WO2019174127A1 - Procédé et kit de détection de plaquettes liquides multiples - Google Patents

Procédé et kit de détection de plaquettes liquides multiples Download PDF

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WO2019174127A1
WO2019174127A1 PCT/CN2018/088904 CN2018088904W WO2019174127A1 WO 2019174127 A1 WO2019174127 A1 WO 2019174127A1 CN 2018088904 W CN2018088904 W CN 2018088904W WO 2019174127 A1 WO2019174127 A1 WO 2019174127A1
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srv
siv
magnetic beads
liquid phase
phase chip
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黄韧
李秀珍
张钰
郭鹏举
丛锋
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广东省实验动物监测所
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Definitions

  • the invention relates to the field of detection of viral diseases in experimental animals, and more particularly to a rapid, accurate and high-throughput detection of monkey herpesvirus, simian immunodeficiency virus, monkey D-type retrovirus and monkey T-lymphocytic leukemia virus. Multiple liquid phase chip detection method and kit.
  • Non-human primates are an ideal animal model for exploring and solving human health and disease problems because of their physiological, biochemical, immunological, and genetic characteristics similar to humans.
  • SPPF pathogen free
  • Internationally, the definition of SPF monkeys mainly refers to monkeys that do not contain specific viruses such as monkey herpesvirus, monkey immunodeficiency virus, monkey retrovirus, and monkey T lymphocytic leukemia virus.
  • Charles River Laboratories (CRL) of the United States has proposed the concept of virus antibody free (VAF), which is recognized by many experimental animal users around the world. This standard meets the requirements of experimental animal breeding and meets the actual needs of scientific research.
  • BV (Monkey B virus, Cercopithecine herpesvirus 1) is classified as a herpes simplex virus of the herpesvirus family. Monkey is the natural host of B virus, the infection rate can reach 10 to 60%, in most cases it is benign, but human infection is mainly manifested as symptoms of cerebrospinal inflammation, often leading to death. To date, the genomic sequence of the monkey B virus has not been completely sequenced, but the sequence obtained has high homology with the sequence of HSV.
  • HSV-1 and HSV-2 antiserum did not react with the epitope, so HSV- was selected in the study.
  • 1 virus culture and gD recombinant protein were coated as microspheres as antigen of B virus single-liquid chip detection technology.
  • SIV seimian immunodeficiency virus
  • SIV belongs to the retrovirus family of lentiviruses and is one of the causative agents of RNA virus and monkey AIDS. It does not cause disease in natural hosts, and only cross-racial infections can cause immunodeficiency symptoms similar to human AIDS.
  • the capsid protein p27 constitutes the inner shell of the double-shell of SIV virion, and its amino acid sequence is relatively conservative. It contains the main antigenic determinants that induce humoral and cellular immunity of the body, and the capsid proteins of the same genus have the same or similar antigenicity. That is, it has a group antigen and is therefore considered to be a marker for SIV antigen detection.
  • SRV Sesimian type D retrovirus
  • SEDV endogenous virus
  • SRV exogenous virus
  • SRV-1, 3, and 5 are mainly prevalent in Asian and Indian rhesus monkeys.
  • SRV-2 is mainly prevalent in Southeast Asian cynomolgus monkeys and porpoise monkeys, and SRV-4 is rarely prevalent.
  • SRV-6 was isolated from Indian long-tailed langurs
  • SRV-7 was isolated from wild Indian rhesus monkeys.
  • SRV-1 ⁇ 3 has been sequenced, and the amino acid sequence of nucleocapsid protein p27 is highly conserved.
  • STLV-1 (simian T-cell lymphotropic virus) belongs to the subfamily of RNA tumor virus, ⁇ retrovirus genus, mainly invading the immune system of monkeys, causing diseases of immune organs or immune function disorder, thus interfering with experimental research.
  • HTLV-1 and STLV-1 (human T lymphocytic leukemia type 1) are inseparable in the evolutionary phylogenetic tree, with 90% to 95% homology in the nucleic acid sequence and complete serological cross-reactivity, with The HTLV-1 antigen detection test antibody of the monkey STLV-1 has been confirmed by the national standard.
  • the liquid phase chip technology is also called flexible multiple-analyte profiling.
  • CRL is the first to use luminex's xMAP technology to monitor the quality of experimental animals.
  • Luminex technology is a new high-throughput macromolecular detection platform based on flow cytometry, ELISA, chemiluminescence, and rapid signal processing technologies. It is the first FDA-approved clinical diagnosis. Biochip technology.
  • one method is to use a cell line to culture the isolated virus, and then collect the secretion as an antigen to detect the content of serum corresponding antibodies in the monkey population. Not only is the cost high and it poses a high threat to the safety of workers; the other method is to use a commercial ELISA kit to detect viral antibodies, which is also the "gold standard" for virus detection, but when there are many detection items, There are problems such as time consuming, laborious, and high cost. Therefore, the flexible application of the luminex xMAP detection technology platform to the monitoring and diagnosis of experimental animal quality can achieve high-throughput, fast, and simultaneous detection, and overcome the above two major problems.
  • the technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies in the prior art, and to provide a multi-liquid phase chip detection method and a kit capable of simultaneously detecting monkey BV, SIV, SRV and STLV antibodies, satisfying fast, accurate and high-pass. Amount of animal disease surveillance requirements.
  • a liquid phase chip kit for simultaneously detecting monkey herpesvirus, simian immunodeficiency virus, monkey D-type retrovirus and monkey T lymphocyte leukemia virus antibody comprising a coupled magnetic bead set and a detection antibody.
  • the coupled magnetic bead group is a coupled magnetic bead coupled with HSV-1 virus culture and BV-gD recombinant protein, SIV-p27 recombinant protein, SRV-p27 recombinant protein and HTLV-1envelope recombinant protein, respectively. .
  • the HSV-1 virus culture used in this study was presented by Guangzhou Lyme Biotechnology Co., Ltd.
  • the signal peptide analysis of BV gD gene was performed using the biological software SignalP 4.1Server to remove the signal peptide of gD, and the region with more epitopes was selected from 26aa to 394aa. Since the GC content of the gD gene has a GC content of more than 75% and the B virus nucleic acid is difficult to obtain, the sequence is sent to Shanghai Biotech for optimization synthesis and construction of the BV-gD-28a expression plasmid to obtain gD inclusion body protein.
  • the gD inclusion body protein was subjected to dialysis renaturation to remove chemical factors affecting the coupling of the recombinant protein and the microsphere.
  • the His tag carried in the vector facilitates the purification operation of the recombinant protein.
  • the expression conditions were as follows: the final concentration of TPTG was 1 mM, the temperature was 20 ° C, and the induction time was 24 h.
  • SIV p27 gene was selected for prokaryotic expression, and the immunogenic SIV-p27-32a recombinant protein was successfully expressed to establish a monkey immunodeficiency virus detection method.
  • the Trx tag in pET-32a vector helps the target protein to form soluble folding protein. .
  • the expression conditions were as follows: the final concentration of TPTG was 1 mM, the temperature was 37 ° C, and the induction time was 5 h.
  • SRV is a single-stranded RNA virus with cross-reactions between different serotypes, mainly occurring in the nucleocapsid protein p27 and the transmembrane protein gp20 to gp22 regions, which are highly conserved sequences in the D-type retroviral genome.
  • the prokaryotic expression vector SRV-p27-4T was constructed by using the p27 gene as a target sequence, and the recombinant SRV-p27-4T recombinant protein was successfully expressed to establish a monkey D-type retrovirus detection method.
  • the GST tag of the pGEX-4T-1 vector has the effect of increasing the solubility of the protein and reducing the formation of inclusion bodies, so that the SRV-p27-4T recombinant protein is expressed in the form of the supernatant.
  • the expression conditions were as follows: the final concentration of TPTG was 1 mM, the temperature was 20 ° C, and the induction time was 24 h.
  • the STLV Env gene encodes a glycosylation protein precursor that can be cleaved into the membrane protein gp46 and the transmembrane protein gp21. These two envelope glycoproteins determine the host range of viral infection and induce an immune response in the body. Detection of antibodies to the experimental monkey STLV-1 using HTLV-1 antigen has been confirmed by national standards. The HTLV-1 Envelop recombinant was used as the antigen coupled to the microspheres of the STLV-1 assay. The purity of the protein was as high as 95%, and the experimental results showed that the STLV positive serum had a strong immune response.
  • the detection antibody is a phycoerythrin-labeled goat anti-monkey IgG antibody (Goat anti-Rhesus monkey IgG H&L antibody).
  • the coupled magnetic beads are prepared by activating four blank magnetic beads of different numbers (28#, 35#, 43#, and 63#), resuspending and dispersing the magnetic field with a coupling buffer. Beads were added with HTLV-1 envelope recombinant protein solution, SRV-p27 recombinant protein solution, HSV-1 virus culture and BV-gD recombinant protein mixture solution, SIV-p27 recombinant protein solution, and mixed at room temperature under light. The reaction was carried out for 2 to 4 hours, and the supernatant was centrifuged to obtain 4 kinds of magnetic beads coated with different antigens, that is, coupled magnetic beads.
  • the coupling buffer is 50 to 100 mM ethanesulfonic acid (MES), and the pH is 5.0 to 6.0.
  • MES mM ethanesulfonic acid
  • blank magnetic bead activation step is as follows:
  • Magnetic bead pretreatment Take 4 blank magnetic beads with different numbers (28#, 35#, 43# and 63#), discard the supernatant, resuspend the magnetic beads with activation buffer, vortex for 10s, then Ultrasonic 10s to break up the magnetic beads, discard the supernatant after centrifugation, repeat 2 to 3 times;
  • the activation buffer was 100 mM NaH 2 PO 4 , pH 6.2; the Sulo-NHS concentration was 50 mg/mL; and the EDC concentration was 50 mg/mL.
  • the concentration of the protein solution used in the study was 1 to 20 ⁇ g/mL.
  • a method for simultaneously detecting monkey herpesvirus and three other retrovirus antibodies is characterized in that the detection is performed by using any of the above liquid phase chips, and the steps are as follows:
  • the coupled magnetic beads are bound to the antibody in the sample: the coupled magnetic beads and the pretreated sample are incubated at room temperature, protected from light and shaking for 1 to 2 hours, and washed 3 times with a washing buffer;
  • the wash buffer in the study was PBS-TBN (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% sodium azide), pH 7.4.
  • the detection method of the invention can simultaneously detect BV, SIV, SRV and STLV antibodies, and the invention has the following beneficial effects compared with the prior art:
  • the invention is based on the fluorescent microsphere immunoassay technology, and the HSV-1 virus culture and the monkey herpes virus envelope protein gD, the monkey immunodeficiency virus nucleocapsid protein p27, and the monkey type D retrovirus nucleocapsid protein p27, respectively.
  • HTLV-1 Env recombinant protein-conjugated microspheres are used as carriers.
  • Based on the single-liquid chip detection methods of BV, SIV, SRV and STLV antibodies it is possible to establish antibodies that can detect a single pathogen separately.
  • the multi-liquid phase chip detection method for detecting 4 antibodies not only provides a more effective and rapid detection method for BV, SIV, SRV and STLV antibody detection, but also saves sample size and human resources.
  • Figure 1 shows the purified recombinant protein of BV-gD-28a, in which M: Thermo pre-stained protein Maker (10-180 kDa); 1: BV-gD-28a inclusion body protein; 2: flow-through; 3-6: protein Washing solution 1-4; 7-14: protein eluent 1-8.
  • M Thermo pre-stained protein Maker (10-180 kDa); 1: BV-gD-28a inclusion body protein; 2: flow-through; 3-6: protein Washing solution 1-4; 7-14: protein eluent 1-8.
  • Figure 2 shows the results of SDS-PAGE detection of HTLV-1 envelope, HSV-1 and BV-g-28 mixtures, SIV-p27-32a and SRV-p27-4T, among which M: Thermo pre-stained protein Maker (10 ⁇ ) 180kDa); 1: HTLV-1envelope protein; 2: HSV-1 and BV-g-28 recombinant protein mixture; 3: SIV-p27-32a recombinant protein; 4: SRV-p27-4T recombinant protein.
  • M Thermo pre-stained protein Maker (10 ⁇ ) 180kDa)
  • 1 HTLV-1envelope protein
  • 2 HSV-1 and BV-g-28 recombinant protein mixture
  • 3 SIV-p27-32a recombinant protein
  • 4 SRV-p27-4T recombinant protein.
  • FIG. 3 shows the results of SDS-p27-32a recombinant protein purification by SDS-PAGE, wherein M: TAKARA low molecular weight protein standard; 1: flow through; 2 ⁇ 8: eluent 1 ⁇ 7.
  • Figure 4 shows the results of purification of SRV-p27-4T recombinant protein, wherein M: Thermo pre-stained protein Maker (10-180 kDa); 1: SRV-p27-4T supernatant protein; 2: flow-through; 3-5: protein wash Liquid 1-3; 6-12: protein eluate 1-7.
  • Figure 5 is a graph showing the optimal coating concentration of the antigen, in which the A: BV antigen optimal coating concentration results; B: SIV antigen optimal coating concentration results; C: SRV antigen optimal coating concentration results ;D: HTLV antigen optimal coating concentration results (Note: Positive: positive serum group; Negative: negative serum; SRV PC: SRV positive control).
  • Figure 6 is the best concentration test results of the secondary antibody, in which the best concentration of A: BV secondary antibody exploration results; B: the optimal concentration of SIV secondary antibody exploration results; C: the best concentration of SRV secondary antibody exploration results; D: STLV The best concentration of secondary antibody exploration results (Note: Positive: positive serum group; Negative: negative serum; SRV PC: SRV positive control).
  • Figure 7 shows the results of specific detection tests for BV, SIV, SRV and STLV liquid phase chip detection methods, wherein A: BV liquid chip detection method specifically detects test results; B: SIV liquid chip detection method specifically detects test results; C: SRV liquid phase chip detection method specific detection test results; D: STLV liquid phase chip detection method specific detection test results (Note: PC: positive control; NC: negative control; BC: blank control).
  • Figure 9 is a comparison of BV, SIV, SRV and STLV liquid protein chip detection methods and ELISA detection methods, in which A: BV liquid protein chip detection method and ELISA detection method comparison test results; B: SIV liquid protein chip detection The method compares the test results with the ELISA method; the C: SRV liquid protein chip detection method and the ELISA detection method compare the test results; D: the STLV liquid protein chip detection method and the ELISA detection method compare the test results.
  • the signal peptide sequence in BV gD was deleted, and the gene sequence corresponding to the region where the epitope was concentrated was selected from 26aa to 394aa, and the sequence was sent to Shanghai Biotech for optimization and synthesis.
  • the BV-gD-28a expression plasmid was constructed.
  • the BV-gD-28a recombinant plasmid was transformed into Transetta (DE3) competent cells (purchased from Beijing Full Gold) to obtain Transetta (DE3) Escherichia coli containing the BV-gD-28a recombinant plasmid.
  • the strain was inoculated into Luria-Bertani (LB) liquid medium containing kanamycin, and when the culture OD value reached 0.6, isopropyl thiogalactoside (IPTG) was added at a final concentration of 1 mM, 37 After incubation for 4 h at ° C, the cells were collected by centrifugation, sonicated in an ice bath, and after centrifugation again, the precipitate and the supernatant were separately subjected to SDS-PAGE to analyze the solubility of the expressed product. The results showed that the BV-gD-28a protein mainly existed in the form of inclusion bodies.
  • IPTG isopropyl thiogalactoside
  • Buffer B containing 8M urea
  • the dissolved inclusion body protein was filtered through a filter (0.45 ⁇ m) and purified by a column using a GE-filled Ni2+ affinity chromatography column (Qiagen).
  • the main steps are as follows: drain 20% ethanol solution from the purification column, rinse the column with 6 column volumes of deionized water; balance the column with 6-10 column volumes of Buffer B; BV-gD-28a
  • the inclusion body protein is added to the chromatography column and mixed with His-tag Resin for 1 to 3 hours; the lid at the bottom of the purification column is opened, and the liquid in the column is discharged under gravity to collect the flow through; 6 to 10 times
  • the column volume of the protein wash (containing 8 M urea, 20 mM imidazole) was washed with a column; finally, the protein of interest was eluted with a protein eluate (containing 8 M urea, 250 mM imidazole), and the protein eluate was collected.
  • the concentration of the target protein collected in stages was measured by a spectrophotometer and subjected to SDS-PAGE purity detection.
  • the SDS-PAGE purity test results are shown in Figure 1.
  • the medium of the refolding buffer is designated as PBS, and the appropriate concentration of urea is added for dialysis and renaturation according to the experiment.
  • the renaturation process is: 6M urea concentration of refolding solution, 4 ° C, dialysis 6 ⁇ 8h; 5M urea concentration of reconstituted solution, 4, dialysis 6 ⁇ 8h; 3M urea concentration of refolding solution, 4 ° C, dialysis 6 ⁇ 8h; 2M urea concentration of refolding solution, 4 ° C, dialysis 6 ⁇ 8h; 1M urea concentration of refolding solution, 4 ° C, dialysis 6 ⁇ 8hh; 0.5M urea concentration of refolding solution, 4 ° C, dialysis 6 ⁇ 8h ; PBS solution, 4 ° C, dialysis for 6-8 h.
  • the protein after dialysis renaturation was collected.
  • the HSV-1 virus culture was mixed with the BV-gD-28a recombinant protein, and the mixed protein solution was measured by spectrophotometer to determine the concentration of the mixed target protein, and the SDS-PAGE purity was detected.
  • the SDS-PAGE purity test results are shown in the figure. 2 is shown.
  • a pair of primers were designed to amplify the SIV-P27 gene fragment according to the SIV viral genome sequence of Genebank accession number KF051800.1, and a gene fragment of about 701 bp was amplified, and restriction enzyme sites EcoRI and XbaI were introduced, and the expression plasmid pET-32a was inserted. +) Corresponding polyclonal cleavage sites.
  • the primers were synthesized by Shanghai Biotech and the sequence is as follows:
  • the PCR reaction system was prepared according to the instructions of the TAKARA reagent Ex-tag enzyme. The reaction procedure was as follows: 94 ° C for 3 min, 1 cycle; 94 ° C for 40 sec, 55 ° C for 40 sec, 72 ° C for 60 sec, 30 cycles; and 72 ° C for 10 min.
  • the PCR product and plasmid pET-32a(+) were digested with EcoRI and XbaI restriction enzymes, respectively, and recovered using a DNA recovery kit.
  • the T4 ligase was ligated overnight at 16 ° C to transform competent DH5 ⁇ competent cells, and positive transformants were picked for PCR identification.
  • the positive transformant SIV-p27-32a-DH5 ⁇ obtained was cultured in LB medium containing ampicillin, and the recombinant plasmid SIV-p27-32a was extracted and identified by double digestion with EcoRI and XbaI endonuclease. Finally, the SIV-p27-32a recombinant plasmid which was positive by PCR and restriction enzyme digestion and which was sequenced correctly by Shanghai Yingjun Company was obtained.
  • the SIV-p27-32a recombinant plasmid was transformed into Transetta (DE3) competent cells to obtain Transetta (DE3) Escherichia coli containing the SIV-p27-32a recombinant plasmid.
  • the strain was inoculated into a liquid medium containing ampicillin LB. When the OD value of the culture reached 0.6, IPTG was added to a final concentration of 1 mM, and cultured at 37 ° C for 4 hours, the cells were collected by centrifugation, ultrasonically disrupted in an ice bath, and centrifuged again. The precipitate and the supernatant were separately subjected to SDS-PAGE to analyze the solubility of the expressed product. The results showed that the SIV-p27-32a protein mainly existed in the form of inclusion bodies.
  • the procedure was identical to the purification of the BV-gD-28a recombinant protein.
  • the concentration of the target protein collected in stages was measured by a spectrophotometer and subjected to SDS-PAGE purity detection.
  • the SDS-PAGE purity test results are shown in Figure 3.
  • the procedure was consistent with the dialysis renaturation of the BV-gD-28a recombinant protein.
  • the concentration of the protein of interest was measured by a spectrophotometer and subjected to SDS-PAGE purity detection.
  • the results of SDS-PAGE purity detection are shown in Fig. 2.
  • the SRV1 viral RNA was extracted using the US-based Viral Total RNA column extraction kit and reverse transcribed into cDNA using random primers.
  • the reverse transcription system was: dNTP (each 10 uM) 1 uL, Radom 6 mers (20 uM) 1 uL, viral RNA 8 uL, total volume 10 uL; reaction procedure: 65 ° C 5 min, 4 ° C for ever.
  • a pair of primers were designed to amplify the SRV-P27 gene fragment according to the SRV viral genome sequence in Genebank accession number M11841.1, and a gene fragment of about 650 bp was amplified.
  • the restriction enzyme sites EcoRI and Xho I were introduced and inserted into the expression plasmid pGEX-4T. The corresponding polyclonal cleavage site of -1.
  • the primers were synthesized by Shanghai Biotech and the sequence is as follows:
  • SRV-p27-R 5'-CTCGAGCATGGCTAAGCCCTGTTGAT-3'
  • the PCR reaction system was prepared according to the TAKARA reagent Ex-tag enzyme instructions. The reaction procedure was as follows: 94 ° C for 3 min, 1 cycle; 94 ° C for 40 sec, 58 ° C for 40 sec, 72 ° C for 60 sec, 35 cycles; and 72 ° C for 10 min.
  • the PCR product and plasmid pGEX-4T-1 were digested with EcoRI and Xho I restriction enzymes, respectively, and recovered using a DNA recovery kit.
  • the T4 ligase was ligated overnight at 16 ° C to transform competent DH5 ⁇ competent cells, and positive transformants were picked for PCR identification.
  • the positive transformant SRV-p27-4T-DH5 ⁇ obtained was cultured in LB medium containing ampicillin, and the recombinant plasmid SRV-p27-4T was extracted and identified by double digestion with EcoRI and Xho I endonuclease. Finally, the SRV-p27-4T recombinant plasmid which was positive by PCR and restriction enzyme digestion and which was sequenced correctly by Shanghai Yingjun Company was obtained.
  • the SRV-p27-4T recombinant plasmid was transformed into Transetta (DE3) competent cells to obtain Transetta (DE3) Escherichia coli containing the SRV-p27-4T recombinant plasmid.
  • the strain was inoculated into a liquid medium containing ampicillin LB. When the OD value of the culture reached 0.6, IPTG was added to a final concentration of 1 mM, cultured at 20 ° C for 24 hours, and the cells were collected by centrifugation, ultrasonically disrupted in an ice bath, and centrifuged again. The precipitate and the supernatant were separately subjected to SDS-PAGE to analyze the solubility of the expressed product. The results showed that the SRV-p27-4T protein mainly existed in the form of soluble protein.
  • the sonicated supernatant solution was filtered through a filter (0.45 um), and the protein was purified using Qiagen GST-tagged purification resin.
  • the main steps are as follows: drain 20% ethanol solution in the purification column, rinse the column with 6 column volumes of deionized water; equilibrate the column with 6-10 column volumes of PBS-EW solution; Add to the column, mix with GST-tag Resin for 1 ⁇ 3h; open the lid at the bottom of the purification column, let the liquid in the column flow out under gravity, collect the flow through the liquid; use 6-10 times the column volume
  • the column was washed with PBS-EW solution; finally, the target protein was eluted with TNGT solution, and the protein eluate was collected.
  • the concentration of the target protein collected in stages was measured by a spectrophotometer and subjected to SDS-PAGE purity detection (as shown in Fig. 4). Since the chemical components in the protein eluate will affect the next magnetic bead coating efficiency, the purified protein is collected and dialyzed several times with PBS to change the buffer. The target protein after dialysis was collected, and the concentration of the target protein was measured by a spectrophotometer, and subjected to SDS-PAGE purity detection (as shown in Fig. 2).
  • Example 1 Method for preparing coupled magnetic beads in a multi-liquid phase chip for simultaneous detection of monkey BV, SIV, SRV and STLV antibodies
  • BV-gD is a recombinant plasmid constructed by Shanghai Shenggong, SIV-p27 and SRV-P27.
  • the recombinant plasmid was constructed by the laboratory, and the above three recombinant plasmids were expressed by E. coli expression system.
  • the antigen information for each package is shown in Table 1.
  • the purpose of activating magnetic beads is achieved by a two-step amide reaction: first, the magnetic beads are activated by Na 2 HPO 4 buffer, Sulfo-NHS and EDC solutions, and secondly BV antigen (HSV-1 culture and BV-gD recombination) Protein mixture), SIV-p27 recombinant protein, SRV-p27 recombinant protein and HTLV-1 Env protein as antigens respectively form covalent amide bond with magnetic beads, and magnetic beads coupled with antigen are resuspended in PBS-TBN solution at 4 ° C Keep away from light.
  • the specific steps are as follows:
  • step (6) Repeat step (6) once.
  • Example 2 Using Example 1 to establish a multi-liquid phase chip detection method for simultaneously detecting monkey BV, SIV, SRV and STLV antibodies
  • the concentration of BV antigen, SIV-p27 recombinant protein, SRVp27 recombinant protein and HLTV-1env protein was measured by BCA (Thermo) protein concentration measurement method, and magnetic bead coating was performed.
  • the concentrations of different antigen types used were not the same, and the detection concentrations of BV antigens were finally determined: 5 ⁇ g, 10 ⁇ g, 15 ⁇ g, and 20 ⁇ g; SIV- The phage concentrations of p27 recombinant protein were: 1 ⁇ g, 2.5 ⁇ g and 5 ⁇ g; the detection concentrations of SRV-p27 recombinant protein were: 5 ⁇ g, 10 ⁇ g, 15 ⁇ g and 20 ⁇ g; the ticking concentrations of HTLV-1env protein were: 5 ⁇ g, 10 ⁇ g and 15 ⁇ g respectively. . 3 replicates per concentration for optimal concentration detection.
  • BV, SIV and STLV positive sera are diluted from 1:50 to 1:400; SRV PC is used directly.
  • the detection concentration of the PE-labeled goat anti-monkey IgG secondary antibody was 1 ⁇ g, and the test was achieved. Too much wasted, so the concentration of the test secondary antibody was 2 ⁇ g, 1 ⁇ g, and 0.5 ⁇ g.
  • the test was performed with PBS-TBN solution as a dilution buffer, with 3 dilutions per dilution, for optimal secondary antibody concentration detection. According to the test results (as shown in Fig. 6), and considering the P/N value and the background value of reducing the negative serum, the concentration of the PE-labeled goat anti-monkey IgG secondary antibody was determined to be 1 ⁇ g.
  • the BV, SIV, SRV and STLV positive controls of common monkey diseases were tested by the liquid phase chip detection method established by the present invention to verify the specificity of the detection method.
  • SIV, SRV and STLV single positive serum were not collected during the experimental study, so the SIV, SRV and STLV positive controls were serum positive for multiple pathogen antibodies. See Table 2 for specific information.
  • the kit used for ELISA detection is BV, SIV, SRV and STLV antibody ELISA test kits of Xishan Biotechnology Co., Ltd. (VRLChina), the detection method is operated according to the test instructions; (2) The result is OD405 ⁇ 0.3 is positive.
  • the test results showed that the BV, SIV, SRV and STLV positive control tests were positive when using the BV liquid chip detection method (Fig. 7A), which was completely consistent with the ELISA test results; SIV positive when using the SIV liquid chip test method The control results were positive, and the BV and SRV positive controls were weakly positive (Fig. 7B). The BV positive control test results showed some slight difference with the ELISA test results.
  • Fig. 7C it is completely consistent with the ELISA test results; when the STLV liquid phase chip detection method is applied, the STLV positive control test results are positive, and the SRV positive control test value is near the critical value (Fig. 7D), which is basically consistent with the ELISA test results. It is indicated that the four liquid protein chip detection methods established in this study have better specificity and almost no cross reaction with other virus positive serum.
  • the positive control was diluted with PBS-TBN solution, BV PC, SIV PC and STLV PC were double-diluted to 1:102400 from 1:50, and SRV PC was double-diluted to 1:128.
  • the liquid phase chip detection method and the ELISA kit established in the above steps 1 to 3 were used to detect the four positive control dilutions, the negative serum control and the PBS-TBN solution blank control, and the dilution was repeated 3 times for each dilution, and the MFI value and OD were recorded. Value, calculate the average.
  • the test results show that for BVPC, the antibody titer detected by the liquid phase chip of the present invention is at least 1:3200, which is consistent with the antibody titer detected by the ELISA kit; for SIV PC, the antibody titer detected by the liquid phase chip of the present invention The lowest is 1:3200, the antibody titer detected by the ELISA kit is at least 1:51200; for SRVPC, the antibody titer detected by the liquid phase chip of the invention is at least 1:8, and the antibody titer detected by the ELISA kit Consistent; for STLV PC, the antibody titer detected by the liquid phase chip of the present invention is at least 1:25600, and the antibody titer detected by the ELISA kit is at least 1:1600.
  • the above results indicate that the specific liquid phase chip prepared by the present invention is comparable to the sensitivity of the existing commercial ELISA kit.
  • the test results are shown in Table 3 and Table 4.
  • the results show that the intra-assay coefficient of variation (CV) of the method for detecting BV, SIV, SRV and STLV liquid protein chips of the present invention is 0.30% to 5.51%, and the average CV is 3.06%. 0.79% to 7.95%, average CV was 2.87%; 0.72% to 8.06%, average CV was 2.75%; 0.43% to 12.03%, and average CV was 4.77% (Table 3).
  • the inter-assay coefficient of variation (CV) was 0.48% to 18.55%, the average CV was 6.23%, 3.75% to 13.88%, the average CV was 8.69%, 4.01% to 11.03%, and the average CV was 6.84%; 2.49% to 113.60.
  • the average CV is 7.15% (as shown in Table 4).
  • the CV in the batch is not more than 10%, and the CV between batches is not more than 20%, indicating that the detection method established in this study has good repeatability.
  • Example 3 The liquid phase protein chip detection method established in Example 2 was compared with the ELISA detection method to detect the monkey field sample.
  • the BV liquid protein chip detection method was used to detect 195 clinical serum samples.
  • the ROC analysis was performed by MedCalc software. The results showed that the sensitivity of the detection method was 97.4%, the specificity was 94.9%, and the area under the curve (Area).
  • the under the ROC curve (AUC) was 99.0%; the SIV liquid protein chip detection method detected 195 clinical serum samples.
  • the ROC analysis by MedCalc software showed that the sensitivity of the detection method was 70.0%.
  • the specificity was 53.5% and the AUC was 60.1%.
  • the SRV liquid protein chip detection method detected 195 clinical serum samples.
  • the ROC analysis by MedCalc software showed that the sensitivity of the detection method was 80%. The specificity was 89.5% and the AUC was 85.6%.
  • the STLV liquid protein chip detection method detected 195 clinical serum samples.
  • the ROC analysis by MedCalc software showed that the sensitivity of the detection method was 85.7%. The specificity was 97% and the AUC was 93.8% (as shown in Table 5 and Figure 9).
  • the detection results of BV, SIV, SRV and STLV quadruple liquid protein chip detection methods show that the method has diagnostic significance, among which BV, STLV and SRV have higher diagnostic accuracy (AUC is greater than 85%); and SIV ELISA Compared with the detection results, the diagnostic accuracy of the SIV liquid protein chip detection method is poor.
  • the reason may be that the detection antigens used in the two detection methods are different, but the detection results do not necessarily indicate the SIV liquid protein detection method established by the research. Not applicable.

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Abstract

L'invention concerne un procédé et un kit de détection de plaquettes liquides multiples, en utilisant une culture virale de HSV-1 et de BV gD, SIV p27, SRV p27 et de protéines recombinantes d'enveloppe HTLV-1 en tant qu'antigènes respectifs du BV, SIV, SRV and STLV pour revêtir des billes magnétiques avec des nombres différents, en utilisant les billes magnétiques revêtues de différents antigènes en tant que porteurs, pour établir un procédé de détection de plaquettes liquides multiples capable de détecter simultanément quatre anticorps, à savoir le BV de singe et trois autres rétrovirus.
PCT/CN2018/088904 2018-03-10 2018-05-29 Procédé et kit de détection de plaquettes liquides multiples WO2019174127A1 (fr)

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CN114470183A (zh) * 2022-02-17 2022-05-13 青岛农业大学 一种右旋糖酐磁珠模拟BoHV-1病毒粒子的制备与应用

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CN111830120B (zh) * 2020-06-10 2023-06-09 北京东西分析仪器有限公司 应用质谱系统鉴定新冠病毒的试剂盒及其使用方法
CN113025753B (zh) * 2021-03-26 2023-05-26 中国人民解放军军事科学院军事医学研究院 一种猴B病毒通用型TaqMan-MGB探针实时荧光定量PCR方法

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