Indirect enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies against Coxsackie B viruses

In tests for IgG antibodies against Coxsackie B viruses in man, the enzyme-linked immunosorbent assay (ELISA) was essentially group-specific and, unlike the type-specific neutralisation test, usually failed to detect rises in antibody titre in paired, acute and convalescent, sera. However, in rabbits immunised against Coxsackie B viruses, ELISA demonstrated both group- and type-specific antibody responses. The lack of type-specificity of ELISA in man is probably because repeated infection with enteroviruses--echoviruses and Coxsackie A as well as Coxsackie B--results in masking of the type-specific antibody response by group-specific antibody.     

Rapid enzyme-linked immunosorbent assay (ELISA) for Aspergillus fumigatus antibodies.

A rapid enzyme-linked immunosorbent assay (ELISA) where component incubation periods were shortened to one hour, was compared with agar gel double diffusion (AGDD) and a standard ELISA procedure for detecting antibodies to Aspergillus fumigatus in 28 asthmatic patients with suspected allergic aspergillosis. Using two A fumigatus antigens the rapid ELISA compared well with AGDD and the standard ELISA method. Eleven sera that reacted with both antigens in AGDD were all positive against antigen 1 in both forms of ELISA, but two failed to react with antigen 2 in the standard ELISA and three failed to react with this antigen in the rapid method. Thirteen AGDD-negative sera were also negative in both forms of ELISA. The rapid ELISA provides a sensitive and reproducible test for routine serological investigation of allergic aspergillosis.     

Development of a semi-quantitative enzyme-linked immunosorbent assay (ELISA) for detection of human IgG subclass antibodies

We have developed a sensitive enzyme-linked immunosorbent assay (ELISA) which measures antibodies to bee venom phospholipase A2 (PLA2) and hyaluronidase (HYAL), horse IgG, bovine casein, and the bacterium Streptococcus mutans in each of the four human IgG subclasses. For this purpose, we have used mouse monoclonal antibodies (McAb) specific for each subclass and one which showed 'pan-IgG' reactivity. Binding to human IgG was similar for all the McAb and dilution of human IgG resulted in similar dilution curves for each subclass. Results were expressed as arbitrary U ml-1 by comparing the optical density obtained with each subclass-specific McAb to a reference curve for total IgG antibody constructed using the 'pan-IgG' McAb. Close agreement was found between the total amount of IgG antibody and the sum of the antibody in each of the four subclasses (PLA2 r = 0.90, horse IgG r = 0.98, bovine casein r = 0.84, S. mutans r = 0.85), confirming that these assays provide semi-quantitative measurements of the amount of subclass-specific antibody.     

A novel enzyme-linked immunosorbent assay (ELISA) for the detection of beryllium antibodies

A novel immunological method has been developed for detecting antibodies (IgG molecules) specific to beryllium, a light metal used in industry and capable of causing chronic beryllium disease. Beryllium metal was vacuum deposited onto commercially available immunological microsticks, which were then exposed to test plasma containing the putative antibodies. Antigen-antibody complexes were located using a biotin-avidin amplification method. One employee diagnosed with chronic beryllium disease and one diagnosed as "sensitized" (lymphocyte transformation positive) exhibited antibody titers graphically and statistically different and higher than a pooled baseline control population. Plasma from these two employees (former beryllium workers) was used in four different approaches to validate the presence of beryllium antibodies. The assay proved to be reproducible.     

Rapid detection of IgG and IgM antibodies for cytomegalovirus by the enzyme linked immunosorbent assay (ELISA)

Summary A simple solid phase enzyme immunoassay for the detection of immunoglobulin G and M to cytomegalovirus (CMV) is described.Using this test IgM antibodies to CMV were detected in 0.7 per cent of newborns and regularly after CMV infection in transplant patients, furthermore in these latter patients IgM production was prolonged for several months. For the determination of IgG the enzyme immunoassay was more sensitive than the complement fixation test (CF) and the antibody titres were 4 to 8 fold higher.Since the ELISA test is rapid, specific and unexpensive it can become an acceptable routine diagnostic procedure.With 1 Figure     

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM anti-idiotypes directed against anti-HBs molecules

A simple and specific enzyme-linked immunosorbent assay (ELISA) has been developed to detect circulating IgG and IgM anti-idiotypic antibodies directed against anti-HBs molecules using 96-well polyvinyl microtitre plates as the solid phase and HRPO-labelled goat anti-HBs as conjugate. Anti-idiotype reactions were observed in the supernatant portion after precipitation of immune complexes from sera with polyethylene glycol 6000 (PEG). Both IgG and IgM with anti-idiotype activity were detected concurrently in HBsAg-positive sera from HBV-infected patients and asymptomatic HBV carriers. Anti-idiotype activity was absent in HBsAg-negative sera from healthy persons, and in patients with non-A, non-B hepatitis and viral hepatitis A. However, such antibodies could be demonstrated in the sera of two out of eight HBsAg vaccine recipients negative for anti-HBs but in none of 11 recipients positive for anti-HBs after receiving a booster immunising dose of HBsAg vaccine. Those sera showing positive anti-idiotype reactions were free from rheumatoid factor and HBsAg/IgM or HBsAg/IgG complex activity. An analysis of anti-idiotype positive sera for anti-HBs, HBeAg and HBV-specific DNA-polymerase activity demonstrated these markers in 20%, 30% and 60% of cases, respectively. The presence of anti-idiotypic antibodies was presumed to permit a more active multiplication of hepatitis B virus.     

Indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Coxsackievirus group B antibodies

An indirect, solid phase, microplate enzyme-linked immunosorbent assay (ELISA) was found to be highly sensitive and reliable for detecting antibodies to the group B Coxsackieviruses and other picornaviruses. Conditions for obtaining maximum sensitivity and reproducibility of the indirect ELISA are described. Antibody titres were comparable to those obtained by the virus neutralization test and over 50 times higher than those obtained by the complement-fixation test. Purified virions used in the indirect ELISA reacted with low levels of cross-reacting heterotypic antibodies elicited by each of the six group B Coxsackieviruses, although homotypic reactions resulted in highest titres.     

Rapid detection of antigen-specific mouse IgE by enzyme-linked immunosorbent assay (ELISA)

A rapid, sensitive, antigen-specific mouse IgE capture ELISA is described. A monoclonal rat anti-mouse IgE was used as the capture antibody, and a DNP-coupled BSA-biotinylated conjugate along with a peroxidase-avidin-biotin complex was utilized as the detection system. The lower detection limit of this assay is 8.5 ng/ml of antigen-specific IgE. With some modifications, this assay can be employed to screen for antigen specific antibodies of other isotypes and subtypes.     

Amplification of the enzyme-linked immunosorbent assay (ELISA) in the detection of class-specific antibodies

A modification of the standard enzyme-linked immunosorbent assay (ELISA) is described which circumvents the requirement for specifically purified antibodies from which antibody-enzyme complexes are made. The assay utilizes the principle of a soluble anti-alkaline phosphatase immune complex (AP-A-AP) and has been called the amplified ELISA. Methods for preparing and evidence for the specificity of rabbit anti-rat γ-FC, IgM (μ) and IgA (α) are presented. These reagents are used to measure anti-DNP antibodies belonging to classes IgG, IgM and IgA in rat serum. Using antiglobulin and anti-enzyme reagents prepared in guinea pigs, anti-ovalbumin antibodies are measured in rabbit serum. Titration curves are similar when the amplified ELISA is compared to the standard ELISA. A change in slope suggesting an effect of saturation of antigen sites, occurs at the same input antibody concentration for both assays. Determination of the anti-DNP concentration of unknown sera by extrapolation from titration graphs of a known serum suggests that the value is overestimated, i.e., amplified when the amplified ELISA is used. In addition, the amplified ELISA has an improved ability to detect low levels of antibody. Evidence is presented which illustrates how the use of optimally conjugated DNP-proteins, age of conjugates, and optimal dilutions of secondary antiglobulins and the AP-A-AP reduce non-specific binding in the amplified ELISA. The amplified ELISA is capable of detecting 2.4 ng of antibody to ovalbumin in a one : one million dilution of rabbit serum with high reproducibility and low background.     

Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Anaplasma phagocytophilum in sheep

An indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Anaplasma phagocytophilum in ovine serum samples was evaluated. The assay used purified A. phagocytophilum grown in tick cell cultures as antigen. Serum samples were diluted 1 in 200 and binding was detected with anti-sheep IgG conjugated to horseradish peroxidase. All tests were carried out in the presence of positive and negative control samples. Optical density (OD) values obtained for each test sample at 490 nm were used to calculate percentage positivity (PP) of each sample based on the ratio of the OD of the test sample that of the positive reference sample. Known negative samples (n=69) obtained from uninfected sheep bred and maintained in a tick-free environment and subsequently shown to be susceptible to A. phagocytophilum were used to establish the cut-off point between negative and positive samples and to establish the specificity of the test. Serum samples obtained from 92 animals 14-21 days after infection were used to establish the sensitivity of the test. Using a cut-off point of 20PP (mean+2 standard deviations of the PP of 69 control samples) the test was shown to have a sensitivity of 84.8% and a specificity of 95.7%. Lowering the cut-off point to 15PP increased the sensitivity to 94.6%, but reduced the specificity to 92.8%.     

An enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of antibodies against swine vesicular disease virus (SVDV)

A liquid phase blocking sandwich ELISA has been compared with virus neutralisation for testing pig sera for antibodies against swine vesicular disease (SVD) virus. Highest infectivity titre of SVD virus was obtained using a multiplicity of infection of 30 pfu/cell and harvesting after 21 h. Titres obtained for 300 clinically normal animals were assessed by ELISA and 89% were found to be 1/6 or less. Results were skewed and spread up to 1/45. Comparison of known positive sera resulted in a correlation between the two methods of 0.68 and showed that a virus neutralisation titre of 1/16 was equivalent to 1/40 (log10 1.61) by ELISA. Variation in results obtained by replicate testing using ELISA and virus neutralisation was almost identical. Overlap between positive and negative sera was shown to be reduced to 1-1/2 fold in ELISA. Therefore, the ELISA correlated well with virus neutralisation and has several advantages over the latter.     

A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus: II. Application

The liquid-phase blocking sandwich ELISA has been evaluated for the serological study of antibodies against foot-and-mouth disease virus (FMDV). The titres recorded for sera from a population of more than 300 British uninfected, unvaccinated cattle which were examined against each of the seven immunologically distinct FMDV types were less than 1 in 40. alternative to the existing VN test for the quantification of antibodies against FMD virus.     

A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. I. Development and method of ELISA

A liquid-phase blocking sandwich ELISA has been developed for the quantification of antibodies against foot-and-mouth disease virus which may replace the virus neutralisation (VN) test. This test employs the incubation of a constant amount of antigen with a range of test serum dilutions in the liquid-phase before being assayed using a trapping ELISA. Thus it does not rely on the availability or growth of tissue culture cells. The assay is rapid and relatively simple to perform, reagents are used economically and results may be recorded within 24 h. The ELISA is sensitive and results are more specific and more reproducible than those obtained by VN. Results are expressed as reciprocal antibody titres which are analogous and of a similar order to those recorded by VN. Individual titres, therefore, may be easily assessed by workers in the field who are already familiar with VN.     

The enzyme-linked immunosorbent assay (ELISA) for determination of IgG and IgM antibodies after infection with mumps virus

Under routine laboratory conditions ELISA was tested for suitability for serological demonstration of specific antibodies of the immunoglobulin classes G and M against mumps virus. Sera from patients with known clinical and virological data were used. The results of ELISA were compared with those of CFT. 45 paired sera were tested in ELISA IgG, 87 first sera in ELISA IgM. Both tests were highly sensitive, antibodies were detected earlier and with higher titers than with the CFT. The ELISA IgM is particularly suitable for early diagnosis of mumps infection with the first serum. In addition 23 paired sera from patients with acute parainfluenza virus infection were examined for cross-reacting antibodies. Low anti-mumps IgG antibody titers were found in some sera. These findings reduced the mumps specificity of the IgG test. In five serum samples from one patient — obtained before, during, and after an infection with mumps — the course of IgG and IgM antibodies could be demonstrated. Advantages and limitations of ELISA IgG and IgM are summarized.Behringwerke AG, Marburg, West Germany     

Quantitative enzyme-linked immunosorbent assay (ELISA) for hirudin

A competitive ELISA assay has been developed that permits reproducible quantitation of the anticoagulant hirudin in buffer and urine. Coupling of peroxidase and hirudin was performed with the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio)-propionate. In both solvents the lower limit of sensitivity was 8 ng hirudin/ml (0.08 AT-U) while the upper limit was 7.7 micrograms/ml (78.45 AT-U).     

Enzyme-linked immunosorbent assay (ELISA) for IgG antibodies in farmers' lung disease

The ELISA was used for detection of specific IgG antibodies to Micropolyspora faeni antigens in 158 farmers with a history of exposure to mouldy hay, eighty-eight of whom had a diagnosis of farmers' lung. The farmers’ lung group had significantly higher values in the ELISA than both the seventy exposed but asymptomatic farmers (P < 0.001) and a group of thirty-one adult controls (P < 0.001). The asymptomatic farmers also had significantly higher values than the control group (P < 0.02). The ELISA correlated better with the clinical diagnosis than the Ouchterlony agar-gel double-diffusion (precipitin) test. None of the control group gave positive reactions in the ELISA or the precipitin tests. The ELISA is therefore a sensitive, specific and quantitative test which is readily available and widely applicable.     

Use of an enzyme-linked immunosorbent assay (ELISA) for screening of hybridoma antibodies against cell surface antigens

A micro-ELISA for screening of antibodies from hybridoma cultures against surface antigens of human melanoma is described. The technique employs alkaline phosphatase-conjugated protein A and target cells attached to poly-l-lysine-coated microtiter plates. The micro-ELISA is equally sensitive as the radioimmunoassay. Mild glutaraldehyde treatment of cells did not lead to an appreciable loss of antigen activity. The fixed cells can be stored at 4°C for at least 6 weeks. It is concluded that the ELISA is superior to the radioimmunoassay in the following aspects: (1) exclusion of radioactive hazards, (2) speed of performance, and (3) lower costs.     

The evaluation of a quantitative enzyme-linked immunosorbent assay (ELISA) for anti-Aspergillus fumigatus IgG

A standard enzyme-linked immunosorbent assay method for anti-Aspergillus fumigatus IgG in human serum was modified to produce a quantitative assay. The resulting assay was reproducible and capable of separating individual precipitin line groups and provided a means of monitoring the variation in antibody levels over long periods in patients with pulmonary aspergillosis.     

An enzyme-linked immunosorbent assay for detection of antibodies against halothane-altered hepatocyte antigens

Patients with massive liver cell necrosis that may follow halothane anaesthesia have a high incidence of circulating antibodies against halothane-induced hepatocyte antigens. In order to provide an objective and quantitative method for the detection of these antibodies, an enzyme-linked immunosorbent assay has been developed. Sera, after absorption with normal rabbit liver microsomal fraction, are tested for binding to microsomal fractions from control and halothane-pretreated rabbits. Those containing antibodies against halothane-induced determinants give significantly enhanced binding to halothane-altered fractions; this specificity was verified by absorption experiments. Using this method, halothane-related antibodies were detected in sera from 16/24 patients with halothane-associated liver failure, at titres ranging from 1:100 to 1:25600. Such antibodies were not detectable in sera from 26 normal blood donors, 5 healthy anaesthetists, 12 patients who had received multiple halothane anaesthetics but had normal liver function tests and 32 patients with a variety of other liver diseases. This rapid and reproducible assay should be of value for the detection of antibodies and for detailed investigation of patient antibody responses, and also for characterization of the route of production and metabolism of the antigen.