WO2012145925A1

WO2012145925A1 - Uses of food allergens and antibodies thereof in iga nephropathy

Info

Publication number: WO2012145925A1
Application number: PCT/CN2011/073545
Authority: WO
Inventors: 刘志刚
Original assignee: 深圳大学
Priority date: 2011-04-29
Filing date: 2011-04-29
Publication date: 2012-11-01

Abstract

The present invention provides uses of food allergens and antibodies thereof for manufacturing the reagents for the detection of IgA nephropathy, and also provides methods for detecting the causes of IgA nephropathy by using food allergens and antibodies thereof.

Description

Application of food allergens and food allergen antibodies in IgA nephropathy

Technical field

The present invention relates to the use of food allergens and food allergen antibodies in IgA nephropathy, namely the use of food allergens and food allergen antibodies in the preparation of IgA nephropathy etiological detection reagents, and methods for detecting the etiology of IgA nephropathy.

Background technique

IgA nephropathy (IgAN) is a group of IgA or IgA-based immune complexes that are mainly characterized by deposition in the mesangial area. Unidentified immune complex glomerulonephritis is the most common worldwide. The glomerular disease accounts for 30% to 40% of glomerular diseases. Even in the United States with a low incidence of IgA nephropathy, IgA nephropathy still accounts for the first place in glomerulopathy among adults aged 20 to 39 years. IgA nephropathy is also the main type of chronic kidney disease in China, accounting for about 40% of glomerular diseases in China. It is now clear that IgA nephropathy is a progressive disease, and about 20% of patients progress to chronic renal failure every 10 years after onset. It is currently believed that about 40% of patients with this disease eventually progress to end-stage renal failure (ESRD). Therefore, IgA nephropathy seriously endangers the health and quality of life of patients, and at the same time, it also imposes a huge economic burden on patients, families and society. At present, the exact etiology and pathogenesis of IgA nephropathy are not yet clear, and there is no uniform and effective treatment. Summary of the invention

The present invention is based on a new study and found that the inventors of the present invention have found through experiments that: the occurrence of IgA nephropathy is directly related to the food allergy, and the food allergen may mediate the occurrence of IgA nephropathy. Therefore, by detecting the presence of food allergens or food allergen antibodies in the sample, it is possible to accurately determine the cause of IgA nephropathy and provide ideas for subsequent treatment. The present invention also provides a novel use of food allergens and food allergen antibodies, i.e., in the preparation of an IgA nephropathy detecting reagent; in addition, the present invention also provides a method for detecting the cause of IgA nephropathy.

Application of food allergen antibodies in the preparation of IgA nephropathy etiological detection reagents.

Preferably, the food allergen is a peanut allergen, a nut allergen, a fish allergen, a crustacean allergen (such as shrimp, crab, etc.), a milk allergen, a hazel allergy, a soybean allergen and an egg. At least one of the class of allergens. The above eight food allergens are the most common of the eight food allergens.

Preferably, the food allergen antibody is milk β _ lactoglobulin monoclonal antibody, egg ovomucin 0VM monoclonal antibody, fish small albumin monoclonal antibody, shrimp myosin monoclonal antibody, peanut Arahl protein monoclonal At least one of an antibody, a scorpion Corhl protein monoclonal antibody, a soybean Gly mBd monoclonal antibody, and a crab tropomyosin monoclonal antibody. Milk β _ lactoglobulin, egg ovomucin 0VM, fish whey protein, shrimp myosin, peanut Arahl protein, scorpion Corhl protein, soybean Gly mBd, sinogen myosin cause an allergic reaction in food allergens of their respective categories The main protein.

Preferably, the food allergen antibody is at least one of a polyclonal antibody and a monoclonal antibody. More preferably, the food allergen antibody is a monoclonal antibody to reduce the false positive rate of the detection reagent during the actual detection process.

Preferably, the food allergen antibody is a fluorescently labeled antibody, an enzyme labeled antibody or a biotinylated antibody. The detection reagent is more sensitive in the actual detection process, the experimental result of the detection is easy to observe, and the accuracy is high.

Preferably, the food allergen antibody is a fluorescein isothiocyanate (FITC)-labeled antibody, a tetramethyl isocyanate rhodamine (TRITC)-labeled antibody, a rhodamine red (RRX)-labeled antibody, a Texas red ( TR) Labeled antibody, cyan dye (Cy2, Cy3, Cy5) labeled antibody, phycoerythrin or phycobiliprotein (PE) labeled antibody or acetic acid (AMCA) labeled antibody.

FITC can be stored in cold and dark dry places for many years and is the most widely used fluorescein. The main advantage of the FITC fluorescent secondary antibody is that the human eye is more sensitive to yellow-green, and the green fluorescence in the sliced specimen is usually less than red.

TRITC is a derivative of rhodamine, which is a purplish red powder and is relatively stable. The maximum absorption wavelength is 550nm, the maximum emission wavelength is 620nm, it is orange-red fluorescence, and it is contrasted with FITC's green fluorescence, which can be used for double labeling or contrast dyeing. Due to its slow fluorescence quenching, it can also be used for separate marking dyeing.

RRX and TR are rhodamine derivative coupling groups with different absorption wavelengths (570, 596 nm) and maximum emission wavelengths (590, 620 nm). Use RRX and TR for better color discrimination.

Cy2 is more stable under UV light than FITC, and Cy3 and Cy5 are brighter, more stable, and weaker than other fluorophore probes.

The phycoerythrin PE has a wide absorption wavelength range, the largest absorption wavelength is 566 nm, and the maximum emission wavelength is 574 nm. As a fluorescent marker, phycoerythrin has the following advantages: Firstly, it has strong long-wavelength excitation and emission fluorescence, which can avoid interference from fluorescence of other biological materials; and has extremely high emission quantum yield; in addition, PE has very high water solubility. It also crosslinks stably with multiple sites in many biological or synthetic materials.

The AMCA signal is relatively weak, and sputum is not recommended for single-label experiments. The fluorescence wavelengths of AMCA and fluorescein have only a small overlap range, and there is little or no overlap with the fluorophore that emits long-wavelength fluorescence, so it is most commonly used in multi-label experiments, such as immunofluorescence microscopy and flow cytometry. instrument.

The present invention also provides a novel use of food allergens, i.e., in the preparation of IgA nephropathy etiological detection reagents.

Preferably, the food allergen is at least one of a peanut allergen, a nut allergen, a fish allergen, a crustacean allergen, a milk allergen, a hazel allergy, a soybean allergen, and an egg allergen. .

Preferably, the food allergen is milk β _ lactoglobulin, egg ovomucin 0VM, fish Xiaoqing At least one of protein, shrimp myosin, peanut Arahl protein, gardenia Corhl protein, soybean Gly mBd, and crab tropomyosin.

Preferably, the food allergen carries a labeling molecule. The detection result by the detection reagent described above is made easier to observe and has high accuracy.

Preferably, the labeling molecule is an enzyme labeling molecule.

The invention also provides a method for detecting the cause of IgA nephropathy, which uses a food allergen antibody to detect a food allergen in a sample by immunohistochemistry, and if one or more food allergens are detected, the food is indicated The allergen or the multi-food allergen is the cause of the patient with the IgA nephropathy.

Preferably, the food allergen is at least one of a peanut allergen, a nut allergen, a fish allergen, a crustacean allergen, a milk allergen, a hazel allergy, a soybean allergen, and an egg allergen. Kind.

Preferably, the food allergen antibody is milk β _ lactoglobulin monoclonal antibody, egg ovomucin 0VM monoclonal antibody, fish small albumin monoclonal antibody, shrimp myosin monoclonal antibody, peanut Arahl protein monoclonal At least one of an antibody, a scorpion Corhl protein monoclonal antibody, a soybean Gly mBd monoclonal antibody, and a crab tropomyosin monoclonal antibody.

Preferably, the food allergen antibody is at least one of a polyclonal antibody and a monoclonal antibody. Preferably, the food allergen antibody is a fluorescently labeled antibody, an enzyme labeled antibody or a biotinylated antibody.

More preferably, the food allergen antibody is fluorescein isothiocyanate (FITC) labeled antibody, tetramethyl isocyanate rhodamine (TRITC) labeled antibody, rhodamine red (RRX) labeled antibody, Texas Red (TR) Labeled antibody, cyan dye (Cy2, Cy3, Cy5) labeled antibody, phycoerythrin or phycobiliprotein (PE) labeled antibody or acetic acid (AMCA) labeled antibody.

More preferably, in any one of the above aspects, the sample is at least one of a tissue paraffin section and a tissue frozen section.

More preferably, the sample is a paraffin section of kidney tissue, comprising the following steps:

A. Deparation of paraffin sections of kidney tissue to water;

B. removing endogenous peroxidase from a sample of paraffin sections of kidney tissue;

C. antigen retrieval, then blocking non-specific sites;

D. Add fluorescently labeled food allergen monoclonal antibody, incubate, wash, seal, and then observe the results by fluorescence microscopy; or add unlabeled food allergen monoclonal antibody, incubate, wash, and then add signal molecule labeled IgA secondary antibody Incubate, wash, seal, and then observe the results by fluorescence microscopy; or add unlabeled food allergen monoclonal antibody, incubate, wash, then add signal molecule labeled IgA secondary antibody, incubate, wash, add signal molecular marker IgG secondary antibody, hatching The cells were incubated, washed, and mounted, and the results were observed by a fluorescence microscope.

Tissue paraffin sections have better localization effect on antigens and are beneficial for long-term preservation. Therefore, the use of kidney paraffin sections makes the experimental results more accurate for food allergens. For samples that need to be stored for a long time, the above methods are used. The test results are more accurate.

Preferably, the sample is a frozen section of kidney tissue, comprising the following steps:

A. removing endogenous peroxidase from frozen sections of kidney tissue;

B. antigen retrieval, then blocking non-specific sites;

C. Add fluorescently labeled food allergen monoclonal antibody, incubate, wash, seal, and observe the results by fluorescence microscopy; or add unlabeled food allergen monoclonal antibody, incubate, wash, and then add signal molecule labeled IgA secondary antibody Incubate, wash, seal, and then observe the results by fluorescence microscopy; or add unlabeled food allergen monoclonal antibody, incubate, wash, then add signal molecule labeled IgA secondary antibody, incubate, wash, add signal molecular marker The IgG secondary antibody was incubated, washed, mounted, and the results were observed by fluorescence microscopy.

Frozen sections have a better fidelity effect on food allergens, while paraffin sections may have an effect on the properties of the antigen during tissue fixation, dehydration, transparency, waxing, embedding, dewaxing, and the like. In addition, the use of frozen sections makes the above method simpler, and the paraffin section requires dewaxing into water, antigen retrieval and other processes, which is cumbersome, and frozen sections can also avoid the interference of paraffin autofluorescence; however, frozen sections can only be preserved after preparation. Shorter time is not conducive to simultaneous detection of a large number of samples. Therefore, the use of kidney tissue paraffin sections and kidney tissue frozen sections have their own advantages and disadvantages.

The invention also provides another method for detecting the cause of IgA nephropathy, which uses a food allergen to detect a food allergen antibody in a sample by an ELISA technique, and if one or more food allergen antibodies are detected, the food is indicated The allergen or the multi-food allergen is the cause of the patient with the IgA nephropathy.

Preferably, the food allergen antibody is milk β _ lactoglobulin, egg ovomucin 0VM, fish small albumin, shrimp myosin, peanut Arah1 protein, scorpion Corhl protein, soybean Gly mBd, crab tropomyosin At least one of them.

Preferably, the food allergen carries a labeling molecule.

More preferably, the labeling molecule is an enzyme labeling molecule.

Preferably, the sample is at least one of serum, plasma, whole blood, and tissue fluid.

Preferably, the sample is a serum sample, comprising the following steps: A. Add a blocking solution to a 96-well plate pre-coated with food allergens, block non-specific sites, and incubate;

B. Washing, then adding the serum sample to be tested and incubating;

C. Wash, add enzyme-labeled IgA secondary antibody, and incubate;

D. After washing, add the color developing solution, develop color in the dark, then add the stop solution, and read the absorbance of each well in the 96-well plate with a microplate reader.

Preferably, the enzyme-labeled molecule of the enzyme-labeled IgA secondary antibody may be selected from horseradish peroxidase (HRP), alkaline phosphatase (AKP) or glucose oxidase (G0D).

When the enzyme-labeled IgA secondary antibody is a goat anti-mouse HRP-IgA secondary antibody, the color developing solution is a TMB color developing solution, and the absorbance value read by the microplate reader is the absorbance at 450 nm of each well in the 96-well plate.

The above methods are used to detect instruments with low requirements, and can be carried out in primary medical institutions and general laboratories, making it possible to screen IgA nephropathy in the general population, and also to facilitate early warning of IgA nephropathy, with good clinical Application prospects.

The above method for detecting the cause of IgA nephropathy using food allergens can be combined with the method for detecting the cause of nephropathy by using food allergen antibodies, so that the detection result of IgA nephropathy is more accurate.

Compared with the prior art, the present invention proposes a new use of food allergens and food allergen antibodies, namely, the application in the preparation of IgA nephropathy detection reagents, so that the cause of IgA nephropathy is conveniently, quickly and accurately determined, thereby The treatment of IgA nephropathy offers new ideas. In addition, the method for detecting the cause of IgA nephropathy provided by the present invention is simple and efficient, and the detection result is accurate.

DRAWINGS

Figure 1 shows the DNA electrophoresis pattern of PCR amplification of 8 food allergen gene fragments;

Figure 2 is a SDS-PAGE electropherogram of the expressed protein of class 8 food allergens;

Figure 3 is a western-blot diagram of monoclonal antibody identification of eight food allergens;

Figure 4A and Figure 4B are immunofluorescence profiles of eight types of food allergens in renal tissue sections of patients with partial IgA nephropathy; Figure 5 is a total of ELISA detection of total antibodies (total IgA and total IgG) and food allergen-specific antibodies in patients with confirmed IgA nephropathy Column chart

Figure 6 is a bar graph showing the common food antigen-specific antibodies in the serum of patients with confirmed IgA nephropathy by ELISA.

detailed description

First, the food allergen and IgA nephropathy directly related to the proof experiment.

1. Cloning and expression of genes for eight food allergens.

Reference NCBI sequence: Milk major allergen BLG gene (GeneBank ID EU883598) (Expressing milk β-lactoglobulin), egg ovomucin OVM (GeneBank ID FJ227543), fish small egg White (GeneBank ID EF591789), Shrimp Myosin (GeneBank ID EF584510), Peanut Ara hi Protein (GeneBank ID AF432231), Gardenia Corhl Protein (GeneBank ID EU195058), Soy GlymBd (GeneBank EU883600) and Cratomyosin Tropomyosin (GeneBank) ID EF143836), 8 pairs of primers were designed separately, see Table 1.

Table 1

1.1 RT-PCR.

Tissues of the above-mentioned eight types of food allergens were taken separately, and total RNA was extracted using the RNasy mini kit (purchased from Qiagen) product description protocol, and RNA was quantified and identified.

The reverse transcription reaction was carried out by using BBI's read reverse transcriptase system, mixing 总μ _§ total RNA with 0.5 μ _§ of OligodT, heating at 70 ° C for 5 minutes, and rapidly cooling to 0 ° C. The buffer was added according to the product instructions and incubated at 42 ° C for 1 hour. The cDNA of the reverse transcription reaction was taken 4 μM, and the genes of the above eight food allergens were amplified as templates for the PCR reaction.

The PCR reaction system was carried out at 95 ° C for 3 min and then amplified by Touchdown. The first cycle was 94 ° C 45S, 65 ° C 45S, 72 ° C 2 min, after which the annealing temperature per cycle decreased by 1 ° C. When the annealing temperature was lowered to 57 ° C, it was operated at 94 ° C for 45 s, 57 ° C for 45 s, and 72 ° C for 2 minutes for 35 cycles, and then extended at 72 ° C for 10 minutes to complete the reaction. The PCR product was subjected to agarose gel electrophoresis.

As a result, as shown in Fig. 1, the amplified fragments of the eight types of food allergens showed bright bands at the predicted positions, indicating that the RT-PCR amplification was successful. In the figure, AH respectively indicates: A: milk β-lactoglobulin 530 bp; B: egg ovomucin 627 bp; C: fish small albumin 230 bp; D: shrimp myosin 850 bp; E: peanut Arahl 130bp, F: scorpion Corhl 432bp _; G: soybean Gly mBd 450bp _; H: crab tropomyosin 855bp ₀

1. 2 Construction of recombinant plasmids.

The PCR product was recovered using the TaKaRa Glueback Kit and recovered according to the product specification protocol. The PCR products recovered in 1. 1 were ligated using the TaKaRa pMD18_T vector kit, respectively. The recombinant plasmid was transformed into a competent cell, and the plate was cultured at 37 ° C overnight, and the positive clone plasmid was screened by PCR amplification and double enzyme digestion. Refer to the third edition of Molecular Cloning, Chapter 1, pages 96-99. The identified positive colonies were sequenced by Shanghai Shenggong Bioengineering Co., Ltd. The correctly sequenced plasmid was digested with the prokaryotic expression vector pET-28a, respectively. The gene fragment obtained by digestion was ligated with the digested product of the prokaryotic expression vector pET-28a (purchased from Qiagen Co., Ltd.) using T4 DNA ligase. The ligation product was transformed into competent Escherichia coli, and the colony PCR and plasmid double enzyme digestion were used to identify the Escherichia coli BL21 (DE3) and E. coli Rosetta (DE3), which were used to induce expression.

1. 3 Induced expression of recombinant food allergen protein.

The 1. 2 transformed expression bacteria were picked up into 20 mL LB (Kanamycin 40 g/mL) tubes and shaken at 37 ° C, 200 rpm overnight. 20 mL of the culture was inoculated into 1 L of LB medium (containing kanamycin 40 g/mL) at a 1:50 inoculation amount. Incubate at 37 ° C, 200 rpm shaker. When the bacterial growth was in the logarithmic growth phase (A600nm=0. 6-0. 8), IPTG was added at a final concentration of 1 匪 ol/L to induce protein expression. After 4 hours, 1 mL of the bacterial solution was taken, and samples were prepared for SDS-PAGE gel electrophoresis to detect the expression. In the above expression product, it was resuspended in 20 mM Tris_HCl (pH 7.5). After three freeze-thaw cycles, the ultrasonic wave (300W, 20min, ice bath) was broken. After centrifugation at 15000 rpm for 15 min at 4 ° C, the supernatant and the precipitate were separately subjected to 15% SDS-PAGE electrophoresis analysis.

As a result, as shown in Fig. 2, the reconstituted class 8 food allergen protein showed a foreign protein band at the theoretical molecular weight, and the recombinant protein was expressed in a large amount in the E. coli expression system. In the figure, AH indicates the expression of eight recombinant food allergens, A: milk β _ lactoglobulin 26kD; B: egg ovomucin 21kD; C: fish small albumin 42kD; D: shrimp myosin 36kD; : peanut Arahl 38kD, F: scorpion Corhl 15. 8kD; G: soybean Gly mBd 16. 5kD _; H: crab tropomyosin 38kD.

1. 4 separation and purification of recombinant protein.

A Ni-NTA column (purchased from Pharmacia Biotech Co., Ltd.) was attached, and the sealing property was examined. Cleaning, the specific cleaning process procedure is carried out according to Ni-NTA (manual operation. Packing with Ni-NTA, 1. 6 X 20cm, column volume 10mL, balance 2-5 bed volumes with buffer 1, flow rate 2mL / Min; 20 mL cell disruption solution (50 mM PBS, pH 7.4, 0.5 mM NaCl) 0. 45 uM filter was filtered, loading, flow rate was 1 mL / min; wash 2 - 5 bed volume with buffer 1 The flow rate is 2 mL/min _{; the} reuse is 20 mM, 50 mM, respectively. 100 mM, 200 mM, 300 mM, 400 mM imidazole eluted at a flow rate of 2 mL/min. The eluted peaks and peaks were collected, subjected to SDS-PAGE electrophoresis, and the eluted fractions containing the target protein were separately dialyzed and lyophilized. Recombinant protein: milk β _ lactoglobulin, egg egg mucin, fish albumin, shrimp myosin, peanut Arahl, scorpion Corhl 15, soy Gly mBd, crab tropomyosin.

1. 5 Immunological characterization.

The purified recombinant protein was subjected to 15% SDS-PAGE electrophoresis, and the voltage was 80 to 100 V for 90 to 120 min, followed by immunoblotting. For the procedure, see Molecular Cloning, and electroporating the protein on SDS-PAGE gel to nitric acid. On the cellulose membrane, the electrorotation conditions were: 300 mA/plate; 350 mA/2 plate, 60 min. Remove the membrane, wash the membrane with TBA, 5 min x 3 . The membrane was placed in a blocking solution and shaken slowly for 30 min. Block overnight at 4 °C. The NC membrane was cut into small strips and placed in a serum pool incubation tank, and incubated with 25 diluted allergic patients (primary antibodies) containing the corresponding specific IgE for 2 h at 37 °C. Recover serum. TBST wash film, 5minx3. A biotinylated goat anti-human IgE antibody (secondary antibody) diluted with a secondary antibody buffer (1:3000) was added and incubated at 37 °C for 2 h. TBST wash film, 5minx3. Horseradish peroxidase (HRP)-labeled streptavidin diluted (1: 1000) with HRP-labeled streptavidin buffer was added. Incubate at 37 ° C for 1. 5 h. TBST wash film, 5 min 3 times. Wash the membrane with TBS, 3 times 3 minutes. The DAB coloring solution was added dropwise to the surface of the film, and the color reaction was terminated by double-distilled water washing.

2. Preparation of monoclonal antibodies to eight food allergens.

2. 1 Preparation of hybridoma cells.

The egg yolk mucin prepared by the preparation of 1.4 was dissolved, and 1 mL of 0.25 g/L and an equal volume of Freund's complete adjuvant (purchased from Sigma) were fully emulsified and subcutaneously injected into 5 Balb/c mice (purchased Since the Guangdong Medical Laboratory Animal Center). The immunization was boosted once every 3 weeks. When the immunization was boosted, the Freund's incomplete adjuvant was used, and the antigen amount and the injection route were unchanged. After the second boost, the tail blood was measured for titer on the 7th day. Three days before the fusion, the same dose of antigen PBS solution was injected through the tail vein and boosted once. The spleen cells of the immunized mice and the NS-1 cells were separately collected in a ratio of about 1: 5 by a conventional method under the action of 50% PEG. The cell pellet after the fusion was gently suspended with 2. 5 mL of the complete culture medium. 2 mL of the semi-solid medium Medium D was mixed and poured into a plate of 3. 5 cm in diameter, each plate was about 2 mL, 37 ° C, 5% CO 2 to cultivate. One week later, the surface of the medium with the visible dish showed white spots on the surface of the plate, which was a cell clonal group under the microscope. Under sterile conditions, the individual cell clusters dispersed in the plate are aspirated and placed in a 96-well plate culture medium, and the culture is continued. In the 96-well plate, the cells were over-injected by indirect ELISA, and the positive wells were subcloned by limiting dilution.

The preparation of hybridoma cells of the other seven food allergens can be carried out by referring to the above method.

2. 2 monoclonal antibody production and titer determination.

The hybridoma cells of the established strain were expanded and the mouse ascites was induced to produce monoclonal antibodies in BALB/c mice. 8毫升每只, 7 days later, 8 weeks old BALB / c mice, intraperitoneal injection of liquid paraffin 0. 5mL / only, after 7 days Hybridoma cells were injected intraperitoneally with 5 X 10 ⁵ -5 X 107 cells. After 7-10 days, the mice were ascites, and the supernatant was taken at 1200 r/min for 10 min. The titer was determined by indirect ELISA. After ascites was diluted 1:100, 1:200, 1:400, 1:800, etc., the recombinant protein was coated, the A450nm value was measured, and the pre-immune mouse serum was used as a parallel negative control test to The maximum dilution of the monoclonal antibody reacting with the recombinant protein target antigen is its potency, and P/N 2. 1 is positive. 8 allergen foods monoclonal antibody titer results are shown in Table 2, 8 antibody titer in 1. 3 X 10- ⁴ to 10 ⁸ or more. Antibody purification was performed by reference to HiTrap protein A affinity chromatography instructions, and HiTrap protein A was purchased from Amersham.

Table 2

2. Identification of 3 monoclonal antibodies.

The recombinant protein prepared in 1.4 was subjected to 12% SDS-PAGE electrophoresis, and the protein on the gel was transferred to a fixed nitrocellulose membrane (300 mA, 50 min) by a protein electrophoresis transfer apparatus, and stained with Lichun red. After the solution was stained with a liquid, the membrane was sheared, and the Ponceau was washed away with ultrapure water, TBST (10TMol/L Tris, 150 匪ol/L NaCl, 0.2% Tween-20, pH 8.0). Add 2% BSA prepared with TBST at 4 °C overnight. After TBST shake, add the monoclonal antibody to incubate for 1 h at room temperature to immunize the pre-immune mouse serum as a negative control; HRP-labeled goat anti-mouse IgG antibody is two Anti-, incubate for 1 h at room temperature, wash the membrane with TBST, and develop color with DAB reagent.

Western blotting analysis showed that the combined effect of multiple monoclonal antibodies with the eight types of food allergens is shown in Figure 3. Thus, the eight monoclonal antibodies were identified to have activity against the class 8 food allergen protein. In the figure, A-H showed that the purified food allergens of the eight types of food all interacted with the mouse anti-food allergen monoclonal antibody IgG, and both had good immunogenicity. A: milk β _ lactoglobulin 26kD; B: egg ovomucin 21kD; C: fish small albumin 42kD; D: shrimp myosin 36kD; E: peanut Arahl 38kD, F: scorpion Corhl 15. 8kD; G: Soy Gly mBd 16. 5kD; H: Crab tropomyosin 38kD.

2. Marking of 4 monoclonal antibodies.

The monoclonal antibody-labeled biotin can be operated by referring to the instructions of the Sulfo-Li S-Biotin kit (purchased from Thermo Corporation). For example: Place Sulfo-NHS-Biotin at room temperature for 10 min. The antibody concentration was adjusted to 3 mg/L, placed in a 30 KD ultrafiltration spin column, centrifuged at 10,000 rpm for 5 min, and Buffer was replaced with PBS buffer. Dissolve 10 mM Sulfo-NHS-Biotin in 50 (μL ultrapure water. Add a pre-calculated volume of Sulfo-NHS-Biotin solution to the antibody solution. Incubate at room temperature in the dark 30min. The reaction was placed in an ultrafiltration spin column, centrifuged at 1000 g for 2 min, and the supernatant was removed, and the filter was returned to the centrifuge tube. LOC^L PBS was added to the filter, centrifuged at 1000 g for 2 min, and the supernatant was discarded and repeated 3 times. The filter was poured into a new centrifuge tube, centrifuged at 1000 g for 2 min, and the filtrate was collected. The filtrate was a purified antibody solution. Store the antibody at -20 °C.

3. 1 Preparation of total protein antigen

Grind the flower with liquid nitrogen to form a powder, soak it in acetone at a ratio of 1:15 for 48 hours, put it in a refrigerator at 4 °C, change the solution once every 12 hours, and stir for 2 times. The acetone was removed, dried and weighed. Each 5 g sample was added with 30 mL of extract (8 mmol/L Tri s-HCl, 25 mmol/L Tricine, pH 8.6) at 4 ° C overnight, 11,000 rpm, centrifuged for 30 min, and the supernatant was collected. The supernatant was taken for discontinuous SDS-PAGE analysis of protein components in the crude extract and determination of molecules

3. 2 Preparation of polyclonal antibodies.

One male New Zealand rabbit weighing 3 kg received blood from the pre-immune Id ear vein, and the blood was separated as a negative control after standing at 4 °C for 1 h. At the time of initial immunization, each New Zealand rabbit was injected subcutaneously with 2 mL of Freund's adjuvant-fermented peanut total protein antigen (protein concentration of 0.5 mg/mL). After 2 weeks, the second immunization was carried out with pure fusion protein emulsified in incomplete Freund's adjuvant, the same method as before. The immunization was repeated once every 2 weeks, and the dose and method were the same as the second immunization. After 3 immunizations, blood was collected from the ear vein for polyclonal antibody ELISA titer analysis. After the titer meets the requirements (ELISA method titer > 1: 1280000), the immunization is boosted once. 3 days after the booster immunization, the common carotid artery was intubated and the blood was collected. The serum was collected by centrifugation and stored at -80 °C for later use.

3. 3 polyclonal antibody assay.

The total protein of peanut was coated with protein to a final concentration of 25 g/mL, and 100 wells per well were coated with 96-well plates (1 well for blank control), incubated at 37 °C for 2 h, and washed by plate washer 3 times. Each time 3 min, PBST-5% BSA was ΙΟΟμ 每 per well and blocked at 37 °C for 2 h. After repeated washing, the polyclonal antibodies (1:5000, 1:2000, 1:80000, 1:320000, 1:1280000) were diluted with PBS, incubated at 37 °C for 2 h, and one duplicate well was selected as a negative control. Three replicate wells were selected for each concentration for comparison. Plates were washed 3 times, and 100 Ll: 5000 TBST-5% BSA diluted HRP-labeled goat anti-rabbit IgG was added to each well, and incubated at 37 ° C for 40 min. After the same method, the plate was washed 3 times, and the substrate was added with TMB 50 μL for 5 min. The reaction was terminated by lmol/LH ₂ S04 stop solution, and the 490 nm single-wavelength ultraviolet light was detected by the microplate reader. The ELISA method for the determination of the polyclonal antibody titer of the peanut was compared with the mean value of the first and second antibody titers after the immunization and the mean value of the three measurements before the immunization. The results were all greater than 2.1 times. Price > 1: 128 000. Serum was stored at -20 °C.

3. 4 purification of polyclonal antibodies

The anti-serum of the prepared polyclonal antibody was diluted with binding buffer (1 mol/L Tri s/HCl, pH 9. 0, 20 mmol/L Na ₃ P04 ), filtered through a filter of 0. 45 μππ, and applied to HiTrapTM. ProteinG HP was purified on a column (purchased from Amersham) and subjected to antibody purification according to the instructions. With elution buffer (100 匪ol / L Glycine-HC1, pH 2. 7) After elution, the elution peak was collected. Western blotting was used to identify anti-peanut polyclonal antibodies.

4. Label food allergen polyclonal antibodies.

For the labeling of polyclonal antibodies, refer to 2. 4 monoclonal antibody labeling.

5. Fluorescence immunohistochemistry detection of renal tissue.

1) 1.7 cases of renal tissue paraffin sections of patients with confirmed IgA nephropathy were routinely dewaxed to water;

2) Incubate with 3% H ₂ 0 ₂ for 20 min, remove endogenous peroxidase, and rinse 3 times with pH 7. 4 0. Olmol/L PBS. 0.25% trypsin (purchased from Invitrogen, Canada) was placed on a paraffin section, placed in a wet box, and incubated at 37 ° C for 15 minutes for antigen retrieval. Dip 3 times with PBST, 5 minutes/time. 5% BSA (purchased from Wuhan Boster Biotech Co., Ltd.) was added dropwise to paraffin sections, and incubated at 37 ° C for 1 hour in a wet box. The blocking solution on the paraffin section was removed, and the mouse monoclonal antibodies of the 8 different food allergens prepared in 2.2 were diluted into different working solutions according to the measured titers (see Table 2), respectively. Add to the paraffin section, each section is about 50 μ 1, subject to the total coverage of the tissue. Place in a wet box, 4 overnight. Dip 3 times with PBST, 5 minutes/time. The tissue surrounding the tissue was blotted in the dark, and the goat anti-human IgA-TRITC secondary antibody (1:3000, purchased from Southern Biotech, USA) was added dropwise, each tissue block was about 50 L, 37 ° C, 45 minutes. Dip 3 times with PBST, 5 minutes/time. The tissue surrounding the tissue was blotted, and rabbit anti-mouse IgG-FITC secondary antibody (1:1000, purchased from Southern Biotech, USA) was added dropwise, each tissue block was about 50 L, 37 ° C, 45 minutes. Wash PBST 5 times, 5 minutes / time. The liquid around the tissue was dried, glycerin was mounted, and the results were observed under a fluorescence microscope. Statistical analysis was performed on the observed experimental results. The results are shown in Table 3, Figure 4A and Figure 4B. Antigen milk BLG for each group Egg egg egg fish small albumin shrimp myosin peanut Am hi type white 0VM protein positive 9 8 4 14 8 negative 6 3 7 4 9 total 15 11 11 18 17 positive rate (%) 60 72. 2 36. 4 72. 2 47 Antigens tested in each group, Corhl egg, soybean Gly Bd, crab primordial ball, egg mixture, total type, white

Positive 13 6 16 27 105 Negative 8 10 8 7 62 Total 21 16 24 34 167 Positive rate (%) 61. 9 37. 5 66. 7 80 62. 8 The mixing described in Table 3 refers to a mixed antibody prepared by mixing the above-mentioned monoclonal antibodies of the eight types of food allergens into eight types of food antigens. Figure 4A and Figure 4B show AH for the detection group of mouse monoclonal antibodies of 8 different food allergens prepared by 2.2: A: milk BLG monoclonal antibody; B: egg egg mucin 0VM Monoclonal antibody; C: fish small albumin monoclonal antibody; D: shrimp myosin monoclonal antibody; E: peanut Ara hi protein monoclonal antibody; F: scorpion Corhl protein monoclonal antibody; G: soybean Gly Bd monoclonal Antibody; H: Crab tropomyosin monoclonal antibody; I: Mixed group (including the above 8 monoclonal antibodies); J: blank control.

As shown in Figures 4A and 4B, renal tissue sections of patients with IgA nephropathy were positive, and food allergens were mainly distributed in glomerular and tubular endothelial cells, while normal controls were negative. The total positive rate of immunofluorescence of food allergens was 62.8% in the kidney tissue sections of all the patients with IgA nephropathy. Among them, IgA nephropathy patients with kidney tissue sections of eggs, milk, wheat, crustaceans (shrimp, crab) food allergens immunofluorescence positive rate of more than 50%; and IgA nephropathy patients with kidney tissue sections of soybeans, peanuts, fish The immunofluorescence localization rate of food allergens is low (less than 50%). The above-mentioned IgA nephropathy patients with positive food allergens improved their condition after fasting the corresponding positive allergen food, indicating that the deposition of the positive food allergen in the glomerulus or renal tubule is the cause of IgA nephropathy.

6. ELISA detection of food allergen antibodies in patients with IgA nephropathy

In the past month, 150 patients with clinically confirmed IgA nephropathy were collected and ELISA was used to detect specific antibodies against eight food allergens in the patient's serum. A certain amount of 8 recombinant food allergens prepared in 1.4 was diluted to a pH of 9.6, to a final concentration of 10 g / mL, added to a 96-well plate, 100 μ ΐ 7 wells, 4 ° C overnight. The plate was washed 5 times with PBST wash (containing 0.5% Tween 20), and the blocking solution was added, 100 μ ΐ 7 well, and incubated at 37 ° C for 2 h. The plate was washed 5 times with PBST wash solution, and 1:3000 patient serum was used in PBST containing 1% calf serum, and then added to the well-coated 96-well microplates, 100 μ ΐ 7 wells, 37°. C was incubated for 2 h. The plate was washed 5 times with PBST wash, 100 μL of diluted goat anti-mouse HRP-IgA (l: 4000) was added to each well, and incubated at 37 ° C for 2 h. Wash the plate 5 times with PBST, add 50 L of coloring solution A and 50 L of coloring solution B, develop color at 37 ° C for 15 min in the dark, add 50 μL of 2M H ₂ S0 _{4 ,} terminate the reaction, and read the absorbance ( A450 value).

The results are shown in Figures 5 and 6. As shown in Figure 5, the total IgA (tlgA) in the serum of patients with IgA nephropathy was significantly higher than that of the serum IgG (tlgG) and the normal human serum control group; the total tlgG in the serum of the patients was significantly lower than that of the normal human serum; The eight types of mixed food-specific IgA (slGA) in the serum of patients with IgA nephropathy were significantly increased; the serum level of slgA in patients with IgA nephropathy was significantly different from that of the patient's own slgG (P<0.01), and the serum level of slgA in patients with IgA nephropathy was higher. There was a significant difference in slgA between the normal human serum control group (P < 0.01). As shown in Figure 6 (1: milk β _ lactoglobulin; 2: egg ovomucin; 3: fish small albumin; 4: shrimp myosin; 5: peanut Arahl protein; 6: scorpion Corhl protein; 7: Soybean Gly mBd protein; 8: crab tropomyosin; 9: blank control), IgA nephropathy patients, the common food allergen slgA increased significantly, and the common food antigen slgG difference is not obvious (except shrimp allergen slgG) Among them, egg, milk, wheat, crustaceans (shrimp, crab) have relatively high slgA, which is consistent with the fluorescence localization detection of food-specific allergens.

The above-mentioned food allergen antibody positive IgA nephropathy patients after the fasting of the corresponding positive allergen food, the condition improved significantly, indicating that the positive food allergen is the cause of IgA nephropathy.

In summary, food allergens and food allergen antibodies are closely related to the occurrence of IgA nephropathy. Food allergens are the cause of some IgA nephropathy. Therefore, it can be tested by samples (such as renal tissue paraffin sections, frozen sections of kidney tissue, serum, all The cause of IgA nephropathy is determined by the presence of food allergens or food allergen antibodies in blood, plasma and/or tissue fluids, etc.

Second, the use of food allergens or food allergen antibodies for the detection of IgA nephropathy.

1. The sample is a paraffin section of the kidney tissue of patients with IgA nephropathy.

A. Deparation of paraffin sections of kidney tissue to water;

C. antigen retrieval, then blocking non-specific sites;

D. Add fluorescently labeled food allergen monoclonal antibody, incubate, wash, seal, and then observe the results by fluorescence microscopy; or add unlabeled food allergen monoclonal antibody, incubate, wash, and then add signal molecule labeled IgA secondary antibody Incubate, wash, seal, and then observe the results by fluorescence microscopy; or add unlabeled food allergen monoclonal antibody, incubate, wash, then add signal molecule labeled IgA secondary antibody, incubate, wash, add signal molecular marker The IgG secondary antibody was incubated, washed, mounted, and the results were observed by fluorescence microscopy.

Preferably, the specific steps are as follows;

A. Deparation of paraffin sections of kidney tissue to water;

B. Remove the endogenous peroxidase from the paraffin section of the kidney tissue: Add 3% hydrogen peroxide (methanol configuration) onto the section, place in a wet box, and incubate for 20 minutes at room temperature;

C. Antigen repair, then block non-specific sites: add 0.25% trypsin (purchased from Invitrogen, Canada) on the sections, place in a wet box, incubate at 37 ° C for 15 minutes; add 5% BSA ( Purchased from Wuhan Boster Bio Company) on the slice, and in the wet box, incubate at 37 ° C for 1 hour;

D1. Add primary antibody: Pour off the blocking solution on the slice, do not wash; add eight different mouse food allergen monoclonal antibodies prepared in 2.2, respectively, to the tissue in the section, each section about 50 L, based on the total coverage of the tissue; in the wet box, overnight at 4 ° C;

D2. Add secondary antibody: (The following procedures need to be protected from light) Soak the liquid around the tissue, add the goat anti-human IgA-TRITC secondary antibody (purchased from Southern Biotech, USA), and incubate each slice about 50 L at 37 °C. 45 points Clock

D3. Add another secondary antibody: blot the tissue around the tissue, add rabbit anti-mouse IgG-FITC secondary antibody (purchased in the United States)

Southern Biotech), about 50 L per slice, incubated at 37 ° C, 45 minutes;

D4. Dry the liquid around the tissue, seal the glycerin, seal the nails; then observe the results under a fluorescence microscope.

2. The sample is a frozen section of kidney tissue from patients with IgA nephropathy.

A. removing endogenous peroxidase from frozen sections of kidney tissue;

B. antigen retrieval, then blocking non-specific sites;

Preferably, the specific steps are as follows;

A. Removal of endogenous peroxidase from frozen sections of kidney tissue: After the frozen section is prepared, it is allowed to stand at room temperature for 30 minutes; then fixed in acetone solution at 4 ° C for 10 minutes; then washed 3 times with PBS solution; Incubate with hydrogen peroxide for 5-10 minutes to eliminate endogenous peroxidase activity;

B. Antigen retrieval, then blocking non-specific sites: 0.25% trypsin (purchased from Invitrogen, Canada) was placed on the sections, placed in a wet box, incubated at 37 ° C for 15 minutes; 5% BSA was added dropwise Purchased from Wuhan Boster Bio Company) on the slice, and in the wet box, incubate at 37 ° C for 1 hour;

C1. Add primary antibody: Pour the blocking solution on the slice, do not wash; add eight different mouse food allergen monoclonal antibodies prepared in 2.2, respectively, to the tissue in the section, each section about 50 L, based on the total coverage of the tissue; in the wet box, overnight at 4 ° C;

C2. Add secondary antibody: (The following procedures need to be protected from light) Absorb the liquid around the tissue, add the goat anti-human IgA-TRITC secondary antibody (purchased from Southern Biotech, USA), and incubate each slice about 50 L at 37 °C. 45 minutes;

C3. Add another secondary antibody: blot the tissue around the tissue, add rabbit anti-mouse IgG-FITC secondary antibody (purchased in the United States)

Southern Biotech), about 50 L per slice, incubated at 37 ° C, 45 minutes;

C4. Wipe the liquid around the tissue, seal the glycerin, seal the nails; then observe the results under a fluorescence microscope.

3. The sample is serum.

A. Add a blocking solution to a 96-well plate pre-coated with food allergens and incubate;

B. Washing, then adding the serum sample to be tested and incubating; C. washing, adding enzyme-labeled IgA secondary antibody, and incubating;

Specific steps are as follows:

A. Pre-coating of food allergens: Dilute a certain amount of 8 recombinant food allergens prepared in 1.4 to the coating solution of PH9.6, to a final concentration of 10 g/mL, and add to 96 wells. In the plate, 100 μ ΐ 7 wells, 4 ° C overnight;

B. Wash the plate with PBST washing solution (containing 0.5% Tween 20) 5 times, add blocking solution, 100 μ ΐ 7 wells, incubate at 37 ° C for 2 h _;

C. Wash the plate again with PBST wash solution 5 times, use 1:3000 patient serum containing PBST containing 1% calf serum, and then add to the well-coated 96-well microtiter wells, 100 μ ΐ 7 wells. Incubate at 37 ° C for 2 h;

D. Wash the plate with PBST wash 5 times, add 100 μL of diluted goat anti-mouse HRP_IgA (l: 4000) per well, incubate at 37 °C for 2 h;

E. Wash the plate 5 times with PBST, add 50 μL of the coloring solution, and 50 μL of the coloring solution. (What are the coloring liquids and Β here? Please confirm), 37 °C in the dark The color was developed for 15 min, 50 μL of ₂ Μ H ₂ S0 _{4 was added} , the reaction was terminated, and the absorbance (A450 value) was read.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments. It is not intended that the specific embodiments of the invention are limited to the description. It will be apparent to those skilled in the art that the present invention may be practiced without departing from the spirit and scope of the invention.

Claims

Claim

1. Application of food allergen antibodies in the preparation of IgA nephropathy etiological detection reagents.

The use of the food allergen antibody according to claim 1 for the preparation of an IgA nephropathy causative detection reagent, characterized in that: the food allergen is a peanut allergen, a nut allergen, a fish allergen, a carapace At least one of an allergen, a milk allergen, a wheat allergen, a soybean allergen, and an egg allergen.

The use of the food allergen antibody according to claim 2, in the preparation of an IgA nephropathy estrogen detecting reagent, characterized in that: the food allergen antibody is milk β-lactoglobulin monoclonal antibody, egg egg mucin 0VM Monoclonal Antibody, Fish Albumin Monoclonal Antibody, Shrimp Myosin Monoclonal Antibody, Peanut Arahl Protein Monoclonal Antibody, Scorpion Corhl Protein Monoclonal Antibody, Soy Gly mBd Monoclonal Antibody and Crab Myosin Monoclonal Antibody At least one.

The use of the food allergen antibody according to claim 1 or 2 or 3, in the preparation of an IgA nephropathy causative detection reagent, characterized in that the food allergen antibody is at least one of a polyclonal antibody and a monoclonal antibody. Kind.

The use of the food allergen antibody according to claim 4, wherein the food allergen antibody is a fluorescently labeled antibody, an enzyme labeled antibody or a biotinylated antibody.

The use of the food allergen antibody according to claim 5, in the preparation of an IgA nephropathy causative detection reagent, characterized in that: the food allergen antibody is a fluorescein isothiocyanate-labeled antibody, tetramethyl isocyanic acid Rhodamine labeled antibody, rhodamine red labeled antibody, Texas Red labeled antibody, cyan dye labeled antibody, phycoerythrin labeled antibody, phycobiliprotein marker antibody or acetate labeled antibody.

7. The application of food allergens in the preparation of IgA nephropathy etiological detection reagents.

The use of the food allergen according to claim 7 for the preparation of an IgA nephropathy causative detection reagent, characterized in that: the food allergen is a peanut allergen, a nut allergen, a fish allergen, a crustacean allergy At least one of an original, a milk allergen, a wheat allergen, a soybean allergen, and an egg allergen.

The use of the food allergen according to claim 8 for preparing an IgA nephropathy causative detecting reagent, characterized in that: the food allergen is milk β _ lactoglobulin, egg ovomucin 0VM, fish small albumin At least one of shrimp myosin, peanut Arahl protein, scorpion Corhl protein, soybean Gly mBd, and crab tropomyosin.

The use of a food allergen according to claim 7 or 8 or 9 for the preparation of an IgA nephropathy causative detection reagent, characterized in that said food allergen carries a labeling molecule.

The use of the food allergen according to claim 10 for the preparation of an IgA nephropathy causative detection reagent, characterized in that the marker molecule is an enzyme labeling molecule.

12. A method for detecting the cause of IgA nephropathy, characterized by: using a food allergen antibody to detect a food allergen in a sample of a patient with IgA nephropathy by immunohistochemistry, and if one or more food allergens are detected, Describe whether the food allergen or the multi-food allergen is the cause of the patient with the IgA nephropathy.

13. The method for detecting the cause of IgA nephropathy according to claim 12, wherein: the food allergen is a peanut allergen, a nut allergen, a fish allergen, a crustacean allergen, a milk allergen At least one of a scorpion allergen, a soybean allergen, and an egg allergen.

The method for detecting the cause of IgA nephropathy according to claim 13, wherein the food allergen antibody is milk β _ lactoglobulin monoclonal antibody, egg ovomucin 0VM monoclonal antibody, fish small albumin At least one of a monoclonal antibody, a shrimp myosin monoclonal antibody, a peanut Arahl protein monoclonal antibody, a scorpion Corhl protein monoclonal antibody, a soybean Gly mBd monoclonal antibody, and a crab tropomyosin monoclonal antibody.

The method for detecting the cause of IgA nephropathy according to claim 12 or 13 or 14, wherein the food allergen antibody is at least one of a polyclonal antibody and a monoclonal antibody.

The method for detecting the cause of IgA nephropathy according to claim 15, wherein the food allergen antibody is a fluorescently labeled antibody, an enzyme labeled antibody or a biotinylated antibody.

The method for detecting the cause of IgA nephropathy according to claim 16, wherein the food allergen antibody is a fluorescein isothiocyanate-labeled antibody, a tetramethyl isocyanate rhodamine-labeled antibody, and a rhodamine red label. An antibody, a Texas Red-labeled antibody, a cyan dye-labeled antibody, a phycoerythrin-labeled antibody, a phycobiliprotein-labeled antibody, or an acetate-labeled antibody.

The method for detecting the cause of IgA nephropathy according to claim 12, wherein the sample is at least one of a tissue paraffin section and a frozen section.

19. The method of detecting the etiology of IgA nephropathy according to claim 18, wherein: the sample is a paraffin section of a kidney tissue, comprising the steps of:

A. Deparation of paraffin sections of kidney tissue to water;

C. antigen retrieval, then blocking non-specific sites;

20. A method for detecting the cause of IgA nephropathy, characterized by: using a food allergen, detecting a food allergen antibody in a sample by an ELISA technique, and detecting one or more food allergen antibodies, indicating the food The allergen or the multi-food allergen is the cause of the patient with the IgA nephropathy.

The method for detecting the cause of IgA nephropathy according to claim 20, wherein: the food allergen is a peanut allergen, a nut allergen, a fish allergen, a crustacean allergen, a milk allergen At least one of a wheat allergen, a soybean allergen, and an egg allergen.

The method for detecting the cause of IgA nephropathy according to claim 21, wherein the food allergen antibody is milk β _ lactoglobulin, egg ovomucin 0VM, fish small albumin, shrimp myosin, At least one of peanut Arah1 protein, gardenia Corhl protein, soybean Gly mBd, and crab tropomyosin.

23. Use of a food allergen according to claim 20 or 21 or 22 for the preparation of an IgA nephropathy causative detection reagent, characterized in that the food allergen carries a labeling molecule.

24. The use of a food allergen according to claim 23 for the preparation of an IgA nephropathy causative detection reagent, characterized in that the marker molecule is an enzyme labeling molecule.

The method for detecting the cause of IgA nephropathy according to claim 20, wherein the sample is at least one of serum, plasma, whole blood, and tissue fluid.

26. The method of detecting the etiology of IgA nephropathy according to claim 25, wherein: said sample is a serum sample, comprising the steps of:

B. Washing, then adding the serum sample to be tested and incubating;

C. Wash, add enzyme-labeled IgA secondary antibody, and incubate;