The development and standardization of an ELISA for ovalbumin determination in influenza vaccines

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of ovalbumin in influenza vaccines has been developed and standardized. Commercially available reagents were used. ELISA was compared to single radial immunodiffusion (SRD) and immunoelectro-osmophoresis (IEOP) techniques. The detection limit by ELISA was 0.5 ng/ml. This method was found to be at least 1000 times more sensitive than SRD and at least 200 times more sensitive than IEOP. It was concluded that ELISA is a specific, sensitive and reproducible method for the determination of the small amounts of ovalbumin found as an impurity in unconcentrated influenza vaccines.     

Enhanced chemiluminescence ELISA for the detection of antibody to hepatitis B virus surface antigen

A chemiluminescent enzyme linked immunosorbent assay (ELISA) for the detection of antibody to hepatitis B virus surface antigen (anti-HBs) in human serum has been developed. Polystyrene microtitre plates were coated with recombinant, yeast-derived hepatitis B surface antigen (rec-HBsAg). Patient serum samples and appropriate controls were added to the rec-HBsAg-coated wells and incubated to bind anti-HBs. The wells were then washed and a fluorescein isothiocyanate (FITC) conjugate of a human plasma-derived hepatitis B surface antigen (HBsAg) was added. Following incubation and further washing the bound FITC-labelled HBsAg was detected after addition of a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody and assaying for the enzyme. The activity of the HRP was measured using luminol and hydrogen peroxide as substrates and iodophenol as a chemiluminescence enhancer. The luminescence was recorded using a camera luminometer. Preliminary tests have shown the assay to be suitable for the detection of antibody in sera from both vaccinees and also from individuals with a past hepatitis B virus infection. The use of the FITC-anti-FITC system together with the measurement of a chemiluminescence signal makes possible the completion of this assay in a few hours. The assay has been shown to be both specific and sensitive and provides a permanent photographic record.     

Follow-up of neurocysticercosis patients after treatment using an antigen detection ELISA

Seven patients with active neurocysticercosis (NCC) received an eight days treatment with albendazole and were followed up using computed tomography (CT-scan) and a monoclonal antibody based ELISA for the detection of circulating antigen (Ag-ELISA). Only three patients were cured as was shown by CT-scan and by the disappearance of circulating antigens one month after treatment. After a second course of albendazole therapy, two other patients became seronegative. CT-scan showed the disappearance of viable cysts in all persons who became seronegative whereas patients who were not cured remained seropositive. These preliminary results show that this Ag-ELISA is a promising technique for monitoring the success of treatment of NCC patients because of the excellent correlation between the presence of circulating antigens and of viable brain cysts.     

应用不同代次兔肾细胞培养风疹病毒松叶株的研究

目的为提高兔肾细胞产量用于增加风疹病毒生产。方法 SPF级家兔肾细胞经传代培养10代,对不同代次兔肾细胞进行遗传稳定性、外源因子及兔脑原虫检测后接种风疹病毒松叶株(Matsuba strain)的试验。结果兔肾细胞产量得到提高,由1对兔肾平均生产1瓶细胞增加为8瓶,细胞核型检查传至第10代的细胞染色体数目与初代细胞一致,细胞培养物均一性提高。第0～第3代细胞病毒培养物滴度间无显著性差异,χ2检验返回相关性的P值均大于0.95。结论第1～第3代细胞培养风疹病毒与第0代相比,可获得相同滴度的病毒培养物,p3代兔肾细胞应用于疫苗生产可显著提高细胞及病毒产量。     

Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus

An indirect enzyme-linked immunosorbent assay (ELISA) method based on a novel multi-epitope antigen of S protein (SE) was developed for antibodies detection against infectious bronchitis virus (IBV). The multi-epitope antigen SE protein was designed by arranging three S gene fragments (166–247 aa, S1 gene; 501–515 aa, S1 gene; 8–30 aa, S2 gene) in tandem. It was identified to be approximately 32 kDa as a His-tagged fusion protein and can bind IBV positive serum by western blot analysis. The conditions of the SE-ELISA method were optimized. The optimal concentration of the coating antigen SE was 3.689 μg/mL and the dilution of the primary antibodies was identified as 1:1000 using a checkerboard titration. The cut-off OD₄₅₀ value was established at 0.332. The relative sensitivity and specificity between the SE-ELISA and IDEXX ELISA kit were 92.38 and 89.83%, respectively, with an accuracy of 91.46%. This assay is sensitive and specific for detection of antibodies against IBV.     

Production of monoclonal antibodies against grouper nervous necrosis virus (GNNV) and development of an antigen capture ELISA

Five (2 IgG, 3 IgM) monoclonal antibodies (MAbs) against the G9508KS strain of grouper nervous necrosis virus (GNNV) were produced and characterized. All 5 MAbs showed positive signals in the retina of GNNV-infected grouper larvae and in the cytoplasm of GNNV-infected GF-1 cells using immunohistochemistry staining. Two MAbs reacted with the denatured capsid protein derived from GNNV-infected GF-1 cells in Western blot analysis, but did not react with the GNNV recombinant capsid protein expressed by E. coli in an indirect immnunosorbent assay (ELISA). All 5 MAbs were able to neutralize GNNV, tiger puffer NNV (TPNNV) and barfin flounder NNV (BFNNV), while only 2 of the MAbs neutralized striped jack NNV (SJNNV). A capture ELISA system based on the use of MAbs for capture and a rabbit polyclonal antibody for detection was developed. When absorbance values higher than 0.5 were judged to be positive, the sensitivity of the capture ELISA system was 2.5 ng per well of purified GNNV protein or 6.5 x 10(4) TCID50 per well of GNNV supernatant from culture cells. This capture ELISA system could become a more specific and sensitive tool for NNV diagnosis in the field and in routine laboratories.     

The distribution of poplar mosaic virus in hybrid poplars and virus detection by ELISA

Purified poplar mosaic virus (PMV) at a concentration of 8 ng/ml was readily detected by enzyme-linked immunosorbent assay (ELISA). Bioassay in Nicotiana megalosiphon was more sensitive (detecting 1–4 ng/ml) and latex flocculation less sensitive (c. 25 ng/ml) than ELISA assays. While the foliar sap of fresh, naturally-infected poplars (e.g. Populus. euramericana cv. Robusta) was not infective at dilutions greater than 2 . 10–², ELISA easily detected PMV antigen when sap was diluted 4 . 10–³ in buffer or when one part of infected tissue was triturated with 99 parts healthy leaf. Furthermore, although sap from poplar leaves stored at -20 °C for 6 months was not infective, PMV was still detectable in ELISA tests. PMV antigen in poplar leaves was not all pelleted after centrifugation for 2.5 h at 130 000 g yet parallel tests using unbuffered sap from systemically infected Nicotiana megalosiphon foliage showed that infectivity was restricted to the pellet. In poplar foliage, the concentration of PMV antigen was generally greatest where symptoms were most obvious; least antigen was detected in the overwintering leaves located at the bases of long shoots. In winter, when root and inner bark tissue in the trunk was an erratic source of PMV, the virus was readily detected in buds, the concentration being greatest in the bases, including the meristem, of terminal buds. Propagation from single node cuttings of P. euramericana cv. Regenerata allowed the selection of clones that consistently showed either ‘severe’ or ‘mild’ foliar symptoms. The associated virus isolates also infected another poplar clone causing symptoms characteristic of their source. ELISA consistently detected less PMV antigen in field-grown cv. Regenerata than in cv. Robusta foliage, but this was reversed when the associated virus isolates were propagated in Nicotiana glutinosa at 24 °C. During 6 yr, 21 out of 127 poplars at a site in Western England, became infected with PMV. By contrast, in Eastern England, none of 46 were infected. The aphids Pterocomma populea and Myzus persicae did not transmit PMV.     

The development and standardization of an enzyme-linked immunosorbent assay for the detection of antibodies to bovine herpesvirus 2

A single dilution enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine herpesvirus 2 has been developed, standardized and compared with the virus neutralization test. The results of the two tests correlated well. A positive/negative threshold was established for the ELISA. The ELISA was reproducible, sensitive, rapid and specific.     

Studies on the development of an ELISA kit for microbiological monitoring. 2. Improvement of the prototype ELISA kit with special references to mouse hepatitis virus antigen

Improvement of the mouse hepatitis virus (MHV) antigen in a prototype ELISA kit was performed. Equivalent divalent antigens of MHV Nu-67 and S strains with a protein concentration of 10 micrograms/ml showed the best sensitivity and specificity for the detection of MHV and sialodacryoadenitis/Parker's rat coronavirus antibodies in mice and rats, respectively. An increase in the reliability of macroscopic evaluation of both antibody tests is expected by using the newly manufactured kit with the improved antigen.     

Expression of recombinant capsid proteins of chitta virus, a genogroup II Norwalk virus, and development of an ELISA to detect the viral antigen

The second open reading frame (ORF2) gene of the Chitta virus (CHV) was cloned to construct a recombinant baculovirus. The CHV ORF2 is predicted to encode a capsid protein of 535 amino acids (aa). CHV showed a high aa identity in the capsid region with genogroup II Norwalk virus (NV) (65-85%), but a low aa identity with genogroup I NV (44-46%). Phylogenetic analysis of the ORF2 gene demonstrated that CHV is genetically closely related to the Hawaii virus included in genogroup II NV. The recombinant capsid protein of CHV (rCHV) self-assembled to form empty virus-like particles (VLPs) when expressed in insect cells with the recombinant baculovirus. An enzyme-linked immunosorbent assay (ELISA) based on antisera to rCHV was developed to detect CHV antigen in stools. The antigen ELISA appeared to be highly specific to both rCHV and CHV-like strains. In addition, combined use of antigen ELISAs using antibodies against two antigenically distinct recombinant VLPs, the recombinant Chiba virus (rCV) and recombinant Seto virus (rSEV), enabled us to determine the genetic as well as antigenic relationship among these three viruses.