Enzyme-linked immunosorbent assay for detection of Pseudomonas aeruginosa Lipopolysaccharides.

A double-antibody sandwich method of enzyme-linked immunosorbent assay was developed to detect lipopolysaccharides (LPS) from the eight most prevalent Pseudomonas aeruginosa serotypes (O1, O2,5,16, O3, O4, O6, O9, O10, and O11). Immunoglobulin M fractions from rabbit antisera were used as the coating antibody and as the antibody to be conjugated to an enzyme. When two fractions of LPS (I and II) obtained by Sepharose 2B column chromatography were assayed, LPS II showed 10 to 100 times more activity than LPS I; the detection level of LPS II was 0.1 ng/ml. When LPS in purified preparations or in culture filtrates was examined with both homologous and heterologous antibody systems, the same specificity pattern was demonstrated, suggesting that, in crude filtrates, antigens other than LPS do not interfere in the assay. The method described can be used to detect LPS in biological fluids.     

Enzyme-linked immunosorbent assay for detection of streptolysin O antibodies.

An enzyme-linked immumosorbent assay (ELISA), based upon the detection of streptolysin O antibodies in human sera, was developed. Disposable polystyrene tubes, sensitized with streptolysin O antigen, were used as the test vehicles. Corresponding antibodies, present in test sera, were detected by binding of the antibodies to goat anti-human immunoglobulin G conjugated to horseradish peroxidase. Demonstration of bound conjugate was accomplished by monitoring peroxidase activity spectrophotometrically at 450 nm, using 5-aminosalicylic acid as the indicator. A total of 97 human sera, previously analyzed by means of the anti-streptolysin O titration technique, were evaluated with the ELISA procedure. A direct quantitative relationship, found to be statistically significant, was demonstrated between Todd units and absorbance values obtained with ELISA.     

Enzyme-linked immunosorbent assay for the detection of influenza type-specific antibodies

An enzyme-linked immunosorbent assay (ELISA) using a purified ribonucleoprotein antigen is described for detection of type-specific anti-influenza virus antibodies. ELISA was found to be more sensitive than indirect immunofluorescent and complement fixation tests. A significant increase in antibody titer could be demonstrated by ELISA between serum specimens collected prior to and during the influenza season. ELISA appears to be useful for rapid and sensitive diagnosis of influenza infections.     

Enzyme-linked immunosorbent assay for detection ofPseudomonas aeruginosa H (Flagellar) antigen

An enzyme-linked immunosorbent assay (ELISA) with goat anti-rabbit IgG conjugated to peroxidase was used to test for the two antigen types ofPseudomonas aeruginosa: b (homogeneous) and a (heterogeneous) which contains the common subantigen (a₀) and combinations of subtypes (a, a₂ a₃ a₄). Preparations of b-type flagellar antigen could be distinguished from a-type by using b-adsorbed antisera titers as reciprocals of endpoint dilutions exceeding one million. Extracts from nonflagellated bacteria or purified lipopolysaccharides from the same strain were used as controls, which showed only background activity. Unknown flagellar antigen was determined using both isolated antigen preparations and formalin-killed bacterial cells. The ELISA procedure proved much more sensitive than the slide agglutination procedure: whereas nine of 18 strains tested did not react in the slide agglutination procedure all 18 strains were definitively typed as a or b strains with the ELISA. The ELISA also revealed the presence of a dominant, cross-reacting epitope (a₀) in the heterogeneous a-type flagella using either isolated antigen or intact cells.     

Enzyme-linked immunosorbent assay for chlamydial antibodies.

An enzyme-linked immunosorbent assay (ELISA) detected chlamydial antibodies in human sera. The assay antigen produced in cell cultures infected with Chlamydia psittaci was Formalin-fixed to microplates. Single convalescent-phase sera positive for chlamydial antibodies by a complement-fixation test were positive at even higher dilutions by ELISA. Paired sera with diagnostic rises in complement-fixing antibody showed seroconversion by ELISA also. Control sera from persons with no history of chlamydial infection were negative by both tests. Sera from patients with psittacosis or lymphogranuloma venereum were ELISA positive, indicating that the assay with the antigen used in this study is genus specific rather than species specific.     

Enzyme-linked immunosorbent assay detection of microcystins using new monoclonal antibodies

New monoclonal antibodies (mAbs) against the microcystin-leucine-arginine variant (microcystin-LR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. Using these mAbs, an enzyme-linked immunosorbent assay (ELISA) experiment was made for the detection of cyanobacterial hepatotoxins, microcystins in water sampled in Soyang Lake, Korea. The performance of the ELISA test with mAbs established in this study was evaluated. The ELISA detection was compared with HPLC detection. Since the detection limit of HPLC is several orders of magnitude higher than with ELISA, attention was also paid to concentration of samples with solid phase extraction cartridges.     

Enzyme-linked immunosorbent assay for detection of antibodies to the unmodified beta-lactam ring

Dose- and pH- dependent carbodiimide-mediated coupling of Penicillin-G to polystyrene microtiter-plates that leaves the beta-lactam ring unchanged is described. A new ELISA method was developed using Penicillin-G coated plates. The binding of 3 different monoclonal antibodies as well as human IgG antibodies of the IgG₁ and IgG₃ subclasses is demonstrated, whereas IgG₂, IgG₄ and IgE antibodies did not bind. Thus, covalently coupled Penicillin-G can be used to study the immune-response to the unchanged β-lactam ring in patients receiving penicillin therapy. The new method is complementary to hitherto described techniques, which generally only allow detection of antibodies binding to penicilloyl-groups.     

Enzyme-linked immunosorbent assay for detection of antibodies to murine hepatitis virus.

An enzyme-linked immunosorbent assay was developed for the detection of antibodies to murine hepatitis virus. A high prevalence of antibody to murine hepatitis virus was found by the enzyme-linked immunosorbent assay in colonies with a low prevalence of complement-fixing antibodies. Murine hepatitis virus strain A59 was found to be broadly reactive as an enzyme-linked immunosorbent assay antigen.     

Enzyme-linked immunosorbent assay for Pseudomonas aeruginosa exotoxin A.

An enzyme-linked immunosorbent assay (ELISA) is described for Pseudomonas aeruginosa exotoxin A. A double antibody sandwich method was used, employing polyvinyl microtiter plates as the solid phase, a primary coat of monospecific rabbit antitoxin serum, an outer layer composed of a horseradish peroxidase-sheep antitoxin immunoglobulin G conjugate, and an ortho-phenylene-diamine substrate. Absorbance (optical density) of hydrolyzed end product was read spectrophotometrically at 492 nm. ELISA detected as little as 30 pg (0.3 ng/ml) of purified toxin, and absorbance was linear over a 20-fold or greater concentration range. Toxin was demonstrated in culture filtrates from 42 of 48 (88%) consecutive clinical P. aeruginosa isolates compared with 37 of 48 (77%) positive by hemagglutination inhibition. Results of the two assays correlated closely (r = 0.82, P less than 0.001). Specificity was confirmed by neutralizability of ELISA activity with monospecific antitoxin. ELISA was thus a sensitive, specific, and quantifiable technique for the assay of P. aeruginosa exotoxin A in both purified and crude culture materials.     

Enzyme-linked immunosorbent assay for detection of specific antibodies to Ureaplasma urealyticum serotypes.

Optimal conditions of a micro-enzyme-linked immunosorbent assay system for the detection of immunoglobulin G antibodies to Ureaplasma urealyticum were established with rabbit antisera. Initially, the antisera, raised against eight U. urealyticum serotypes grown on medium containing horse serum, displayed nonspecific reactions with our enzyme-linked immunosorbent assay antigens. Substitution of fetal bovine serum in the medium eliminated this nonspecificity. The assay was then serotype-specific for the original eight U. urealyticum serotypes. The prominent homologous reaction was easily differentiated from the heterologous reactions. A one-way cross-reaction was observed with serotype 2 antiserum and serotype 5 antigen. The results were reproducible and could be obtained in 4 h with only 10 microliters of serum for eight serotypes. Optimal antigen concentrations for the U. urealyticum serotypes ranged from 0.40 to 1.60 micrograms/ml. Our results indicated that enzyme-linked immunosorbent assay has the potential for the detection of antibodies to specific serotypes of U. urealyticum.     

Enzyme-linked immunosorbent assay for detection of antibodies against Streptococcus pneumoniae capsular polysaccharides.

The development of an assay to measure the human immune response to pneumococcal capsular polysaccharides is described.     

Enzyme-linked immunosorbent assay for detection of immunoglobulin A and G antibodies toCampylobacter pylori

An enzyme-linked immunosorbent assay (ELISA) employing an acid-glycine extract was used to detect IgG and IgA antibodies toCampylobacter pylori in sera from 179 patients with upper gastrointestinal disease, 174 blood donors and 65 children. The incidence of positive ELISA results clearly increased with the severity of histopathologic findings in the antrum mucosa and was also high in patients with peptic ulcers and gastric cancer. The incidence in blood donors and children was much lower and increased with age. The results achieved with the ELISA were similar to those observed previously using the immunoblot method. Differences between whole cell preparation and acid-glycine extract with respect to their protein profiles and immunoblot reactivities were minor. IgM titres were very low and could not be related to histopathological findings, peptic lesions or culture findings. The ELISA may be particularly useful for monitoring the outcome of therapy aimed at eradication ofCampylobacter pylori.     

Enzyme-linked immunosorbent assay for quantitation of islet cell antibodies

We have developed an enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of islet antibodies (ICA) in sera from insulin-dependent diabetic (IDD) subjects or from Bio-Breeding/Worcester (BB/W) rats. Whole rat or mouse islet cells, either glutaraldehyde-fixed or desiccated, which can be stored and used over a long period, were used as antigens. The amount of antibody bound to the cells is quantitated by the addition of sheep anti-human or anti-rat IgG conjugated with beta-galactosidase. This quantitative ELISA compared favourably with other assays for ICA detection.     

Enzyme-linked immunosorbent assay for detection of human antibodies to Salmonella typhi Vi antigen

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to Salmonella typhi Vi antigen in human serum, and the results were compared with those from a previously described hemagglutination assay (HA). The ELISA detected Vi antibodies at a titer of greater than or equal to 20 in 40 (52%) of 77 sera from typhoid fever patients, whereas the HA gave titers of greater than or equal to 20 in 35 (47%). Determination of titers of serum specimens from 170 persons without typhoid fever revealed Vi antibody titers of greater than or equal to 20 in 4 (2.3%) by the ELISA and 3 (1.7%) by the HA. Unlike the sensitized erythrocytes used in the HA, the ELISA reagents have a shelf life of greater than or equal to 1 year. The ELISA may be preferred by some laboratories, especially those already performing other ELISA tests.     

Enzyme-linked immunosorbent assay for streptococcal M protein antibodies.

The enzyme-linked immunosorbent assay (ELISA) described by Engvall and Perlmann, which uses antigen-coated tubes and enzyme-labeled anti-immunoglobulin, has been used for the detection of antibodies against streptococcal M protein. The antigen used in the assay was obtained by guanidine extraction of type M-12 streptococcal cell walls followed by hydroxyapatite chromatography. This antigen has the capacity to elicit bactericidal antibodies in rabbits. The results show that the ELISA is specific and highly sensitive for the detection of antibodies in rabbit and human antisera. Preliminary results suggest that, when M-12 antigen is used, the antibodies detected by ELISA are the same antibodies detected in the bactericidal test. The assay has been performed with human and rabbit sera. There was a 96% agreement between bactericidal and ELISA results with rabbit sera and 97.5% agreement with human sera. All bactericidal antibody-positive sera tested thus far yielded positive ELISA results.     

Enzyme-linked immunosorbent assay using three different antigen preparations for detection of antibodies toChlamydia trachomatis

The ability of an enzyme-linked immunosorbent assay (ELISA) to detect antibodies toChlamydia trachomatis was evaluated in 100 sera using three different antigen preparations as substrates (sonicated organisms, Triton X solubilized antigen and SDS solubilized antigen). The results were compared to those obtained by a standard microimmunofluorescence assay. The results obtained by the three ELISA techniques and the microimmunofluorescence method were in relatively good agreement (76%); some discrepant results were observed in sera with a low antibody titer. There was good agreement of results obtained by the three ELISA techniques (84%). The microimmunofluorescence method showed the greatest sensitivity. Assuming the microimmunofluorescence method accurately demonstrates antibodies, the ELISA using Triton X solubilized antigen showed the highest degree of specificity (97%), and the ELISA with sonicated organisms the greatest sensitivity (82%) and accuracy (86%).     

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.