Enzyme linked immunosorbent assay (ELISA) for paraquat

An Enzyme Linked Immunosorbent Assay (ELISA) has been developed for the estimation of paraquat. The amount of paraquat present in samples of human plasma was estimated in terms of the degree to which it combined with a rabbit antibody raised to a conjugate to paraquat with bovine serum albumin. The amount of residual, uncombined, antibody after being allowed to react with a conjugate of paraquat with keyhole-limpet haemocyanin bound to the surface of a polystyrene micro-titre plate, was estimated by the addition of an enzyme-labelled anti-rabbit IgG, followed after washing by addition of substrate and subsequent determination of optical density. Concentrations of paraquat in the range 0.3-10 ng/ml could be measured and the antibody showed a high degree of specificity. Results correlated well with those of a widely-validated radio-immunoassay but the ELISA was simpler and more sensitive.     

Enzyme linked immunosorbent assay (ELISA) for cytomegalovirus antibody in donor plasma.

An adaptation of an enzyme immunoassay technique was developed to screen donor plasma for high titres of antibodies to cytomegalovirus (CMV). The technique uses microtitre plates treated with glutaraldehyde and coated with CMV antigen and an anti-IgG alkaline phosphatase conjugate to detect the captured antibody. Using an anti-CMV standard with a 1/64 titre by complement fixation, 34 (6.8%) of 500 sera were shown to have an antibody titre that was acceptable to the Blood Products Laboratory in England for anti-CMV immunoglobulin production.     

Enzyme linked immunosorbent assay (ELISA) for IgG and IgE antibodies to protein and polysaccharide antigens of Aspergillus fumigatus

ELISA has emerged as a useful alternative to other more costly and complex tests. Polystyrene microhaemagglutination plates have been used as solid phase to absorb Aspergillus fumigatus protein and polysaccharide components for detection of specific antibodies in patients with various forms of pulmonary aspergillosis. IgG and IgE antibodies to the polysaccharide as well as the protein allergens have been found. For the IgE test a double antibody technique has been developed, which is more sensitive than the conventional indirect ELISA.     

Enzyme linked immunosorbent assay (ELISA) for detection of meningococcal antibodies in north India.

Serological response was studied in 16-30 years high risk age group, during the period December 1986-April, 1988, following meningococcal vaccine (Biomerieux, France). A total of 200 serum samples were collected from 50 individuals before vaccination and at 3 intervals of 1, 3 and 6 months post-vaccination respectively. Antibody response was measured by Enzyme Linked Immuno-Sorbent Assay (ELISA) and indirect haemagglutination assay (IHA). In the vaccinees antibody response by IHA test showed 56%, 82%, 78% and 74% positivity in the pre-vaccination, 1 month, 3 months and 6 months post vaccination samples. By ELISA 2%, 80%, 74% and 66% of the above groups showed serological response. Difference in the pre-vaccination and titres/O.D. of various post-vaccination groups was found to be statistically significant (p less than 0.001). There was, however, no significant difference (p greater than 0.05) amongst the titres/O.D. of three post-vaccination groups. Similarly acute and convalescent blood samples of 25 patients and one sample each of 31 contacts was studied for antibody response. In general, higher antibody titres are produced with systemic infection than with local nasopharyngeal infection or following vaccination.     

Validation and long term performance characteristics of a quantitative enzyme linked immunosorbent assay (ELISA) for human anti-PA IgG

Accurate, reliable and standardized quantification of anti-protective antigen (PA) IgG antibody levels is essential for comparative analyses of anti-toxin immune responses in anthrax cases, recipients of PA-based anthrax vaccines and for evaluation of anti-PA based immunotherapies. We have previously reported the early performance characteristics and application of a quantitative anti-PA IgG enzyme linked immunosorbent assay. The principal application of this assay was in a Phase 4 human clinical trial of anthrax vaccine adsorbed (AVA, BioThrax), the central component of the CDC Anthrax Vaccine Research Program (AVRP) and in humans following bioterrorism associated Bacillus anthracis infection (Quinn et al., 2002; Quinn et al., 2004; Marano et al., 2008). The objective of the AVRP was to determine the feasibility of reducing the number of priming series and booster doses of the licensed Anthrax Vaccine Adsorbed (AVA) (BioThrax?; Emergent BioSolutions, Lansing, MI) and changing the route of administration from subcutaneous (SC) to intramuscular (IM) (Marano et al., 2008). In this paper we report the validation and long term performance characteristics of the assay during its six year application in the AVRP (2002-2008). The critical features are 1) extensive validation of the assay using two standard reference sera; 2) long term stability and 3) consistency of the data for quantitative analysis of human long term anti-PA IgG responses. The reportable value (RV) of the assay was expressed as anti-PA IgG concentration (μg/ml). Accuracy of the assay was high with a percent error (%ER) range of 1.6-11.4%. Overall intra-operator and intermediate precision were high with Coefficients of Variation (%CVs) of 2.5-15.4% and 6.3-13.2%, respectively. The assay demonstrated excellent dilutional linearity for human sera using log(10) transformed data with the slope=0.95 to 0.99, intercept=0.02 to 0.06 and r(2)=0.980-0.987. The assay was robust, tolerating changes in serum incubation temperatures from 35 to 39°C, serum incubation times from 55 to 65min and changes in key reagents. The long-term assay stability over 6years using consecutive reference sera AVR414 and AVR801 demonstrated sustained high accuracy and precision for the assay, confirming its suitability for long term studies of PA protein-based anthrax vaccines.     

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     

Determination of human IgG and IgM class antibodies to West Nile virus by enzyme linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed and used for the detection of IgG and IgM antibodies to West Nile virus in human sera. Thirteen paired sera of clinical cases and 24 control sera taken randomly from a blood bank repository were tested. The sera were reacted in microtiter plates coated with PEG-treated WNV antigen. IgG or IgM antibodies were quantitated by the use of alkaline-phosphatase-conjugated anti-human IgG or IgM antibodies. Of the 24 randomly collected serum samples, 7 were positive in the IgG-ELISA test. One positive by the IgM-ELISA was found to contain rheumatoid factor. In 12 of 13 paired sera of clinical cases, IgM as well as IgG antibodies were detected in the second serum sample taken about 3 wk after the onset of clinical signs. The IgM positive sera were screened for rheumatoid factor (RF) on IgG-coated plates. None of them contained RF. Antibody titers obtained by ELISA showed a good correlation with titers obtained by hemagglutination inhibition, complement fixation, and neutralization tests. The ELISA tests for detection of IgM and IgG antibodies to WNV therefore can replace the other serological methods for epidemiological surveillance and diagnostic purposes.     

Enzyme linked immunosorbent assay for milk progesterone

A direct antigen heterologous enzyme linked immunosorbent assay (ELISA) for milk progesterone has been developed using progesterone-3-O-carboxymethyloxime-bovine serum albumin (P-3-O-CMO-BSA) antiserum and 17-α-hydroxy-progesterone-3-O-carboxymethyloxime-horseradish peroxidase (17-α-OH-P-3-O-CMO-HRP) enzyme conjugate. The data of the present study reveal that the homologous assay, which employed P-3-O-CMO-HRP as the label, showed no displacement. On the contrary, replacement of P-3-O-CMO-HRP with 17-α-OH-P-3-O-CMO-HRP as the label showed significant displacement and led to the development of a sensitive and specific assay. The recovery of the exogenously spiked progesterone from milk pools was in the range of 94.3-97.88% for toned milk and 97.6-101% for full-cream milk. The intra-assay and interassay coefficients of variation (CVs) ranged from 4.1-7.8% and 4.4-7.0%, respectively. A high ionic strength buffer was used to obtain released progesterone from binding protein/fat. The progesterone values measured in toned and full-cream milk ranged from 1.198-9.745 ng/mL and 6.949-14.923 ng/mL, respectively. The milk progesterone values obtained by this method correlated well with those obtained by radioimmunoassay; r = 0.95 (n = 65).     

Rapid indirect enzyme linked immunosorbent assay (ELISA) for detecting antitoxoplasma IgG: comparison with dye test.

A rapid and simple enzyme linked immunosorbent assay (ELISA) for the detection of specific IgG against Toxoplasma gondii was compared with the dye test on 533 serum samples. In general, results were comparable but not with sera that contained high concentrations of toxoplasma specific IgM or that had been heated at 56 degrees C. There were no false positive results with sera containing rheumatoid factor or anti-nuclear factor. It is concluded that if a dye test is not to be performed then the serum should be tested for both toxoplasma specific IgG and IgM to avoid misleading results. Heat inactivated serum should also not be tested in this type of specific IgG assay.     

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.     

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.     

Rapid enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies against

A rapid ELISA was developed for the detection of specific IgG against Micropolyspora faeni and Thermoactinomyces vulgaris and compared with counterimmunoelectrophoresis (CIE) in twenty-seven patients with suspected farmer's lung disease (FLD). Seventeen patients had precipitins to M. faeni or T. vulgaris or both, and ten had no precipitins. The optimum conditions for each step in the ELISA were determined: pre-equilibration of reagents at 37⁰C and vigorous agitation during incubation enabled the total test time required for the procedure to he reduced to 4 hr. A serum dilution of 10-fold produced good differentiation between CIE-positive and -negative sera. Little correlation was seen between CIE and ELISA for either M. faeni or T. vulgaris antigens in tests with sera from patients with precipitins: high readings were often recorded in ELISA where no precipitins had been detected with the same antigen. Positive- negative discrimination of unknown sera in ELISA was achieved through the inclusion of CIE-positive and -negative reference sera in each test run. Thirteen CIE-positive sera were classed as positive in ELISA with the M. faeni antigen while eight of thirteen CIE-positive sera were positive in ELISA with the T. vulgaris antigen. For both antigens, four CIE-negative sera were recorded as positive in ELISA.     

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     

Enzyme linked immunosorbent assay for measuring antithrombin III.

A competitive ELISA for the measurement of antithrombin III antigen was developed using a pure preparation of antithrombin. This two step immunoassay requires only one antibody binding step and avoids the binding of anti-antithrombin antibody to the solid phase. Plasma samples measured by this ELISA correlated well with measurements taken with immune electrophoresis measurements (r = 0.933); the ELISA was also shown to be both sensitive and precise.     

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.     

A specific enzyme-linked immunosorbent assay (ELISA) for the determination of human C5a antigen

An enzyme-linked immunosorbent assay has been developed to detect human C5a antigen. This ELISA methodology has been shown to be a highly sensitive technique capable of detecting C5a antigen concentrations below 10 ng/ml. The microELISA technique used in this study is specific for human C5a and C5a des arg (C5a antigen) but not for human C5. Conditions to establish sensitivity and specificity are outlined in ths report.     

Enzyme-linked immunosorbent assay (ELISA) and colorimetric determination of cow gamma-glutamyltransferase

Using specific antibodies of anti-heavy and light forms of bovine kidney gamma-glutamyltransferase, both enzyme forms were determined in some cow body fluids and tissue preparations by enzyme linked immunosorbent assay (ELISA). The mean activity of the light form of gamma-glutamyltransferase in cow sera, new-born calf sera and in cow colostra was 29.2, 612 and 2630 mU/ml respectively. Good correlation was noted between the results obtained by ELISA and by colorimetric method. In supernatants from cow kidney and liver homogenate after incubation at 37 degrees C, a marked increase of the heavy form assayed by ELISA was noted.