Production of subgroup-specific monoclonal antibodies against human rotaviruses and their application to an enzyme-linked immunosorbent assay for subgroup determination

Nonneutralizing monoclonal antibodies were prepared against two strains, S2 and YO, of human rotaviruses isolated in cell culture. S2-37 and YO-5 antibodies had subgroup I and subgroup II specificities, respectively. The remaining antibodies (S2-65, YO-71, YO-89, and YO-156) reacted commonly with all the rotaviruses examined. All of the monoclonal antibodies agglutinated exclusively single-shelled particles and immunoprecipitated 42,000-dalton protein, a major component of inner capsid. Using the three monoclonal antibodies (S2-37, YO-5, and YO-156), an enzyme-linked immunosorbent assay was developed for detecting and subgrouping human rotavirus isolates.     

Development of an enzyme-linked immunosorbent assay for octylphenol

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative analysis of octylphenol. A linear carboxylated analogue of 4-nonylphenol was synthesised and characterised as hapten. The hapten was coupled to the carrier protein to produce a specific antibody. An indirect competitive ELISA method was established based on the polyclonal antibody, which exhibited an IC₅₀ value of 51 ng/mL for octylphenol. A monoclonal antibody was produced with an IC₅₀ value of 76 ng/mL for octylphenol. The immunoassay used with the monoclonal antibodies was applied to analyse water samples from Tai Lake in China.     

Factors influencing the sensitivity of herpes simplex virus detection in clinical specimens in a simultaneous enzyme-linked immunosorbent assay using monoclonal antibodies

A rapid simultaneous enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was investigated for herpes simplex virus (HSV) detection. All HSV isolated (n = 127) were detected, whereas no response was obtained with HSV negative preparations. Equivalent results were obtained from 275 of 277 clinical specimens in the monoclonal ELISA and in an ELISA using polyclonal antibodies, confirming that appropriately selected monoclonal antibodies may be as efficacious as polyclonal antibodies in antibody-based assays. In clinical specimens, the rate of HSV detection (sensitivity) relative to tissue culture isolation was low for both assays, and the major factor responsible for this was the low concentration of virus present in some specimens. The sensitivity of ELISA obtained in routine use varied with different panels of unselected specimens and was related to the speed of development of the cytopathic effect. These results emphasise the need for caution in assigning a definitive sensitivity level to ELISA tests evaluated on different panels of specimens.     

Detection of herpes simplex virus type-specific antibodies by an enzyme-linked immunosorbent assay based on glycoprotein G

In order to develop a simple and quantitative method to detect herpes simplex virus (HSV) type-specific antibodies, the usefulness of an enzyme-linked immunosorbent assay (ELISA) using HSV glycoprotein G (gG) captured on a plate by monoclonal antibodies as antigen was studied. The gG1- and gG2-specific IgG antibody activities were measured by the ELISA for 54 sera which had been collected from culture-proven genital herpes patients and pre-characterized by an immunodot assay using purified gG antigens. Thirty control sera without antibodies against the HSV whole antigens were also included. In comparison with the immunodot assay as standard, the sensitivities of the ELISA were 88.9% (32/36) for HSV-1 antibody and 89.2% (33/37) for HSV-2 antibody and the specificities were both 100%. Sera taken within a few months after primary infection tended to give false negative results. The HSV type-specific ELISA based on easy-to-prepare gG antigens might be useful to help improve the serological assessment of HSV infections. J. Med. Virol. 53:319–323, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of monoclonal antibodies to Marburg virus (strain Musoke) glycoprotein and identification of two protective epitopes

Monoclonal antibodies (MAbs) reactive with Marburg virus (strain Musoke) were evaluated for both biological activity and specificity. Several of the Marburg virus- (MBGV) specific MAbs reduced the size and/or number of MBGV plaques in vitro. The ability of the MAbs to affect plaque formation in vitro was demonstrated to be specific for the glycoprotein (GP) of the strain of MBGV used for vaccination. Using deletion analysis and peptide mapping, the binding epitopes of several of these neutralizing MAbs were identified. Not unexpectedly, the epitopes were shown to lie in the most hypervariable and highly glycosylated region of MBGV GP. An analysis of the in vivo activity of several MAbs revealed that some antibodies provided substantial but incomplete protection of naive guinea pigs by passive transfer. These data suggest that neutralizing epitopes exist within MBGV GP but that induction of antibodies to these neutralizing epitopes may not be sufficient for protection from lethal infection.     

A highly sensitive enzyme-linked immunosorbent assay to quantitate antibodies to Epstein-Barr virus membrane antigen gp340

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of antibodies to Epstein-Barr (EB) virus membrane antigen (MA) glycoprotein, gp340, in tamarins. The assay was found to be a thousand-fold more sensitive than conventional indirect immunofluorescence tests and consequently it was possible to follow accurately the sequential production of specific antibodies to gp340 by tamarins during a course of immunization.     

Enzyme-linked immunosorbent assay (ELISA) for mumps virus antibodies

An ELISA for the detection of mumps virus-specific IgG and IgM antibodies was developed. Three different antigen preparations were compared. Equally good results were obtained with virus concentrated by ultracentrifugation and with virus that was further purified by sucrose gradient centrifugation. Crude infected allantoic fluid was unsuitable for use as antigen. The variability and reproducibility of the tests were satisfactory. When the ELISA was compared to conventional serological methods, a good correlation of IgG absorbance values with complement fixation (CF) antibody titers was found (r = 0.574), the ELISA being more sensitive in detecting antibodies in acute-phase sera. For the determination of immunity, ELISA IgG values were compared with results obtained in hemagglutination-inhibition (HI) and hemolysis-in-gel (HIG) tests. Again there was a good correlation with both tests (r_HI = 0.528, r_HIG = 0.667). The ELISA was more sensitive than the HI and HIG test for the detection of low levels of antibodies.     

Use of monoclonal antibody in an ELISA test for the detection of antibodies to bovine leukaemia virus

A variant of the ELISA technique, involving a monoclonal anti-gp51 antibody yields a highly sensitive method for the detection of bovine leukaemia virus (BLV) antibodies. The gp51 antigen-coated microtitre plates are obtained by incubation of plastic-adsorbed monoclonal antibodies with a non-purified mixture of BLV antigens. Sera to be tested are incubated in the wells of the gp51-coated plates and bound antibodies are revealed by an enzyme-linked antibovine immunoglobulin reagent. This test is as sensitive as liquid phase radioimmunoassay using the same gp51 antigen and thus appears as a highly sensitive, practical, rapid and cheap method for the detection of BLV antibodies.     

A highly sensitive enzyme-linked immunosorbent assay for idiotype-bearing antibodies

A sensitive and specific immunoenzyme assay (ELISA) for quantitation of total and cross-reactive idiotype-bearing (CRI) anti-ABA antibodies is described. Total anti-ABA antibodies are directly assessed in ABA-BGG coated polyvinyl wells with enzyme-labelled rabbit anti-mouse immunoglobulins. By interpolation on a standard curve absorbance values give the concentration of anti-ABA antibodies with a sensitivity of 30 ng/ml. CRI+ antibodies are quantitated by inhibition of enzyme-labelled monoclonal CRI+ antibody binding to solid-phase coated rabbit anti-CRI immunoglobulins. The concentration of CRI+ antibodies, evaluated by interpolation on a standard inhibition curve, can be measured at the level of 10 ng/ml. This highly sensitive, rapid, specific and reproducible assay is easily used, with minor modifications, to detect specific antibodies in any idiotype system.     

Use of monoclonal antibody in a blocking ELISA to detect group specific antibodies to bluetongue virus

An enzyme-linked immunosorbent assay (ELISA) in the form of a blocking test is described for the detection of group specific antibodies to bluetongue virus (BTV). The test relies upon interruption of the reaction between BTV antigen and a group specific murine monoclonal antibody against BTV by addition of serial dilutions of bovine or ovine test sera containing specific antibodies to BTV which inhibit binding of the monoclonal antibody to the BTV antigen. This is detected as a reduction in the optical density (O.D.) reading obtained with the monoclonal antibody alone. The test is capable of specific detection of antibodies to all 22 serotypes of BTV but, unlike the agar gel precipitin (AGP) test, does not show cross-reactions with antibodies to epizootic haemorrhagic disease of deer (EHD) viruses. Furthermore, antibodies to cellular proteins which complicate interpretation of the AGP test and the indirect ELISA are not detected in the blocking ELISA. The high sensitivity and specificity of the blocking ELISA make it an ideal alternative to the AGP test. The use of a monoclonal antibody would facilitate standardisation of diagnostic testing between laboratories.     

Rapid enzyme-linked immunosorbent assay of whole blood for detection of antibodies to japanese encephalitis virus

The application of the rapid system of enzyme-linked immunosorbent assay (ELISA) was studied to quantify antibodies to Japanese encephalitis virus in large-scale epidemiological surveys, especially by testing under field conditions. The assay system, with 15 min for the first reaction and 30 min each for the second and the third reactions, was highly reproducible (coefficients of variation with swine positive sera were less than 5.8%) and was significantly correlated with the routine assay system with 1 h for each reaction (correlation coefficient was 0.960). Compared with the haemagglutination inhibition test, the rapid system gave a correlation coefficient of 0.916 and qualitative agreeement of 96.1%. The substitution of whole blood for serum in the first reaction was also examined not only to avoid serum separation but also to apply this system to antibody quantification in animals from which sufficient amounts of sera cannot be easily obtained: only 2 μl were needed for the test. The results obtained with 51-fold diluted whole blood had a linear relationship to those obtained with 100-fold diluted sera in swine and humans.     

Characterization and potential diagnostic application of monoclonal antibodies specific to rabies virus

Objective

Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem. It is necessary to develop standard rabies virus diagnostic tools, especially for diagnosing the strains prevalent in China.

Methods

Monoclonal antibodies (MAbs) specific to rabies virus were produced and characterized by enzyme linked immunosorbent assay (ELISA), isotyping, affinity assay, immunofluorescence assay (IFA), and immunocytochemistry. The MAb, whose affinity was higher for antigen, was used to establish an antigen capture-ELISA (AC-ELISA) detection system and test the efficiency by using clinical samples.

Results

The heavy chain subclasses of two MAbs were all determined to be IgG2a. The 3C7 MAb showed stronger reactivity with rabies virus protein than the 2C5 MAb in an ELISA analysis, whereas the 3C7 MAb showed the highest affinity for antigen. IFA and immunocytochemistry results also indicated that the two MAbs could recognize rabies virus protein in its native form in cell samples. Data obtained using clinical samples showed that rabies virus could be detected by AC-ELISA detection system using the 3C7 MAb.

Conclusion

It was potentially useful for the further development of highly sensitive, easily handled, and relatively rapid detection kits/tools for rabies surveillance in those areas where rabies is endemic, especially in China.     

The induction and characterization of monoclonal antibodies specific to GP of Ebola virus

The Ebola virus is highly infectious and characterized by hemorrhagic fever, headache, and so on with a high mortality rate. Currently, there are neither therapeutic drugs or vaccines against the Ebola virus nor fast diagnostic methods for the detection of Ebola virus infection. This study reported the induction and isolation of two monoclonal antibodies that specifically recognized the glycoprotein (GP) and secreted glycoprotein (sGP) of the Ebola virus. Plasmids encoding either GP or sGP were constructed and immunized BALB/c mice, accordingly purified sGP was boosted. The antisera were analyzed for binding activity against sGP protein in enzyme-linked immunosorbent assay (ELISA) and neutralization activity in a pseudotyped virus neutralization assay. A number of reactive clones were isolated and two monoclonal antibodies T231 and T242 were identified to react with both GP and sGP. Western blot and ELISA assays showed that the monoclonal antibodies could react with GP and sGP, respectively. Moreover, they could recognize Ebola pseudovirus by cellular immunochemistry assay. We labeled the monoclonal antibody T231 with biotin and analyzed the competitiveness of the two antibodies by the ELISA test. The results showed that the binding epitopes of the two monoclonal antibodies to sGP were partially overlapped. In summary, two GP-specific mAbs were identified, which will be used to detect the Ebola virus or investigate GP.     

Development of an enzyme-linked immunosorbent assay for the detection of florfenicol in fish feed

A sensitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody was developed for routine screening of florfenicol (FF) in fish feed. FF succinate was synthesised and conjugated to human serum albumin by N-hydroxysuccinimide-activated ester method as immunogen, while it was conjugated to ovalbumin by mixed anhydride reaction as coating antigen. The ELISA for FF showed an IC₅₀ value of 2.5 ng/mL, and negligible cross-reactivities with other amphenicol family of antibiotics. The inter-assay recoveries of FF from fortified fish feed at levels of 2–20 mg/kg ranged from 98.8 to 117.6% with coefficients of variation (CV) of 9.3–14.2%, intra-assay recoveries ranged from 102.5 to 121.2% with CV of 10.9–13.8%. The developed ELISA were then validated by liquid chromatography method and liquid chromatography–tandem mass spectrometry method, the results indicated this ELISA could be used as convenient method for detection of FF in fish feed.     

The use of human antigen-specific monoclonal antibodies in an enzyme-linked immunosorbent assay for the determination of anti-HBsAg antibody subclasses

A human monoclonal anti-HBsAg antibody (IgGl, λ) was used as a reference for an ELISA determination of the serum levels of anti-HBsAg antibodies in human volunteers after vaccination with H-B vax. The IgG subclass distribution of specific antibodies showed a marked dominance of IgGl antibodies. In addition, small amounts of specific IgG4 antibodies were occasionally found, suggesting a different pattern from that found after natural disease. The novel use of a human monoclonal antibody to measure specific antibodies may give a more accurate determination of specific IgG subclass levels than previously available methods.     

Ⅰ型登革病毒NS1抗原捕获ELISA的建立和初步临床诊断应用

目的以登革病毒特异性非结构蛋白1（NS1）单克隆抗体为基础建立Ⅰ型登革病毒（DEN1）抗原检测的酶联免疫吸附（ELISA）法，并探索从病人早期血清样品中检测DEN1-NS1的可行性。方法利用已制备的抗DEN1-NS1单克隆抗体（单抗），进行多种抗体组合配对优化模式的分析，建立双抗体夹心抗原捕获ELISA，以469份健康人血清样品确定cut off值，检测DEN1感染患者急性期血清样品。结果对多种抗体组合反复筛选，最终确立了最佳的包被单抗和酶标测定单抗，建立了抗体夹心捕获DEN1-NS1抗原的酶联免疫测定方法，能特异检测DEN1，与其他血清型登革病毒不发生交叉反应。检测16例临床确诊DEN1感染病人急性期血清样品，15例呈特异的抗原反应阳性。结论成功建立了DEN1-NS1抗原捕获ELISA并应用于临床血清样品的检测，为登革热的早期实验室诊断提供技术方法。     

Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays

Stereospecific monoclonal antibodies (McAb) have been prepared against the tobacco alkaloid (S)-(-)-nicotine and its major metabolite (S)-(-)-cotinine. Nine anti-nicotine and 4 anti-cotinine hybridomas, selected by a screening procedure that utilized immunoprecipitation of the ³H-labeled natural isomers of nicotine or cotinine, were grown in the ascites fluid of pristane-primed syngeneic BALB/c mice. Antibodies in concentrations up to 7.5 mg/ml ascites and with binding affinities that generally exceeded 10⁸ M⁻¹ were obtained. Enzyme-linked immunosorbent assays (ELISAs) were developed in which nicotine or cotinine derivatives bound covalently to poly- -lysine werecoated onto wells of polyvinyl chloride microtiter plates. Coated wells were incubated sequentially with McAb in the presence or absence of inhibitor, rabbit anti-mouse immunoglobulin, then horseradish peroxidase-labeled protein A (HRP-SpA) before addition of substrate. The antibodies are highly specific and show minimal cross-reactivity with several nicotine metabolites and other structurally related compounds. In the respective assays, only 0.25 ng (S)-(-)-nicotine and 0.12 ng (S)-(-)-cotinine are required to give 50% inhibition of antibody binding, and as little as 0.05 ng nicotine and 0.02 ng cotinine give 15% inhibition. These assays are 5–10 times more sensitive than analogous ELISAs developed with rabbit antisera and HRP-SpA or conventional radioimmunoassays (RIAs) that utilize the rabbit antisera and ³H-labeled ligands. There was good correlation between the levels of nicotine (r = 0.967) found in saliva samples from smokers and non-smokers assayed by McAb-based ELISAs and conventional RIAs.     

A sensitive and rapid enzyme-linked immunosorbent assay using monoclonal antibodies for simultaneous quantitation of free and IgA-complexed protein HC

A sensitive and rapid enzyme-linked immunosorbent assay for simultaneous quantitation of free and IgA-complexed human protein HC was developed with monoclonal murine antibodies. The total amount of protein HC (free plus IgA-complexed) was measured by a competitive procedure while the protein HC-IgA complex was quantitated by a sandwich enzyme immunoassay. The amount of free protein HC was then obtained as the difference between the 2 measured values. The sensitivity of the procedure was 70 micrograms/1 for the total amount of protein HC and 80 micrograms/1 for the protein HC-IgA complex. At ordinary human serum levels of free protein HC and protein HC-IgA complexes the coefficient of variation for the procedure was about 5%. One worker could determine the concentrations of free and IgA-complexed protein HC in up to 400 serum samples in a working day.     

An indirect competitive enzyme-linked immunosorbent assay for acrylamide detection based on a monoclonal antibody

Acrylamide (AA) is formed spontaneously in heated foodstuffs and is a focus of concern in many people due to safety. In this study, we developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on a monoclonal antibody (MAb) to detect a derivative, which was generated from 4-mercaptobenzoic acid (4-MBA). As AA is a very small molecule (71.08?Da) and cannot elicit a homologous monoclonal antibody, we coupled the AA derivative (AA-4-MBA) to a carrier protein such as bovine serum albumin (BSA) and ovalbumin (OVA). The conjugates were used as the immunogen and coating antigen. A rapid and sensitive icELISA against AA-4-MBA was obtained by optimizing the experimental parameters. The MAb which had no specificity for AA or 4-MBA, but had high affinity for AA-4-MBA, had a satisfactory IC₅₀ of 32?ng/ml and a limit of detection of 8.87?ng/ml. The quantitative working range was 8.87–112.92?ng/ml (IC₂₀ to IC₈₀). Cross-reactivity with other analogues was lower than 10%. These results indicated that the developed icELISA was a fast and efficient method for detecting AA in food.