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 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 for subclass distribution of human IgG and IgA antigen-specific antibodies

In this study we describe an ELISA using monoclonal antibodies to IgG 1, 2, 3, 4, IgA1 and IgA2 for determining the subclass distribution of human-specific antibodies. No cross-reactivity of the subclass-specific reagents under the conditions used was observed. The sensitivity was 0.5 ng/ml for IgG1, 3, 4; 1.5 ng/ml for IgG2 and 50 ng/ml for IgA1 and IgA2. The reproducibility as described by the coefficient of variation calculated on repeated runs was 8-26% if the data were obtained by relating the absorbance values to a positive serum run in the assay, 17-58% when relating the OD figures to those of a standard myeloma plate. The method may be considered semiquantitative with high sensitivity and specificity, easy to handle and with small day-to-day variation. The assay has been applied to a number of antigens of protein and polysaccharide nature.     

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) 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) 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.     

Determination of IgA antibodies to human cytomegalovirus by enzyme-linked immunosorbent assay (ELISA)

A sensitive enzyme-linked immunosorbent assay (ELISA) is described for detection of IgA antibodies cytomegalovirus (CMV). The antigen consisted of a sonicated extract of CMV infected human embryo cells. The tested sera were absorbed with staphylococcus aureus (strain Cowan 1) before analysis. Rabbit antihuman IgA peroxidase conjugate was used to detect human IgA bound to viral antigen. In parallel, Igm and IgG antibodies to CMV were studied by ELISA and by the immunoperoxidase antibody to membrane antigen (IPAMA) technique, respectively. CMV IgA was detected in high titers by ELISA in eight of nine CMV patients. The maximal IgA titers were generally lower than the IgM titers detected by ELISA. No CMV IgA antibodies (titer less than 20) were detected by ELISA in 57 control sera (healthy adults, hospitalized patients with various other diseases), paired sera of five patients with acute herpes simplex, infection, two patients with Epstein-Barr infections, one patients with varicella, and one with zoster infections. The potential application of the ELISA CMV IgA technique in serodiagnosis of CMV 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 for IgG antibodies to Epstein-Barr virus-associated early antigens and viral capsid antigen

An enzyme-linked immunosorbent assay has been developed for the detection of IgG antibodies to Epstein-Barr virus-associated early antigens and late antigens including the viral capsid antigen. The antibody titers of human sera determined in this way correlate well with those by indirect immunofluorescence. ELISA was more sensitive than the IF method. The assays described may be used for rapid and sensitive diagnosis of EBV-related diseases. In addition, the ELISA will be useful for the determination of antibody titers to isolated EBV-associated antigens, e.g., purified components of the EA complex.     

Enzyme-linked immunosorbent assay (ELISA) for the detection and differentiation of Clostridium botulinum toxins type A and B

Affinity chromatography has been used for a two-step purification of commercial horse botulinum antitoxic globulins type A and B. The first step performed using CH-Sepharose 4B conjugated to toxin type A (or B), permitted the removal of non-botulinal antibodies from antitoxic globulin type A (or B). The anti-botulinal antibodies obtained from the first step were cross-absorbed in the second affinity chromatography using CH-Sepharose 4B conjugated to toxin B (for the purification of antibodies to type A) or to toxin A (for antibodies to type B). The antibodies obtained were used to coat polystyrene wells in an ELISA for the detection of botulinum toxin type A and type B. The first purification step increases the sensitivity of such an ELISA whereas the second step improved the specificity of the test. Only slight cross-reactions were observed between the type A and type B detection systems. The sensitivity achieved with ELISA was 100 and 300 DLM (dosis lethalis minima) for type A and B respectively.     

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 immunoassay for detection ofClostridium difficile toxins a and b in patients with antibiotic-associated diarrhoea and colitis

A sandwich enzyme immunoassay (ELISA) was developed to detectClostridium difficile toxins A and B in stools from patients with antibiotic associated diarrhoea and colitis. Immune serum to crudeClostridium difficile toxin and non-immune serum were coated onto polystyrene microtiter plates to act as capture antibodies; toxins A and B in human stools were detected by antibodies from rabbits immunized with purified toxins A and B. The ELISA for toxin B showed cross-reactions withClostridium bifermentans andClostridium sordellii and lacked diagnostic sensitivity in clinical samples. The ELISA for toxin A showed no cross-reactions with other clostridiae investigated and was positive in 33 % (62/189) of patients with antibiotic associated diarrhoea. This compared with 38 % (71/189) positive in the tissue culture assay forClostridium difficile cytotoxin. With a predictive value of 96 % in clinical specimens, the ELISA for toxin A constitutes a sensitive and specific tool for diagnosis ofClostridium difficile associated diarrhoea and colitis.     

Enzyme-linked immunosorbent assay of allergen-specific IgG antibodies in bee sting allergic patients hyposensitized with pure bee venom

Summary An ELISA is presented for detection of IgG antibodies to bee venom. By this method, sera of 11 bee sting allergic patients, who were treated with rapid hyposensitization with pure bee venom, were tested. The highest antibody titers were observed after 30 days of treatment, a maximum rise of 7.4±1.5 log 2-titer steps. Pure bee venom is shown to be more potent immunologically than whole body bee extract. Prediction of the clinical success, measured by tolerance to a bee sting challenge, is not yet possible using venom specific IgG determinations.This work was supported by the Deutsche Forschungsgemeinschaft, grant UR 12/2     

Enzyme-linked immunosorbent assay to monitor colorectal carcinoma patients treated with a monoclonal antibody (17-1A)

An enzyme-linked immunosorbent assay (ELISA) was compared to a radioimmunoassay (RIA) for the detection and quantification of mouse monoclonal antibody MoAb 17-1A and for measurement of the host response (i.e. anti-mouse immunoglobulin in sera from patients receiving immunotherapy with MoAb 17-1A. Comparable sensitivity and reproducibility were noted with RIA and ELISA but ELISA was more rapid to perform than RIA. Thus quantitative ELISA compared favorably with the RIA for MoAb detection.     

Development of a competitive enzyme-linked immunosorbent assay (ELISA) for Escherichia coli heat-stable enterotoxin (STa)

A sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the low molecular weight heat-stable enterotoxin (ST_a) in culture supernatant fluids of enterotoxigenic Escherichia coli (ETEC). Competitive inhibition was observed between ST_a in solution and a glutaraldehyde-coupled ST_a-human serum albumin (HSA) conjugate bound to microtiter wells when antiserum raised against a glutaraldehyde-coupled ST_a-bovine serum albumin (BSA) conjugate was used as detecting antibody. No competition was observed with conjugates prepared using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or dimethyl suberimidate and antisera raised against each conjugate. A biotin/avidin system increased the sensitivity of the assay such that 133 pg/ml of purified ST_a can be detected in less than 4 h. The assay was used to detect and quantify ST_a in culture supernatant fluids from human, porcine, and bovine ETEC isolates. No cross-reactivity was observed with the heat-labile enterotoxin (LT) or the form of ST with biological activity only in piglets (ST_a). Results from the quantitative ST_a ELISA showed good correlation (0.87) with the suckling mouse bioassay and a previously described radioimmunoassay. The quantitative assay was modified to reduce the total incubation time to less than 2 h. The qualitative ST_a ELISA provides a rapid and sensitive assay for clinical isolates of ETEC and should facilitate epidemiological studies on the incidence of ST_a-producing ETEC.     

Detection of BK virus IgM antibodies by two enzyme-linked immunosorbent assays (ELISA) and a hemagglutination inhibition method

We have used an antigen solid-phase enzyme-linked immunosorbent assay (SP-ELISA) and an IgM antibody capture ELISA (MACELISA) for detecting IgM antibodies to human polyomavirus BK (BKV). These tests were compared with the standard hemagglutination inhibition test (HAI) of IgM serum fractions following sucrose density gradient fractionation. The SP- and MACELISA were not influenced by concomitant BKV-IgG, but high levels of both BKV-IgG and rheumatoid factor could cause false positive results by SPELISA, but not by MACELISA. The MACELISA gave much higher positive to negative ratios than the SPELISA. The sensitivity and specificity of the two tests were high compared to the IgM-HAI method. The sera could be tested in a single dilution (1:160), and thus the ELISA-tests are useful for testing large numbers of sera.     

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.     

Comparison of ELISA for tissue transglutaminase autoantibodies with antiendomysium antibodies in pediatric and adult patients with celiac disease

BACKGROUND: Tissue transglutaminase (t-TG) is the main autoantigen recognized by the endomysium antibodies (EMA) observed in patients with celiac disease (CD). The aim of the study was to assess an ELISA method for t-TG antibodies (t-TGA) with respect to EMA IF assay in pediatric and adult patients. METHODS: t-TGA were analyzed by ELISA in 220 sera samples: 82 patients with biopsy-proven untreated CD (23 adults and 59 children), 14 CD children on gluten-free diet, 18 asymptomatic relatives of CD patients, and 106 age-matched control patients with gluten-unrelated gastrointestinal diseases (58 adults and 48 children). Serum IgA EMA were tested on umbilical cord sections in all patients. RESULTS: The great majority (92.7%) of untreated CD patients (both adults and children) were t-TGA positive (values ranging from 20.1 to > 300 AU). None of the child control patients and only two out of 58 (3.4%) of the adults with unrelated gastrointestinal diseases had serum t-TGA positivity; two out of 18 first-degree relatives with biopsy-proved silent CD were t-TGA (as well as EMA) positive. Finally, two out of 14 CD children, assuming a gluten-free diet, had serum t-TGA (as well as EMA). A highly significant correlation (P < 0.001) was observed between t-TGA concentrations and EMA. t-TGA showed a sensitivity of 87% and 95%, a specificity of 97% and 100% for adults and children, respectively. CONCLUSION: The method is highly sensitive and specific in the diagnosis of CD and is promising as a tool for routine diagnostic use and population screening, especially in children.     

Comparison of a radioallergosorbent (RAST) inhibition method and a monoclonal enzyme linked immunosorbent assay (ELISA) for aeroallergen measurement

Background Mouse and rat urinary proteins are potent occupational allergens for exposed personnel. Methods of measuring airborne allergens differ greatly, and reported levels of allergens vary considerably between laboratories. Objectives To compare the values obtained using two different methods of allergen detection. Methods Air samples were collected in rat rooms in Sweden and the United Kingdom at 2 L/min on to polytetrafluoroethylene (PTFE) filters and extracted in buffer containing 0.5% v/v Tween 20. Airborne rat urinary allergen (RUA) was measured in all samples by both RAST inhibition using a polyclonal human serum pool (UK) and a two monoclonal antibody sandwich ELISA employing antibodies specific for Rat n 1.02 (α_2u-globulin) (Sweden). Results The two methods gave values which were correlated (r² log values = 0.72, P < 0.0001), but differed by several orders of magnitude (median [range] ratio of RAST inhibition/ELISA = 316 [7-26₈₀]. There was a systematic bias; as the absolute values increased, the difference in the measurements increased. The rat urine standards used were antigenically similar. Conclusions A large contrast in RUA values obtained from the two assays was observed in this study. This may be primarily due to methodological differences, but variations in antibody specificities or composition of allergenic epitopes in the air samples may contribute. The results demonstrate that standardization of methods and antibodies is necessary before interlaboratory comparisons can be made.