Enzyme-linked immunosorbent assay (ELISA) (author's transl)]

The development of enzymo-immunological technics and the synthesis of efficacious immuno-adsorbants has given rise to several immuno-enzymometric methods of which one, ELISA (enzyme-linked immunosorbent assay) has proved particularly practical and sensitive for the estimation of antibodies.     

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.     

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.     

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.     

Enzyme-linked immunosorbent assay (ELISA) for detection of herpes simplex virus-specific IgM antibodies

Herpes simplex virus (HSV)-specific IgM in human serum could be detected by a microplate enzyme-linked immunosorbent assay, using extracts of HSV-infected cells as antigen. Peroxidase-conjugated anti-human IgM was used to detect human IgM bound to viral antigen. Pretreatment of sera with protein A-bearing staphylococcus or with aggregated human IgG was necessary to eliminate false-positive results caused by the presence of rheumatoid factor. Specificity controls included sera of patients with other herpes group virus infections.     

Enzyme-linked immunosorbent assay (ELISA) for HIV antibody by a glass slide technique

An enzyme-linked immunosorbent assay (ELISA) technique is described which utilizes a commercially available glass microscope slide coated with hydrophobic teflon in such a pattern as to give 30 small circular wells, each of which has a glass bottom. Each well serves as a solid phase, analogous to a microtiter well for adsorption of purified human immunodeficiency virus (HIV) antigens. Since only 5-10 microliter volumes of reagents are used and rinsing processing is simple, the cost per test is much less than most other ELISA technologies. HIV antigen is stable for over 1 year at 37 degrees C when dried on the glass slides. The sensitivity and specificity of the micro slide immunoenzymatic assay (Micro-SIA) was studied by testing randomly selected, known HIV-seropositive and seronegative plasma. Results compare well with microtiter and Western blot assays. A simple vertical-beam colorimeter is described (useful in the Micro-SIA) which can be easily assembled by the user from commonly available components.     

Enzyme-linked immunosorbent assay (ELISA) to detect antibodies in gonorrhea using whole cells

The choice of an antigen that will adequately differentiate between infected and non-infected patients has been a problem in detecting gonococcal antibodies for diagnosis. We have used the sensitive technique of ELISA to test various serotypes of Neisseria gonorrhoeae for their suitability as antigens. Whole cells of each serotype were attached to polystyrene plates using poly-L-lysine, N gonorrhoeae, strain H1 type 1 was used to detect antibodies in patients with known clinical history and then as a standard to evaluate the ability of different serotypes to differentiate between infected and non-infected groups.     

Enzyme-linked immunosorbent assay (ELISA) for detection of specific IgA antibodies to mumps virus

A sensitive enzyme-linked immunosorbent assay (ELISA) is described for detection of IgA antibodies to mumps virus. Specific mumps IgA antibodies could be demonstrated in 10 patients with mumps virus infections. No specific mumps IgA antibodies (titres <1/40) were detected by ELISA in 46 control sera (healthy adults; hospitalised patients with various other diseases). The potential application of the ELISA mumps IgA technique in serodiagnosis of mumps infections is discussed.     

Enzyme-linked immunosorbent assay (ELISA) for detecting infectious laryngotracheitis viral antibodies in chicken serum

The conditions of an indirect-enzyme linked immunosorbent assay for infectious laryngotracheitis virus (ILT) antibodies have been established. The specificity of the reaction was demonstrated. The method offers a simple and specific antibody assay for the detection of antibodies to ILT virus arising from vaccination or challenge infection.     

Enzyme-linked immunosorbent assay (ELISA) for detecting antisperm antibodies in mice with testicular autoimmunity

We developed an ELISA for measuring antisperm antibodies in the mouse by using serum samples obtained from mice immunized with murine testicular antigens in complete Freund's adjuvant (CFA) as well as from mice rendered vasectomized. Sperm antigens used were syngeneic epididymal spermatozoa and two types of soluble, murine testicular antigens prepared in our laboratory. This study deals with a) the sequential changes of antisperm antibody levels following immunization; b) determination of immunoglobulin classes of these antibodies; c) a correlation between the absorbance values and the endpoint titers of antisperm antibodies; and d) comparison of endpoint titers of antisperm antibodies detected by ELISA with those by immunoperoxidase staining method in immune and nonimmune sera. It is suggested that serum dilution as high as 1/800 or more is required for detecting antibody titers of immune sera, because nonimmune mouse sera reveal a definite, although low, level of absorbance value at a serum dilution of 1/400 or less.     

Enzyme-linked immunosorbent assay (ELISA) for detecting antibodies in sera of patients with adenovirus infection

The 41 distinct antigenic types of adenoviruses (Ads) are responsible for a broad spectrum of diseases in humans. We have developed an enzyme-linked immunosorbent assay (ELISA) using adenovirus (Ad) infected MRC-5 cells for detecting IgG and IgM antibodies to Ads. Using the ELISA, we detected IgG antibodies in 100% (20/20) of sera from normal adults (geometric mean titer, GMT = 1840.8, range = 40-20,480) and IgM antibodies in 3 of 20 sera (15%) with a GMT of 25.1. Our indirect immunofluorescence (IF) technique also detected IgG antibodies in 100% of these sera (GMT = 248.3, range = 40-5,120) and IgM antibodies in the 3 samples reactive in ELISA (GMT = 20.0, range = less than 5-40). In contrast, the complement fixation (CF) test detected antibodies to Ads in only 65% (13/20) of these sera (GMT = 10.9, range = less than 4-32). Moreover, IgG and IgM responses could not be distinguished using CF. Thus the sensitivity of these three techniques is greatest for ELISA. Additionally, a study of sequential sera from 3 patients with acute Ad infection disclosed seroconversion using all three methods. Both the ELISA and IF techniques permit the detection of transition from IgM to IgG, whereas CF only detects conversion from seronegativity to seropositivity. Finally, preliminary data suggest that the IgM response as measured by ELISA is specific for subgroups or types of Ad. This newly devised ELISA may be useful for detecting Ad infections.     

Enzyme-linked immunosorbent assay (ELISA) using antibody class capture for the detection of antitoxoplasma IgM.

Sera from 180 patients with suspected toxoplasmic lymphadenopathy were examined for antitoxoplasma IgM by an enzyme-linked immunosorbent assay (ELISA), using antibody class capture (ACCA). Of 82 positive ACCA results, 78 were confirmed by testing the IgM fractions of the sera, obtained by sucrose density gradient centrifugation (SDGC). The four positive results which could not be confirmed were all from patients with at least a year''s history of lymphadenopathy. Sera from 10 patients with low Sabin Feldman dye test (DT) titers gave positive ACCA results and subsequent specimens from them showed a rise in antibody concentration, confirming the diagnosis of acute toxoplasmosis. The antitoxoplasma IgM immunofluorescent antibody test (IgM-IFA) on whole serum was relatively insensitive and gave false-positive results with sera containing rheumatoid factor (RF) and antinuclear factor (ANF). There were no false-positive ACCA results with such sera, probably because the conjugates were prepared from F(ab'')2 fragments of antitoxoplasma serum. The ACCA proved to be sensitive, specific and easily automated enabling examination of large numbers of specimens.     

Enzyme-linked immunosorbent assay (ELISA) and blocking with bovine serum albumin (BSA)-not all BSAs are alike

The enzyme-linked immunosorbent assay (ELISA) is an extremely common and powerful laboratory technique for detecting proteins by antibodies. Researchers frequently use bovine serum albumin (BSA) as a blocking agent to prevent non-specific binding of antigens and antibodies to the microtiter well. While studying the interactions of the vaccinia virus complement control protein (VCP) with complement, we found non-specific binding of VCP to BSA and identify a BSA preparation that did not result in non-specific binding. This work draws attention to the fact that not all BSA preparations are alike. It also highlights the need to perform critical controls to ensure that ELISA reactants do not inappropriately bind to the blocking agent.     

Enzyme-linked immunosorbent assay (ELISA) of changes in specific IgG antibodies to five venoms during venom immunotherapy

An enzyme-linked immunosorbent assay (ELISA) was developed that could measure titres of human IgG antibodies to five different venoms (honeybee, yellow jacket, yellow hornet, white-faced hornet, and wasp), and to honeybee phospholipase A. Changes in specific IgG anti-venom titres were measured in twenty patients that had systemic anaphylactic reactions to insect stings, and ten non-allergic controls. After being stung and prior to treatment all patients had anti-venom IgG titres greater than controls. Treatment with small doses of venom over 1–2 months resulted in prompt rises in anti-venom IgG titres that may represent secondary anemnestic responses primed by prior slings. All patients undergoing venom immunotherapy showed at least 2-fold increases in IgG antibody lo the venoms they were treated with by the time maintenance doses of 100 meg were achieved, with one exception. Significant cross-reactive increases in anti-vespid IgG antibodies to venoms not used for treatment occurred in nine of eighteen treated patients. Overall, ELISA of IgG antibodies lo five venoms allowed clear evaluation of the considerable variation of IgG responses among different patients. We conclude that serial determination of venom-specific IgG titres by ELISA offers an important adjunct to evaluating the results of venom immunotherapy.     

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.     

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).