An enzyme-linked immunosorbent assay (ELISA) for human corticosteroid-binding globulin

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) of the "sandwich-type" for human corticosteroid-binding globulin was developed. A polyclonal rabbit anti-CBG antibody is immobilised to a microtitre plate. Following incubation of standards and samples a second monospecific rabbit anti-CBG antibody, labelled with alkaline phosphatase, is added. After colour development the microtitre plate is read at 405 nm wavelength. The assay shows good agreement to CBG binding capacity assay and commercially available RIA.     

An enzyme-linked immunosorbent assay (ELISA) for human sex hormone-binding globulin

An enzyme-linked immunosorbent assay (ELISA) of the "sandwich-type" for sex hormone binding globulin (SHBG) has been developed. A rabbit anti-SHBG antibody (RAb) is immobilized to the microtitre plate. After incubation with standards and samples a second monospecific rabbit anti-SHBG antibody, labelled with alkaline phosphatase is added (RAb). Following further washing substrate is added, colour developed and the plate read at 405 nm wavelength on a standard ELISA plate reader. The assay is not influenced by the presence of steroids at the binding site, and shows good agreement to SHBG binding capacity assay and commercially available IRMA. Its sensitivity, specificity and precision allows its use in the routine laboratory. The SHBG ELISA has been used to measure SHBG concentrations in sera of normal men, women, pregnant women, and women receiving high-dose medroxyprogesterone acetate as a treatment of metastatic breast cancer.     

Optimality assessment in the enzyme-linked immunosorbent assay (ELISA)

An optimality criterion is proposed for evaluating the precision of alternative designs in the enzyme-linked immunosorbent assay. Assay profiles are represented as four-parameter logistic functions with parameter estimation based on either a weighted nonlinear regression or a simple nonlinear regression after a logarithmic transformation. Assay design changes are characterized in terms of their effects on parameters in the four-parameter logistic model. General optimality results are derived for the variance of relative potency estimates in routine assay applications.     

An enzyme-linked immunosorbent assay (ELISA) for plasma testosterone

A rapid, single extraction ELISA for testosterone in plasma is described, using a standard 96 well microtitre plate. Testosterone is covalently bonded to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to the ELISA plate, giving an immobilised antigen approach which simplifies subsequent assay standardisation for steroid hormone assays. The addition of standard, sample and first antibody (rabbit anti-testosterone), which is unique for each different assay, is followed by a general procedure which includes washing, addition of peroxidase labelled goat antirabbit IgG, further washing and finally, addition of o-phenylenediamine substrate with colour development and reading of the plate at 492 nm on an automatic ELISA processor. The ELISA assay is compared to a testosterone RIA with 125I-label and has similar specificity and precision to the latter with a quicker processing time, and is more cost effective. The added advantages that ELISA assays confer over RIAs in terms of isotope purchase and disposal make this an ideal procedure for use in a routine steroid laboratory.     

An enzyme-linked immunosorbent assay (ELISA) for plasma cortisol

A direct ELISA for plasma cortisol is described which is carried out in a standard 96 well microtitre plate. In this ELISA cortisol-thyroglobulin conjugate is immobilised to the microtitre plate and competes with cortisol in the standard or plasma sample for antibody binding sites. Following washing the rabbit cortisol antibody bound to immobilised cortisol is incubated with peroxidase labelled goat antirabbit IgG. Following further washing o-phenylenediamine is added, colour developed, and the plate read at 492 nm on a standard ELISA plate reader. This ELISA shows good agreement with RIA and its sensitivity, specificity and precision allow its use in the routine steroid laboratory.     

Improved canine and human visceral leishmaniasis immunodiagnosis using combinations of synthetic peptides in enzyme-linked immunosorbent assay

Background

Zoonotic visceral leishmaniasis (VL) is a severe infectious disease caused by protozoan parasites of the genus Leishmania and the domestic dogs are the main urban parasite reservoir hosts. In Brazil, indirect fluorescence antibody tests (IFAT) and indirect enzyme linked immunosorbent assay (ELISA) using promastigote extracts are widely used in epidemiological surveys. However, their sensitivity and specificity have often been compromised by the use of complex mixtures of antigens, which reduces their accuracy allowing the maintenance of infected animals that favors transmission to humans. In this context, the use of combinations of defined peptides appears favorable. Therefore, they were tested by combinations of five peptides derived from the previously described Leishmania diagnostic antigens A2, NH, LACK and K39.

Methodology/Principal Findings

Combinations of peptides derived A2, NH, LACK and K39 antigens were used in ELISA with sera from 44 human patients and 106 dogs. Improved sensitivities and specificities, close to 100%, were obtained for both sera of patients and dogs. Moreover, high sensitivity and specificity were observed even for canine sera presenting low IFAT anti-Leishmania antibody titers or from asymptomatic animals.

Conclusions/Significance

The use of combinations of B cell predicted synthetic peptides derived from antigens A2, NH, LACK and K39 may provide an alternative for improved sensitivities and specificities for immunodiagnostic assays of VL.     

Use of heterologous antigens for the immunodiagnosis of abdominal angiostrongyliasis by an enzyme-linked immunosorbent assay

Angiostrongylus costaricensis has a broad geographic distribution spanning from North to South America and the infections of vertebrates with this nematode can result in abdominal complications. Human infections are diagnosed by histological or serological methods because the isolation of larvae from feces is not feasible, as most parasites become trapped in intestinal tissues due to intense eosinophilic inflammation. Because A. costaricensis is difficult to maintain in the laboratory, an immunodiagnostic IgG enzyme-linked immunosorbent assay (ELISA) using antigens from the congeneric Angiostrongylus cantonensis species was evaluated against a panel of serum samples from patients who were histologically diagnosed with A. costaricensis infections. Sera from uninfected individuals and individuals infected with other parasites were used as controls. The sensitivity and specificity of the assay were estimated at 88.4% and 78.7%, respectively. Because the use of purified or cloned antigens has not been established as a reliable diagnostic tool, the use of heterologous antigens may provide a viable alternative for the development of an ELISA-based immunodetection system for the diagnosis of abdominal angiostrongyliasis.     

Anti-galactocerebroside antibodiesin human cerebrospinal fluids determined by enzyme-linked immunosorbent assay (ELISA)

The standard ELISA technique was improved for the detection of antigalactocerebroside antibody in biological fluid. Mouse monoclonal antigalactocerebroside antibody was used to demonstrate specificity and sensitivity of the technique. After optimization of the assay, the usefulness of this measurement for the evaluation of patients with multiple sclerosis was assessed. The presence of antigalactocerebroside antibodies in the cerebrospinal fluid of 20 patients with multiple sclerosis, 10 with other neurological diseases and 10 normal individuals was determined. All the CSF samples from normal individuals were negative. In patients with multiple sclerosis 14 of the 20 samples had elevated levels of antigalactocerebroside antibody, whereas with other neurological diseases 5 out of 10 were positive. Antigalactocerebroside levels were lower in samples from patients during an acute relapse than in those from more chronic cases. These results indicate that the presence of anti-galactocerebroside antibody in cerebrospinal fluid is not specific to MS but may reflect previous damage to myelin.Abbreviations and trivial names used ELISA Enzyme-Linked Immunosorbent Assay - CSF cerebrospinal fluid; galacto- or glucocerebroside, ceramide-1-0-beta-galactoside or-glucoside     

Enumeration of rhizobia by enzyme-linked immunosorbent assay (ELISA)

The use of the enzyme-linked immunosorbent assay (ELISA) to enumerate rhizobia in peat carrier and in soil has been investigated. The ELISA technique takes less time than the conventional plant infection technique often used to enumerate rhizobia present in the presence of other micro-organisms. A minimum of 10₂–10₃ cells are required for a detectable ELISA reaction, limiting the use of this technique when the number of rhizobia is low.     

An enzyme-linked immunosorbent assay (ELISA) for measurement of heterophile antibody

An enzyme-linked immunosorbent assay (ELISA) test has been developed for measurement of heterophile antibody. The microtiter test utilizes a bovine erythrocyte monolayer as antigen and anti-human IgM antiserum conjugated with horseradish peroxidase to measure the degree of binding of the heterophile antibody in the test serum with the erythrocytes. A single serum dilution yields quantitative results when read in a spectrophotometer. The ELISA test showed a sensitivity comparable with the immune adherence hemagglutination assay (IAHA) and other heterophile tests, good reproducibility, and high specificity.     

The use of dacron plates for dot enzyme-linked immunosorbent assay (dot-ELISA).

Dacron (polyethylenetherephthalate) is proposed as a matrix for dot-ELISA procedures, as an alternative to nitrocellulose. Plates of dacron were partially hydrazinolyzed and hydrazide groups introduced were converted to azide groups. The derivative dacron-antigen was covalently linked on to the plates through these azide groups. The derivative dacron-antigen was exaustively washed according to CROOK and antigen was still fixed onto the plates. Protein F1A purified from Yersinia pestis was used as a model. Titration of sera from immunized and non immunized rabbits against this protein was carried out by employing the dot-ELISA method. No significant difference was observed using dacron-antigen and nitrocellulose-antigen preparations. However, both procedures showed to have a significant better performance in comparison with the passive hemagglutination method. The specificity and reproductibility of the dot-ELISA assay using both preparations showed a similar behaviour. Nitrocellulose preparation was stable at 4 degrees C, 28 degrees C and -20 degrees C for 90 days, whereas the dacron-antigen derivative was stable only when stored at 4 degrees C. Dacron-antigen derivative could be re-used when the spot developing was proceeded using 4-chloro-1-naphtol as substrate.     

Evaluation of the enzyme-linked immunosorbent assay for the serodiagnosis of tularemia

The usefulness of the ELISA using as antigen prepared in our laboratory supernatant obtained after centrifugation of sonicated F. tularensis cell suspension was compared with the tube agglutination test with commercial available antigen. Paired serum specimens obtained from 6 patients with ulceroglandular syndrome of tularemia were tested in both tests. The cut-off limit of serum antibodies was set at mean antibody titre determined in the sera of 115 blood donors exceeded by three standard deviations. Antibodies to F. tularensis in diagnostically significant titre were detected in all 12 serum samples by both tests. However the titres obtained in ELISA were several times higher than in tube agglutination test. In the second serum sample the level of IgA and IgM was lower but the level of IgG higher than in the first sample. We could not observe any difference in the level of antibodies between paired serum specimens in tube agglutination test.     

Evaluation of an enzyme-linked immunosorbent Raji cell assay (ELISA) in the investigation of gastrointestinal cancer

Summary The levels of circulating immune complexes (CICs) have been estimated in a group of patients with colorectal cancer and gastric cancer, in addition to which a normal range has been established in a group of patients with benign gastrointestinal disease. A newly developed enzyme-linked immunosorbent Raji cell assay has been used in this study. Overall only 30% of patients with gastrointestinal cancer showed elevation of CIC levels outside the normal range. Elevated levels correlated with tumour differentiation bud did not correlate with site of disease or with the presence of metastases. In an attempt to define the specificity of CIC estimation, soluble tumour extract was added to sera from tumour-bearing patients. Specific IC elevations were produced by addition of allogeneic tumour extract of colon cancer in patients with colorectal cancer; this phenomenon was not seen when the same extract was added to the sera of patients with gastric cancer.     

Evaluation of an enzyme-linked immunosorbent Raji cell assay (ELISA) in the investigation of gastrointestinal cancer

The levels of circulating immune complexes (CICs) have been estimated in a group of patients with colorectal cancer and gastric cancer, in addition to which a normal range has been established in a group of patients with benign gastrointestinal disease. A newly developed enzyme-linked immunosorbent Raji cell assay has been used in this study. Overall only 30% of patients with gastrointestinal cancer showed elevation of CIC levels outside the normal range. Elevated levels correlated with tumour differentiation bud did not correlate with site of disease or with the presence of metastases. In an attempt to define the specificity of CIC estimation, soluble tumour extract was added to sera from tumour-bearing patients. Specific IC elevations were produced by addition of allogeneic tumour extract of colon cancer in patients with colorectal cancer; this phenomenon was not seen when the same extract was added to the sera of patients with gastric cancer.     

Detection of Renibacterium salmoninarum by the enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection and identification of Renibacterium salmoninarum. The immune γ-globulin used in the assay was absorbed with two species of cross-reacting bacteria to make a specific test system. R. salmoninarum could be detected in clinically-diseased fish within 30 minutes of preparing a kidney sample, and thus because of its ease of use, the ELISA could be employed as a rapid field test for bacterial kidney disease (BKD), although isolation of R. salmoninarum was more sensitive than the ELISA for detecting individual carrier fish.     

Serodiagnosis of extrapulmonary tuberculosis by enzyme-linked immunosorbent assay (ELISA)

Antibodies against Mycobacterium tuberculosis antigenic glycolipids were determined by enzyme-linked immunosorbent assay (ELISA). The 720 sera were collected from adult patients under investigation, suspected with extrapulmonary tuberculosis. The test performance was estimated according to definitive diagnosis in terms of specificity, sensitivity, positive predictive value and negative predictive value. These parameters calculated on 142 sera from patients with extrapulmonary tuberculosis and on 578 sera from patients with different nontuberculosis diseases were 92%, 81.6%, 70.9% and 95.1%, respectively. The specificity decreased to 85% when tuberculosis was associated with cancer or hepatic cirrhosis. In reactivated tuberculosis the sensitivity and the positive predictive value were 86.9% and 83.3%, respectively. Our results showed that ELISA was conclusive for patients with active tuberculosis, before the initiation of the treatment. The sensitivity decreased to 30% in inactive forms. It was demonstrated that ELISA was positive in cases with negative microscopy genitourinary tuberculosis. ELISA could be used as a supporting test in the laboratory diagnosis of active extrapulmonary tuberculosis in adults, disregarding the site involved.     

A dot-immunobinding assay for the laboratory diagnosis of tuberculous meningitis and its comparison with enzyme-linked immunosorbent assay

V.V. RADHAKRISHNAN AND A. MATHAI. 1991. In an attempt to establish an alternative to standard bacteriological methods in the laboratory diagnosis of tuberculous meningitis (TBM), a simple dot-immunobinding assay (Dot-Iba) was standardized to detect Mycobacterium tuberculosis antigen 5 and antimycobacterial antibody in cerebrospinal fluid (CSF) specimens of patients with TBM. Sensitivity and specificity of Dot-Iba was compared with conventional enzyme-linked immunosorbent assay (ELISA) and standard bacteriological techniques. The Dot-Iba showed excellent correlation with indirect ELISA for the detection of antimycobacterial antibody in CSF and showed 60% sensitivity and 100% specificity in culture-negative patients with TBM. However Dot-Iba was less sensitive for the detection of antigen 5 in CSFs and showed false negative results (60%) in culture-positive patients with TBM.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection ofCampylobacter jejuni antibodies,and comparison with a complement fixation test (CFT)

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of total anti-Campylobacter immunoglobulins in human sera. In this assay disintegratedCampylobacter bacteria were used as the antigen. Absorption tests including other possibly enteropathogenic bacterial species showed that the ELISA system displayed a high immunological specificity forCampylobacter. Using this ELISA it was found that in about 80% ofCampylobacter patients theseCampylobacter antibodies are produced to almost maximal levels within 8 days after onset of disease, and that they may persist for at least 4 months. Indeed,Campylobacter antibodies were demonstrated at low levels in a large number of control sera. However, accepting an antibody titre of 1: 640 as indicative ofCampylobacter infection, the statistical sensitivity of the ELISA system was 77% and the specificity 95%. In an epidemiological survey a high association was demonstrated between the severity ofCampylobacter-related symptoms and antibody titre values. Assessment ofCampylobacter antibody titres by means of this ELISA and by a complement fixation test in 92 sera from index patients and contacts with and without symptoms showed a high association of results.