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CN-121975869-A - Method for detecting neutralizing antibody of animal anti-rabies virus

CN121975869ACN 121975869 ACN121975869 ACN 121975869ACN-121975869-A

Abstract

The invention belongs to the technical field of biological detection, provides a method for detecting an animal anti-rabies virus neutralizing antibody, discloses a replication-defective recombinant human adenovirus type 5 (Ad 5-RABV-GG) for expressing rabies virus disaccharide protein, and has the advantages of high virus titer, wide coverage and the like. The invention also discloses an improved fluorescent antibody virus neutralization test detection method (IFAVNT), which solves the problem that the rabies strong virus CVS strain in the traditional FAVNT method can be detected only by a three-stage biosafety laboratory, improves the safety of the experiment, reduces the threshold and the requirement of rabies virus neutralization antibody detection, and has the advantages that the positive and negative coincidence rates of the IFAVNT method and the ELISA kit detection result are 100%, the sensitivity is high, and the cost is low, so that the method can be used for detecting animal rabies virus neutralization antibodies.

Inventors

  • ZHAO HONGJIN
  • SHENG WENWEI
  • CHEN QI
  • XIA LUMING
  • CHANG XIAOJING
  • LU JUN
  • TANG CONGSHENG
  • LIU JIAN
  • XIA QIQI
  • GUI YAPING
  • JIAO YUZHOU
  • WANG JIAN
  • YANG XIANCHAO
  • Feng Guidan
  • XIANG MENG

Assignees

  • 上海市动物疫病预防控制中心(上海市兽药饲料检测所、上海市畜牧技术推广中心)

Dates

Publication Date
20260505
Application Date
20260127

Claims (8)

  1. 1. The preparation method of the replication-defective recombinant human adenovirus 5 is characterized by comprising the steps of selecting a double-copy G protein mode to replace E1 and E3 genes of the human adenovirus 5 respectively, synthesizing a rabies virus Shanghai isolate RVSH strain G gene and a rabies virus Shanghai isolate RVSH strain G gene, loading the rabies virus Shanghai isolate RVSH strain G gene into an Ad5 replication-defective adenovirus vector to obtain a recombinant adenovirus plasmid, co-transfecting 293A cells with an adenovirus shuttle plasmid cloned with an exogenous gene and a skeleton plasmid carrying most of adenovirus genome through a AdMax adenovirus packaging system, and collecting cell supernatants, namely the replication-defective recombinant human adenovirus Ad5-RABV-GG of rabies virus disaccharide protein when 80% of cells have pathological effects.
  2. 2. A replication-defective recombinant human adenovirus type 5 expressing rabies virus disaccharide protein, characterized by being produced by the production method of claim 1.
  3. 3. The method of claim 2, wherein the method of identifying replication-defective recombinant human adenovirus type 5 comprises, but is not limited to, PCR, IFA, electron microscopy, and Western blot.
  4. 4. A method for detecting an animal anti-rabies virus neutralizing antibody, said method comprising in particular: (1) The Ad5-RABV-GG is prepared by amplifying and culturing the Ad5-RABV-GG virus prepared in claim 2, filtering after checking to confirm sterility, obtaining the Ad5-RABV-GG virus liquid with cells and fragments removed, and low-temperature preserving for standby after measuring the virus titer; (2) Collecting and treating animal serum to be detected, namely aseptically collecting animal blood, standing at room temperature or 37 ℃ for 1h, then standing at 4 ℃ for 1-24 hours, centrifugally collecting supernatant, and inactivating at 56 ℃ for 30 min later use; (3) The method comprises the steps of diluting the Ad5-RABV-GG virus with a measured concentration to 200 TCIDs 50 , treating 30 min in a 56 ℃ water bath after diluting serum to be detected for 20 times, then carrying out 2 -1 ～2 -11 times serial dilution, mixing the serial diluted serum and the Ad5-RABV-GG virus according to the volume of 1:1, incubating to 37 ℃ for 1.5 h, adding 100 mu L of the incubated mixed solution into a 96-well plate, adsorbing 1.5 h at 37 ℃, removing the supernatant, washing 2 times by using DMEM, adding a maintenance solution, continuously culturing 5 d in a 37 ℃ CO 2 incubator, carrying out IFA detection on the 96-well plate, and recording the serum dilution corresponding to the negative result, namely the maximum serum dilution capable of neutralizing virus strains, wherein the negative result is negative result when no fluorescence is generated; (4) Positive judgment, namely judging that the maximum dilution multiple of serum is more than or equal to 40 as positive.
  5. 5. The method of claim 4, wherein the centrifugation parameter is 6 000 r/min centrifugation 5 min.
  6. 6. The method of claim 4, wherein the maintenance fluid is dmem+2% FBS.
  7. 7. Use of the method of preparation according to claim 1 or the replication defective recombinant human adenovirus type 5 according to claim 2 for the detection of animal anti-rabies virus neutralizing antibodies.
  8. 8. Use of the method according to claim 4 for detecting animal anti-rabies virus neutralizing antibodies.

Description

Method for detecting neutralizing antibody of animal anti-rabies virus Technical Field The invention belongs to the technical field of biological detection, relates to application of replication-defective recombinant human adenovirus type 5 Ad5-RABV-GG, and in particular relates to a method for detecting an animal anti-rabies virus neutralizing antibody. Background Rabies is one of the oldest diseases which endanger human health, is caused by rabies virus, is an acute infectious disease with the highest death rate of human, is one of epidemic diseases which cannot be effectively controlled by human, and rabies vaccine is the only effective method for preventing rabies. Detection of antibodies in serum often evaluates the immune effects of rabies vaccine or monitoring of antibody levels after rabies infection, and can also be used in epidemiological investigation, including mouse neutralization assay (mice neutralizing test, MNT), rapid fluorescence focus inhibition assay (rapid fluorescent focus inhibition test, RFFIT), fluorescent antibody virus neutralization assay (fluorescent antibody virus neutralization test, FAVNT), enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA), and the like. MNT was established by Webster and Dawson in 1935, and developed later, but has not been recommended by WHO and WOAH. Its advantage is quantitative detection of the neutralizing antibody titer of RABV. The method has the defects of (1) high cost, more test mice, (2) long time, two weeks for obtaining results, unfavorable for rapid diagnosis, (3) complex operation, special technical training, and (4) influence on the test results due to individual differences among animals. RFFIT is a standard procedure recommended by the WHO rabies expert committee, commonly used to evaluate the content of RABV neutralizing antibodies. The RFFIT only needs 20 h in the whole course, the sensitivity is slightly higher than MNT, and the RFFIT is an alternative test of MNT. However, laboratory personnel need to be trained, cell culture and fluorescence microscopy equipment, and have sufficient safety facilities to handle live viruses. RFFIT is sensitive to cytotoxicity in poor quality serum, where unspecific viral inhibitors may produce false positive results. FAVNT established in 1998 that the results correlated well with RFFIT and MNT are WOAH-recognized methods. The method can be used for tracking and investigating orally-immunized animals, but cells are sensitive to toxins and can generate false positives due to poor quality of serum collected in the field. Muhamuda et al established ELISA using monoclonal antibodies directed against RABV N protein and G protein to detect specific immune complexes in human CSF. The second generation ELISA detects G protein antibodies in human serum or cerebrospinal fluid, and the method has been evaluated by multiple centers, has good correlation with RFFIT, and can be used in laboratories without virus and cell culturing equipment. ELISA was less sensitive than the virus neutralizing antibody assay. Nishizono et al used the principle of immunochromatography for qualitative and semi-quantitative tests of neutralizing antibodies in humans or dogs, established a rapid neutralizing antibody detection test (RAPINA). Nishizono and the like prove that compared with virus neutralization tests and ELISA, the method is simple and quick, has better positive and negative predictive values, is suitable for screening large-scale specimens, and is recommended by WHO and WOAH. As early as 2003, madhusudana et al established a rapid, simple latex agglutination method for detecting RABV-specific antibodies. Adenovirus as an effective tool for gene transfer and expression satisfies a plurality of conditions as a recombinant vector, and is currently used in a large number of fields of vaccine development, clinical gene therapy and neutralizing antibody detection, wherein the most widely studied application is human adenovirus type 5 (Ad 5). Rabies virus glycoprotein is the only protein exposed on the surface of the virus particle and is the only virus antigen capable of inducing the organism to generate protective neutralizing antibodies. Wright et al in 2008 proposed that pseudo-lentiviral vector is used to detect the level of neutralizing antibody of RABV, the pseudo-lentiviral vector is the core where the genome carrying one virus is located and the envelope of the other virus, and the method has high safety and can be used as a substitution test for detecting rabies virus. The human adenovirus has the advantages of wide cell types infected by the adenovirus, light clinical symptoms, few naturally occurring antibodies in animals, large exogenous gene capacity and the like, and is widely used for vaccine development, clinical gene therapy and detection and research of neutralizing antibodies. The Ad5-RABV-GG belongs to recombinant replication defective human adenovirus type 5 live vector virus, and