Use of ELISA forin vitroPotency Test of Rabies Vaccines for Animal Use

Gamoh, Koichiro; Senda, M.; Itoh, Osamu; Muramatsu, Masatake; Hirayama, Norio; Koike, Ryuji; Endoh, Y. S.; Minamoto, Nobuyuki

doi:10.1006/biol.1996.0012

Cited by 34 publications

(18 citation statements)

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“…The serum obtained showed a neutralizing antibody titer of [5,13,18,20,23,25]. ELISA in particular is an easy to use and highly sensitive method.…”

Section: Purification Of Rvmentioning

confidence: 95%

“…Examples of the former are plaque assay [2,15,24], TCID 50 method, rapid fluorescent antigen test (RFAT) [1] and immunoperoxidase test [17]. Antigen assays include hemagglutination test [7], hemadsorption test [16], singleradial-immunodiffusion and enzyme-linked immunosorbent assay (ELISA) [5,13,18,20,23,25]. ELISA in particular is an easy to use and highly sensitive method.…”

Section: Purification Of Rvmentioning

confidence: 99%

“…This method was carried out according to the protocol of Gamoh et al [5] except that the samples used were culture supernatants and were not gel filtrated to eliminate Gs protein.…”

Section: G-elisamentioning

confidence: 99%

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A New Quantitative Method for Rabies Virus by Detection of Nucleoprotein in Virion Using ELISA.

Katayama

Yamanaka

Ota

et al. 1999

The Journal of Veterinary Medical Science

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ABSTRACT. We have developed a new quantitative method for rabies virus (RV) detection using enzyme-linked immunosorbent assay (ELISA). The method named N-ELISA was based on the quantitation of nucleoprotein (N) in RV virions captured by RV-specific polyclonal antibodies on an ELISA plate. Both infective and defective interfering (DI) particles of RV could be detected by this method. When viruses were propagated in a medium of pH 7.4 adjusted with 7% NaHCO 3 , N-ELISA could detect them with titers of more than 10 6 pfu/ml, though the result did not correlate highly with that of the infectivity assay. The reason for this was considered to be that RVs included spikeless and damaged particles which were produced under conditions of low or high pH. However, in the time course of virus yield, titers of N-ELISA correlated well with those of the infectivity assay.-KEY WORDS: ELISA, G protein, N protein, rabies virus.J. Vet. Med. Sci. 61(4): 411-416, 1999 HmLu-1 cells were cultured in Eagle's minimum essential medium (Eagle's MEM) supplemented with 5% fetal calf serum (FCS).Virus propagation: RV was inoculated on HmLu-1 cells at a m.o.i. (multiplicity of infection) of 0.01 pfu/cell. After adsorption for 1 hr at 37°C, the cells were added Eagle's MEM of pH 7.4 supplemented with 1% FCS. The pH of the medium was adjusted with 7% NaHCO 3 . The cells were cultured at 34°C without CO 2 .In the test of virus-growth, the pH of the medium was controlled by addition of 20 mM HEPES buffer (pH 6.5, 7.0 or 7.5) into Eagle's MEM supplemented with 1% FCS and 1.4 mM NaHCO 3 . After adsorption of the virus, the cells were added to each medium and incubated at 34°C in air. Ten ml aliquots of culture fluids were sampled every day for 6 days, and their pH was measured. The samples were concentrated to 10 µl by ultracentrifugation (100,000 × g, 1 hr, 4°C). Purification of RV:Purified RV was prepared according to the method of Kawai et al. [10]. The fractions with infective RV were collected as purified virion and then dialyzed against PBS.Antibodies: To prepare an anti-N serum, N protein derived from purified RV was resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The band of N protein in the gel was cut, and then homogenized in PBS. The homogenate was emulsified with oil-adjuvant ISA70 (Seppic, France) at a ratio of 3:7. Three guinea pigs were injected intramuscularly three times with the emulsion at three week-intervals. One week after final injection, the animals were bled to collect sera. Antibody titers of the sera measured by the indirect fluorescent antibody method were 1,280 to 2,560.To prepare RV-specific antiserum, two rabbits were immunized with an emulsion of purified RV and oiladjuvant ISA70 three times at three week-intervals. The serum obtained showed a neutralizing antibody titer of [5,13,18,20,23,25]. ELISA in particular is an easy to use and highly sensitive method.Several investigators have developed ELISA methods for the quantitation of RV glycoprotein (G) [13,25]. These methods have been used to test in...

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“…The serum obtained showed a neutralizing antibody titer of [5,13,18,20,23,25]. ELISA in particular is an easy to use and highly sensitive method.…”

Section: Purification Of Rvmentioning

confidence: 95%

Section: Purification Of Rvmentioning

confidence: 99%

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A New Quantitative Method for Rabies Virus by Detection of Nucleoprotein in Virion Using ELISA.

Katayama

Yamanaka

Ota

et al. 1999

The Journal of Veterinary Medical Science

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“…Though some laboratories have used enzyme-linked immunosorbent assay (ELISA) to assess RV GP content for determination of the potencies of inactivated vaccines, variable correlation between ELISA and the NIH test (7,8,9,13,14,17,18,20) has been reported. Essentially, all these ELISAs incorporate the use of either polyclonal antibodies or hybridomaderived monoclonal antibodies (MAbs).…”

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confidence: 99%

Recombinant Diabody-Based Immunocapture Enzyme-Linked Immunosorbent Assay for Quantification of Rabies Virus Glycoprotein

Nimmagadda

Aavula

Biradhar

et al. 2010

Clin Vaccine Immunol

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The potency of rabies vaccines, determined using the NIH mouse protection test, can be directly correlated to the amount of rabies virus glycoprotein (RV GP) present in the vaccine. In an effort to develop a simple and sensitive enzyme-linked immunosorbent assay (ELISA) using recombinant diabody for quantification of RV GP, the variable heavy (V H ) and light chain (V L ) domains of an RV GP-specific human monoclonal antibody (MAb) secreted by a human ؋ mouse heterohybridoma (human MAb R16E5) was amplified, linked using splicing by overlap extension PCR (SOE PCR), and expressed as a recombinant diabody (D06) in the pET28a bacterial expression system. The diabody D06 was purified by immobilized metal affinity chromatography on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The purified diabody was used in combination with a well-characterized RV GP-specific mouse MAb, M5B4, to develop an immunocapture ELISA (IC-ELISA) for the quantification of RV GP in human rabies vaccine preparations. The maximum detection limit of the IC-ELISA using the M5B4-D06 combination was up to 31.25 ng/ml of RV GP. The specificity of the diabody was established by its nonreactivity toward other human viral antigens as determined by ELISA and toward RV GP as determined by immunoblot transfer assay and competitive ELISA with the parent human MAb R16E5 and MAb M5B4. The adjusted r 2 value obtained by the regression through the origin model was 0.902, and the equation for predicted potency values for M5B4-D06-based IC-ELISA and MAb M5B4 IC-ELISA were 0.5651x and 0.8044x, respectively, where x is the estimate of RV GP from the IC-ELISA in micrograms. Analysis of variance (ANOVA) results showed the estimates of the two methods differed significantly (P < 0.001), while the predicted potencies by the two tests did not differ significantly (P > 0.05). The IC-ELISA can be readily adapted to measure the RV GP content in purified antigen, and a vaccine can be formulated based on the estimated GP.Rabies is a fatal viral infection of the nervous system affecting all mammals, including humans through bite wounds from a rabid animal, which can be prevented by vaccination coupled with administration of anti-rabies virus serum (6, 11). Rabies transmission from nonbite exposures is rare. Scratches, abrasions, open wounds, or mucous membranes contaminated with saliva or other potentially infectious material (such as brain tissue) from a rabid animal constitute nonbite exposures. Occasionally reports of nonbite exposure are such that postexposure prophylaxis is given. Inhalation of aerosolized rabies virus is also a potential nonbite route of exposure, but with the exception of laboratory workers, most people are unlikely to encounter an aerosol version of the rabies virus (5). Organ transplantations have also been credited with nonbite transmission of rabies from human to human (3). Despite significant scientific progress, rabies remains an important zoonotic disease globally. Annually, 20,000 deaths are reported in India, making ra...

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“…The results were analyzed to determine relative sensitivity and specificity, predictive values and accuracy (Gamoh et al 1996, Rooijakkers et al 1996, Strady et al 2000. Sensitivity was defined as the proportion of positive results obtained by standard technique that were correctly identified by the two serological methods applied in this study.…”

mentioning

confidence: 99%

Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

Cardoso¹,

Silva²,

Albas³

et al. 2004

Mem. Inst. Oswaldo Cruz

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Use of ELISA forin vitroPotency Test of Rabies Vaccines for Animal Use

Cited by 34 publications

References 0 publications

A New Quantitative Method for Rabies Virus by Detection of Nucleoprotein in Virion Using ELISA.

A New Quantitative Method for Rabies Virus by Detection of Nucleoprotein in Virion Using ELISA.

Recombinant Diabody-Based Immunocapture Enzyme-Linked Immunosorbent Assay for Quantification of Rabies Virus Glycoprotein

Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

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