like.

Wherein, the amino acid sequences of the light chain and heavy chain variable regions of the mouse anti-goose IgY monoclonal antibody (2F 2) are respectively shown as SEQ ID NO. 7 and 8. The heavy chain 3 CDR regions of antibody 2F2 are: SYGMS, TINSVGTYTHYVDSVKG and HADYVGFAY (SEQ ID NOS: 18-20), the 3 CDR regions of the light chain are: KASQDVISAVA, SASYRYT and QQHYNTPYT (SEQ ID NOS: 15-17).

Further, the antibody is immobilized on a solid support, which may be selected from an ELISA plate, a microsphere, a nitrocellulose membrane, a glass cellulose membrane, a nylon membrane, etc., preferably an ELISA plate.

Further, the label may be an enzyme label, a biotin label, a fluorescein label, or the like, preferably an enzyme label.

The enzyme-labeled enzyme may be selected from any one of the following: horseradish peroxidase (HRP), alkaline Phosphatase (AP), glucose oxidase, β -galactosidase, lysozyme, malate dehydrogenase, and the like, with horseradish peroxidase being preferred.

Common substrates include: o-phenylenediamine (OPD), tetramethylbenzidine (TMB), and ABTS [2,2' -azino-di- (3-ethylbenziazobine sulfonate-6) ] or the like, TMB being preferred.

Further, the kit also comprises a washing buffer (1×pbst) with the following components: na (Na) ₂ HPO ₄ 8 mM、NaCl 0.136M、KH ₂ PO ₄ 2 mM、KCL 2.6 mM、Tween-20 0.05%v/v。

Further, the concentration of the antibody used for coating the ELISA plate is 1-10. Mu.g/mL.

Further, the concentration of the horseradish peroxidase-labeled mouse anti-goose IgY monoclonal antibody is 0.5-20 mg/mL.

Further, the stop solution is a 1.5-2.5M (preferably 2M) sulfuric acid solution.

In a seventh aspect, the invention provides a composition comprising said antibody (2H 6) and said mouse anti-goose IgY mab (2F 2).

In an eighth aspect, the invention provides a goose IgY enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit prepared from the composition.

In a ninth aspect, the invention provides the use of said antibody, or said kit or said composition, in goose IgY enzyme-linked immunosorbent assay (including qualitative and quantitative assays), including for non-disease diagnostic purposes.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

the invention has the advantages of high sensitivity, strong specificity, high accuracy, simple experimental operation, short time and the like in the detection of goose meat products, serum, plasma and other biological samples of geese by using the double-antibody sandwich ELISA method established by the two monoclonal antibodies with strong specificity. The detection range of the goose IgY ELISA kit constructed by the monoclonal antibody is 0-40ng/ml, and the sensitivity is 87pg/ml.

And secondly, the kit is subjected to an accelerated stability experiment at 37 ℃, the kit has no obvious change within 13 days, and the stability is high.

And thirdly, carrying out a similar protein cross experiment on the kit, wherein the kit shows good specificity and does not recognize duck IgY, chicken IgY, rabbit IgG, cow IgG, goat IgG, pig IgG, human IgG, mouse IgG, rat IgG, duck IgA, goose IgA, duck IgM and goose IgM.

Fourthly, the kit can conveniently and accurately detect the IgY content in the goose meat, the reaction time/detection time is 3 hours, the reaction can be carried out only by standing at room temperature, a constant temperature incubator or a shake incubator is not needed, and the kit does not have cross reaction with meat of other economic animals such as ducks, chickens, pigs, sheep, cattle and the like. Compared with foreign antibody pairs of the same variety, the method has higher measurement accuracy and can replace the foreign imported kit.

Drawings

FIG. 1 shows the result of SDS-PAGE for identifying the purity of an antibody in the preferred embodiment 1 of the present invention. Wherein M is Marker;2, a mouse anti-goose IgY monoclonal antibody 2F2;3 mouse anti-goose IgY monoclonal antibody 2H6.

FIG. 2 is a standard curve established by the double antibody sandwich ELISA method of the preferred embodiment 2 of the invention.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.

EXAMPLE 1 preparation of anti-goose IgY monoclonal antibodies

1. Immunization of animals

Female Balb/c mice with the age of 6-8 weeks are selected, the purified goose IgY is emulsified with an equal volume of Freund's adjuvant for immunization, the immunization period is two weeks, the blood is taken for measuring the titer after 3 times of immunization, and the immunization is boosted again three days before fusion.

2. Cell fusion

Mice were sacrificed by cervical scission, spleens were removed by aseptic manipulation, and spleen cell suspensions were prepared by squeeze milling in a plate. The prepared syngeneic myeloma cells and the spleen cells of the mice are mixed according to a certain proportion, and a fusogenic agent polyethylene glycol (PEG) is added. Under the action of polyethylene glycol, various lymphocytes can be fused with myeloma cells to form hybridoma cells. The specific operation is as follows:

myeloma (SP 2/0) cell activation:

thawing and resuscitating commercial SP2/0 cells, and then re-suspending in nutrient solution (RPMI-1640, added calf serum) at 37deg.C in 5% CO ₂ Culturing in an incubator under the condition; passaging is carried out after 3-5 d; the cells were collected and suspended in 1640 basal medium, and 0.5X10 after counting ⁶ ~1×10 ⁶ The mice were injected subcutaneously into the back of BALB/c mice and cultured continuously for 9-10 days. After the tumor volume on the back increased to about 0.8cm in diameter, mice were sacrificed by cervical stretchingSoaking in 75% alcohol for 5min, and performing aseptic operation to obtain tumor. Cutting off tumor blocks, placing the cut tumor blocks in a sterilized homogenizer, adding 1640 basal medium, fully grinding, adding 10mL1640 basal medium, standing for 2min, sucking the cell suspension on the upper layer, placing in another centrifuge tube, adding 10mL1640 basal medium, and repeatedly grinding twice; the cell suspension obtained above was centrifuged at 1000 r/min for 10min to remove the supernatant, followed by resuspension in 30 mL1640 basal medium. Adding 15mL of lymphocyte separation liquid into another centrifuge tube, and carefully placing the cell suspension on the separation liquid; then, the mixture is centrifuged for 15min at 1200 r/min, white cell layers at the interface are sucked by a suction tube, the cells are washed by 1640 basal medium for 2 times and then resuspended in 10mL1640 basal medium, and the cells are counted for later use.

Preparation of immune spleen cells:

taking one BALB/c mouse with enhanced immunity, killing the eye socket by bleeding (collecting serum, namely positive serum), soaking in 75% alcohol for 5-10 min for sterilization, then fixing the BALB/c mouse on an dissecting plate for dissection, taking out spleen, shearing the spleen, and placing the mouse in a sterilized homogenizer; grinding and cell suspension preparation method are as described in SP2/0, and counting for later use.

Preparation of feeder cells:

one non-immunized BALB/c mouse was taken, the orbit was exsanguinated, and serum was collected as negative serum. Injecting 2-3 mL1640 basic culture medium into the abdominal cavity of the mouse, sucking out the basic culture medium after blowing, and placing the basic culture medium into another centrifuge tube for standby, wherein the basic culture medium contains abdominal macrophages. Spleen cell suspensions were prepared and placed into the peritoneal macrophage tube in the same manner as above. 1000 Centrifuging at r/min for 10min to remove supernatant, suspending cells in HAT medium, standing at 37deg.C, and 5% CO ₂ And (5) placing the mixture in an incubator for later use.

Fusion:

will be 1X 10 ⁷ -2×10 ⁷ SP2/0 and 10 ⁸ The individual immunocytes were mixed well in a 50mL centrifuge tube and centrifuged at 1000 r/min for 8 min. After discarding the supernatant, the centrifuge tube containing the cell mixture was placed in a 37℃water bath, followed by addition of 50% PEG 0.8 mL (sigma) pre-warmed to 37℃and allowed to stand for 30s after stirring. After standing, 10ml of a base 1640 solution preheated at 37℃was added. Centrifuging at 1000 r/min for 5min, removing supernatant, standing at 37deg.C for 5-8min. Subsequently mixed with feeder cell suspension, seeded in 96-well plates at 250 ul/well, at 37℃with 5% CO ₂ Culturing in an incubator. HT medium was changed for continued culture on day 4 after fusion. And (4) when the colony of the fused cells grows to 1/4 of the culture hole and the culture medium turns yellow slightly, detecting the antibody.

3. Selection of hybridoma positive clones and cloning of cells

The purpose of the selective culture is to screen the fused hybridoma cells using HAT selective medium. In HAT medium, unfused myeloma cells lack hypoxanthine-guanine-phosphoribosyl transferase and cannot synthesize DNA by salvage pathways to die. Unfused lymphocytes have hypoxanthine-guanine-phosphoribosyl transferase, but do not survive in vitro for long periods and die. Only fused hybridoma cells survive and proliferate in HAT medium due to the hypoxanthine guanine phosphoribosyl transferase obtained from spleen cells and the unlimited proliferation of myeloma cells. The specific operation is as follows:

positive hybridoma cells were screened by indirect ELISA as follows:

coating known antigens: diluting the purified coating antigen to 1-10 mug/ml with coating buffer; 100 μl of each well is added to the microwells, gently shaken, and incubated at 4deg.C overnight in a refrigerator or 37℃for 1h; the liquid in the hole is thrown away (the liquid in the hollow is beaten as much as possible); washing for 2-3 min for 3 times.

Blocking the positions of the enzyme-labeled wells, which are not coated by antigen, by adding 200 μl of blocking solution (5% skimmed milk powder or 0.1% BSA) to each well, and gently shaking at 37deg.C for 1 hr; throwing away the liquid in the hole; washing buffer solution according to Kong Jiaman, standing for 2-3 min, throwing away liquid in the hole, beating to dry, and washing 3 times with the washing buffer solution. And (3) sample adding, namely taking 50 mu l of supernatant from each hole of the hybridoma to be detected, sequentially adding the supernatant into the enzyme-labeled holes, and gently shaking the mixture. Incubate for 1h at 37 ℃, wash and pat dry.

Adding enzyme-labeled anti-antibody: diluting the enzyme-labeled secondary antibody to a proper working concentration according to instructions by using a diluent, adding 100 mu l of the enzyme-labeled secondary antibody into each hole, gently shaking the mixture, and placing the mixture at 37 ℃ for 1h; then washing and beating to dry. Adding a color development liquid: each well was added with 100. Mu.l of freshly prepared color development solution, gently shaken well, and incubated at 37℃for 10min. Terminating the reaction: mu.l of stop solution (2M sulfuric acid solution) was added to each well.

Determination result: enzyme label instrument OD ₄₅₀ Readings were taken 3 times larger than the negative wells and were judged positive.

Cloning of hybridoma cells (limiting dilution method)

Preparing a mouse feeder cell layer before cloning; gently blowing the hybridoma cells to be cloned from the culture well, and counting the number of living cells by using a blood cell counting plate; diluting cells to 1 cell/ml with complete medium; the cell suspensions were added to 96-well plates of prepared feeder cells, 100. Mu.l/well, respectively, so that 1 cell was contained in each well. Culturing until 4 days of fluid infusion is one drop, carefully observing the growth condition of cells in each hole on 5-6 days, and recording;

detection of specific antibodies: the cell clone can be detected when 1/3-1/2 field of vision is full of the cell clone on the 7 th to 9 th days after the cloning; cells in the positive holes can be transferred to a 24-hole culture plate, and when the cells in the 24-hole plate grow well, the mice can be inoculated in the abdominal cavity to collect ascites.

4. Monoclonal antibodies 2H6 and 2F2 variable region sequencing

Collecting hybridoma cell number greater than 10 ⁶ : extracting total RNA of two strains of cells respectively by adopting a total RNA extraction kit of Soy Bao technology Co., ltd according to the operation of a specification; synthesizing a first strand of cDNA according to a Soxhaust reverse transcription kit; and (5) sending the sample to a qing department organism for subsequent construction and sequencing to obtain a gene sequencing result.

The amino acid sequences of the light chain and heavy chain variable regions of the monoclonal antibody 2H6 are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4, and the coding gene sequences are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2.

The amino acid sequences of the light chain and heavy chain variable regions of the monoclonal antibody 2F2 are shown as SEQ ID NO. 7 and 8 respectively, and the coding gene sequences are shown as SEQ ID NO. 5 and 6 respectively.

5. Large-scale preparation of monoclonal antibodies 2H6 and 2F2

The hybridoma cells after the strain establishment are injected into the abdominal cavity of the mice, and ascites is collected about 7 days. Subjecting collected ascites to low-temperature centrifugation:4000rpm，4 ^o c was centrifuged for 20 minutes and the supernatant was collected. The ascites supernatant was filtered with a disposable 0.45 [ mu ] m filter, the treated ascites was diluted with an equal volume of equilibration buffer, 0.5ml of protein G was loaded into the column, the column was returned to room temperature, peristaltic pump, recorder and detector were turned on, the column equilibrated with equilibration buffer, then loaded, the effluent was collected, the column was continued to be washed with equilibration buffer to thoroughly remove the contaminating proteins, the elution buffer was replaced, and the elution peak was collected with a collection tube containing an appropriate amount of neutralization buffer. Purified antibodies were dialyzed against an excess of 0.1M PBS, pH 7.4. Ultrafiltering and concentrating dialyzed antibody>1mg/ml. After concentration, the antibody was subjected to 10000rpm,4 ^o C centrifugation for 10min to remove the precipitate. The concentrated antibody concentration was determined by a disposable 0.22 μm filter for sterilization and an ultraviolet-visible spectrophotometer. The purity of the antibodies was identified by SDS-PAGE and the results are shown in FIG. 1.

Example 2 establishment of double antibody sandwich ELISA detection method

1. Relative affinity constant determination

Diluting goose IgY to 2 mug/ml by using coating buffer solution; 100 μl of each well was added to the microwells and coated overnight in a refrigerator at 4deg.C; throwing away the liquid in the hole, adding 200 mu l of sealing liquid into each hole in the micropore, gently shaking, and incubating for 2 hours at room temperature; throwing away the liquid in the hole; washing buffer solution Kong Jiaman, standing for 1-2 min, throwing away liquid in the hole, and drying; the method is performed by washing 3 times with washing buffer. And (3) sample adding, namely diluting the monoclonal antibody to be detected to a saturation concentration, adding 100 mu l of the monoclonal antibody into the enzyme-labeled wells, and incubating for 2 hours at room temperature. After washing the plate with the washing solution, 60 μl/well of sodium thiocyanate solution of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 and 0mol/L was added sequentially, and the plate was left standing at room temperature for 15min, and washed with the washing buffer solution for 3 times. HRP-labeled goat anti-mouse IgG was added, washed after incubation, and patted dry. TMB color development solution is added to 100 μl/well, and color development is performed at room temperature for 10min. Mu.l of stop solution was added to each well. OD was measured with a microplate reader at a wavelength of 450 nm. And (3) result judgment: OD after elution _450nm The concentration of sodium thiocyanate corresponding to the reduction to 50% without elution is the relative affinity constant of the antibody, expressed in mol/l. The results in Table 1 show that all monoclonal antibodies have a high affinity (> 1.5 mol/L).

TABLE 1

2. HRP labelling of monoclonal antibody 2F2

Monoclonal antibody 2F2 was added to a dialysis bag and dialyzed overnight at 4 ℃ in 0.01M CB buffer. 10mg of HRP was dissolved in 2ml of water. Preparation of fresh NaIO ₄ Solution, 0.1M, 0.4ml of 0.1M NaIO was added ₄ In 2ml HRP solution, mix well, keep out light for 45min at room temperature, soak in 10mM NaAc buffer overnight at 4deg.C for dialysis. The overnight dialyzed antibody and HRP solution were removed and the pH was adjusted by adding 0.1ml of 0.2M carbonate buffer, pH 9.5. The antibody that had been removed was then immediately added to the HRP solution and gently stirred at room temperature for 2 hours in the dark. Weigh 0.04g NaBH ₄ Dissolving in 10ml of water, adding 0.4ml into the reaction solution, mixing, and standing at 4 ℃ for reaction for 2 hours. Taking out, placing in a dialysis bag, dialyzing in 0.01M PBS, changing the solution once after 2 hours, and dialyzing at 4deg.C overnight. Taking out the overnight dialyzed labeling solution, adding equal volume of glycerol, and storing at-20deg.C.

3. Preparation of monoclonal antibody 2H6 ELISA plate

And diluting the monoclonal antibody 2H6 purified by Protein G affinity chromatography to 2 mug/mL by using a 96-hole flat-bottom polystyrene ELISA plate as a solid-phase carrier, adding the diluted antibody into micropores of the ELISA plate, 100 mug/hole, coating the ELISA plate at 4 ℃ overnight after sealing a plate membrane, taking out the coated ELISA plate, discarding the liquid in the holes, washing the plate 3 times by using ELISA washing liquid, adding a sealing liquid, 250 mug/hole, sealing the plate hole by using a sealing plate membrane, sealing the plate hole at room temperature for 2H, and drying the plate hole in a dry room for 16-18H. Placing into aluminum foil bag containing drying agent, vacuum sealing, and preserving at 4deg.C.

4. Establishment of double antibody sandwich ELISA method

And taking out the ELISA plate coated with the monoclonal antibody 2H 630 min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. 100 μl of goose IgY standard with different dilution concentrations of 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0.625ng/ml was added and blank control was set. After closing the plate, the plate is at room temperatureIncubating the incubator for 2 hours, washing the plate for 4 times and spin-drying. Adding 100 μl enzyme-labeled antibody HRP-2F2 working solution into the reaction well, sealing, incubating in a room temperature incubator for 45min, washing the plate for 4 times, and spin-drying. 100. Mu.l of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.l of stop solution was added, dual wavelength detection was performed using an ELISA reader, the OD at the 450nm maximum absorption wavelength and 630nm reference wavelength was determined, and the OD at 630nm was subtracted from the OD at 450 nm. Standard curves (using four parameter fitting) were plotted with standard concentration on the abscissa (ng/ml) and absorbance OD on the ordinate (450 nm-630 nm) using ELISA Calc software. The result shows that the detection range is 0.625-40ng/ml, R ² 0.99998 (fig. 2).

5. Double-antibody sandwich ELISA sensitivity detection

The average of 20 zero standard concentrations OD was measured plus two standard deviations, and the corresponding detectable concentration was calculated with a sensitivity of 87pg/ml (Table 2).

TABLE 2

6. Double-antibody sandwich ELISA specific detection

And taking out the ELISA plate coated with the monoclonal antibody 2H 630 min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. 100 μl of goose IgY standard or goose IgY structural similar proteins with different dilution factors including duck IgY, chicken IgY, rabbit IgG, cow IgG, goat IgG, pig IgG, human IgG, mouse IgG, rat IgG, duck IgA, goose IgA, duck IgM, and goose IgM are added. And (3) incubating the plates for 2 hours in a room temperature incubator, washing the plates for 4 times and spin-drying. Adding 100 μl enzyme-labeled antibody HRP-2F2 working solution into the reaction well, sealing, incubating in a room temperature incubator for 45min, washing the plate for 4 times, and spin-drying. 100. Mu.l of chromogenic substrate TMB was added to the reaction wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.l of stop solution was added, the dual wavelength detection was performed using an ELISA reader, the OD at the 450nm maximum absorption wavelength and the 630nm reference wavelength was determined, and the OD at 630nm was subtracted from the OD at 450 nm. And (5) establishing a standard curve and calculating a result. The results showed that the antibody pair did not react with other similar proteins (table 3).

TABLE 3 Table 3

7. Double-antibody sandwich ELISA stability detection

a. Stability investigation by 37℃acceleration experiments

The monoclonal antibody 2H6 coated ELISA plate, HRP labeled monoclonal antibody 2F2, and goose IgY standard were placed at 37deg.C for accelerated stability test and allowed to stand for 13 days (equivalent to about 19.5 months at 4deg.C). And then taking out for detection, wherein the detection method is that the ELISA plate is taken out for washing the plate for 3 times and spin-drying. 100 μl of standard at various dilution concentrations of 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0.625ng/ml was added and a blank was placed. After sealing the plates, incubating for 2h at room temperature, washing the plates for 4 times and spin-drying. 100 μl of enzyme-labeled antibody HRP-2F2 working solution was added to the wells, the plates were incubated at room temperature for 45min after sealing plates, washed 4 times and spin-dried. 100. Mu.l of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.l of stop solution was added, dual wavelength detection was performed using an ELISA reader, the OD at the 450nm maximum absorption wavelength and 630nm reference wavelength was determined, and the OD at 630nm was subtracted from the OD at 450 nm. And drawing a standard curve by taking standard substances with different concentrations as an abscissa and corresponding OD values as an ordinate, and establishing a regression equation. The results show that on day 10, the high point (at 40 ng/ml) OD values of the standard curve were still greater than 2.0 and the standard curve OD gradient was good, indicating good stability of the kit (table 4).

TABLE 4 Table 4

b. The components of the kit are subjected to stability assessment under the storage condition of 4 ℃/-20 ℃:

and (3) placing the ELISA plate coated with the monoclonal antibody 2H6 and the goose IgY standard at 4 ℃, placing the HRP-labeled monoclonal antibody 2F2 at-20 ℃ for 25 months. And then taking out and detecting, wherein the detection method is to take out the ELISA plate for washing the plate for 3 times and spin-drying. 100 μl of standard at various dilution concentrations of 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0.625ng/ml was added and a blank was placed. Sealing the reaction holes by using a sealing plate film, incubating for 2 hours at room temperature, washing the plate for 4 times, and spin-drying. Adding 100 μl enzyme-labeled antibody HRP-2F2 working solution into the reaction well, sealing the reaction well with a sealing plate membrane, incubating at room temperature for 45min, washing the plate for 4 times, and spin-drying. 100. Mu.l of chromogenic substrate TMB was added to the reaction well, the reaction well was sealed with a sealing plate membrane and developed at room temperature in the dark for 15min, 50. Mu.l of stop solution was added, dual wavelength detection was performed using an enzyme-labeled instrument, the OD values at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm were determined, and the OD measurement value of 630nm was subtracted from the OD measurement value of 450 nm. And drawing a standard curve by taking standard substances with different concentrations as an abscissa and corresponding OD values as an ordinate, and establishing a regression equation. The results show that the OD value at the high point (40 ng/ml) of the standard curve is still greater than 2.0 after being placed for 25 months, and the gradient of the OD value of the standard curve is good, which indicates that the stability of the components of the kit is good, and the shelf life can reach two years (Table 4).

EXAMPLE 3 determination of IgY content in goose serum and detection of Cross-reactivity of chicken and Duck serum

And taking out the ELISA plate coated with the monoclonal antibody 2H 630 min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. The chicken, duck and goose serum are selected to be 4 parts respectively, serum samples with different dilution factors and standard substances are prepared, and 100 mu l of serum samples are added into each hole. And (3) incubating the plates for 2 hours in a room temperature incubator, washing the plates for 4 times and spin-drying. Adding HRP-2F2 working solution into the reaction holes, sealing the plates, incubating for 30min in an incubator at room temperature, washing the plates for 4 times, and spin-drying. 100. Mu.l of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.l of stop solution was added, dual wavelength detection was performed using an ELISA reader, the OD at the 450nm maximum absorption wavelength and 630nm reference wavelength was determined, and the OD at 630nm was subtracted from the OD at 450 nm. According to the literature report, the content range of IgY in normal chicken serum is (5.29+/-1.35) mg/ml (according to different ages and individual contents), and the content of IgY in goose serum is not much different from that of chicken. The measurement result shows that the measurement content of the kit is consistent with the report, and the detection result shows that the method can accurately measure the content of IgY in goose serum. And has no cross reaction with chicken serum; because of the high homology of ducks to geese, there was cross-reactivity, but the cross-reactivity of the present antibodies to the assay was very low (< 1%) (Table 5). The data of the content of the original chicken serum IgY and the duck serum IgY are derived from ELISA kits (product number: SEKCN-0126, SEKD-0119) produced by Beijing Soy treasure technology Co.

TABLE 5

Example 4 comparison with a foreign Brand antibody control sample

An analogy test was performed by selecting a foreign brand a (generise company, usa) antibody pair, and experiments were performed in a manner optimal for each brand (operating fully according to their instructions). By measuring the sample content, result comparison is performed: 34 goose serum samples are randomly selected and simultaneously measured, and the content range of IgY in normal goose serum is similar to that of IgY (5.29+/-1.35) mg/ml in chicken serum. The measurement results show that the measurement content of the kit is consistent with literature data, and the content measured by the brand A is low (Table 6).

TABLE 6

Example 5 determination of goose IgY content in meat

And taking out the ELISA plate coated with the monoclonal antibody 2H 630 min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. Serum samples of different dilution and different treatments of extracted meat total protein were added at 100 μl per well. And (3) incubating the plates for 2 hours in a room temperature incubator, washing the plates for 4 times and spin-drying. Adding HRP-2F2 working solution into the reaction holes, sealing the plates, incubating for 45min in an incubator at room temperature, washing the plates for 4 times, and spin-drying. 100. Mu.l of chromogenic substrate TMB was added to the wells, the wells were sealed, developed at room temperature in the absence of light for 15min, 50. Mu.l of stop solution was added to perform dual wavelength detection using a microplate reader, the OD at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm was determined, and the OD at 630nm was subtracted from the OD at 450 nm. From the results, it can be seen that the kit can specifically recognize IgY in highly diluted (500-fold dilution) goose tissues without reacting with proteins of other species (table 7).

TABLE 7

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. The mouse anti-goose IgY monoclonal antibody is characterized in that the heavy chain 3 CDR regions of the antibody are respectively: DYYMK, DIIPNTGDTFFNRKFKD and SIVTAYRDY, the 3 CDR regions of the light chain are: KASRDIDQYLA, YTSTLQP and LQYDSLYT.

2. The antibody of claim 1, wherein the light chain variable region of the antibody is: an amino acid sequence shown in SEQ ID NO. 3; or polypeptide with the same function obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 3;

3. A nucleic acid molecule encoding the antibody of claim 1 or 2.

4. A biological material comprising the nucleic acid molecule of claim 3, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, or an engineering bacterium.

5. A goose IgY detection reagent or kit prepared from the antibody of claim 1 or 2.

6. The goose IgY enzyme-linked immunosorbent assay kit is characterized by comprising the following components: the antibody of claim 1 or 2 and a labeled mouse anti-goose IgY mab, the kit further comprising at least one of a substrate chromogenic solution, a blocking solution, or a stop solution;

wherein, the amino acid sequences of the light chain and heavy chain variable regions of the mouse anti-goose IgY monoclonal antibody are respectively shown as SEQ ID NO. 7 and 8.

7. The kit of claim 6, wherein the antibody of claim 1 or 2 is immobilized on a solid support selected from the group consisting of an elisa plate, a microsphere, a nitrocellulose membrane, a glass cellulose membrane, and a nylon membrane;

the label is an enzyme label, a biotin label or a fluorescein label;

the enzyme-labeled enzyme is selected from any one of the following: horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, lysozyme, malate dehydrogenase.

8. A composition comprising the antibody of claim 1 or 2 and a mouse anti-goose IgY mab;

9. A goose IgY enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit prepared from the composition of claim 8.

10. Use of the antibody of claim 1 or 2, or the kit of any one of claims 5 to 7, or the composition of claim 8 in a goose IgY enzyme-linked immunosorbent assay;

the applications include qualitative and quantitative detection, and the applications are for non-disease diagnostic purposes.