CN113325176A - Double-antibody sandwich direct ELISA (enzyme-Linked immuno sorbent assay) method for detecting avian leukosis P27 - Google Patents
Double-antibody sandwich direct ELISA (enzyme-Linked immuno sorbent assay) method for detecting avian leukosis P27 Download PDFInfo
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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Abstract
The invention discloses a double-antibody sandwich direct ELISA method for detecting avian leukosis P27, which comprises the following steps: 1) designing a pair of primers by using an avian leukosis virus RSA strain p27 gene sequence to amplify a p27 gene, constructing a recombinant expression vector, and obtaining the supernatant expression of a recombinant expression protein through induced expression, wherein the supernatant expression is used as an immunogen after being purified to prepare a rabbit polyclonal antibody; 2) washing the ELISA plate coated with the rabbit polyclonal antibody, sealing for 1h at 37 ℃, and washing the plate; 3) adding the serum to be detected for incubation, and washing the plate; 4) adding enzyme-labeled antibody for incubation, and washing the plate; 5) adding TMB color developing liquid for color development, and finallyAdding a chromogenic termination solution to terminate the reaction; 6) measuring the OD of the liquid in the plate hole of the enzyme label in the step 5) by using an enzyme label instrument450And (5) determining the negative and positive according to the threshold value by the nm value. The rabbit polyclonal antibody prepared by the ALV p27 protein has the advantages of short preparation period, low cost, good specificity and repeatability, high sensitivity, good reliability and the like.
Description
Technical Field
The invention relates to a double-antibody sandwich direct ELISA method for detecting avian leukosis P27, belonging to the technical field of avian leukosis detection.
Background
Avian Leukemia (AL), one of the neoplastic diseases of chickens caused by Avian Leukemia Virus (ALV), has been common to poultry as a result of the fact that rooff reported in 1968 that one example of chicken 'lymphosarcoma' occurs in europe, causing great economic loss to the poultry industry. The p27 protein is a group-specific antigen of avian leukosis virus encoded by the gag gene, is highly conserved, is a major component of the ALV core capsid protein, and has many viral antigenic sites that are easily detected. Homology between the various subgroups of exogenous ALV (A, B, C, D, J) is up to 90%. The p27 protein content is high, which accounts for more than 30% of the total virus protein component, and is the first choice antigen for preparing detection antibody.
Disclosure of Invention
Based on the above, the invention provides the double-antibody sandwich direct ELISA method for detecting the avian leukosis P27, and the method has the advantages of good specificity and repeatability, high sensitivity and good reliability.
The technical scheme of the invention is as follows: a double-antibody sandwich direct ELISA method for detecting avian leukosis P27 comprises the following steps:
1) designing a pair of primers by using an avian leukosis virus RSA strain p27 gene sequence to amplify a p27 gene, constructing a recombinant expression vector, and obtaining the supernatant expression of a recombinant expression protein through induced expression, wherein the supernatant expression is used as an immunogen after being purified to prepare a rabbit polyclonal antibody;
2) washing the ELISA plate coated with the rabbit polyclonal antibody, sealing for 1h at 37 ℃, and washing the plate;
3) adding the serum to be detected for incubation, and washing the plate;
4) adding enzyme-labeled antibody for incubation, and washing the plate;
5) adding TMB color development liquid for color development, and finally adding color development termination liquid to terminate the reaction;
6) measuring the OD of the liquid in the plate hole of the enzyme label in the step 5) by using an enzyme label instrument450And (5) determining the negative and positive according to the threshold value by the nm value.
Optionally, the avian leukemia virus is the viral RSA strain.
Optionally, the primer sequences of the pair of primers are as follows:
p27-EcoR I-F：CGGAATTCATGCCTGTAGTGATTAAGACAG；
p27-Xba I-R：GCTCTAGACCTAGGGCTGGATAGCAGAC。
optionally, the concentration of the coated rabbit polyclonal antibody is 4 μ g/mL, the coating condition is 4 ℃ overnight after 2h at 37 ℃, and the blocking condition is 2h at 37 ℃.
Optionally, the reaction condition of the serum to be detected is that the serum acts for 0.5h at 37 ℃.
Optionally, the dilution multiple of the enzyme-labeled antibody is 1:10000, and the reaction time is 1h at 37 ℃.
Optionally, the conditions shown are 37 ℃ for 15min.
Optionally, the criterion for determining positivity and negativity of the critical value is as follows: when OD is reached450nm greater than 0.222 is positive and less than 0.222 is negative.
The invention has the beneficial effects that: the double-antibody sandwich direct ELISA method for detecting the avian leukosis P27, which is established by the invention, adopts the ALV P27 protein to prepare the rabbit-derived polyclonal antibody, has the advantages of short preparation period, low cost, good specificity and repeatability, high sensitivity, good reliability and the like, can detect the avian leukosis group specific antigen, does not react with common avian viruses, and lays a foundation for diagnosis, prevention and control of the avian leukosis.
Drawings
FIG. 1 shows the PCR amplification of cell supernatant (A), the PCR identification of plasmid bacterial liquid (B) and the double restriction enzyme identification (C), wherein A: 1,2: cell pellet, 3: negative control; b: 1,2,3,4: plasmid bacterial liquid, 5: negative control; c: 1,2: recombinant plasmids;
figure 2 SDS-PAGE identification of recombinant p27 protein, M: prestained protein molecular weight standards; 1: supernatant of the bacterial liquid; 2: precipitating thalli;
fig. 3ALV p27 protein purification results, M: prestained protein molecular weight standards; 1-5: 50mmol/L imidazole-eluted heteroproteins; 6-7: purified protein eluted with 400mmol/L imidazole; 8-9: protein eluted by 500mmol/L imidazole;
fig. 4 his mab validation of p27 recombinant protein, M: prestained protein molecular weight standards; 1 and 2 are respectively: purified, unpurified ALV p27 protein; 3: pCold I;
FIG. 5 Western blot validation of rabbit anti-ALV p27 positive sera, M: prestained protein molecular weight standards; 1: ALV p27 protein; 2: blank control;
fig. 6 rabbit anti-p 27 polyclonal antibody purification results, M: prestained protein molecular weight standards; 1: rabbit anti-p 27 polyclonal antibody purification results;
FIG. 7 Western blot validation of purified rabbit anti-ALV p27 polyclonal antibody, M: prestained protein molecular weight standards; 1: ALV p27 protein; 2: blank control;
FIG. 8 Western blot validation of HRP-labeled rabbit anti-ALV p27 polyclonal antibody, M: prestained protein molecular weight standards; 1: ALV p27 protein; 2: blank control.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
1. Method of producing a composite material
1.1 design of primers for P27 Gene
According to the P27 gene sequence of ALV RSA (GenBank accession number: M37980) strain in GenBank, a pair of primers for specifically amplifying the P27 gene are designed, and the primer sequence is as follows:
p27-EcoR I-F：CGGAATTCATGCCTGTAGTGATTAAGACAG；
p27-Xba I-R：GCTCTAGACCTAGGGCTGGATAGCAGAC；
the upstream and downstream primers are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, the target gene amplification fragment is 757bp, and the underlined parts are EcoR I enzyme cutting sites and Xba I enzyme cutting sites respectively.
1.2 construction of prokaryotic expression vector pCold I-ALV-A-P27
Taking DF-1 cell sediment inoculated with RSA strain virus to extract virus RNA, carrying out reverse transcription by taking the virus RNA as a template to obtain cDNA to carry out PCR amplification of ALV-A P27 gene, carrying out gel electrophoresis identification on the PCR product, purifying and recovering the PCR product, carrying out double enzyme digestion on the recovered product and a prokaryotic expression vector pCold I by using EcoR I and Xba I respectively, connecting for 4h at 16 ℃ (ligase Solution I), then transforming the product into a competent cell of Escherichia coli DH5 alpha, coating the competent cell on an LB plate containing ampicillin (Amp), culturing for 16-24 h at 37 ℃, selecting single colony shake bacteria for 12-14 h, identifying the cells as positive by bacterial liquid PCR, extracting plasmids to carry out double enzyme digestion verification, and naming the plasmids verified as positive as pCold I-ALV-A-P27.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 deg.C for 1 min; annealing at 56 ℃ for 30 s; extension for 2min at 72 ℃ for 30 cycles; finally, the extension is carried out for 10min at 72 ℃.
The ALV-inoculated cell pellet is amplified by the designed specific primer, and the product is detected by electrophoresis to amplify a band with the size of about 750bp (figure 1-A). After purification, ligation and transformation of the product, a band of the expected size was also present as identified by PCR in the bacterial solution (FIG. 1-B). Extracting the plasmid of the bacterial liquid with positive identification result, carrying out double enzyme digestion identification on the recombinant plasmid, and obtaining two specific bands (figure 1-C) of about 750bp and 4700bp, and naming the recombinant plasmid as pCold I-ALV-A-P27.
1.3 Induction expression of recombinant proteins
BL21 competent cells were transformed with the recombinant plasmid pCold I-ALV-A-P27, plated with Amp resistant plates, and single clones were picked up at 37 ℃ overnight and then treated as follows: 100 to 5mL of fresh LB liquid medium (containing Amp 50. mu.g/mL),shaking the strain to OD at 37 ℃600Adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mmol/L (0.6-1.0), then shaking the bacteria at 18 ℃ for 18-24 h, taking 8000r/min of bacteria liquid, centrifuging for 10min, removing supernatant, and adding PBS (phosphate buffer solution) for heavy suspension and precipitation. And (3) placing the bacterial liquid on ice for ultrasonic crushing for 20min, and fully cracking. Then, the mixture is centrifuged at 10000r/min for 15min, and supernatant and sediment are collected respectively.
The supernatant and the precipitate after ultrasonic lysis and centrifugation were subjected to SDS-PAGE analysis, and the results are shown in FIG. 2, wherein the supernatant and the precipitate have a specific protein band at 26kD, which is consistent with the expected results. The recombinant protein was expressed in both supernatant and pellet, but was expressed in inclusion body form. Many foreign proteins, when expressed at high levels, tend to exist as insoluble, biologically inactive inclusion bodies. Inclusion bodies are insoluble materials formed by the undesired interaction of folded intermediate molecules as nascent peptide chains are formed, causing the intermediates to clump together. The P27 protein is expressed in both supernatant and precipitate after ultrasonic disruption, but is expressed in the form of inclusion bodies, which is contrary to the research result of the prior literature, and the reason for this is probably because the induction temperature is lower and the cold shock expression vector pCold I is used in the invention. The growth of the thalli is slow under the low-temperature condition, the protein synthesis rate is correspondingly slowed, so that the newly synthesized protein has sufficient time for folding, the degradation rate of the recombinant protein is reduced at the lower culture temperature, and the stability of the recombinant protein is improved. However, too low a culture temperature may result in substantial cessation of expression or minimal expression of the recombinant protein. The cold shock expression vector pCold I-IV is a high-efficiency protein expression vector designed by utilizing a cold shock gene cspA promoter. CspA is a cold shock protein, which is hardly expressed at a culture temperature of 37 ℃ and can efficiently regulate the expression of recombinant proteins when cultured at a low temperature.
1.4 purification of ALV p27 protein
Washing and purifying by using imidazole PBS, and specifically comprising the following steps:
(1) 3mL of nickel column is loaded into a chromatographic column, and 5mL of 50mM PBS is used for balancing the nickel column;
(2) adding the ultrasonically cracked sample into a chromatographic column, and repeatedly loading the sample for 5 times;
(3) washing the nickel column with 5mL of 50mM imidazole PBS (8 mol urea is required to be added to the imidazole PBS if the precipitation is expressed, the same is applied below), and collecting 1mL of the nickel column with 1.5mL centrifuge tubes per tube to collect 5 tubes;
(4) washing the nickel column with 5mL of 400mM imidazole PBS, and collecting 2.5mL of each tube with 4mL centrifuge tubes and 2 tubes;
(5) washing the nickel column with 5mL of 500mM imidazole PBS, and collecting 2.5mL of each tube with 4mL centrifuge tubes and 2 tubes;
(6) the washes were verified by SDS-PAGE.
After the expression form of ALV p27 protein was identified, the supernatant of the bacterial suspension was selected and purified, and the results after purification are shown in FIG. 3, which shows that ALV p27 protein has the largest elution amount and a single band at 400mmol/L imidazole concentration.
1.5 Western blot identification of ALV p27 protein
After SDS-PAGE electrophoresis of ALV p27, proteins were transferred to PVDF membrane using a protein wet-transfer machine as follows:
(1) after SDS-PAGE electrophoresis is finished, removing redundant glue, shearing a PVDF membrane and filter paper with the same size as the glue, activating the PVDF membrane in methanol for 1min, and then putting the activated PVDF membrane into a membrane transfer buffer solution for later use. Rotating the membrane for 2h at a constant voltage of 30v by an electric rotating instrument according to the sequence of the negative electrode, the cushion, the gel, the PVDF membrane, the cushion and the positive electrode;
(2) after the electrotransformation is finished, putting the membrane into TBST containing 5% skimmed milk powder, and sealing for 1h at the constant temperature of 37 ℃;
(3) removing skimmed milk powder, washing membrane with TBST for 3 times, each for 5-10 min;
(4) dividing into three membranes, respectively dropwise adding 1: 1000-fold diluted His monoclonal antibody, 1:1000 rabbit anti-p 27 positive serum and 1:1000 purified rabbit anti-p 27 polyclonal antibody, and incubating overnight at 4 ℃ in a shaking table;
(5) recovering primary antibody, washing the membrane for 3 times by TBST, 5-10min each time;
(6) respectively dropwise adding HRP-labeled goat anti-mouse diluted by 1:1000 times, and incubating for 1h at 37 ℃;
(7) discarding the secondary antibody, washing the membrane for 3-5 times by TBST, 5-10min each time;
(8) ECL color development: diluting the solution A and the solution B in the ECL hypersensitive color development kit according to the same proportion, dripping the diluted solutions on a PVDF membrane, developing in the dark for 2min, sucking the edges dry, then placing the solution in an instrument for developing and shooting.
His monoclonal antibody was used as the primary antibody, and goat anti-mouse HRP-labeled antibody was used as the secondary antibody, and the results are shown in FIG. 4. The results in the figure show that ALV p27 recombinant protein can be specifically combined with his monoclonal antibody and the band size is consistent.
1.6 determination and identification of ALV p27 protein concentration
The protein concentration determination is carried out by using a Byunnan BCA protein concentration determination kit, and the specific steps are as follows:
(1) adding standard substance into 96-well plate at ratio of 0, 1, 2, 4, 8, 12, 16, 20. mu.L, adding diluent at ratio of 20, 19, 18, 16, 12, 8, 4 to make up to 20. mu.L, that is, standard substance concentration is 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/mL;
(2) adding 20 mu L of sample into each detection hole;
(3) adding 200 mu L of BCA working solution into each well, and incubating for 30min at 37 ℃;
(4) measuring the absorbance between the wavelength of 540 and 595nm by using a microplate reader;
(5) and drawing a standard curve to calculate the concentration of the measured sample.
The determination is carried out according to the BCA kit instruction, a standard curve is drawn, and the purified rabbit source p27 polyclonal antibody concentration is calculated to be 2mg/mL according to the formula of the standard curve.
1.7 preparation of rabbit antiserum of ALV p27 protein
New Zealand white rabbits were immunized with 500. mu.g/rabbit of ALV p27 pure protein as immunogen. Preparing rabbit-derived and mouse-derived polyclonal antibodies, comprising the following steps:
(1) adding equivalent volume of Freund's complete adjuvant (Freund's complete adjuvant is used for the first immunization) into the required protein amount, mixing the protein amount and the Freund's complete adjuvant, and then carrying out back subcutaneous multipoint injection immunization on the thoroughly emulsified immunogen by using a 1mL disposable sterile syringe;
(2) after the interval of the primary immunization for 14d, carrying out secondary immunization, and operating the same as the above;
(3) after the second immunization for 14d, carrying out the third immunization, and the operation is the same as the above;
(4) after the third immunization interval of 7d, the fourth immunization is carried out;
(5) collecting whole blood from ear vein of white rabbit in New Zealand 7d after the fourth immunization, collecting blood from mouse eyeball, centrifuging at 4 deg.C and 3500r/min for 10min to separate serum, repeating the operation if complete separation is not found, and detecting titer of hyperimmune serum;
(6) when the titer of the hyperimmune serum meets the requirement, the blood can be collected from New Zealand white rabbits through the heart, and the blood can be collected from eyeballs of mice.
1.8 determination of Rabbit-derived immune serum titer
The immune effect can be judged by the titer of hyperimmune serum, the titer of the polyclonal antibody is usually determined by an indirect ELISA method, and the specific operation steps are as follows:
(1) coating: diluting the coating antigen to 1 mu g/mL, coating 100 mu L/well of the coating antigen on a 96-well enzyme label plate, and incubating for 2 hours at 4 ℃ overnight or in an incubator at 37 ℃;
(2) washing: pouring off the solution in the enzyme-labeled pores, spin-drying, and washing with PBST for 3 times, each time for 3 min;
(3) and (3) sealing: adding 200 mu L of sealing liquid PBST into each hole, and sealing for 2h at 37 ℃ in an incubator;
(4) primary antibody multiple dilution;
(5) adding a primary antibody: after the ELISA plate is washed, adding a primary antibody diluted in a gradient way, wherein the primary antibody is 100 mu L/hole, and incubating for 30min in an incubator at 37 ℃;
(6) adding a secondary antibody: after the ELISA plate is washed, diluting a goat anti-rabbit IgG antibody (rabbit secondary antibody) marked by HRP (horse radish peroxidase) by 2500 times, adding the sample, incubating the sample in a constant-temperature oven at the temperature of 37 ℃ for 30min at a concentration of 100 mu L/hole;
(7) color development: after the ELISA plate is washed, adding TMB developing solution, incubating for 15min in a constant-temperature oven at 37 ℃ in a 100 mu L/well manner;
(8) and (4) terminating: 2M H2S04 stop the reaction, 50. mu.L/well;
(9) reading a plate: the OD450 values were read at the microplate reader.
The titer of rabbit and mouse antiserum is determined by the dilution ratio of the antibody when P/N (positive/negative) is more than or equal to 2.1.
The titer of rabbit-derived and mouse-derived ALV p27 hyperimmune serum was determined by indirect ELISA, and it was found from Table 1 that the titer of rabbit anti-p 27 polyclonal antiserum reached 1: 320000.
TABLE 1 measurement results of rabbit-derived p27 immune serum titer
1.9 Western blot identification of rabbit anti-ALV p27 protein polyclonal antiserum
The method is similar to the Western blot identification of ALV p27 protein. A1: 1000-fold dilution of rabbit anti-ALV p27 positive serum was used as the primary antibody, and HRP-labeled goat anti-rabbit antibody was used as the secondary antibody, and the results are shown in FIG. 5.
1.10 purification of Rabbit-derived Multi-antiserum
The specific operation steps are as follows:
(1) accurately sucking 5mL of prepared rabbit antiserum, centrifuging at 4 ℃ and 10000r for 10min, sucking out the supernatant, adding the supernatant into a 50mL centrifuge tube, adding 10mL of 60mmol/L sodium acetate buffer solution with pH of 4.8, and uniformly stirring;
(2) stirring at room temperature for 30min, adding octanoic acid dropwise during the stirring, adding 33 μ L octanoic acid into 1mL diluted serum, and standing for 5hr (more than or equal to 2 hr) to precipitate completely;
(3) centrifuging the obtained mixed solution at the temperature of 4 ℃ and at 9500r/min for 30min, discarding the precipitate, retaining the supernatant, filtering the supernatant by using filter paper, adding 1/10-volume 10 XPBS buffer solution into the supernatant, and adjusting the pH value to 7-8;
(4) slowly stirring saturated ammonium sulfate at 4 deg.C, adding into the above mixed solution to make the solution saturation degree to 50%, stirring for at least 30min, and standing at 4 deg.C for more than 1 hr;
(5) centrifuging the mixed solution at 4 ℃ and 9500r/min for 30min, discarding the supernatant and storing the precipitate;
(6) dissolving the precipitate with a proper amount of PBS, filling the solution into a dialysis bag, putting the dialysis bag into large-volume PBS, dialyzing for 72 hours at 4 ℃, and replacing PBS buffer solution for 3-5 times during dialysis;
(7) after dialysis, centrifuging at 4 deg.C and 10000r for 10min, discarding insoluble precipitate protein, and packaging the antibody at-20 deg.C for storage.
The purification results are shown in FIG. 6, and according to the same method, using acetic acid-sodium acetate buffer, adding 33. mu.L/mL of mixed solution to octanoic acid in the first step, centrifuging and precipitating at the bottom, adding 1/10 volumes of 10 XPBS buffer to adjust the pH to 7-8, and using saturated ammonium sulfate to precipitate at the bottom in the second step. The results were confirmed by SDS-PAGE after dialysis and shown in FIG. 6. Only two bands, one heavy and one light chain of IgG, are present, approximately at 50kD and 25kD, indicating successful purification of the antibody.
1.11 determination of Rabbit-derived polyclonal antibody purity
The procedure was followed by prokaryotic SDS-PAGE. The purified polyclonal antibody concentration was calculated to be 4.58mg/mL, as determined according to the BCA kit instructions.
1.12 determination of Rabbit-derived anti-p 27 polyclonal antibody concentration
The determination of the concentration of the polyclonal antibody herein employs the BCA method.
1.13 Western blot identification of rabbit anti-ALV p27 polyclonal antibody purified by caprylic-ammonium sulfate
The method is similar to the Western blot identification of ALV p27 protein. Purified rabbit anti-ALV p27 polyclonal antibody diluted 1:600 times was used as the primary antibody, and HRP-labeled goat anti-rabbit antibody was used as the secondary antibody, and the results are shown in FIG. 7, and purified rabbit-derived ALV p27 polyclonal antibody has good specificity.
1.14 HRP labeling of Rabbit anti-p 27 polyclonal antibody
The experiment is marked by adopting a sodium periodate method, and the specific operation is as follows:
(1) 5mg of HRP was weighed and dissolved in 1mL of ddH2In O, shaking and mixing uniformly;
(2) compounding NaIO40.2mL of the solution having a concentration of 0.1mol/L was added to the above HRP solution. Shaking at room temperature (25 deg.C) in dark for 20 min;
(3) boiling and activating dialysis bag, filling the above solution, and sealing;
(4) the dialysis bag was placed in a beaker containing 1L of 1mmol/L NaAc dialysis buffer pH4.4 and dialyzed overnight at 4 ℃;
(5) adding 20 mu L of 0.2mol/L pH9.5 carbonate buffer solution, adjusting the pH value to 9.0-9.5, immediately adding 5mg of antibody to be marked, and lightly shaking for 2 hours at room temperature in a dark place;
(6) 0.1mL of freshly prepared 4mg/mL NaBH was added4Shaking and mixing the solution uniformly, and standing for 2h at 4 ℃;
(7) putting the mixed solution into an activated dialysis bag, putting the bag into a beaker containing 0.15mol/L PBS buffer solution with pH7.4, and dialyzing the bag overnight at 4 ℃;
(8) equal volume of saturated (NH) was added dropwise with stirring4)2SO4Standing the solution at 4 ℃ for 1 h;
(9) centrifuging at 3000r/min at 4 deg.C for 30min, and discarding the supernatant. The precipitate was half-saturated with (NH)4)2SO4Resuspending the solution, centrifuging at 3000r/min and 4 deg.C for 30min, discarding the supernatant, and repeating the operation once;
(10) dissolving the precipitate in 500. mu.L of 0.15mol/L PBS (pH7.4);
(11) the solution was dialyzed against PBS at 4 ℃ to completely remove ammonium ions, centrifuged at 10000r/min for 30min, and the supernatant (enzyme conjugate) was collected.
1.15 Western blot identification of HRP-labeled Rabbit anti-p 27 polyclonal antibodies
The method is similar to the Western blot identification of ALV p27 protein.
Western blot identification is carried out by using HRP-labeled rabbit anti-ALV p27 polyclonal antibody as a primary antibody, the result is shown in figure 8, and the HRP-labeled rabbit anti-ALV p27 polyclonal antibody can be well combined with ALV p27 protein, which indicates that the HRP is successfully labeled on the rabbit anti-ALV p27 polyclonal antibody.
2. Optimization of double-antibody sandwich direct ELISA method
2.1 determination of optimal working concentration of coating antibody and enzyme-labeled antibody
The optimal working concentration of the coated antibody and the enzyme-labeled antibody is determined by a square matrix test:
1) the purified polyclonal antibody was diluted with carbonate buffer to 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625. mu.g/mL 8 dilution gradients, coated at 100. mu.L per well, coated 12 wells laterally per dilution, and coated overnight at 4 ℃. Throwing out the liquid in the hole, washing for 3 times by PBST oscillation, 3min each time, and finally drying on absorbent paper;
2) adding PBST 200 μ L containing 5% skimmed milk powder into each well, sealing at 37 deg.C for 2 hr, washing for three times as above, and patting to dry;
3) adding His-p27 protein diluted by PBS in odd number columns and 2 mug/mL in 100 mug/well into an ELISA plate, setting negative control in even number columns, incubating for 1h at 37 ℃, washing for three times as above, and patting to dry;
4) enzyme-labeled antibodies were treated with PBS 1:1000,1: 2000, l: 4000,1: 8000,1: 10000, 1:15000 times and 6 gradient dilutions, adding into an enzyme label plate, performing two rows per dilution with 100 mu L/hole, performing action for 1h at 37 ℃, washing for three times as above, and patting to dry;
5) adding a ready-prepared TMB color development solution, performing incubation at 37 ℃ for 15min at a concentration of 100 mu L/hole;
6) add 2M H2S04 stop solution to each well at 50. mu.L/well and read OD450 on a microplate reader. When the positive hole OD value is close to l and the P/N value is maximum, the concentration of the coating antibody and the concentration of the enzyme-labeled antibody are combined to be the optimal working concentration.
As shown in Table 2, the concentration of the selected coated antibody was 4. mu.g/mL, and the dilution ratio of the enzyme-labeled antibody was 1:10000, which was the optimum concentration of the coated antibody and the optimum dilution ratio of the enzyme-labeled antibody.
TABLE 2 determination of optimal working concentrations of coating antibody and enzyme-labeled antibody
2.2 determination of coating Condition
3 coating conditions were selected at 37 ℃ for 2h, 4 ℃ overnight, and 2h at 37 ℃ and 4 ℃ overnight, each of which was repeated 3 times, and the other procedures were as described in 2.1. And calculating the P/N value, and taking the envelope condition of the maximum P/N value as the optimal envelope condition.
As is clear from Table 3, the optimal conditions were 4 ℃ overnight after 2 hours at 37 ℃ among the 3 coating conditions.
TABLE 3 determination of the coating time
2.4 determination of the closure time
Selecting 5% skimmed milk powder, performing sealed time exploration for 60min, 90min and 120min, and performing other operation steps as 2.1. And calculating the value of P/N, and taking the maximum value of P/N as the optimal optimization condition.
As is clear from Table 4, the blocking at 37 ℃ for 2 hours out of 3 blocking conditions was the most preferable.
TABLE 4 determination of the blocking time
2.5 determination of optimal action time of the test serum
Selecting 30min, 60min and 90min for searching the optimal action time of serum, and performing the same other operation steps as 2.1. Measuring OD450 value, calculating P/N value, and taking the maximum value of P/N as the optimal action time of the antigen.
As is clear from Table 4, the optimal time is 0.5 hour at 37 ℃ among the 3 antigen-acting time conditions.
TABLE 4 determination of serum action time
2.6 determination of optimal action time of enzyme-labeled antibody
And (3) selecting the optimal dilution of the enzyme-labeled antibody for 30min, 60min and 90min to search the optimal secondary antibody action time, and performing other operation steps as 2.1. And calculating the value of P/N, and taking the maximum value of P/N as the optimal optimization condition.
As is clear from Table 5, the optimal conditions were 37 ℃ for 1 hour out of the 3 enzyme-labeled antibody reaction time conditions.
TABLE 5 determination of the duration of action of the enzyme-labeled antibody
2.7 determination of optimal color development time
Selecting 10min, 15min and 20min for searching for the optimal color development time, and performing the same other operation steps as 2.1. And calculating the value of P/N, and taking the maximum value of P/N as the optimal color development time.
As is clear from Table 6, the optimal condition was 37 ℃ for 15min out of 3 development time conditions.
TABLE 6 determination of color development time
2.8 determination of Positive and negative cut-off values
80 parts of IDEXX kit is used for detecting the determined negative serum, the indirect ELISA method is used for detecting, and the OD is measured450Value, calculate 80 sample OD450Mean (X) and Standard Deviation (SD) of the values, the measured OD being the off-line of the positive cut-off value of the method according to the statistical principle X +3SD450Positive when not less than X +3SD, OD450When the detected signal is less than or equal to X +3SD, the signal is negative and needs to be repeatedly detected.
TABLE 7 ALV p27 double antibody Sandwich ELISA reaction conditions
3. The double-antibody sandwich direct ELISA method for detecting the avian leukosis P27 comprises the following specific steps:
1) designing a pair of primers by using an avian leukosis virus RSA strain p27 gene sequence to amplify a p27 gene, constructing a recombinant expression vector, and obtaining the supernatant expression of a recombinant expression protein through induced expression, wherein the supernatant expression is used as an immunogen after being purified to prepare a rabbit polyclonal antibody;
2) washing the ELISA plate coated with the rabbit polyclonal antibody, sealing for 1h at 37 ℃, and washing the plate;
3) adding the serum to be detected for incubation, and washing the plate;
4) adding enzyme-labeled antibody for incubation, and washing the plate;
5) adding TMB color development liquid for color development, and finally adding color development termination liquid to terminate the reaction;
6) measuring the OD of the liquid in the plate hole of the enzyme label in the step 5) by using an enzyme label instrument450And (5) determining the negative and positive according to the threshold value by the nm value.
The optimization conditions in each step are as described above.
4. Test verification
4.1 specificity assay
The established ELISA method is used, and Newcastle disease virus, Ankara virus, Marek's disease virus and avian leukosis virus are used as the antigens to be detected, and negative and positive controls are set for detection.
The results of the specificity test are shown in Table 8, and only ALV detected positive in the tested samples, indicating that the method has good specificity.
TABLE 8 ALV p27 Dual antibody Sandwich ELISA specificity
4.2 sensitivity test
Taking a positive serum determined by the detection of the IDEXX kit to perform multiple-fold dilution from 1:125, performing parallel experiments on diluted samples by using the IDEXX kit and an ELISA method established in the experiments, and determining the sensitivity of the method.
The ELISA method established in the experiment is used for carrying out parallel experiments on diluted samples, whether the diluted samples are positive or not at the dilution ratio of 1:320 is shown in a table 9, and the test results show that the method has stronger sensitivity.
TABLE 9 ALV p27 double antibody Sandwich ELISA sensitivity
4.3 compliance experiments
Taking a cotton swab of a cloaca to be detected and cell supernatant, carrying out parallel experiments by using an established ELISA method and an IDEXX kit, and comparing the coincidence rate of the two.
92 cell supernatants of 61 cloaca cotton swabs to be detected are taken, 16 positive cloaca cotton swabs are detected by the IDEXX kit, and the positive rate is 26.2 percent (16/61); 4 positive cells were detected in 92 cell supernatants with a positive rate of 4.35% (4/92). 8 positive cloacal cotton swabs are detected by the established ALV p27 double-antibody sandwich ELISA method, the positive rate is 13.1 percent (8/61), 6 of the positive cloacal cotton swabs accord with the IDEXX kit, and the coincidence rate is 37.5 percent (6/16); 4 positive results were detected in 92 cell supernatants with a positive rate of 4.35% (4/92), and 4 were in compliance with the IDEXX kit with a 100% compliance rate.
4.4 repeatability test
Taking 3 parts of negative and positive serum respectively, carrying out a test by using the enzyme label plate coated in the same batch, repeatedly detecting 3 holes in each serum, and calculating the variation coefficient of repeated experiments in batches according to the detection result; 3 coating plates of different batches are taken according to the same method to carry out experiments according to an in-batch experiment method, and the variation coefficient of the repeatability among the batches is calculated. The Coefficient of Variation (CV) is calculated as: CV (%) ═ SD/X × 100%. Wherein: SD is standard deviation; x is the average number.
ALV p27 double antibody sandwich ELISA method was performed as an in-batch and an inter-batch differential assay under optimized reaction conditions, and the results of the in-batch differential assay are shown in Table 10. The table shows that the ALV p27 double antibody sandwich ELISA method has the difference in batch less than 15 percent and has good repeatability.
TABLE 10 ALV p27 double antibody Sandwich ELISA in-batch differences
The results of the inter-lot variation tests are shown in Table 11. The table shows that the ALV p27 double antibody sandwich ELISA method has the batch difference of less than 15 percent and good repeatability.
TABLE 11 ALV p27 double antibody Sandwich ELISA inter-batch differences
SEQUENCE LISTING
Sequence listing
<110> Guizhou university
<120> double-antibody sandwich direct ELISA method for detecting avian leukosis P27
<160> 2
<210> 1
<211> DNA
<212> 30
<213> Artificial sequence
<400> 1
CGGAATTCAT GCCTGTAGTG ATTAAGACAG 30
<210> 2
<211> DNA
<212> 28
<213> Artificial sequence
<400> 2
GCTCTAGACC TAGGGCTGGA TAGCAGAC 28
Claims (8)
1. A double-antibody sandwich direct ELISA method for detecting avian leukosis P27 is characterized by comprising the following steps:
1) designing a pair of primers by using an avian leukosis virus RSA strain p27 gene sequence to amplify a p27 gene, constructing a recombinant expression vector, and obtaining the supernatant expression of a recombinant expression protein through induced expression, wherein the supernatant expression is used as an immunogen after being purified to prepare a rabbit polyclonal antibody;
2) washing the ELISA plate coated with the rabbit polyclonal antibody, sealing for 1h at 37 ℃, and washing the plate;
3) adding the serum to be detected for incubation, and washing the plate;
4) adding enzyme-labeled antibody for incubation, and washing the plate;
5) adding TMB color development liquid for color development, and finally adding color development termination liquid to terminate the reaction;
6) measuring the OD of the liquid in the plate hole of the enzyme label in the step 5) by using an enzyme label instrument450And (5) determining the negative and positive according to the threshold value by the nm value.
2. The double antibody sandwich direct ELISA method for detecting avian leukemia P27 according to claim 1, wherein the avian leukemia virus is virus RSA strain.
3. The double antibody sandwich direct ELISA method for detecting avian leukemia P27 according to claim 1, wherein the primer sequences of the pair of primers are as follows:
p27-EcoR I-F：CGGAATTCATGCCTGTAGTGATTAAGACAG；
p27-Xba I-R：GCTCTAGACCTAGGGCTGGATAGCAGAC。
4. the double antibody sandwich direct ELISA method for detecting avian leukosis P27 according to claim 1, wherein the concentration of the coated rabbit polyclonal antibody is 4 μ g/mL, the coating condition is 4 ℃ overnight after 2h at 37 ℃, and the blocking condition is 2h at 37 ℃.
5. The double antibody sandwich direct ELISA method for detecting avian leukosis P27 according to claim 1, wherein the reaction condition of the serum to be detected is 0.5h at 37 ℃.
6. The double-antibody sandwich direct ELISA method for detecting avian leukosis P27 as claimed in claim 1, wherein the dilution factor of the enzyme-labeled antibody is 1:10000, and the reaction time is 1h at 37 ℃.
7. The double antibody sandwich direct ELISA method for detecting avian leukosis P27 according to claim 1 wherein the conditions shown are 37 ℃ for 15min.
8. The double antibody sandwich direct ELISA method for detecting avian leukosis P27 of claim 1 wherein the cut-off value determines the negative and positive markersThe method comprises the following steps: when OD is reached450nm greater than 0.222 is positive and less than 0.222 is negative.
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| CN116735888A (en) * | 2022-11-18 | 2023-09-12 | 昆明医科大学第一附属医院 | Indirect ELISA method for detecting COG5 by specific polyclonal antibody |
| CN116735888B (en) * | 2022-11-18 | 2024-01-12 | 昆明医科大学第一附属医院 | Indirect ELISA method for detecting COG5 by specific polyclonal antibody |
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