CN111537736A - Indirect ELISA (enzyme-linked immunosorbent assay) detection kit and detection method for mycoplasma gallisepticum antibody - Google Patents
Indirect ELISA (enzyme-linked immunosorbent assay) detection kit and detection method for mycoplasma gallisepticum antibody Download PDFInfo
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Abstract
The invention discloses an indirect ELISA detection kit and a detection method for mycoplasma gallisepticum antibodies. The invention discloses an indirect ELISA detection kit for mycoplasma gallisepticum antibodies, which comprises PVPA protein, 2.5% skimmed milk powder, 5% BSA, a coating stabilizer, MG positive serum, MG negative serum, 0.01M PBS (PH7.2-7.4), horseradish peroxidase-labeled rabbit anti-chicken IgY, a microporous plate, a washing solution, a chromogenic substrate and a stop solution, wherein the PVPA protein is protein of which the amino acid sequence is 35-408 th site of a sequence 2 or the sequence 2. The kit can detect whether serum of an animal to be detected contains mycoplasma gallisepticum antibodies or not, and judge whether the animal to be detected is infected with mycoplasma gallisepticum or not, thereby providing a reliable detection method for identifying MG infection and providing scientific guidance for MG epidemic prevention and control.
Description
Technical Field
The invention relates to the field of biotechnology, and discloses an indirect ELISA detection kit and a detection method for mycoplasma gallisepticum antibodies.
Background
Mycoplasma Gallisepticum (MG) is the etiological agent of Chronic Respiratory Disease (CRD) of chickens, can cause damages to respiratory tracts, air sacs and infraorbital sinuses of the chickens, reduces laying rate of the layers, reduces quality of broilers, increases abandonment rate, and is one of infectious diseases seriously harmed by poultry industry. MG can be vertically spread through eggs, so that the hatchability and the fertility rate are reduced, the mortality of chicks is increased, the growth and development are hindered, the offspring of the breeding hens becomes MG-infected positive chicken flocks, the disease is very common and important reason, and meanwhile, great difficulty is brought to the control and purification of the pathogen, so that whether MG exists in hatching eggs is detected, and the method has important significance for cutting off the vertical spread of the eggs, purifying the chicken flocks and improving the production benefit of the offspring chicken flocks. At present, detection kits of companies such as IDEXX are generally used for MG serological monitoring, so that the detection cost is high and the popularization range is narrow. Therefore, research and development of the MG detection kit with independent intellectual property rights can reduce the MG detection cost, enlarge the chicken flock detection range and have important practical significance for MG prevention and control.
The PVPA protein is a species-specific protein which is present on the surface of MG cell membranes and can be recognized by the immune system of chickens.
Disclosure of Invention
The technical problem to be solved by the invention is how to detect whether the serum of the animal to be detected contains the MG antibody.
In order to solve the technical problems, the invention firstly provides a kit, which comprises a PVPA protein and P1, wherein the PVPA protein is a1), a2) or A3 as follows:
A1) the amino acid sequence is the protein at 35 th-408 th position of the sequence 2;
A2) the amino acid sequence is the protein of sequence 2;
A3) the protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the 35 th to 408 th sites of the sequence 2 or the sequence 2 in the sequence table;
A4) a fusion protein obtained by connecting labels at the N terminal or/and the C terminal of A1) or A2) or A3);
the P1 is 2.5% skimmed milk powder and/or 5% BSA and/or coating stabilizer;
the 2.5% skimmed milk powder consists of 0.01M PBS (PH7.2-7.4) and skimmed milk powder, and the concentration of the skimmed milk powder in the 2.5% skimmed milk powder is 2.5g/100 mL;
the 5% BSA consisted of 0.01M PBS (pH7.2-7.4) and bovine serum albumin at a concentration of 5g/100mL in the 5% BSA;
the coating is stableThe fixing agent consists of a solvent and a solute, wherein the solvent is water, and the solute and the concentration of the solute in the coating stabilizer are respectively HEPES 20mM, NaCl 120mM, KCl 5mM and CaCl21mM、MgCl21mM, Tween-200.05% (volume percent), sucrose 3g/100mL, PEG40000.1g/100 mL, and Proclin 3000.05% (volume percent).
The 0.01M PBS (Ph7.2-7.4) is a product of Beijing Sorley Tech Co.
In order to facilitate the purification of the protein in A1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in the 35 th to 408 th positions of the sequence 2 or the sequence 2 in the sequence table is linked with the tags shown in the following table.
Table: sequence of tags
| Label (R) | Residue of | Sequence of |
| Poly-Arg | 5-6 (typically 5) | RRRRR |
| Poly-His | 2-10 (generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
The PVPA protein in A2) is a protein having 75% or more identity with the amino acid sequence of the protein shown in 35-408 th position of SEQ ID NO. 2 or SEQ ID NO. 2 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.
The PVPA protein in A2) can be synthesized artificially, or can be obtained by synthesizing the coding gene and then carrying out biological expression.
The gene encoding the PVPA protein of A2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in positions 9-1133 of SEQ ID NO. 1, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching to its 5 'end and/or 3' end a coding sequence for the tag shown in the above table. Wherein, the DNA molecule shown in the 9 th to 1133 rd positions of the sequence 1 codes the PVPA protein shown in the 35 th to 408 th positions of the sequence 2.
The kit may further comprise MG positive serum and/or MG negative serum.
The MG positive serum may be chicken serum infected with MG; the MG can be chicken serum that is not infected with MG.
The kit also comprises the 0.01M PBS (PH7.2-7.4), the rabbit anti-chicken IgY marked by horseradish peroxidase, a microplate, a washing solution, a chromogenic substrate and/or a stop solution.
The wash solution can be PBST. The PBST can be obtained by adding Tween-20 to 0.01M PBS, and the concentration of Tween-20 is 0.05% (volume percentage content).
The chromogenic substrate may be TMB.
The stop solution can be 2mol of H2SO4An aqueous solution.
The kit may consist of the PVPA protein and the P1, or at least one of the PVPA protein, the P1, and the MG-positive serum and the MG-negative serum, or at least one of the PVPA protein, the P1, the MG-positive serum, the MG-negative serum, and: the 0.01M PBS (PH7.2-7.4), the rabbit anti-chicken IgY marked by horseradish peroxidase, the micropore plate, the washing solution, the chromogenic substrate and the stop solution.
The kit may have any of the following uses:
(c1) detecting whether the serum of the animal to be detected contains mycoplasma gallisepticum antibodies;
(c2) preparing a kit for detecting whether the serum of an animal to be detected contains mycoplasma gallisepticum antibodies;
(c3) detecting the content of mycoplasma gallisepticum antibodies in the serum of an animal to be detected;
(c4) preparing a kit for detecting the content of mycoplasma gallisepticum antibodies in the serum of an animal to be detected;
(c5) judging whether the animal to be detected is infected with mycoplasma gallisepticum;
(c6) preparing a kit for determining whether the animal to be detected is infected with mycoplasma gallisepticum.
Any of the following uses of the kit also fall within the scope of the invention:
(c1) detecting whether the serum of the animal to be detected contains mycoplasma gallisepticum antibodies;
(c2) preparing a kit for detecting whether the serum of an animal to be detected contains mycoplasma gallisepticum antibodies;
(c3) detecting the content of mycoplasma gallisepticum antibodies in the serum of an animal to be detected;
(c4) preparing a kit for detecting the content of mycoplasma gallisepticum antibodies in the serum of an animal to be detected;
(c5) judging whether the animal to be detected is infected with mycoplasma gallisepticum;
(c6) preparing a kit for determining whether the animal to be detected is infected with mycoplasma gallisepticum.
In the above application, the animal may be poultry. Further, the poultry may be chickens.
The invention also provides an enzyme linked immunosorbent assay kit, which contains the kit.
The kit may have any of the following uses:
(a1) detecting whether the serum of the animal to be detected contains mycoplasma gallisepticum antibodies;
(a3) detecting the content of mycoplasma gallisepticum antibodies in the serum of an animal to be detected;
(a5) and judging whether the animal to be detected is infected with mycoplasma gallisepticum.
In the above kit, the animal may be poultry. Further, the poultry may be chickens.
Any one of the following applications of the kit also belongs to the protection scope of the invention:
(c1) detecting whether the serum of the animal to be detected contains mycoplasma gallisepticum antibodies;
(c2) preparing a product for detecting whether the serum of an animal to be detected contains mycoplasma gallisepticum antibodies;
(c3) detecting the content of mycoplasma gallisepticum antibodies in the serum of an animal to be detected;
(c4) preparing a product for detecting the content of mycoplasma gallisepticum antibodies in the serum of an animal to be detected;
(c5) judging whether the animal to be detected is infected with mycoplasma gallisepticum;
(c6) preparing a product for determining whether the animal to be tested is infected with mycoplasma gallisepticum.
In the above application, the animal may be poultry. Further, the poultry may be chickens.
The product may be a kit.
The invention also provides a method for detecting whether the serum of an animal to be detected contains mycoplasma gallisepticum antibodies, which is realized by adopting the kit to carry out indirect ELISA and comprises the following steps: using the PVPA protein as an antigen, using the 2.5% skimmed milk powder as a blocking solution, using the 5% BSA as a sample diluent to dilute the serum of an animal to be detected, using the MG positive serum and the MG negative serum as a positive standard and a negative standard respectively, sequentially performing coating and blocking of the antigen, binding with the sample to be detected, binding with the horseradish peroxidase-labeled rabbit anti-chicken IgY, developing, stopping and reading the OD450nm value of a reaction system, obtaining the OD450nm values of the sample to be detected, the positive standard and the negative standard, sequentially recording the values as an S value, a P value and an N value, and determining whether the serum of the animal to be detected contains the mycoplasma gallisepticum antibody according to the following method:
when the P value is more than 1.0 and the N value is less than 0.2, judging that the experiment is established, and calculating S/P (S value-N value)/(P value-N value);
when the S/P is more than or equal to 0.67, the serum of the animal to be detected contains or is candidate to contain mycoplasma gallisepticum antibody; when S/P is less than 0.67, the serum of the animal to be tested does not contain or does not contain the mycoplasma gallisepticum antibody as a candidate.
The invention also provides a method for detecting whether an animal to be detected is infected with mycoplasma gallisepticum by indirect ELISA using the kit of reagents according to any one of claims 1 to 3, the method comprising: using the PVPA protein as an antigen, using the 2.5% skimmed milk powder as a blocking solution, using the 5% BSA as a sample diluent to dilute the serum of an animal to be detected, using the MG positive serum and the MG negative serum as a positive standard and a negative standard respectively, sequentially performing coating and blocking of the antigen, binding with the sample to be detected, binding with the horseradish peroxidase-labeled rabbit anti-chicken IgY, developing, stopping and reading the OD450nm value of a reaction system, obtaining the OD450nm values of the sample to be detected, the positive standard and the negative standard, sequentially recording the values as an S value, a P value and an N value, and determining whether the animal to be detected is infected with the mycoplasma gallisepticum according to the following method:
when the P value is more than 1.0 and the N value is less than 0.2, judging that the experiment is established, and calculating S/P (S value-N value)/(P value-N value);
when the S/P is more than or equal to 0.67, the animal to be detected is infected or candidate infected with mycoplasma gallisepticum; and when the S/P is less than 0.67, the animal to be tested is not infected or candidate is not infected with mycoplasma gallisepticum.
The above method may further comprise the step of treating with the coated stabilizer after blocking.
In the above method, the antigen is coated at a concentration of 1. mu.g/mL. The antigen can be diluted or solubilized using the 0.01M PBS (pH 7.9).
The time for the blocking may be 1 hour.
Both the MG-positive and MG-negative sera can be diluted with the 5% BSA. The MG-positive serum and the MG-negative serum may be diluted 500-fold.
The serum of the test animal can be diluted with the 5% BSA. The dilution factor of the serum of the animal to be tested can be 500 times.
The binding time with the sample to be tested may be 60 minutes.
The rabbit anti-chicken IgY marked by the horseradish peroxidase can be diluted and then added into a reaction system, and the dilution multiple can be 10000 times. The binding time with the horseradish peroxidase-labeled rabbit anti-chicken IgY may be 60 minutes.
The indirect ELISA may specifically comprise:
A. coating of antigen: diluting the PVPA protein by using the 0.01M (Ph7.9) PBS to obtain a protein diluent with the protein concentration of 1 mu g/mL, respectively coating a microporous plate by using the obtained protein diluent, wherein each hole is 100 mu l/hole, sealing the microporous plate by using a sealing tape, and coating for 10-12h at 4 ℃; after coating, throwing off liquid in the holes, adding PBST into each hole, washing for three times, and discarding the liquid in the micropores;
B. and (3) sealing: after the step A is finished, adding 200 mul of 2.5 percent skimmed milk powder into each hole of the microporous plate, sealing the microporous plate by using a sealing paste, and acting for 1h at 37 ℃; after the sealing is finished, throwing off liquid in the holes, adding PBST into each hole for washing three times, and discarding the liquid in the micropores;
C. sample adding: after the step B is finished, diluting the MG positive serum and the MG negative serum respectively by 1:500 by using 5% BSA, adding the diluted serum into a microporous plate, diluting the animal serum to be detected by using the 5% BSA, and adding the diluted animal serum into the microporous plate, wherein each hole is 100 mu l; after the sample adding is finished, sealing the microporous plate by using a sealing paste, shaking for 30s by using a micro-oscillator or manually slightly shaking left and right in parallel for 60s on an experiment operating platform for uniformly mixing, and then placing the microporous plate at 37 ℃ for incubation for 60 min; after incubation, throwing off liquid in the pores, adding PBST into each pore for washing three times, and discarding the liquid in the micropores;
D. adding a secondary antibody: after the step C is finished, diluting the rabbit anti-chicken IgY marked by the horseradish peroxidase by using 0.01M PBS (Ph7.2-7.4) by 1:10000 times, and adding the diluted IgY into a micropore plate, wherein each hole is 100 mu l; after the sample is added, sealing the microporous plate by using a sealing patch, and acting for 60 minutes at 37 ℃; after incubation, throwing off liquid in the pores, adding PBST into each pore for washing three times, and discarding the liquid in the micropores;
E.TMB color development: after the step D is finished, adding a chromogenic substrate into each hole of the microplate, standing for 10min at 37 ℃ in a dark place for color development, and then adding 50 mu L of 2mol/L H into each hole2SO4The reaction was terminated.
F. Detection of OD450nm values: placing the microplate which has terminated the reaction in a reading groove of an enzyme-labeling instrument, and measuring and reading the absorbance value of each hole by using the detection wavelength of 450 nm; and finishing data reading within 10min to obtain an S value, a P value and an N value.
In the above method, the animal may be poultry. Further, the poultry may be chickens.
The reagent set and the kit can detect whether serum of an animal to be detected contains mycoplasma gallisepticum antibodies or not, judge whether the animal to be detected is infected with the mycoplasma gallisepticum or not, have higher coincidence rate, better sensitivity, specificity and stability, better specificity to other viruses and convenient and quick operation compared with similar commercial products, and can carry out quick detection on the mycoplasma gallisepticum antibodies for a large number of samples in the field. The invention can effectively detect the generation of the antibody after MG infection, provides a reliable detection method for identifying MG infection, and also provides scientific guidance for MG epidemic prevention and control.
Drawings
FIG. 1 shows the expression of PVPA recombinant protein detected by SDS-PAGE. Lane M is the protein molecular weight standard, lane 1 is the detection of uninduced control bacteria, and lane 2 is the detection of IPTG-induced bacteria. The right arrow shows the PVPA recombinant protein.
FIG. 2 shows the expression of PVPA recombinant protein in IPTG-induced bacteria detected by SDS-PAGE. Lane M is the protein molecular weight standard, lane 1 is IPTG induced bacterial precipitation, and lane 2 is IPTG induced bacterial supernatant. The long arrow indicates the PVPA recombinant protein.
FIG. 3 shows the purification of PVPA recombinant protein detected by SDS-PAGE. Lane M is a protein molecular weight standard, and lane 1 is a purified recombinant protein.
FIG. 4 shows the Western Blot detection of purified recombinant protein using murine anti-His antibody as the primary antibody.
FIG. 5 shows the results of 262 ROC fits of field sera.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.
The MG strain in the following examples is mycoplasma gallisepticum BG44T strain, which is a product of the institute of veterinary medicine (also called the chinese veterinary microbial strain collection management center) and has a strain number of CVCC 350.
Example 1 detection of Mycoplasma gallisepticum antibodies Using PVPA recombinant proteins
Preparation of PVPA recombinant protein
1.1 Gene preparation
Carrying out PCR amplification on the genomic DNA of the MG strain by using a primer pair consisting of Mqpvpa-F and Mqpvpa-R, wherein the Mqpvpa-F and the Mqpvpa-R are two single-stranded DNAs shown as sequences 3 and 4 in a sequence table respectively, sequencing the obtained PCR product, and displaying that the sequence is sequence 1 in the sequence table, the 9 th to 1133 rd sites of the sequence 1 are the PVPA gene sequence of the MG strain, and encoding the PVPA protein shown as 35 th to 408 th sites of the sequence 2 in the sequence table.
1.2 construction of recombinant vectors
Carrying out enzyme digestion on the PCR product obtained in the step 1.1 by using restriction enzymes BamHI-HF and XhoI to obtain a PCR product enzyme digestion product; carrying out enzyme digestion on the PET-28a (+) vector by using restriction enzymes BamHI-HF and XhoI, recovering a large vector fragment, connecting a PCR product enzyme digestion product with the large vector fragment to obtain a recombinant vector, and naming the recombinant vector with a correct sequence as PET-PVPA. The PET-PVPA is a recombinant vector obtained by replacing a DNA fragment between BamHI and XhoI recognition sequences of a PET-28a (+) vector with a PVPA gene shown in the 9 th to 1133 rd sites of a sequence 1 in a sequence table, the recombinant vector can express a fusion protein shown as a sequence 2 in the sequence table and is marked as PVPA recombinant protein, and the expression of the PVPA recombinant protein is driven by a T7 promoter.
Wherein, the 1 st to 34 th sites of the sequence 2 in the sequence table are the amino acid sequence coded by the DNA sequence on the PET-28a (+) carrier, the 5 th to 10 th sites are His labels, and the 35 th to 408 th sites are PVPA protein sequences.
1.3 PVPA recombinant protein expression, purification and identification
1.3.1. Prokaryotic expression of recombinant protein:
and (3) introducing the recombinant vector PET-PVPA obtained in the step (1.2) into escherichia coli BL21(DE3) to obtain a recombinant bacterium, and marking the recombinant bacterium as BL 21-PET-PVPA.
BL21-PET-PVPA is inoculated into 5mL LB liquid culture, and shake culture is carried out at 220r/min at 37 ℃ for 8-12h to obtain seed liquid; respectively inoculating the seed solution into 4 triangular flasks containing 50ml of new LB liquid culture medium according to the proportion of 1:100, culturing at 37 ℃ for about 3h until OD600nm is approximately equal to 0.5-0.6, adding IPTG into 2 of the triangular flasks until the final concentration of the IPTG is 1mmol/L, taking the rest 2 of the triangular flasks as non-induced control, continuously culturing for 12h in a shaker at 25 ℃ under the oscillation of 200r/min, after the induction culture is finished, centrifuging at 12000rpm for 2min, harvesting thalli, and respectively obtaining IPTG induced thalli and non-induced control thalli, wherein each thalli is repeated twice.
SDS-PAGE identification:
the thalli obtained in the step 1.3.1 are respectively operated according to the following steps:
adding 1mL of PBS buffer solution with pH7.2 into the thallus for resuspension, then carrying out ultrasonic disruption on the thallus to obtain an ultrasonic product, centrifuging the ultrasonic product at 10000r/min for 15min, and collecting supernatant and precipitate. The resulting supernatant was subjected to SDS-PAGE.
The results of IPTG induction of the supernatant of the thallus and non-induction of the supernatant of the control thallus are shown in figure 1, and compared with the control, the protein with the obvious size which is consistent with the expected size and is about 40kDa appears in the induced bacteria sample sediment under the condition of 25 ℃, which indicates that the PVPA recombinant protein is expressed.
The results of IPTG induction of the supernatant and the precipitate of the cells are shown in FIG. 2, and the IPTG-induced PVPA recombinant protein is expressed as inclusion bodies.
1.3.3. And (3) purifying the recombinant protein:
sequentially adding washing solutions I and II into the IPTG induced thallus precipitate obtained in the step 1.3.2, and purifying the washing solutions I and II by His-tagged protein purification resin (QIAGEN company, product number: 30210) according to the operation instruction to obtain purified recombinant protein; the obtained purified recombinant protein was detected by SDS-PAGE, and the results showed that the size of the obtained purified recombinant protein was consistent with that expected and the protein band was single, indicating that the recombinant protein PVPA was purified, as shown in FIG. 3.
Wash I (pH 7.9): 100mM NaCl, 50mM Tris, 10mM EDTA, 0.5% Triton X-100, HCl to pH7.9, and the balance water. Wash II (pH7.9): 2M Urea, 100mM NaCl, 50mM Tris, 10mM EDTA, 0.5% Triton X-100, HCl to pH7.9, balance water.
Western Blot identification:
identifying the purified recombinant protein obtained in the step 1.3.3 by using Western Blot, using MG positive chicken serum AS a first antibody, and using HRP marked rabbit anti-chicken (ABClonal, cat # AS030) AS a second antibody to verify the immunogenicity of the protein; meanwhile, a mouse anti-His antibody (ABClonal, cat # AE003) was used AS the primary antibody, and goat anti-mouse IgG (ABClonal, cat # AS003) was used AS the secondary antibody to verify the correct expression of the protein.
The results show that target bands consistent with the size of the target protein are generated, and the results show that the PVPA recombinant protein is correctly expressed, can be recognized by MG positive chicken serum and has antibody binding activity. The results of the murine anti-His antibody are shown in FIG. 4.
Wherein, the MG positive chicken serum is prepared in step 2.
Preparation of MG-positive chicken serum
1) The method comprises the steps of feeding SPF white leghorn chickens (Beijing Meiliya Viton laboratory animal technology Co., Ltd.) with the age of 4 weeks, collecting blood, separating serum, and detecting by an IDEXX avian mycoplasma septicum antibody detection kit (Beijing Romansia technology Co., Ltd., product number: 99-06729) and determining that no MG infection exists.
2) And (3) taking the chicken which is not subjected to MG infection identification in the step 1), and taking blood to separate serum to obtain standard negative control serum.
3) Taking the chicken which is identified to have no MG infection in the step 1), immunizing by adopting MG bacterium mycoplasma gallisepticum BG44T strain (China veterinary medicine institute (also called China veterinary medicine microbial strain preservation management center), CVCC350), and specifically comprising the following steps:
(1) the concentration is 1x108After being concentrated by 20 times by using CCU/ml MG strain mycoplasma gallisepticum BG44T, the MG strain mycoplasma gallisepticum is mixed with Freund's complete adjuvant according to the volume ratio of 1:1, and then the mixture is injected and immunized (0.4 ml/mouse) through neck subcutaneous injection and leg muscle injection;
(2) two weeks after completion of step (1), the concentration was 1X108After being concentrated by 20 times by using CCU/ml MG strain mycoplasma gallisepticum BG44T, the MG strain mycoplasma gallisepticum is mixed with Freund incomplete adjuvant according to the volume ratio of 1:1, and then the mixture is injected and immunized (0.8 ml/mouse) through neck subcutaneous injection and leg muscle injection;
(3) two weeks after completion of step (2), the concentration was 1X108After being concentrated 66 times by CCU/ml MG strain mycoplasma gallisepticum BG44T, the MG strain mycoplasma gallisepticum is mixed with Freund incomplete adjuvant according to the volume ratio of 1:1, and then the mixture is injected by neck subcutaneous injection and leg muscle injection (0.5 ml/mouse);
(4) two weeks after completion of step (3), the concentration was 1 × 108After being concentrated by 30 times by using CCU/ml MG strain mycoplasma gallisepticum BG44T, the vaccine is injected subcutaneously and intramuscularly in the neck and the legs (0.5 ml/mouse);
(5) and (3) after 10 days of the step (4), collecting blood by a fin root vein, separating serum, detecting that S/P (S/P ═ S value-N value)/(P value-N value)) reaches more than 2.0 by using a mycoplasma gallisepticum antibody detection kit of IDEXX company, so that blood can be collected, and preparing MG positive chicken serum after blood collection.
3. Establishment of indirect ELISA method for detecting MG antibody
3.1 selection of PVPA antigen coating concentration and optimal dilution factor of serum
The coating concentration and the optimal dilution multiple of serum of the PVPA recombinant protein are optimized by a chessboard method, and the steps are as follows:
3.1.1 coating of antigen: the purified PVPA recombinant protein was diluted with 0.01M PBS (pH7.9, product of Beijing Solebao technologies, Ltd., P1010) to obtain protein dilutions having protein concentrations of 0.125. mu.g/mL, 0.25. mu.g/mL, 0.5. mu.g/mL, 1.0. mu.g/mL and 2.0. mu.g/mL, respectively, and the microplate was coated with each protein dilution at 100. mu.l/well for 10-12h at 4 ℃. After coating, the wells were drained and 300. mu.l PBST was added to each well and washed three times.
PBST: adding Tween-20 into 0.01M PBS (ph7.2-7.4) (Beijing Sorleibao science and technology Co., Ltd., product number: P1020), wherein the concentration of Tween-20 is 0.05% (volume percentage content).
3.1.2 blocking: after washing, 200. mu.l of blocking solution (5% SBA, obtained by dissolving BSA (bovine serum Albumin, VWR Co.) in 0.01M PBS at a concentration of 5g/100mL) was added to each well of the plate, the plate was allowed to react at 37 ℃ for 2 hours, the liquid in the wells was discarded, and PBST was washed 3 times.
3.1.3 sample application: and (3) diluting the MG positive chicken serum and the standard negative control serum in the step (2) by 1:125, 1:250, 1:500, 1:1000 and 1:2000 times respectively by using sample diluent, adding the diluted samples into a microplate, wherein each well is 100 mu l, and the corresponding serum dilution of each protein diluent is set to be three times. After the sample application was completed, the plate was sealed with a sealing patch and then incubated at 37 ℃ for 60min (i.e., serum exposure time). After incubation, the wells were discarded and the PBST washed three times.
Wherein, the sample diluent: 0.01M PBS (ph7.2-7.4) (Beijing Soilebao Tech., Ltd., cat # P1020).
3.1.4 Add Secondary antibody: a secondary antibody (horseradish peroxidase-labeled rabbit anti-chicken IgY (ABClonal, cat # AS 030)) is diluted by 0.01M PBS (ph7.2-7.4) by 1:10000 times and then added into a washed microplate, the reaction is carried out for 60 minutes at 37 ℃, liquid in a hole is removed after the incubation is finished, and PBST is washed for three times.
3.1.5TMB color development: adding 100 μ L substrate reaction solution (ready to use, light shielding) into each well, standing at 37 deg.C for 10min, taking out, and adding 50 μ L2 mol/L H into each well2SO4The reaction was terminated.
Wherein, the substrate reaction solution: i.e., TMB substrate: beijing Solaibao Tech Co., Ltd., Cat #: PR 1210. When in use, the solution A and the solution B are sucked and mixed uniformly at a ratio of 1: 1.
3.1.6 detection of OD450nm values: the OD450nm value of each well was measured. P/N values were calculated for each group, i.e. the ratio of MG positive chicken serum well OD450nm values (P values) to standard negative control serum well OD450nm values (N values).
The P value is about 1.0, the P/N value is the maximum and the N value is the lower, and the P/N value is used as the optimal antigen coating concentration and the optimal dilution factor of serum. The results showed that the OD450nm value of MG-positive serum well diluted 1:500 times was 0.85 at 1. mu.g/ml, the OD450nm value of standard negative control serum well diluted 1:500 times was 0.1 at the maximum P/N value, which was 8.30. This condition is therefore the optimum condition. The results are shown in Table 1.
TABLE 1 determination of Indirect ELISA antigen coating concentration and serum dilution factor
3.2 optimized sealing liquid
According to the steps 3.1.1-3.1.6, the blocking solution is replaced by 5% BSA, 2.5% BSA, 5% horse serum, 10% horse serum, 5% pig serum, 10% pig serum, 5% skimmed milk powder and 2.5% skimmed milk powder respectively, the influence of each blocking solution on the result is detected under the optimal conditions, the optimal blocking solution is selected, and other steps are not changed.
5% BSA: BSA was dissolved in 0.01M (ph7.2-7.4) PBS at a concentration of 5g/100 mL.
2.5% BSA: BSA was dissolved in 0.01M (ph7.2-7.4) PBS at a concentration of 2.5g/100 mL.
5% horse serum: 5ml of horse serum (Beijing Soilebao Tech Co., Ltd.) was added to 95ml of 0.01M PBS.
10% horse serum: 10ml of horse serum was added to 90ml of 0.01M PBS.
5% pig serum: 5ml of pig serum (Biotopped Life sciences) was added to 95ml of 0.01 MPBS.
10% pig serum: 10ml of pig serum was added to 90ml of 0.01M PBS.
5% of skimmed milk powder: it was obtained by dissolving skimmed milk powder (BD Co., USA, Cat. No. 232100) in 0.01M (ph7.2-7.4) PBS at a concentration of 5g/100 mL.
2.5% skimmed milk powder: is prepared by dissolving skimmed milk powder in 0.01M (ph7.2-7.4) PBS (2.5 g/100 mL).
The results show that the P/N value is maximal at 20.79 when blocked with 2.5% skim milk powder, and the N value is lower. Therefore 2.5% skimmed milk powder was used as the optimal blocking liquid. The results are shown in Table 2.
TABLE 2 determination of indirect ELISA blocking solutions
3.3 optimizing the seal time
And (3) according to the steps 3.1.1-3.1.6, replacing the sealing time from 2h to 1h, 2h and 3h respectively, detecting the influence of each sealing time on the result under the optimal condition, and keeping other steps unchanged.
The results show that the P/N values do not differ much after 1 and 2 hours of blocking at 37 ℃ and that 1h is chosen as the optimum blocking time, taking time saving into account. The results are shown in Table 3.
TABLE 3 determination of blocking time by indirect ELISA
3.4 optimization of Primary antibody Diluent
The sample dilutions were replaced with 0.01M (ph7.2-7.4) PBS, 5% BSA, 2.5% BSA, 5% horse serum, 2.5% horse serum, 5% rabbit serum, 2.5% skim milk powder, respectively, according to the above steps 3.1.1-3.1.6, and the effect of each sample dilution on the results was examined under the optimal conditions described above, with no change in the other steps.
2.5% horse serum: 2.5ml of horse serum was added to 97.5ml of 0.01M (ph7.2-7.4) PBS.
5% rabbit serum: 5ml of rabbit serum (Beijing Soilebao Tech Co., Ltd.) was added to 95ml of 0.01M
(ph7.2-7.4) in PBS.
2.5% rabbit serum: 2.5ml of rabbit serum was added to 97.5ml of 0.01M (ph7.2-7.4) PBS.
The results show that when 5% BSA was used as the sample diluent, the N value was low and the P/N value was maximal at 17.83. Thus 5% BSA is the optimal sample dilution. The results are shown in Table 4.
TABLE 4 determination of the Indirect ELISA-anti dilution
3.5 optimization of serum action time
According to the steps 3.1.1-3.1.6, the serum action time is respectively replaced by 30min, 60min and 90min from 60min, the influence of each serum action time on the result is detected under the optimal condition, and other steps are not changed.
The results show that the P/N value is maximal at 15.82 when the serum exposure time is 60 minutes, so 60min was selected as the optimal serum exposure time. The results are shown in Table 5.
TABLE 5 determination of the serum action time by indirect ELISA
3.6 optimization of the dilution of the Secondary antibody
The effect of each secondary antibody dilution on the results was examined under the optimal conditions described above, with all other steps unchanged, following the above steps 3.1.1-3.1.6, replacing the secondary antibody dilution from 1:10000 to 1:5000, 1:10000, 1:20000, 1:40000, respectively.
The results showed that the P/N value was maximal at 11.42 when the secondary antibody dilution was 1: 10000. The optimal secondary antibody dilution was chosen to be 1: 10000. The results are shown in Table 6.
TABLE 6 determination of dilution of enzyme-labeled secondary antibody for indirect ELISA
3.7 optimization of the second antibody action time
According to the steps 3.1.1-3.1.6, the action time of the secondary antibody is respectively replaced by 30min, 60min and 90min from 60min, the influence of the action time of each secondary antibody on the result is detected under the optimal condition, and other steps are not changed.
The result shows that when the second antibody action time is 60min, the P/N value is maximum, and 60min is selected as the optimal second antibody action time. The results are shown in Table 7.
TABLE 7 determination of the duration of the action of the enzyme-labeled secondary antibody in the indirect ELISA
3.8 determination of Indirect ELISA method for detecting MG antibodies
According to the optimization of the above conditions, the indirect ELISA method for detecting MG antibody is obtained by the following steps:
A. coating of antigen: diluting the purified PVPA recombinant protein by using 0.01M (ph7.9) PBS to obtain a protein diluent with the protein concentration of 1 mu g/mL, respectively coating a micropore plate by using the obtained protein diluent, sealing the micropore plate by using a seal, and coating for 10-12h at 4 ℃. After coating, the liquid in the wells was spun off, 300. mu.l PBST was added to each well, washed three times, and the liquid in the wells was discarded.
B. And (3) sealing: and (C) after the step A is finished, adding 200 mul of 2.5% skimmed milk powder into each hole of the microporous plate, sealing the microporous plate by using a sealing paste, and acting for 1h at 37 ℃. After the sealing is finished, the liquid in the holes is thrown off, 300 mul PBST is added into each hole for washing three times, and the liquid in the micropores is discarded.
C. Sample adding: after the step B is finished, MG positive chicken serum (positive standard substance) and standard negative control serum (negative standard substance) in the step 2 are diluted by 1:500 respectively by using 5% BSA, and then added into a microplate, and each well is 100 mul. Then, the serum to be tested was diluted with 5% BSA and added to a microplate in an amount of 100. mu.l per well. After the sample application was completed, the microplate was sealed with a sealing patch and then incubated at 37 ℃ for 60 min. After incubation, the wells were drained, and 300. mu.l PBST was added to each well and washed three times, and the wells were drained.
D. Adding a secondary antibody: after the step C is completed, a secondary antibody (rabbit anti-chicken IgY marked by horseradish peroxidase) is diluted by 0.01MPBS (ph7.2-7.4) by 1:10000 times and then added into the micropore plate, and each hole is 100 mul. Sealing the microporous plate with a sealing tape, and acting at 37 ℃ for 60 minutes. After incubation, the liquid in the wells was spun off, 300. mu.l PBST was added to each well and washed three times, and the liquid in the wells was discarded.
E.TMB color development: after the step D is finished, adding 100 mul of substrate reaction solution (which is used as prepared) into each hole of the microporous plate, standing for 10min at 37 ℃ in the dark for color development, and then adding 50 mul of 2mol/L H into each hole2SO4The reaction was terminated.
F. Detection of OD450nm values: placing the microplate which has terminated the reaction in a reading groove of an enzyme-labeling instrument, and measuring and reading the absorbance value of each hole by using the detection wavelength of 450 nm; data reading is completed within 10min, a serum well OD450nm value (S value) of a serum well to be tested, an MG positive chicken serum well OD450nm value (P value) and a standard negative control serum well OD450nm value (N value) are obtained, and S/P is calculated and is (S value-N value)/(P value-N value).
3.9 determination of Positive and negative cutoff values
262 parts of chicken serum (from the animal epidemic disease prevention and control center in Beijing) is used as serum to be detected, and detection is respectively carried out by using a Mycoplasma gallisepticum antibody detection kit of IDEXX company and an indirect ELISA method for detecting MG antibody in the step 3.8, so as to obtain an OD450nm value (S value) of a serum hole to be detected, an OD450nm value (P value) of an MG positive chicken serum hole and an OD450nm value (N value) of a standard negative control serum hole, and S/P is calculated and is (S value-N value)/(P value-N value).
The results were input to Medcalc software to obtain the ROC curve (FIG. 5).
The results showed that sensitivity and specificity were both better when S/P was 0.67, at which time sensitivity was 91.6, specificity was 94.7, and area under the curve (AUC) was 0.971. Therefore, S/P is 0.67 and is determined as a critical value. The results are shown in FIG. 5.
Specifically, the standard for determining whether the serum to be detected is positive serum is as follows:
when the positive standard OD450nm (namely P value) is more than 1.0 and the negative standard OD450nm (namely N value) is less than 0.2, the test is judged to be established, and S/P is calculated; when the S/P is more than or equal to 0.67, the serum to be detected is judged to be positive serum (containing MG antibody), namely MG infection serum; when S/P is less than 0.67, the serum to be tested is judged to be negative serum (without MG antibody), namely the serum not infected by MG.
3.10 specificity test
And (3) taking the IBV positive serum, the IBDV positive serum, the NDV positive serum, the MS positive serum, the avian influenza H5, H7 and H9 subtype positive serum as the serum to be detected, taking the MG positive chicken serum and the standard negative control serum in the step (2) as controls, respectively detecting by using the indirect ELISA method for detecting the MG antibody in the step (3.8), judging whether the serum to be detected is the positive serum by using the judgment standard of 3.9, and checking the specificity of the indirect ELISA method for detecting the MG antibody, which is established by the invention.
The IBV positive serum, the IBDV positive serum, the NDV positive serum and the MS positive serum are all products of Chinese veterinary medicine inspection, and are not MG infection serum.
The avian influenza H5, H7 and H9 subtype positive serum is a product of Harbin national biotechnology GmbH, and is not MG infection serum.
The results (table 8) show that the positive serum of IBV, IBDV, NDV, MS, avian influenza H5, H7 and H9 subtypes and the negative serum are detected as negative serum, and the positive serum is detected as positive serum, which indicates that the method of the invention has good specificity.
TABLE 8 specificity test
3.11 sensitivity test
HI (hemagglutination inhibition titer) prepared according to the method of step 2 was 1: and (3) diluting the 128 MG-positive chicken serum in an equal-fold ratio of 1:1, 1:2, 1:4, 1:8 and 1:16, respectively detecting by using the indirect ELISA method for detecting the MG antibody in the step 3.8, and checking the sensitivity of the indirect ELISA method for detecting the MG antibody established by the invention, wherein positive control and negative control are the MG-positive chicken serum in the step 2 and standard negative control serum. As can be seen from the data in Table 9, the lowest limit that can be detected by the method is 1: 16.
TABLE 9 results of sensitivity test
3.12 compliance test
301 parts of chicken serum (from the animal epidemic prevention and control center in Beijing) is used as serum to be detected, and is respectively detected by using a Mycoplasma gallisepticum antibody detection kit of IDEXX company and the indirect ELISA method for detecting MG antibody, and the results of the two methods are compared and analyzed, and are shown in Table 10.
The results show that the total compliance of both methods is 88.04%.
TABLE 10 test results of 301 clinical specimens with the kit and the IDEXX kit
3.13 stability test
3.13.1. Preparation of coating stabilizer
The coated stabilizer was prepared by preparing a solution of shmpkt according to the recipe of table 11, then weighing a certain amount of sucrose and PEG4000, adding them, stirring well to make the concentration of sucrose 3g/100mL and PEG4000 0.1g/100mL, and finally adding a certain amount of Proclin300 to make the concentration (v/v) 0.05%. Wherein, Proclin300 is a product of Beijing Sorley Tech technologies GmbH, and the product number is P6840.
TABLE 11 SHMCKT solution formulation
3.13.2. Preparation of antigen coated plates
The purified PVPA recombinant protein solution obtained above is diluted to 1 mu g/ml by 0.01M (ph7.9) PBS, coated on an enzyme label plate, each well is L00 mu L, and the antigen coating solution is discarded after the solution is coated for 10-12h at 4 ℃. Washing was repeated 3 times with washing solution (PBST). 200. mu.L of 2.5% skimmed milk powder was added to each well and blocked at 37 ℃ for 1 hour. The blocking solution was then discarded and the washing was repeated 3 times with washing solution (PBST). Adding 200 μ L of the coating stabilizer obtained in step 3.13.1 into each well, incubating at 37 deg.C for 2 hr, removing liquid from the wells, beating to dry, placing the coated plate in a clean bench, drying at room temperature for 4 hr, packaging the antigen coated plate in aluminum foil bags, simultaneously placing a bag of desiccant, bagging, and sealing.
Destructive test at 3.13.337 deg.C
The antigen-coated plate prepared in step 3.13.2 was left at 37 ℃ for 0 day, 1 day, 3 days and 7 days, respectively, and the effect of the coating stabilizer was examined according to C-F of step 3.8. The serum used is 1-4, wherein, the serum 1-3 is MG positive chicken serum obtained by different individuals according to the method of step 2, and the serum 4 is standard negative control serum of step 2. Antigen-coated plates not treated with the coating stabilizer were used as controls.
TABLE 1137 ℃ destructive testing (OD450nm values)
As can be seen from the data in the table, the 7-day test results of the antigen-coated plate did not fluctuate much at 37 ℃, indicating that the antigen-coated plate could be stably stored at 37 ℃ for at least 7 days. Whereas antigen-coated plates without coating stabilizer can only be stored for 0 day.
Comparing chinese patent application 201910226908.7 with chinese patent 201110094758.2:
the Chinese patent application 201910226908.7 requires the use of aptamer, and the synthesis cost of aptamer is high. In addition, the method has more operation steps and long reaction time.
The stability test of the kit is not mentioned in the Chinese patent 201110094758.2. The formulation of the coating stabilizer is supplemented, and destructive test data at 37 ℃ are provided, so that the coating stabilizer can be stably stored for at least 7 days at 37 ℃.
Therefore, in a comprehensive view, the method has the advantages of low cost, simple reaction steps and good stability.
<110> university of agriculture in China
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Claims (10)
1. A kit comprising a PVPA protein and P1, said PVPA protein being a1), a2), or A3):
A1) the amino acid sequence is the protein at 35 th-408 th position of the sequence 2;
A2) the amino acid sequence is the protein of sequence 2;
A3) the protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the 35 th to 408 th sites of the sequence 2 or the sequence 2 in the sequence table;
A4) a fusion protein obtained by connecting labels at the N terminal or/and the C terminal of A1) or A2) or A3);
the P1 is 2.5% skimmed milk powder and/or 5% BSA and/or coating stabilizer;
the 2.5% skimmed milk powder consists of 0.01M PBS (PH7.2-7.4) and skimmed milk powder, and the concentration of the skimmed milk powder in the 2.5% skimmed milk powder is 2.5g/100 mL;
the 5% BSA consisted of 0.01M PBS (pH7.2-7.4) and bovine serum albumin at a concentration of 5g/100mL in the 5% BSA;
the coating stabilizer consists of a solvent and a solute, wherein the solvent is water, and the solute and the concentration of the solute in the coating stabilizer are respectively HEPES 20mM, NaCl 120mM, KCl 5mM and CaCl21mM、MgCl21mM, Tween-200.05% (volume percent), sucrose 3g/100mL, PEG40000.1g/100 mL, and Proclin 3000.05% (volume percent).
2. The kit of claim 1, wherein: the kit further comprises MG-positive serum and/or MG-negative serum.
3. The kit of claim 1 or 2, wherein: the kit also comprises 0.01M PBS (PH7.2-7.4), rabbit anti-chicken IgY marked by horseradish peroxidase, a micropore plate, a washing solution, a chromogenic substrate and/or a stop solution.
4. Use of any one of the following kits of claims 1-3:
(c1) detecting whether the serum of the animal to be detected contains mycoplasma gallisepticum antibodies;
(c2) preparing a kit for detecting whether the serum of an animal to be detected contains mycoplasma gallisepticum antibodies;
(c3) detecting the content of mycoplasma gallisepticum antibodies in the serum of an animal to be detected;
(c4) preparing a kit for detecting the content of mycoplasma gallisepticum antibodies in the serum of an animal to be detected;
(c5) judging whether the animal to be detected is infected with mycoplasma gallisepticum;
(c6) preparing a kit for determining whether the animal to be detected is infected with mycoplasma gallisepticum.
5. Use according to claim 4, characterized in that: the animal is poultry;
further, the poultry is chicken.
6. An enzyme linked immunoassay kit comprising the kit of any one of claims 1 to 3.
7. The kit of claim 6 for any one of the following uses:
(c1) detecting whether the serum of the animal to be detected contains mycoplasma gallisepticum antibodies;
(c2) preparing a product for detecting whether the serum of an animal to be detected contains mycoplasma gallisepticum antibodies;
(c3) detecting the content of mycoplasma gallisepticum antibodies in the serum of an animal to be detected;
(c4) preparing a product for detecting the content of mycoplasma gallisepticum antibodies in the serum of an animal to be detected;
(c5) judging whether the animal to be detected is infected with mycoplasma gallisepticum;
(c6) preparing a product for determining whether the animal to be tested is infected with mycoplasma gallisepticum.
8. Use according to claim 7, characterized in that: the animal is poultry;
further, the poultry is chicken.
9. A method for detecting whether serum of an animal to be tested contains mycoplasma gallisepticum antibodies by indirect ELISA using a kit according to any one of claims 1 to 3, comprising: using the PVPA protein of claim 1 as an antigen, using 2.5% skimmed milk powder of claim 1 as a blocking solution, using 5% BSA of claim 1 as a sample diluent to dilute the serum of an animal to be tested, using the MG-positive serum and the MG-negative serum of claim 2 as a positive standard and a negative standard, respectively, sequentially performing coating, blocking, binding to a sample to be tested, binding to horseradish peroxidase-labeled rabbit anti-chicken IgY, developing, terminating, and reading the OD450nm value of the reaction system to obtain the OD450nm values of the sample to be tested, the positive standard, and the negative standard, sequentially recording the values as S value, P value, and N value, and determining whether the serum of the animal to be tested contains mycoplasma gallisepticum antibody according to the following method:
when the P value is more than 1.0 and the N value is less than 0.2, judging that the experiment is established, and calculating S/P (S value-N value)/(P value-N value);
when the S/P is more than or equal to 0.67, the serum of the animal to be detected contains or is candidate to contain mycoplasma gallisepticum antibody; when S/P is less than 0.67, the serum of the animal to be tested does not contain or does not contain the mycoplasma gallisepticum antibody as a candidate.
10. A method for detecting whether a test animal is infected with mycoplasma gallisepticum by indirect ELISA with a kit of reagents according to any one of claims 1-3, comprising: using the PVPA protein of claim 1 as an antigen, using 2.5% skimmed milk powder of claim 1 as a blocking solution, using 5% BSA of claim 1 as a sample diluent to dilute the serum of an animal to be tested, using the MG-positive serum and the MG-negative serum of claim 2 as a positive standard and a negative standard, respectively, sequentially performing coating, blocking, binding to a sample to be tested, binding to horseradish peroxidase-labeled rabbit anti-chicken IgY, developing, stopping and reading the OD450nm value of a reaction system to obtain the OD450nm values of the sample to be tested, the positive standard and the negative standard, sequentially recording the values as an S value, a P value and an N value, and determining whether the animal to be tested is infected with mycoplasma gallisepticum according to the following method:
when the P value is more than 1.0 and the N value is less than 0.2, judging that the experiment is established, and calculating S/P (S value-N value)/(P value-N value);
when the S/P is more than or equal to 0.67, the animal to be detected is infected or candidate infected with mycoplasma gallisepticum; and when the S/P is less than 0.67, the animal to be tested is not infected or candidate is not infected with mycoplasma gallisepticum.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010419178.5A CN111537736A (en) | 2020-05-18 | 2020-05-18 | Indirect ELISA (enzyme-linked immunosorbent assay) detection kit and detection method for mycoplasma gallisepticum antibody |
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| CN117491657A (en) * | 2023-11-03 | 2024-02-02 | 中国兽医药品监察所 | Mycoplasma gallisepticum positive serum and negative serum standard substance and preparation method thereof |
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