Indirect ELISA method for detecting yolk antibody against cat allergen Fel d1
The detection of yolk extraction antibodies in eggs through indirect ELISA method solves the problem of low detection efficiency of cat allergen Fel d1 yolk antibody in the prior art, and realizes efficient monitoring and prevention of cat allergens, providing fast and simple tool.
Patent Information
- Application Number
- CN202210094240.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-01-26
- Publication Date
- 2024-12-13
- Estimated Expiration
- 2042-01-26
AI Technical Summary
The existing methods for detecting cat allergen Fel d1 yolk antibody have problems such as low efficiency and difficulty in large-scale production, resulting in poor monitoring and prevention of cat allergens.
By using indirect ELISA method, immunoassays were performed on the yolk extraction antibody of eggs, the coating buffer was coated with cat allergen Fel d1, the non-specific antibody binding site was blocked, and the yolk antibody and HRP-labeled secondary antibody were added for incubation, and the The color-developing solution and the stop solution undergo a color-developing reaction, combining specific amino acid sequences and preparation methods to achieve effective monitoring of the Fel d1 protein content.
This method is simple and effective, and can quickly monitor cat allergen Fel d1, providing tools for screening allergens, disease diagnosis and vaccine immunity evaluation, and improving the detection efficiency and accuracy of cat allergens.
Smart Images
Figure CN114487439B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological antibodies, and in particular to an indirect ELISA method for detecting cat allergen Fel d1 egg yolk antibodies. Background Art
[0002] It is estimated that the prevalence of cat sensitization in Europe ranges from 15% in the south to 30% in northern Europe; in the United States, 5% of children and 12% of adults have specific cat IgE; from 2005 to 2014, the rate of pet allergy sensitivity in my country increased from 16.5% to 28.5%, which fully demonstrates the importance of taking more effective prevention and treatment measures. Cats, as one of the most popular pets in the world, have close contact with humans and have become an indispensable member of more and more human families, but they increase the risk of cat-allergen-related diseases such as allergic rhinoconjunctivitis and asthma in those who are allergic to cats. In this case, abandoning the favorite pet to avoid exposure to cat allergens or not keeping pets for fear of allergies is usually a less acceptable solution. Studies have shown that in 90% to 95% of cat allergy patients, the cat allergen Fel d1 is the most common indoor allergen that causes IgE-mediated allergic reactions, accounting for 60% to 90% of the total sensitizing activity of cat extracts. A new approach to reducing Fel d1 allergens is to use the interaction between antibodies and allergens to neutralize feld1 after it is produced by cats and before it spreads into the environment. A recent pilot study showed that adding egg products containing anti-fel d1 IgY to the diet of cats can lead to a significant reduction in active feld1 in cat saliva. This provides a new idea and method for effectively preventing diseases caused by cat allergens in humans from the source.
[0003] Clarifying the cause of the disease is to do a more effective job of prevention and treatment. The most urgent task is to cut off the source of cat allergens, which is a difficult challenge facing scientific researchers, clinical veterinarians and pet owners. Establishing a systematic and complete detection method for cat allergen Feld1 yolk antibody and effectively monitoring the main cat allergen Fel d1 provides a powerful tool for allergen screening, disease diagnosis and vaccine immune effect evaluation. Summary of the invention
[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide an indirect ELISA method for detecting cat allergen Fel d1 yolk antibodies. The method extracts antibodies from the yolk of eggs laid by chickens and performs immune detection, so that the cat allergen Fel d1 protein content can be effectively monitored and the immune effect can be judged. The method is simple and easy to implement and can be used for large-scale production of cat allergen Fel d1 yolk antibodies, thereby reducing losses and improving benefits.
[0005] In order to achieve the above object, the present invention provides an indirect ELISA method for detecting cat allergen Fel d 1 yolk antibody, the method comprising the following steps:
[0006] 1. coating cat allergen Fel d 1 in the coating buffer;
[0007] 2. blocking the non-specific antibody binding sites of the cat allergen Fel d1 with a blocking solution;
[0008] 3. Add egg yolk antibody for incubation;
[0009] 4. Add HRP-labeled secondary antibody for incubation;
[0010] 5. After adding the color developing solution for color development, add the stop solution to terminate the reaction;
[0011] The amino acid sequence of the cat allergen FeI d1 is shown in SEQ ID.NO.1.
[0012] The above-mentioned indirect ELISA method, further, the coating buffer comprises 1.59 g / L Na 2 CO 3 and 2.93 g / L NaHCO 3 ; The blocking solution is 5% BSA.
[0013] The color developing solution preparation method: according to the ELISATMB color developing solution instruction manual of Nanjing Jiancheng Biotechnology Co., Ltd., item number B019-1-2, A:B:C was prepared in a volume ratio of 5000:5000:4.
[0014] In the above-mentioned indirect ELISA method, further, the coating concentration of cat allergen Fel d1 is 40-160 ng / μL; and the dilution of the secondary antibody is 1:4000-6000.
[0015] In the above-mentioned indirect ELISA method, further, the cat allergen Fel d 1 is prepared by the following method:
[0016] S1, mixing the cat allergen Fel d 1 recombinant antigen with a primary immune adjuvant to obtain a mixture;
[0017] S2. Inject the mixture subcutaneously into the neck of laying hens at the peak of egg-laying period, and collect eggs; the yolk of the eggs is enriched with yolk antibodies specific to cat allergen Fel d 1.
[0018] In the above-mentioned indirect ELISA method, further, the primary immune adjuvant is Freund's complete adjuvant; or, a mixture of aluminum hydroxide, CpG-ODN and BSF-1 in a mass ratio of 1-3:2-4:4-7;
[0019] And / or, the cat allergen Fel d 1 recombinant antigen is mixed with the primary immune adjuvant in a volume ratio of 1:1.
[0020] The above-mentioned indirect ELISA method, further, the preparation method also includes the following steps:
[0021] S3. Three weeks later, booster immunization is performed. During the booster immunization, the cat allergen Fel d1 recombinant antigen is mixed with a booster immunization adjuvant;
[0022] S4. After that, booster immunization is performed every two weeks according to the method of S3.
[0023] In the above-mentioned indirect ELISA method, further, the primary immunization adjuvant and the booster immunization adjuvant are one of the following A or B:
[0024] A: The adjuvant for the first immunization is Freund's complete adjuvant, and the adjuvant for the booster immunization is Freund's incomplete adjuvant;
[0025] B: The primary immunization adjuvant is a mixture of aluminum hydroxide, CpG-ODN and BSF-1 in a mass ratio of 1~3:2~4:4~7, and the booster immunization adjuvant is a mixture of aluminum hydroxide, CpG-ODN and BSF-1 in a mass ratio of 1~3:2~4:4~7.
[0026] In the above-mentioned indirect ELISA method, further, the cat allergen Fel d 1 recombinant antigen is mixed with the primary immunization adjuvant in a volume ratio of 1:1; the cat allergen Fel d 1 recombinant antigen is mixed with the booster immunization adjuvant in a volume ratio of 1:1.
[0027] In the above-mentioned indirect ELISA method, further, in S1, the cat allergen Fel d 1 recombinant antigen is prepared by the following method:
[0028] S1-1, using the nucleotide sequence shown in SEQ ID NO.2 as an upstream primer and the nucleotide sequence shown in SEQ ID NO.3 as a downstream primer to perform PCR amplification to obtain a PCR amplification product;
[0029] S1-2, connecting the PCR amplification product PET-30a (+) to construct a recombinant plasmid;
[0030] S1-3, transforming the recombinant plasmid into Top10 competent cells to obtain transformants;
[0031] S1-4, extracting the protein in the transformant to obtain the cat allergen Fel d 1 recombinant antigen.
[0032] The above-mentioned indirect ELISA method, further, said S1-4 specifically comprises the following steps:
[0033] (1) Pick a positive single colony from the transformant and culture it in LB liquid medium containing kanamycin until the OD value reaches 0.6-0.8, add IPTG, and induce at 37°C for 4 h;
[0034] (2) Collect the precipitate by centrifugation, add Buffer A and perform ultrasonication for 50 min at an ultrasonic power of 35% to 45%, with each ultrasonication for 5 s and an interval of 5 s; after the ultrasonication, centrifuge at 10000 r / min and 4°C for 40 min to obtain precipitate 1; the components of the Buffer A include: 20 mM Tris-HCl, 150 mM NaCl;
[0035] (3) The precipitate 1 was resuspended in Buffer B, stirred at 4°C for 40 min, and centrifuged at 10,000 r / min for 30 min at 4°C to obtain precipitate 2; the components of Buffer B include: 20 mM Tris-HCl, 0.3% Triten X-100;
[0036] (4) Precipitate 2 was resuspended in Buffer C, stirred at 4°C for 40 min, and centrifuged at 10,000 r / min at 4°C for 30 min to obtain Precipitate 3; the components of Buffer C included: 20 mM Tris-HCl, 1 M NaCl;
[0037] (5) Precipitate 3 was resuspended in 2 M urea, stirred at 4°C for 60 min, and centrifuged at 10,000 r / min at 4°C for 30 min to obtain Precipitate 4.
[0038] (6) The precipitate was resuspended in Buffer D, stirred at 4°C overnight, and centrifuged at 10,000 r / min at 20°C for 30 min. The supernatant was retained as the protein extract; the components of Buffer D included: 50 mM Tris-HCl, 150 mM NaCl, 20% glycerol, and 6 M urea.
[0039] Compared with the prior art, the advantages of the present invention are:
[0040] (1) The present invention provides an established indirect ELISA method for detecting cat allergen Fel d1 yolk antibody, which can effectively monitor the major cat allergen Fel d1 and provide a rapid and simple tool for allergen screening, disease diagnosis and vaccine immune effect evaluation. BRIEF DESCRIPTION OF THE DRAWINGS
[0041] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.
[0042] Figure 1 The purification effect of Fel d1 recombinant protein was detected by SDS-PAGE. M1 and M2 are protein molecular weight standards, lane 1 is the bacterial solution before column purification, lane 2 and lane 3 are samples dissolved in Buffer D after bacterial solution ultrasound, lane 4 is the sample flowing out when the protein is attached to the column (indicating that the protein is bound to the Ni column), lane 5 is the sample of the impurity protein washed from the protein purification column, lane 5 is the first 5 mL sample of the eluted target protein, lane 7 is the sample after protein purification, and lane 8 is the sample of the last column washing. The only band between 17 kDa and 25 kDa is Fel d1, indicating that the purification effect is good and the concentration is very high.
[0043] Figure 2 This is a specific test for the Fel d1 yolk antibody test method. It can be seen that the samples immunized with other vaccines cannot be separated from the negative serum, and the P / N value is always less than 2, while the P / N value of the samples injected with Fel d1 is always greater than 2. DETAILED DESCRIPTION
[0044] The present invention is further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby.
[0045] The materials and instruments used in the following examples are all commercially available. Embodiment 1:
[0046] A cat Fel d1 recombinant protein, whose amino acid sequence is shown in SEQ ID.NO.1, specifically: MHHHHHHEICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSVLDKIYTSPLCGSGSSGSGVKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTAMKKIQDCYVENGLISRVLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGR.
[0047] The feline Fel d1 recombinant protein of this example was assembled to form a complete protein sequence based on the amino acid sequence of the feline Fel d1 protein in the Uniprot database (the accession numbers of Fel d1 peptide chain I and peptide chain II are P30438 and P30440, respectively).
[0048] Cat Fel d1 antigen is prepared by the method of prokaryotic expression of recombinant protein. The specific steps are as follows:
[0049] (1) PCR amplification: A pair of PCR primers was designed using the molecular biology primer design software DNAMANV6. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, specifically: catatgcatcaccaccaccaccac; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3, specifically: taccctgggccgttaatgaaagctt.
[0050] PCR reaction system (20 μL): dd H 2 O 9 μL, LA Taq 0.5 μL, cDNA 3 μL, upstream and downstream primers (10 μL / L) 1 μL each, Buffer 2 μL, d NTP 3.5 μL.
[0051] PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 34 cycles; extension at 72°C for 10 min.
[0052] The PCR product was obtained by PCR amplification, and the PCR product was identified by 1.5% agarose gel electrophoresis (electrophoresis at a constant voltage of 80 V for about 40 min, observation with UV light, and photography with an automatic gel imaging scanner) and the target fragment was recovered.
[0053] (2) Cloning of target fragment:
[0054] The target fragment was connected with PET-30a (+) to construct a recombinant plasmid, and the ligation product was identified by double digestion with Ndel and HindⅢ. The ligation product was transformed into Top10 competent cells and cultured at 37℃ for 10 h. The single clone colony was picked and identified by PCR and double digestion, and then transformed into Escherichia coli BL21 (DE3) competent cells to obtain transformants.
[0055] (3) Prokaryotic expression and purification of Fel d1:
[0056] 3.1. Cultivate the transformants to obtain bacterial liquid.
[0057] 3.2. Inoculate the obtained bacterial liquid into the screening medium overnight, pick the positive single colony into LB liquid medium containing kanamycin, and culture at 37℃ and 200 rpm until the OD value reaches 0.6~0.8, then add IPTG (the final concentration of IPTG is 0.5 mM~1.0 mM) and induce at 37℃ for 4 h to achieve high-abundance expression of the protein.
[0058] 3.3. Centrifuge at 8000 r / min and 4℃ for 5 min to collect the precipitate, which contains E. coli expressing Fel d1 protein. Add the precipitate to Buffer A (20 mM Tris-HCl; 150 mM NaCl; pH 8.0) for ultrasonication (ultrasonication time is 50 min, ultrasonication for 5 s, interval of 5 s, ultrasonication power is 35%~45%), centrifuge at 10000 r / min and 4℃ for 40 min after the ultrasonication, discard the supernatant and keep the precipitate.
[0059] 3.4. Resuspend the above precipitate in Buffer B (20 mM Tris-HCl; 0.3% Triten X-100; pH 8.0), stir at 4℃ for 40 min, centrifuge at 10000 r / min, 4℃ for 30 min, discard the supernatant and keep the precipitate.
[0060] 3.5. Resuspend the above precipitate in Buffer C (20 mM Tris-HCl; 1 M NaCl; pH 8.0), stir at 4℃ for 40 min, centrifuge at 10,000 r / min, 4℃ for 30 min, discard the supernatant and keep the precipitate.
[0061] 3.6. Resuspend the above precipitate with 2 M urea, stir at 4°C for 60 min, centrifuge at 10,000 r / min, 4°C for 30 min, discard the supernatant and keep the precipitate.
[0062] 3.7. Finally, the precipitate was resuspended in Buffer D (50 mM Tris-HCl; 150 mM NaCl; 20% glycerol; 6 M urea; pH 8.0), stirred at 4°C overnight, and then centrifuged at 10,000 r / min, 20°C for 30 min, discarding the precipitate and retaining the supernatant. The supernatant is the protein extract and is stored at -80°C.
[0063] The supernatant was purified using a Ni-NTA column and the purity was determined by 15% SDS-PAGE gel electrophoresis. Figure 1 .
[0064] Figure 1The purification effect of Fel d1 recombinant protein was detected by SDS-PAGE. M1 and M2 are protein molecular weight standards, lane 1 is the bacterial solution before column purification, lane 2 and lane 3 are samples dissolved in Buffer D after bacterial solution ultrasound, lane 4 is the sample flowing out when the protein is attached to the column (indicating that the protein is bound to the Ni column), lane 5 is the sample of the impurity protein washed from the protein purification column, lane 5 is the first 5 mL sample of the eluted target protein, lane 7 is the sample after protein purification, and lane 8 is the sample of the last column washing. The only band between 17 kDa and 25 kDa is Fel d1, indicating that the purification effect is good and the concentration is very high.
[0065] Embodiment 2:
[0066] A method for producing yolk antibodies by immunizing chickens with the antigen of Example 1, the specific steps of which are:
[0067] The purified Fel d1 recombinant protein of Example 1 was diluted with autoclaved PBS and injected subcutaneously in the neck to the laying hens at the peak of egg laying, and the laying hens were immunized at 100 μg / feather, 200 μg / feather and 300 μg / feather. During the first immunization, the Fel d1 recombinant protein was mixed with Freund's complete adjuvant at a volume ratio of 1:1. A second immunization was performed three weeks later, and a third immunization was performed two weeks later. During the second and third booster immunizations, the Fel d1 recombinant protein was mixed with Freund's incomplete adjuvant at a volume ratio of 1:1, and the eggs laid by the immunized laying hens were regularly collected to extract yolk antibodies.
[0068] Experiment 1: Investigate the titer of yolk antibody produced by laying hens immunized with different doses of antigen. The results of the comparison of the titer of yolk antibody produced by laying hens immunized with different doses of antigen are shown in Table 1.
[0069] Table 1: Changes in specific yolk antibody titers produced by different immunization doses of cat recombinant protein Fel d1 antigen (Log2X) (n=4)
[0070]
[0071] From the results in Table 1, we can see that in the first week after the first vaccination, the difference in the yolk antibody titer of each dose group was small. In the 2nd to 8th week after the first vaccination, the difference in the antibody titer level between the 100 μg / feather, 200 μg / feather, and 300 μg / feather dose groups increased, while there was no significant difference in the yolk antibody titer between the 200 μg / feather and 300 μg / feather dose groups. Eggs were collected and the yolk antibody titer was detected to be above 1:65536. Embodiment 3:
[0072] A method for producing yolk antibodies by immunizing chickens with the antigen of Example 1, the specific steps of which are:
[0073] The purified Fel d1 recombinant protein of Example 1 was used to immunize laying hens at 200 μg / feather. During the first immunization, the Fel d1 recombinant protein was mixed with adjuvant B (aluminum hydroxide + CpG-ODN + BSF-1 (1:2:7)) at a volume ratio of 1:1. During the booster immunization, the Fel d1 recombinant protein was mixed with adjuvant B (aluminum hydroxide + CpG-ODN + BSF-1 (1:2:7)) at a volume ratio of 1:1. Eggs laid by the immunized laying hens were collected regularly to extract yolk antibodies. The remaining parameters were the same as those of Example 2. Embodiment 4:
[0074] A method for producing yolk antibodies by immunizing chickens with the antigen of Example 1, the specific steps of which are:
[0075] The purified Fel d1 recombinant protein of Example 1 was used to immunize laying hens at 200 μg / feather. During the first immunization, the Fel d1 recombinant protein was mixed with adjuvant C (aluminum hydroxide + CpG-ODN + BSF-1 (2:4:4)) at a volume ratio of 1:1. During the booster immunization, the Fel d1 recombinant protein was mixed with adjuvant B (aluminum hydroxide + CpG-ODN + BSF-1 (2:4:4)) at a volume ratio of 1:1. Eggs produced by the immunized laying hens were collected regularly to extract yolk antibodies. The remaining parameters were the same as those of Example 2. Embodiment 5:
[0076] A method for producing yolk antibodies by immunizing chickens with the antigen of Example 1, the specific steps of which are:
[0077] The purified Fel d1 recombinant protein of Example 1 was used to immunize laying hens at 200 μg / feather. During the first immunization, the Fel d1 recombinant protein was mixed with adjuvant D (aluminum hydroxide + CpG-ODN + BSF-1 (3:3:4)) at a volume ratio of 1:1. During the booster immunization, the Fel d1 recombinant protein was mixed with adjuvant D (aluminum hydroxide + CpG-ODN + BSF-1 (3:3:4)) at a volume ratio of 1:1. Eggs laid by the immunized laying hens were collected regularly to extract yolk antibodies. The remaining parameters were the same as those of Example 2.
[0078] Experiment 2: Comparison of the titers of yolk antibodies produced by immunizing laying hens with different immune adjuvant combinations in Examples 2 to 5
[0079] After the cat Fel d1 recombinant protein was purified, it was fully mixed with different adjuvant combinations at a ratio of 1:1 (volume ratio), and then immunized with 200 μg / feather (calculated according to the amount of immunogen) according to the same immunization procedure. Eggs produced by immunized laying hens were collected regularly, and yolk antibodies were extracted and the antibody titer was tested. Table 2 shows the combination of immune adjuvants used in different test groups; Table 3 shows the changes in the titer of specific yolk antibodies produced by different immune adjuvant combinations of cat recombinant protein Fel d1 antigen.
[0080] Table 2: Combinations of immune adjuvants used in different trial groups
[0081]
[0082] Table 3: Changes in specific egg yolk antibody titers produced by different immune adjuvant combinations of cat recombinant protein Fel d1 antigen (Log2X) (n=4)
[0083]
[0084] From the results of yolk antibody titers produced after immunization of laying hens with different immune adjuvant combinations in Table 3, it can be seen that: in the first week after the first immunization, the differences in yolk antibody titers of each immune adjuvant group were small. Three weeks later, with the injection of enhanced immunity, the antibody titer of group D was significantly higher than that of groups A, B and C, and this titer advantage continued until the 8th week.
[0085] Comparative Example 1:
[0086] A method for preparing negative yolk antibodies comprises the following steps:
[0087] Collect eggs laid by healthy chickens that have not been immunized with feline Fel d1 vaccine, open the eggs in a clean bench, aspirate 0.5 mL of yolk liquid, mix it thoroughly with 0.5 mL of sterile saline, then add 1 mL of chloroform, mix it thoroughly, centrifuge it at 10,000 r / min, 4°C for 20 min, collect the supernatant and set aside. Embodiment 6:
[0088] A method for detecting the optimal antigen-antibody concentration by the cat allergen Fel d1 egg yolk antibody indirect ELISA method of the present invention:
[0089] (1) Determination by matrix method. Use coating buffer (weigh 1.59 g Na2CO3, 2.93 g NaHCO3, add 800 mL distilled water, adjust pH to 9.6 with 1M NaOH and 1M HCL, and make up to 1 L with double distilled water to make coating buffer) to dilute the purified Fel d1 by 20, 40, 80, 160, 320, and 640 times. Add 100 μL of the dilution to each well of a 96-well ELISA plate in two vertical rows, incubate at 37°C for 2 h, and then at 4°C overnight. Discard the liquid in the wells, wash with washing solution, 300 μL per well, wash three times, 3 min each time, and pat dry.
[0090] (2) Add 200 μL of blocking solution (5% BSA: weigh 5 g of fetal bovine serum (BSA) and dissolve it in 100 mL of PBST washing solution) to each well and incubate at 37°C for 3 h. Discard the liquid in the wells and wash with washing solution (300 μL per well), wash three times, 3 min each time, and pat dry.
[0091] (3) Dilute the positive egg yolk antibody of Example 5 with the negative egg yolk antibody sample of Comparative Example 1 by 25, 50, 100, and 200 times, respectively. Add 100 μL of the 25-fold diluted positive egg yolk antibody to the first row, 100 μL of the 25-fold diluted negative egg yolk antibody to the second row, 100 μL of the 50-fold diluted positive egg yolk antibody to the third row, and 100 μL of the 50-fold diluted negative egg yolk antibody to the fourth row, and so on. After adding all the egg yolk antibody samples, incubate at 37°C for 1 hour. Discard the liquid in the wells, wash with washing solution, 300 μL per well, wash three times, 3 minutes each time, and pat dry.
[0092] (4) Add HRP-labeled donkey anti-chicken IgY secondary antibody (1:5000 dilution, solvent is blocking solution), 100 μL per well, incubate at 37°C for 1 h. Discard the liquid in the wells, wash with washing solution, 300 μL per well, wash three times, 3 min each time, and pat dry.
[0093] (5) Add TMB substrate for color development (prepare A:B:C in a volume ratio of 5000:5000:4 according to the instructions of ELISA TMB color development solution (Cat. No. B019-1-2, Nanjing Jiancheng Biotechnology Co., Ltd.), 100 μL per well, and develop at 37°C in the dark for 10 min to 15 min.
[0094] (6) After taking out, add stop solution (2 mol / L H 2 SO 4 After the reaction was terminated, the OD value at 450 nm was immediately read using a multifunctional microplate reader.
[0095] The antigen concentration and yolk antibody dilution corresponding to the hole with the largest P / N value are respectively taken as the optimal antigen coating concentration and yolk antibody dilution. P is the OD450 value of positive yolk antibody, and N is the OD450 value of negative yolk antibody. Table 4 is the test results.
[0096] Table 4: Determination of indirect ELISA antigen coating concentration and serum dilution multiple
[0097]
[0098] According to the above Example 2 (1), the purified FeI d 1 was diluted 20, 40, 80, 160, 320, and 640 times with coating buffer, and the positive and negative serum was diluted 25, 50, 100, and 200 times, and ELISA detection was performed. The optimal antigen coating concentration and the optimal serum dilution multiple were determined by taking the P value of about 1.0, the maximum P / N value, and the lower N value. The results showed that when the antigen coating concentration was 80 ng / μL, the P / N value was the largest at 1:200 dilution of the positive chicken serum, which was 3.48. Therefore, this condition was the best. Embodiment 7:
[0099] A method for detecting the concentration and critical value of enzyme-labeled secondary antibody by indirect ELISA method of cat allergen Fel d1 yolk antibody of the present invention:
[0100] (1) Dilute the purified antigen to the optimal dilution multiple with coating buffer (prepared as above); then add the diluted antigen solution to a 96-well ELISA plate, 100 μL per well, incubate at 37°C for 2 h, and then overnight at 4°C. Discard the liquid in the wells, wash with washing solution, 300 μL per well, wash three times, 3 min each time, and pat dry.
[0101] (2) Add 200 μL of blocking solution (prepared as above) to each well and incubate at 37°C for 3 h. Discard the liquid in the wells and wash with washing solution (300 μL per well), wash three times, 3 min each time, and pat dry.
[0102] (3) Dilute the negative and positive egg yolk antibodies 50 times with PBS, add 100 μL to each well of the ELISA plate, and incubate at 37°C for 1 h. Discard the liquid in the wells, wash with washing solution, 300 μL per well, wash three times, 3 min each time, and pat dry.
[0103] (4) HRP-labeled donkey anti-chicken IgY secondary antibody was diluted at a mass ratio of 1:500, 1:1000, 1:2000, 1:5000, 1:10000, and 1:20000, respectively, and 100 μL was added to each well and incubated at 37°C for 1 h. The liquid in the wells was discarded, and the wells were washed with washing solution, 300 μL per well, for three washes, each for 3 min, and patted dry.
[0104] (5) Add TMB substrate for color development (Nanjing Jiancheng, B019-1-2, preparation method as specified in the instruction manual: A:B:C = 5000:5000:4), 100 μL per well, and incubate at 37°C in the dark for 10–15 min.
[0105] (6) After taking out, add stop solution (2 mol / L H 2 SO 4 Solution), 100 μL per well. After the reaction is terminated, the OD value at a wavelength of 450 nm is immediately read using a multifunctional microplate reader. The antigen concentration and yolk antibody dilution corresponding to the well with the positive yolk antibody OD450 close to 1 and the largest P / N value are respectively taken as the optimal antigen coating concentration and yolk antibody dilution.
[0106] According to the above Example 2(2), the HRP-labeled donkey anti-chicken IgY secondary antibody was diluted 1:500, 1:1000, 1:2000, 1:5000, 1:10000, and 1:20000. The effect of each secondary antibody dilution concentration on the results was detected under the above optimal conditions, and the other steps remained unchanged.
[0107] The dilution of the above enzyme-labeled secondary antibody was tested. The test results are shown in Table 5.
[0108] Table 5: Determination of dilution of enzyme-labeled secondary antibody for indirect ELISA
[0109]
[0110] The results in Table 5 show that when the dilution of the secondary antibody is 1:5000, the P / N value is the largest, which is 4.56. 1:5000 is selected as the optimal dilution of the secondary antibody. Embodiment 8:
[0111] A method for detecting specificity of the cat allergen Fel d1 yolk antibody indirect ELISA method of the present invention:
[0112] (1) Specificity test of yolk antibodies in eggs produced by chickens immunized with other types of vaccines. Extract yolk antibodies according to the above method and set aside.
[0113] (2) Dilute the purified feline Fel d1 antigen to 50 times with coating buffer and coat it in a 96-well plate, 100 μL per well, incubate at 37°C for 2 h, and then overnight at 4°C. Discard the liquid in the wells, wash with washing buffer, 300 μL per well, wash three times, 3 min each time, and pat dry.
[0114] (3) Add 200 μL of blocking solution to each well and incubate at 37℃ for 3 h. Discard the liquid in the wells, wash with washing solution, 300 μL per well, wash three times, 3 min each time, and pat dry. Dilute the positive and negative yolk antibodies of other types of vaccine chickens 50 times, add 100 μL per well to the ELISA plate in turn, and incubate at 37℃ for 1 h. Discard the liquid in the wells, wash with washing solution, 300 μL per well, wash three times, 3 min each time, and pat dry.
[0115] (4) HRP-labeled donkey anti-chicken IgY secondary antibody was diluted 1000 times, 100 μL per well, added to the positive and negative wells respectively, and incubated at 37°C for 1 h. The liquid in the wells was discarded, and the wells were washed with washing solution, 300 μL per well, three times, 3 min each time, and patted dry.
[0116] (5) Add TMB substrate for color development (preparation method as described in the instruction manual: A:B:C = 5000:5000:4), 100 μL per well, incubate at 37°C in the dark for 10 min to 15 min, remove from the well and add stop solution (2 mol / L H 2 SO 4 After the reaction was terminated, the OD value at 450 nm was immediately read using a multifunctional microplate reader.
[0117] Yolk antibodies of mice immunized with other vaccines and Fel d1 vaccine were detected under the above optimal conditions, and other steps remained unchanged.
[0118] Results Figure 2 As can be seen from the figure: only the P / N value of the samples of chickens immunized with Fel d1 vaccine can be greater than 2.
[0119] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as above in the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.
Claims
1. An indirect ELISA method for detecting cat allergen Fel d1 yolk antibodies, characterized in that: The method comprises the following steps:
1. coating cat allergen Fel d1 in the coating buffer with a coating buffer; the coating concentration of cat allergen Fel d1 is 40-160 ng / μL; 2. blocking the non-specific antibody binding sites of the cat allergen Fel d1 with a blocking solution; 3. Add yolk antibody for incubation; 4. Add HRP-labeled donkey anti-chicken IgY secondary antibody for incubation, wherein the dilution of the donkey anti-chicken IgY secondary antibody is 1:4000-6000; 5. After adding the color developing solution for color development, add the stop solution to terminate the reaction; The amino acid sequence of the cat allergen Fel d1 is shown in SEQ ID.NO.1; The cat allergen Fel d1 is prepared by the following method: S1, mixing the cat allergen Fel d 1 recombinant antigen with a primary immune adjuvant to obtain a mixture; S2, injecting the mixture subcutaneously into the neck of laying hens at the peak of egg-laying period, and collecting eggs; the yolk of the eggs is enriched with yolk antibodies specific to cat allergen Fel d1; S3. Three weeks later, booster immunization is performed. During the booster immunization, the cat allergen Fel d1 recombinant antigen is mixed with a booster immunization adjuvant; S4, and then perform booster immunization every two weeks according to the method of S3; The primary immune adjuvant is a mixture of aluminum hydroxide, CpG-ODN and BSF-1 in a mass ratio of 3:3:4; the booster immune adjuvant is a mixture of aluminum hydroxide, CpG-ODN and BSF-1 in a mass ratio of 3:3:4; The cat allergen Fel d 1 recombinant antigen is mixed with the primary immunization adjuvant in a volume ratio of 1:1; the cat allergen Fel d 1 recombinant antigen is mixed with the booster immunization adjuvant in a volume ratio of 1:
1.
2. The indirect ELISA method according to claim 1, characterized in that The coating buffer includes 1.59 g / L Na2CO3 and 2.93 g / L NaHCO3; the blocking solution is 5% BSA.
3. The indirect ELISA method according to claim 1, characterized in that In S1, the cat allergen Feld1 recombinant antigen is prepared by the following method: S1-1, using the nucleotide sequence shown in SEQ ID NO.2 as an upstream primer and the nucleotide sequence shown in SEQ ID NO.3 as a downstream primer to perform PCR amplification to obtain a PCR amplification product; S1-2, connecting the PCR amplification product PET-30a (+) to construct a recombinant plasmid; S1-3, transforming the recombinant plasmid into Top10 competent cells to obtain transformants; S1-4, extracting the protein in the transformant to obtain the cat allergen Fel d 1 recombinant antigen.
4. The indirect ELISA method according to claim 3, characterized in that The S1-4 specifically includes the following steps: (1) Pick a positive single colony from the transformant and culture it in LB liquid medium containing kanamycin until the OD value reaches 0.6-0.8, add IPTG, and induce at 37°C for 4 h; (2) Collect the precipitate by centrifugation, add Buffer A and perform ultrasonication for 50 min at an ultrasonic power of 35-45%, with each ultrasonication for 5 s and an interval of 5 s; after the ultrasonication, centrifuge at 10000 r / min and 4°C for 40 min to obtain precipitate 1; the components of the Buffer A include: 20 mM Tris-HCl, 150 mM NaCl; (3) The precipitate 1 was resuspended in Buffer B, stirred at 4°C for 40 min, and centrifuged at 10,000 r / min for 30 min at 4°C to obtain precipitate 2; the components of Buffer B include: 20 mM Tris-HCl, 0.3% Triten X-100; (4) Precipitate 2 was resuspended in Buffer C, stirred at 4°C for 40 min, and centrifuged at 10,000 r / min for 30 min at 4°C to obtain Precipitate 3; the components of Buffer C included: 20 mM Tris-HCl, 1 M NaCl; (5) Precipitate 3 was resuspended in 2 M urea, stirred at 4°C for 60 min, and centrifuged at 10,000 r / min at 4°C for 30 min to obtain Precipitate 4. (6) The precipitate was resuspended in Buffer D, stirred at 4°C overnight, and centrifuged at 10,000 r / min at 20°C for 30 min. The supernatant was retained as the protein extract; the components of Buffer D included: 50 mM Tris-HCl, 150 mM NaCl, 20% glycerol, and 6 M urea.