Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a detection reagent for feline herpesvirus based on a double antibody sandwich ELISA.
The ELISA detection reagent for detecting the I-type feline herpesvirus provided by the invention has three CDR areas of the light chain of the antibody, and the three CDR areas have the amino acid sequence shown in SEQ ID NO: 1-3, wherein the three CDR regions of the heavy chain have the amino acid sequences shown in SEQ ID NO: 4-6;
three CDR regions of the light chain of the capture antibody have the sequences shown in SEQ ID NO: 1-3, wherein the three CDR regions of the heavy chain have the amino acid sequences shown in SEQ ID NO:4 to 6.
Wherein, SEQ ID NO:1 is as follows: x is XL1 SXL2 XL3 XL4 XL5 NXL6 The method comprises the steps of carrying out a first treatment on the surface of the Wherein X isL1 Q or E, XL2 Is V or I, XL3 Is E or Y or none, XL4 S or none, XL5 Is N or S, XL6 Is N or Y;
SEQ ID NO:2 is as follows: x is XL7 AS; wherein X isL7 F, A or G;
SEQ ID NO:3 is as follows: x is X8 XL9 XL10 YXL11 XL12 XL13 XL14 XL15 XL16 XL17 XL18 XL19 The method comprises the steps of carrying out a first treatment on the surface of the Wherein X isL8 Is V or H or L, XL9 Is G or Q, XL10 Y or T or E, XL11 Is Y or S, XL12 Is D or Y, XL13 Is G or N or T, XL14 Is S or N or K, XL15 Is D or V or A, XL16 Is T or G or none, XL17 Is F or N or none, XL18 Is F or G or none, XL19 A or none;
the three CDR regions of its heavy chain have in sequence the amino acid sequence shown in SEQ ID NO: 4-6;
SEQ ID NO:4 is as follows: GXH1 XH2 LXH3 XH4 XH5 XH6 XH7 The method comprises the steps of carrying out a first treatment on the surface of the Wherein X isH1 Is I or F, XH2 Is D or S, XH3 Is S or I, XH4 R or N or S, XH5 Y or N or G or S, XH6 G or D, XH7 Is none or W;
SEQ ID NO:5 is as follows: IX (IX)H8 XH9 XH10 XH11 XH12
XH13 XH14 XH15 XH16 XH17 RDXH18 XH19 XH20 XH21 XH22 XH23 XH24 XH25 S XH26 XH27 T is a T; wherein X isH8 Is none or D, XH9 Is none or A, XH10 Is none or G, XH11 Is none or T or S, XH12 Is none or S, XH13 Is none or G, XH14 Is none or S, XH15 Is none or T, XH16 Is none or W, XH17 Is none or Y, XH18 Is none or A, XH19 Is none, A or T, XH20 Is none or W, XH21 Is none or V, XH22 Is none or N, XH23 Is none or A, XH24 Is S or I or V or G, XH25 Is N or S, XH26 Is A or G, XH27 Is N or S;
SEQ ID NO:6 is as follows: x is XH28 RXH29 XH30 XH31 XH32 XH33 XH34 XH35 XH36 XH37 XH38 The method comprises the steps of carrying out a first treatment on the surface of the Wherein X isH28 Is G or A, XH29 Is none or W, XH30 Is none or S, XH31 Is T or G, XH32 Is I or V or T or S, XH33 Is T or Y, XH34 Is G or A, XH35 Is S or T or Y, XH36 Is G or L or Y, XH37 Is N or D, XH38 Is I or L.
In the detection reagent of the present invention, the capture antibody or the detection antibody may be the same or different.
In some embodiments of the present invention,
the three CDR regions of the light chain of the capture antibody are QSVESNN, FAS and VGYYYDGSDTFFA respectively; the three CDR regions of the heavy chain are GIDLSRYG, ISNSANT and GRTITGSGNI, respectively;
the three CDR regions of the light chain of the detection antibody are ESINNY, AAS and HQTYSYNNVGNG respectively; the three CDR regions of its heavy chain are GFSLSNNG, IISSGST and ARGVYATLDI, respectively.
Still further, the method comprises the steps of,
the amino acid sequence of the light chain of the capture antibody is shown as SEQ ID NO. 7; the amino acid sequence of the heavy chain is shown as SEQ ID NO. 12;
the amino acid sequence of the light chain of the capture antibody is shown as SEQ ID NO. 9; the amino acid sequence of the heavy chain is shown as SEQ ID NO. 14.
In the invention, the capture antibody is coated on a solid-phase medium, and the detection antibody is labeled with a chemical label or a biological label.
The chemical label is an isotope, an immunotoxin and/or a chemical drug; the biomarker is biotin, avidin or an enzyme label. The enzyme label is preferably horseradish peroxidase or alkaline phosphatase. The immunotoxin is preferably aflatoxin, diphtheria toxin, pseudomonas aeruginosa exotoxin, ricin, abrin, mistletoe lectin, podophyllotoxin, PAP, sequoyins, gelonin or luffa toxin.
The solid or semi-solid medium refers to any support to which the recombinant antibodies, labeled recombinant antibodies, of the present invention can be attached, including but not limited to nitrocellulose membranes, polyvinylidene difluoride (PVDF) membranes, iPDMS chips, microwell plates, polystyrene plates, microparticles, microcarriers, gels, and the like.
The capture antibody is coated on an ELISA plate, and the detection antibody is marked with HRP.
In the detection reagent, the mass ratio of the capture antibody to the detection antibody is 10:1.
The detection reagent also comprises a coating buffer solution, a washing solution, a sealing solution, TMB color development solution and a termination solution; wherein:
the coating buffer solution is PBS buffer solution with pH of 7.2-7.4,
the washing liquid is a PBST solution containing 0.05 percent of Tween-20 by mass percent,
the sealing liquid is a skimmed milk powder solution with the mass fraction of 2%;
the termination liquid is 2mol/L H2 SO4 A solution.
Further, the invention also provides a detection method of the type I feline herpesvirus, which comprises detecting a sample by the detection reagent.
The detection method of the present invention may be for diagnostic purposes or non-diagnostic purposes, and the present invention is not limited thereto. Among them, in the detection method for diagnostic purposes, the sample is derived from an animal body, and in the detection method for non-diagnostic purposes, the sample is derived from an environment such as food, water, laboratory culture, or a swab on the surface of an implement.
The detection method comprises the following steps: after capturing the sample by the capture antibody, adding the detection antibody for incubation, wherein the mass ratio of the capture antibody to the detection antibody is 10:1.
In the invention, the detection method specifically comprises the following steps: after capturing the sample by the capture antibody, adding the detection antibody for incubation; the capturing condition is 37 ℃ for 1h; the incubation conditions were 37℃for 1h.
After the incubation, the steps of TMB development, termination of development and reading of absorbance at 450nm are also included.
The detection method can be a qualitative detection method or a quantitative detection method.
And the qualitative detection is based on whether fluorescence judgment results are generated, and the sample generating fluorescence is a positive sample.
And the quantitative detection is carried out according to the judgment result of the fluorescence intensity, and the virus load in the sample is high if the fluorescence intensity is high.
According to the invention, a phage single-chain antibody library is constructed by phage display technology, and 5 single-chain antibodies capable of specifically binding with FHV-I are screened through enrichment and sequencing. According to the pairing situation, the reagent for detecting the I-type feline herpesvirus by the double-antibody sandwich enzyme-linked immunosorbent assay is developed, and the reagent has the advantages of high specificity, high sensitivity, good stability and the like.
Detailed Description
The invention provides ELISA detection reagents for feline herpesvirus, and one skilled in the art can use the content of the invention to properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
Strains, plasmids, test animals and reagents:
adult New Zealand rabbits were purchased from Yongkangqing source farms in the county of Anhui province; FHV gB protein is purchased from Nanjing Jinsri biotechnology Co., ltd; FHV-1 virus was saved by the present laboratory (GenBank: NC-013590.2); vectors pTT5-V5 and pTT5-V6 containing rabbit antibody frameworks were stored for this laboratory (FIG. 6); HRP-labeled recombinant protein a was purchased from engineering bioengineering (Shanghai) stock, inc; HRP-labeled murine anti-M13K 07 phage were purchased from adzuki biotechnology limited; HRP-labeled goat anti-rabbit IgG was purchased from beginner Mo De biotechnology limited; pComb3XSS plasmid, e.g. dh5α competent cells were purchased from shandong hertz biotechnology limited; M13K07 helper phage, E.coli ER2738 competent cells were purchased from Nanjing He Ding Biotechnology Co., ltd; SDS-PAGE protein gel kit was purchased from Yase Biotechnology Co., ltd; penicillin-streptomycin solutions were purchased from GE company (for cell culture); freund's adjuvant was purchased from Sigma company; clonExpress II One Step Cloning Kit from Nanjinouzan Biotechnology Co., ltd; premix taq, plasmid mini kit, gel Extraction and transfection reagents were all purchased from TaKaRa.
Heavy chain variable region amino acid sequences of single chain antibody FHV gB-scFv-1 and recombinant antibody Ab-FHV gB-1:
light chain variable region amino acid sequences of single chain antibody FHV gB-scFv-1 and recombinant antibody Ab-FHV gB-1:
heavy chain variable region amino acid sequences of single chain antibody FHV gB-scFv-2 and recombinant antibody Ab-FHV gB-2:
light chain variable region amino acid sequences of single chain antibody FHV gB-scFv-2 and recombinant antibody Ab-FHV gB-2:
heavy chain variable region amino acid sequences of single chain antibody FHV gB-scFv-3 and recombinant antibody Ab-FHV gB-3:
light chain variable region amino acid sequences of single chain antibody FHV gB-scFv-3 and recombinant antibody Ab-FHV gB-3:
heavy chain variable region amino acid sequences of single chain antibody FHV gB-scFv-4 and recombinant antibody Ab-FHV gB-4:
light chain variable region amino acid sequences of single chain antibody FHV gB-scFv-4 and recombinant antibody Ab-FHV gB-4:
heavy chain variable region amino acid sequences of single chain antibody FHV gB-scFv-5 and recombinant antibody Ab-FHV gB-5:
light chain variable region amino acid sequences of single chain antibody FHV gB-scFv-5 and recombinant antibody Ab-FHV gB-5:
amino acid sequence of linker peptide linker:
primer sequence:
library construction of VH-scFv-F1
Library construction of VH-scFv-F2
Library construction of VH-scFv-F3
Library construction of VH-scFv-F4
Library-building VH-scFv-R1
Library VH-scFv-R2
Library construction VL-scFv-F1
Library construction VL-scFv-F2
Library construction VL-scFv-F3
Library construction VL-scFv-F4
Library construction VL-scFv-R1
Library construction VL-scFv-R2
Overlapping PCR-FP
Overlapping PCR-RP
Verification of pComb3XSS-FP
Verification of pComb3XSS-RP
The invention is further illustrated by the following examples:
EXAMPLE 1 animal immunization
Healthy adult New Zealand rabbits are selected for the experiment, vein blood collection is carried out before immunization, and serum is separated to serve as a negative control. Taking 1×105 TCID50 FHV virus/mL was injected on the back neck of New Zealand rabbits, immunized once a week for a total of 4 times, and 0.5mL of the isolated serum was collected intravenously before each immunization on days 0, 7, 14, 21 for storage. 7 days after the 4 th immunization (i.e. day 28), 0.5mL of the isolated serum was collected intravenously and stored. ELISA results show that the serum antibody titer is obviously increased after primary immunization, and the secondary antibody titer is close to 9×104 Three-free up to 2X 105 The titer of the tetraimmune serum is greatly improved to be more than 5 multiplied by 105 The serum antibody titers before immunization are all significantly improved (1-A). New Zealand rabbits were sacrificed on day 28, 0.5mL of blood was collected and isolated serum was saved, the spleen was dissected and removed for lymphocyte separation.
EXAMPLE 2 extraction of Total lymphocyte RNA and amplification of antibody light-heavy chain Gene
Total RNA of lymphocytes is extracted by Trizol reagent, and cDNA is synthesized by reverse transcription using Oligo (dT) as a primer with the total RNA as a template. Primer sequence combinations are designed according to light and heavy chain framework regions of rabbit antibody sequences, and coding regions of antibody VH and VL are amplified by PCR. The PCR reaction conditions were: the pre-denatured product was 5min at 95℃for 30s, annealed at 55℃for 30s, extended at 72℃for 60s, and after 35 cycles, extended at 72℃for another 5min. The PCR product was confirmed by 1% agarose gel electrophoresis to have a band of interest of about 400bp, and was purified and recovered for use (1-B).
EXAMPLE 3 construction of FHV gB-scFv antibody library
The purified VH and VL are used as templates, a linker (with a sequence of SEQ ID NO. 17) is inserted between the VH and the VL after overlapping PCR, and the VL-linker-VH of the scFv fragment is synthesized, and the two ends of the sequence contain sfiI restriction enzyme sites.
Overlapping extension first round PCR: without primer, 1min was denatured at 98 ℃, annealed at 45 ℃ for 30s, extended at 72 ℃ for 1min, repeated 10 cycles, and finally acted at 72 ℃ for 5min. The primer overlap PCR-FP and overlap PCR-RP were added and denatured at 98℃for 30s, then renatured at 58℃for 30s, extended at 72℃for 1min, and extended at 72℃for another 5min after 30 cycles. The amplified product was recovered by gel recovery of the PCR product by 1% agarose gel electrophoresis, and the band was about 800bp (1-B) and stored at-20 ℃. The overlapping PCR primer sequences are shown as follows.
And respectively carrying out enzyme digestion on the PCR product and the pComb3XSS vector after gum recovery by using restriction enzyme sfiat 50 ℃ for 30min, and carrying out column recovery and purification after 1% agarose gel electrophoresis verification on the enzyme digestion product, and connecting the enzyme digestion product with T4 DNA ligase to obtain the FHV gB-scFv-pComb3XSS library. The ligation products were transformed into ER2738 competent cells, and positive clones were screened with tetracycline and ampicillin resistant 2YT (2X Yeast extract and Tryptone) solid medium. 20 monoclonal antibodies in the random selection library were identified by PCR, and the recombination rate of the antibody library was calculated to be 50% (2-A). All clones on solid medium were washed off with 2YT medium and stored at 4 ℃.
Example 4 amplification of phages
2mL of the library bacterial solution of all clones washed by the 2YT medium is inoculated into 50mL of the 2YT liquid medium, and activated to OD at 37 ℃ at 200rpm600 Reaching 0.8-1.0, adding into the mixture with the final concentration of 1 multiplied by 1012 The pfu/mL M13K07 helper phage was infected, allowed to stand for 30min, kanamycin (final concentration 50. Mu.g/mL) was added, and incubated overnight at 30℃at 200 rpm. Centrifugation was performed for 20min at 10000rpm4℃the next day, the supernatant was retained and 1/2 volume of PEG8000 was added thereto, and the mixture was allowed to stand on ice for 2 hours to precipitate phage. Centrifuge at 10000rpm at 4℃for 20min, discard supernatant. The pellet was resuspended in 5mL PBS, 3mL PEG8000 was added for further precipitation, centrifugation was repeated, and after resuspension of the pellet with 4mL PBS and sterilization by filtration, the pellet was stored at 4℃for further use.
EXAMPLE 5FHV gB-scFv screening
The FHV gB protein is biotinylated in advance. After the amplified FHV gB-scFv library phage, dynabeads M-280 magnetic beads marked with FHV gB protein and 2% nonfat milk powder sealing liquid are mixed, incubating for 1h at 25+/-5 ℃, and washing the magnetic beads for 5-10 times by using PBST. The bead surface phage was eluted with 0.1M Glycine-HCl (ph=2.0) and after elution neutralized with 1M Tris to pH 7.0. ER2738 competent cells in the logarithmic growth phase are infected with the phage neutralized after elution, enriched by culturing on a 2YT solid medium, and colonies are harvested for the next round of screening.
The above experiment was repeated twice. From the two rounds of enrichment affinity screening library, 20 monoclonal antibodies are randomly selected for PCR verification, and the result shows that the positive rate of the first round of elutriation of the established FHV gB-scFv library reaches 80%, and the positive rate of the second round of elutriation reaches 85% (2-B and 2-C).
EXAMPLE 6Phage ELISA
FHV gB antigen was coated on the ELISA plate at a concentration of 50. Mu.g/mL per well at 4℃overnight. The cells were washed 5 times with PBST solution containing 0.05% Tween-20, PBST was discarded, and 2% nonfat dry milk blocking solution was added and blocked at 37℃for 1 hour. Washing with PBST solution for 5 times, beating the residual liquid in the hole, and mixing the supernatant of the positive clone antibody to be screened and 2% skim milk powder sealing liquid according to the following steps of 3: mixing in proportion, standing at 25+/-5 ℃ for 10min to remove interference, adding into an ELISA plate, and incubating at 37 ℃ for 1h. Wash 5 times with PBST solution, dry the wells with residual liquid, add HRP-labeled murine anti-M13K 07 antibody (diluted 1:3000 with blocking solution), incubate for 1h at 37 ℃. Washing with PBST solution for 5 times, drying the residual liquid in the wells, adding TMB color development liquid, standing in dark for 15min, and adding 2mol/L H2 SO4 The color development was stopped and the absorbance at 450nm was read (Table 1). As shown in 2-D in FIG. 2, select OD450 And sequencing 15 antibodies with high values, and finally obtaining 5 antibodies with different sequences for subsequent experiments.
TABLE 1 affinity detection of FHV gB-scFv with antigen FHV gB
EXAMPLE 7 recombinant antibody Ab-FHV gB, expression and purification of FHV gB
Amplifying and purifying VH and VL sequences of 5 single chain antibody FHV gB-scFv, double digestion of pTT5-V5 and pTT5-V6 carrier containing rabbit antibody skeleton with EcoRI and HindIII restriction endonuclease, and homologous recombination to obtain antibody expression plasmid VH-pTT 5-V5-1-VH-pTT 5-V5-5 and VL-pTT 5-V6-1-VL-pTT 5-V6-5. The recombinant plasmids VH-pTT 5-V5-1-VH-pTT 5-V5-5 and VL-pTT 5-V6-1-VL-pTT 5-V6-5 are in one-to-one correspondence and are transfected into HEK293f cells through PEI for eukaryotic expression. After 48h, the culture supernatant was collected by centrifugation at 10000rpm for 20min at 4℃and the antibody was purified by Protein A affinity chromatography, and finally the fusion proteins were stored in PBS solution, designated Ab-FHV gB-1, ab-FHV gB-2, ab-FHV gB-3, ab-FHV gB-4, ab-FHV gB-5, respectively.
The purified sample was added to a reduced protein loading buffer, denatured, and subjected to SDS-PAGE electrophoresis, and the result was shown in FIG. 3, wherein disulfide bonds between the heavy chain and the light chain of the antibody were reduced, and the target bands were detected at about 50kDa and 25kDa, consistent with the expected results.
Example 8 detection of binding of recombinant antibody Ab-FHV gB to FHV gB antigen by indirect ELISA
The affinity of Ab-FHV gB to antigen FHV gB was determined by indirect ELISA. Antigen was added to the elisa plate for incubation overnight at 4 ℃ and blocked with 2% nonfat dry milk blocking solution at 37 ℃ for 2h. The antibodies were diluted 12 gradients with 2% nonfat milk powder and added to the wells and incubated for 1h at 37 ℃. HRP-labeled goat anti-rabbit IgG secondary antibody was added, color development reaction was performed with TMB, and absorbance at a wavelength of 450nm was detected in a microplate reader (tables 2 to 6).
The detection of the binding effect of the antibody Ab-FHV gB is shown as 3-B-3-F in figure 3, the dose response curve is successfully fitted with a regression model, all 5 antibodies of Ab-FHV gB can effectively bind FHV gB protein, the antibody Ab-FHV gB-3 has the highest affinity, and the EC combined with recombinant expression FHV gB protein50 1.113 ×10-1 Mu g/mL, the recombinant antibody has activity and good affinity, and Ab-FHV gB-3 is used for subsequent experiments.
TABLE 2 Ab-FHV gB-1 EC binding to antigen FHV gB50
TABLE 3 EC50 of Ab-FHV gB-2 binding to antigen FHV gB
TABLE 4 EC of Ab-FHV gB-3 binding to antigen FHV gB50
TABLE 5 EC of Ab-FHV gB-4 binding to antigen FHV gB50
TABLE 6 EC of Ab-FHV gB-5 binding to antigen FHV gB50
Example 9 Indirect immunofluorescence detection of recombinant antibodies
Resuscitating CRFK adherent cells with EMEM containing 10% FBS, inoculating FHV venom with MOI of 0.1 when cell fusion degree reaches 80-90%, and inoculating 5% CO at 37deg.C2 The incubator was co-infected and incubated while virus negative groups were set as controls. An equal volume of 2% FBS EMEM was added and cultured in a cell incubator, after 24h the cells were washed 3 times with PBS. Cells were fixed by adding 500. Mu.L of 4% paraformaldehyde, and after 30min, the cells were washed 3 times with PBS. mu.L of 0.1% TritonX100 was added, and after 15min the cells were washed 3 times with PBS. Blocking with 3% sodium caseinate in PBS at 37℃for 1h and washing with PBS 3 times. The recombinant antibody Ab-FHV gB-3 is used as a primary antibody of indirect immunofluorescence, the concentration is 0.5 mug/mL, the secondary antibody is goat anti-rabbit FITC, the secondary antibody is incubated for 1h at 37 ℃, the plate is washed 3 times, 500uL DAPI is added for dyeing 20min, and the PBS is used for washing the plate 3 times for observation by using an inverted fluorescence microscope. As shown in FIG. 4, the recombinant antibody Ab-FHV gB-3 showed a distinct specific green fluorescent signal with FHV virus positive samples, whereas no fluorescent signal was seen in the virus negative control group. Proved that the recombinant antibody Ab-FHV gB-3 can be specifically combined with FHV virus and can be applied toThe method is used for specific detection of the feline herpesvirus FHV.
Example 10 antibody specificity validation
Respectively 1X 104 TCID50 The concentration of the per mL FPV, FCV, FHV cell venom supernatant coating coated the ELISA plate at 4℃overnight. The cells were washed 5 times with PBST solution containing 0.05% Tween-20, PBST was discarded, and 2% nonfat dry milk blocking solution was added and blocked at 37℃for 1 hour. Washing with PBST solution for 5 times, drying the residual liquid in the wells, and mixing the recombinant antibody Ab-FHV gB-3 with 2% skim milk powder blocking solution according to the formula 3: mixing in proportion, standing at 25+/-5 ℃ for 10min to remove interference, adding into an ELISA plate, and incubating at 37 ℃ for 1h. Wash 5 times with PBST solution, dry the remaining liquid in the wells, add HRP-labeled goat anti-rabbit IgG secondary antibody, incubate for 1h at 37 ℃. Washing with PBST solution for 5 times, drying the residual liquid in the wells, adding TMB color development liquid, standing in dark for 15min, and adding 2mol/L H2 SO4 The color development was stopped and the absorbance at 450nm was read (Table 7). As shown in FIG. 5, the recombinant antibody Ab-FHV gB-3 shows good affinity with FHV virus, has binding level difference with FPV and FCV virus, shows FHV virus specificity, proves that the recombinant antibody Ab-FHV gB-3 can be specifically combined with FHV virus, can be applied to specific detection of feline herpesvirus FHV, is not confused with specific detection of feline parvovirus and feline calicivirus, and is an excellent feline herpesvirus detection antibody.
TABLE 7 verification of specific binding of Ab-FCV VP1-4 to FCV, FPV, FHV
| 1 | 2 | 3 |
| FHV | 2.3106 | 2.1627 | 2.3135 |
| FPV | 0.0461 | 0.0397 | 0.0472 |
| FCV | 0.0502 | 0.0542 | 0.0407 |
Example 11 method for establishing double-antibody sandwich ELISA based on anti-feline herpesvirus rabbit single-chain recombinant antibody
Horseradish peroxidase (HRP) labeling treatment method: the procedure was as described for HRP-labeled kit (6012-1).
The specific steps of the double-antibody sandwich method ELISA method are as follows: (1) Diluting anti-feline herpesvirus rabbit single-chain recombinant antibodies Ab-FHV gB-1, ab-FHV gB-2, ab-FHV gB-3, ab-FHV gB-4 and Ab-FHV gB-5 to 5 mug/mL by using PBS buffer solution (pH=7.2-7.4) for coating, incubating overnight at 4 ℃, and washing the plates by using washing solution; (2) blocking with 2% skim milk powder blocking solution at 37deg.C for 2 hr; (3) Adding 50ng/mL standard sample (FHV GB protein), incubating for 1h at 37 ℃ in 100 mu L of each well, and washing the plate with washing liquid; (4) Adding Ab-FHV gB-1-HRP, ab-FHV gB-2-HRP, ab-FHV gB-3-HRP, ab-FHV gB-4-HRP and Ab-FHV gB-5-HRP detection antibodies which are diluted 2000 times respectively, incubating for 1h at 37 ℃ in each hole with 100 mu L of each hole, and washing the plates with washing liquid; (5) Adding TMB color development liquid, developing at normal temperature in dark for 10min, adding 2mol/L H2 SO4 The color development was stopped, and the absorbance at 450nm was read to determine the optimal pairing antibody.
TABLE 8 results of antibody pairing experiments
As can be seen from the data in Table 8, when the capturing antibody was Ab-FHV gB-1 and the detecting antibody was Ab-FHV gB-3 after pairing 5 antibodies, the OD was measured for the same concentration sample450 The method has the highest value, so that the rabbit single-chain recombinant antibody Ab-FHV gB-1 is selected as a capture antibody, and the rabbit single-chain recombinant antibody Ab-FHV gB-3 marked by horseradish peroxidase is used as a detection antibody, and the two antibodies are combined to be used for enzyme-linked immunosorbent assay by a double antibody sandwich method, so that the method has the advantages of high specificity, high sensitivity, good stability and the like, and has important significance in clinical diagnosis and scientific research application.
Example 12 establishment of double antibody sandwich ELISA detection method
1 determination of optimal Capture antibody and detection antibody use concentration
The optimal capture antibody Ab-FHV gB-1 was coated on the ELISA plate at a concentration of 0.5. Mu.g/mL, 1. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL, and the optimal detection antibody Ab-FHV gB-3 was incubated at a gradient concentration of 0.25. Mu.g/mL, 0.125. Mu.g/mL, 0.083. Mu.g/mL. The results in Table 9 show that the P/N value was the most optimal reaction condition when the coating concentration of the capture antibody Ab-FHV gB-1 was 4. Mu.g/mL and the dilution concentration of the detection antibody Ab-FHV gB-3 was 0.25. Mu.g/mL.
TABLE 9
Determination of antibody incubation time
ELISA assays were performed at a coating concentration of 4. Mu.g/mL for each of 2h, 4h and 4h at 37℃in an incubator, and 12h at 4℃with 4℃incubation at 12h and the maximum P/N value, as shown in FIG. 7A, thus the optimal coating conditions were obtained when the capture antibody was incubated at 4℃for 12 h.
3. Determination of closure time
The blocking conditions were optimized using the determined optimal coating parameters, i.e. 3% BSA was added, 100. Mu.L per well, incubated at 37℃for 30, 60, 90, 120, 150min, respectively, ELISA assays were performed, and the results showed that as shown in FIG. 7B, the P/N values were maximum when incubated at 37℃for 120min, and therefore, the optimal blocking conditions were reached at 37℃for 120 min.
Determination of optimal incubation time for 4TMB
Using the experimental conditions determined above, ELISA assays were performed by adding TMB color development solution to ELISA plates at 37℃for 5, 10, 15, and 20min, respectively, with 100. Mu.L per well, and the results in FIG. 7C showing that the P/N value was maximum when incubated at 37℃for 10min, and therefore, the optimal color development time when incubated at 37℃for 10 min.
5 sensitivity detection
The optimized sandwich ELISA method is adopted as a reaction condition, and antigen gB proteins are diluted by 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.125ng/mL and 1.5625ng/mL for detection, and the result shows that the P/N value is still greater than 2 when the antigen gB protein amount is 12.5ng/mL, which indicates that the method has good sensitivity.
Table 10
6 specific detection
The experimental laboratory-maintained FCV, FPV, FHV-1 virus was tested and negative controls were established using the optimized sandwich ELISA method reaction conditions, and the results showed that the established ELISA specifically recognized FHV-1 as shown in FIG. 7D. Therefore, the double-antibody sandwich ELISA detection kit has good specificity for FHV-1 pathogen.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.