Enzyme-linked immunosorbent assay of antibody to group A Streptococcus-specific C carbohydrate with trypsin-pronase-treated whole cells as antigen

We describe the measurement by enzyme-linked immunosorbent assay of antibody to group A Streptococcus C carbohydrate in immunized rabbits and human sera, with trypsin-pronase-treated group A streptococcal whole cells used as the antigen. The optimal concentration of the enzyme-treated whole cells used to coat the wells was 2 x 10(7) cells per well. Rabbit antiserum diluted to 1:12,800 and human serum diluted to 1:1,000 were found to be the optimal concentrations for antibody measurement. Antibody that reacted with enzyme-treated whole cells in rabbit antiserum was absorbed with group A streptococcal whole cells, purified C carbohydrate, and N-acetylglucosamine only. Enzyme-treated whole cells did not react with anti-lipoteichoic acid antibody, and rabbit antiserum did not react with lipoteichoic acid. There was a highly significant correlation between the anti-C carbohydrate antibody titrated with enzyme-treated whole cells and that with purified C carbohydrate as antigen. The correlation coefficient for the immunoglobulin M (IgM) antibodies was r = 0.75, and for the IgG antibodies it was r = 0.77. When the IgG antibody titers to the enzyme-treated whole cells of the sera of patients with acute poststreptococcal glomerulonephritis and rheumatic fever were compared with those of sera of healthy individuals, the sera of patients with poststreptococcal sequelae had significantly higher titers than did healthy individuals. Although anti-C carbohydrate antibody in human sera mostly belonged to the IgG2 subclass, there was anti-C carbohydrate antibody that belonged to the IgG3 subclass in a certain percentage of patients with rheumatic fever and acute poststreptococcal glomerulonephritis.     

Enzyme-linked immunosorbent assay for detection of measles antibody.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of measles immunoglobulin G antibody (MEASELISA). This assay was found to be comparable to the measles hemagglutination inhibition (HAI) test. Approximately 500 sera from three centers were tested by MEASELISA and the HAI test. MEASELISA demonstrated values of greater than 99% for sensitivity, specificity, and accuracy. Values were very precise, with a mean coefficient of variation of 5.4%. MEASELISA values were shown by linear regression analysis to increase as HAI titers increased. A coefficient of determination of 1.00 was obtained from test center three. MEASELISA values were found to be linearly related (r2 greater than 0.97) to MEASELISA titers, thus enabling quantitation of measles antibody from a single value. Also, data are presented that show MEASELISA to be equivalent to complement fixation for evaluating paired sera for the presence of a significant increase in antibody levels to measles virus.     

Enzyme-linked immunosorbent assay employing a recombinant antigen for detection of protective antibody against swine erysipelas

The specificities and sensitivities of five recombinant proteins of the surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae were examined by indirect enzyme-linked immunosorbent assay (ELISA) with the aim of developing a reliable serological test for the detection of protective antibody against E. rhusiopathiae. Fully mature protein and the N-terminal 416 amino acids (SpaA416) showed sufficient antigenicities, and further examination was done with SpaA416 because of its higher yield. The antibody titers of pigs experimentally immunized with commercial live vaccine and two types of inactivated vaccines clearly increased after immunization, and all pigs were completely protected against challenge with virulent strains. On the other hand, the antibody titers of nonimmunized control pigs remained very low until they were challenged, and all showed severe symptoms or subsequently died. Interference with the production of antibody against live vaccine by maternal antibody or porcine respiratory and reproductive syndrome virus infection 1 week after vaccination was also clearly detected. Because the ELISA titer correlated well with the protection results, the specificity and sensitivity of the ELISA were further evaluated with sera collected from pigs reared on 1 farm on which animals had acute septicemia, 2 farms on which the animals were infected or free from infection, and 10 farms on which the animals were vaccinated with live vaccine, among others. The ELISA titers clearly revealed the conditions of the herds. These results indicate that the SpaA416 ELISA is an effective method not only for evaluating pigs for the presence of protective antibody levels resulting from vaccination or maternal antibody but also for detecting antibody produced by natural infection. This test has important potential for the effective control of swine erysipelas.     

Enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody to hepatitis B core antigen.

An enzyme-linked immunosorbent assay for detection of specific immunoglobulin M (IgM) antibodies against the core antigen of the hepatitis B virus (anti-HBc IgM) is described. The interference of IgM rheumatoid factor was evaluated quantitatively. In the anti-HBc IgM test, the rheumatoid factor gave false-positive results when the concentration exceeded 20 IU/ml. The rheumatoid-positive sera were disclosed by a control and retested for anti-HBc IgM after absorption of rheumatoid factor with latex particles aggregated with human IgG. In five of seven selected patients with acute hepatitis B followed to biochemical and clinical recovery, anti-HBc IgM was present transiently until antibodies against hepatitis B surface antigen (anti-HBs) appeared. Two patients had persistent anti-HBc IgM during the follow-up period. Four patients with hepatitis B surface antigenemia and progression to chronic liver disease did not clear their anti-HBc IgM in the period of observation (11 to 24 months). Anti-HBc IgM could not be demonstrated in 223 of 225 Danish blood donors. The two donors found positive for anti-HBc IgM also had anti-HBs. Twenty patients with acute A or non-A non-B hepatitis were negative for anti-HBc IgM. The enzyme-linked immunosorbent assay for anti-HBc IgM described here has a high specificity and sensitivity. The diagnostic relevance needs further evaluation, including quantitation of anti-HBc IgM, but the results presented indicate that anti-HBc IgM may be helpful in differentiating between prior and recent or ongoing hepatitis B infection.     

Enzyme-linked immunosorbent assay for detection of Streptococcus pneumoniae antigen.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Streptococcus pneumoniae polysaccharide antigen in cerebrospinal fluid and serum. Sensitivity and specificity were determined for purified antigen preparations. Specificity was also evaluated in the rabbit meningitis model, and the sensitivity was compared to counterimmunoelectrophoresis, using the infected rabbits' cerebrospinal fluid and serum. The ELISA was a specific technique for detecting S. pneumoniae antigen. ELISA was 25 times more sensitive than counterimmunoelectrophoresis for purified antigen and resulted in an increased positivity of the cerebrospinal fluid and serum from infected rabbits. ELISA should prove very useful in the diagnosis of pneumococcal infections.     

Enzyme-linked immunosorbent assay for immunoglobulin G antibody to encephalomyocarditis virus

An enzyme-linked immunosorbent assay for immunoglobulin G antibody to encephalomyocarditis virus was developed. This assay was comparable to antibody assay by neutralization. Its adaptability should be useful for laboratory and epidemiological studies of infections due to encephalomyocarditis virus.     

Enzyme-linked immunosorbent assay for detection of antibody to the hepatitis B surface antigen-associated delta antigen.

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for specific detection of antibody to the hepatitis B surface antigen-associated delta antigen. The sensitivity of ELISA was intermediate between that of previously described immunofluorescence and radioimmunological assays for anti-delta. Performance of ELISA was simple and required only ordinary and inexpensive laboratory equipment.     

Enzyme-linked immunosorbent assay for detection of equine infectious anemia virus p26 antigen and antibody.

A sensitive specific enzyme-linked immunosorbent assay utilizing purified p26 antigen was developed for the detection of antibodies to equine infectious anemia virus in naturally and experimentally infected horses. Generally, antibodies to the virus could be detected by the enzyme-linked immunosorbent assay 3 to 4 days earlier than by the standard agar gel immunodiffusion test, and they could be detected more reliably in horses with weak or equivocal agar gel immunodiffusion test reactions. The enzyme-linked immunosorbent assay was also successfully applied to the detection of p26 antigen in tissue culture fluids.     

Enzyme-linked immunosorbent assay for detection of antibody to varicella-zoster virus.

Primary varicella-zoster virus (VZV) infection is a serious illness in immunocompromised individuals, and a rapid, sensitive, and reliable assay to identify high-risk VZV-susceptible patients would be clinically useful. An enzyme-linked immunosorbent assay (ELISA) for antibody to VZV was compared with the fluorescent antibody-to-membrane antigen (FAMA) assay and found to be similar in both sensitivity and specificity. The antibody titers determined by both assays were also similar. The absence of antibody detected by ELISA correlated with susceptibility to VZV infection. Because it is simple to perform and has equivalent sensitivity to FAMA, ELISA should be useful for VZV antibody testing in diagnostic and research laboratories.     

Enzyme-linked immunosorbent assay for detection of Salmonella typhi protein antigen.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was designed for the detection of Salmonella typhi protein antigen. The optimal concentration of antibody for coating the plate was found to be 50 micrograms/ml. The optimum conditions for antibody coating and antigen and conjugate incubation were 37 degrees C for 3 h, 37 degrees C for 2 h, and 4 degrees C overnight, respectively. The enzyme-substrate reaction was allowed to take place at 30 degrees C for 1 h. The established ELISA was found to be reproducible, with an inter-run coefficient of variation of less than 12% for the detection of an S. typhi protein antigen concentration of 0.5 to 50 micrograms/ml. The minimal detectable level of the antigen was 0.5 micrograms/ml. Cross-reactions were observed with the high level (50 micrograms/ml) of protein antigens obtained from Salmonella typhimurium, Escherichia coli, Salmonella paratyphi A, and Salmonella enteritidis. The ELISA established was used for the detection of S. typhi protein antigen in serum from 62 patients with typhoid, 30 patients with clinically diagnosed typhoid fever, 21 patients with paratyphoid, 17 patients with pyrexia caused by other bacteria, and 160 normal, healthy individuals. It was found that the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of this assay were 83.87, 89.04, 87.93, 67.53, and 95.31%, respectively.     

Enzyme-linked immunosorbent assay for detection of antibody to leucocytozoon cauller yi

An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies to Leucocytozoon caulleryi in chicken sera. The assay was standardised in terms of antigen coating conditions, optimum antigen concentration, serum and conjugate dilutions. The schizont antigen showed a strong affinity for binding or absorbing to the surface of the polystyrene wells of the microplate. Nonspecific binding of antigen and cross-reactions were not observed with sera from specific-pathogen-free chickens and antisera against various chicken disease agents. The ELISA was more sensitive than the indirect immuno-fluorescent antibody test and the agar gel precipitation test.     

Use of a monoclonal antibody enzyme-linked immunosorbent assay to measure human respiratory glycoprotein production in vitro

High-molecular-weight glycoprotein from human airway cultures was used to generate murine monoclonal antibodies, one of which recognizes a high-molecular-weight, hyaluronidase-resistant glycoprotein localized by immunofluorescent microscopy and immunogold electron microscopy to the secretory granules of human airway submucosal gland mucous cells and goblet cells. This monoclonal antibody was used to develop an enzyme-linked immunosorbent assay (ELISA) that was adapted to the study of respiratory glycoprotein secretion from human airways in vitro. Using the assay, the effect of a known mucus secretagogue, the cholinergic agonist methacholine, was studied on explant cultures of tissue from human bronchus or from human nasal mucosa. In studies of human bronchus explants, methacholine, 100 and 10 microM, stimulated increased secretion of respiratory glycoprotein (RGP) by 109 +/- 8% (n = 14; P less than 0.001) and 96 +/- 14% (n = 9; P less than 0.001), respectively, above control values. In studies of human nasal turbinate mucosal explants, methacholine, 100 and 10 microM, stimulated increased secretion of RGP by 75 +/- 28% (n = 7; P less than 0.01) and 70 +/- 21% (n = 4; P less than 0.01) above control values. An ELISA for the measurement of RGP secretion may provide a sensitive and more specific method for the performance of in vitro studies of RGP secretion from human tissues.     

Enzyme-linked immunosorbent assay with a monoclonal antibody for detecting group A meningococcal antigens in cerebrospinal fluid.

Hybridomas were produced from spleen cells of BALB/c mice immunized with a membrane preparation from Neisseria meningitidis group A strain 4402 and S194/5.XXOBU.14 myeloma cells. The hybridomas were screened for secretion of antibodies suitable for an enzyme-linked immunosorbent assay (ELISA) diagnostic for group A meningococcal meningitis. One hybridoma antibody, 3G7, was directed against the pilus protein. This antibody bound to all six lipopolysaccharide and protein group A meningococcal serotyping strains, as well as to meningococcal strains from serogroups C, W135, and Y, but not to a strain of Escherichia coli, Haemophilus influenzae type b, or to two or more strains of Streptococcus pneumoniae, Neisseria gonorrhoeae, and Salmonella typhi. The ELISA used on antibody, antigen, antibody-conjugate sandwich. Rabbit anti-meningococcal serum was the coating antibody for the antibody sandwich, cerebrospinal fluids contained the bacterial antigens, and 3G7-alkaline phosphatase conjugate was the detecting antibody. The monoclonal antibody conjugate ELISA system was able to detect group A meningococcal antigens in 21 of 25 cerebrospinal fluid specimens that were positive in an immune rabbit serum conjugate ELISA; cerebrospinal fluid samples from patients with Haemophilus meningitis served as the controls. Counterimmunoelectrophoresis detected meningococcal antigens in 16 of the same 25 cerebrospinal fluid samples.     

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 mumps IgM antibody: comparison of IgM capture and indirect IgM assay

We have established two enzyme-linked immunosorbent assays (ELISAs) for detection of mumps IgM antibody, i.e., indirect IgM ELISA and IgM capture ELISA, for serodiagnosis of recent mumps infection. In the latter method, peroxidase-conjugated monoclonal antibody to mumps virus was employed. Both methods detected mumps antibody of IgM class only in serum fractions separated by centrifugation through a sucrose density gradient. Optical density values given by both ELISAs were correlated for most sera examined. Indirect IgM ELISA, however, gave a false positive reaction for sera containing both rheumatoid factor and mumps IgG antibody, while giving a false negative reaction for sera containing high titers of mumps IgG antibody. This technique was, therefore, less reliable than IgM capture ELISA. IgM antibody detectable by IgM capture ELISA was present in all patients with mumps by the fifth day of illness and persisted for up to 3 mth in most and up to 5 mth in same cases.     

Enzyme-linked immunosorbent assay for detection of solubleToxoplasma gondii antigen in acute-phase toxoplasmosis

Sixty-two sera from 51 patients with lymphadenopathy presumed to be due to acute-phase toxoplasmosis were tested for specific IgM class antibodies by both the immunofluorescence antibody toToxoplasma gondii in sera were first dissociated in 3M NaSCN. Antigen attached to the solid phase was detected with enzyme-coupled IgG antibody toToxoplasma antibody toToxoplasma gondii in sera were first dissociated in 3M NaSCN. Antigen attached to the solid phase was detected with enzyme-coupled IgG antibody toToxoplasma gondii. Neither hepatitis B surface antigen nor antigen ofMycoplasma pneumoniae, rubella, cytomegalovirus or herpes simplex virus interfered with this ELISA. Soluble antigen was detected in 13(30%) of 42 IgM-positive acute-phase toxoplasmosis patients and in only one of 20 sera cleared of IgM. None of an additional 44 IgM-negative patients with low IgG titres had a positive result in the antigen ELISA. Follow-up studies in four acute-phase toxoplasmosis patients showed that the soluble antigen cleared in all cases before the specific IgM antibodies. Simultaneous detection of IgM antibodies toToxoplasma gondii and soluble antigen would thus seem to indicate an early stage of the infection.     

Enzyme-linked immunosorbent assay for detection of antibody to Treponema hyodysenteriae antigens.

The enzyme-linked immunosorbent assay (ELISA) was evaluated and compared with the microtitration agglutination test for the detection of swine antibody to Treponema hyodysenteriae lipopolysaccharide antigens. Cells of T. hyodysenteriae serotypes 1 and 2 were extracted with hot phenol-water (68 degrees C). The lipopolysaccharide fraction from the aqueous phase was coated on plastic wells at concentrations of 1 micrograms (serotype 1) and 10 micrograms (serotype 2) of carbohydrate per ml. The ELISA was serotype specific when lipopolysaccharide antigens were reacted against sera from convalescent swine. Seroconversion of infected pigs was detectable with the ELISA within 1 to 2 weeks postinoculation and with the microtitration agglutination test 2 to 3 weeks postinoculation. Antibody titers could be detected in convalescent pigs as long as 19 weeks postinoculation by the ELISA and 12 to 13 weeks postinoculation by the microtitration agglutination test. Therefore, the ELISA may be useful for the detection of asymptomatic carriers.     

Enzyme-linked immunosorbent assay for detection of antibody to human herpesvirus 6.

The results obtained with an enzyme-linked immunosorbent assay (ELISA) for detection of human herpesvirus 6 (HHV-6) immunoglobulin G using a single 1:100 dilution of serum correlated well with those found by an indirect fluorescence microscopic assay (IFA) (r = 0.71). Concordant results were found in all 7 paired serum samples obtained from patients with acute primary infections and in 37 of 41 (90.24%) single serum samples. Fourteen serum samples (25%) which yielded nonspecific results by IFA were evaluable by ELISA. In a serologic survey using the ELISA, a disproportionate number of 12-month-old infants had low difference-of-optical-density values, suggesting that maternal antibody might persist beyond a year of age. This finding and the rises in antibody to HHV-6 found in patients with primary cytomegalovirus infections might lead to overestimation of HHV-6 infection rates in young children in seroprevalence studies. Other herpesvirus infections produced lesser effects on anti-HHV-6.     

Enzyme-linked immunosorbent assay for detection of IgG antibody to human herpesvirus 6

A lysate of human herpesvirus 6 (HHV-6)-infected cord blood mononuclear cells was used as antigen for enzyme-linked immunosorbent assay for the detection of IgG antibody to HHV-6. Antibody responses after exanthem subitum were well correlated with clinical recovery from the disease and the level of antibody activities was well correlated with indirect immunofluorescence assay and the neutralization test. Seroconversion to other human herpesvirus, including cytomegalovirus, was not observed in infants with exanthem subitum. All of the infants had by the age of 1 month antibodies to HHV-6, which decreased with age to the lowest level at the age of 3 to 6 months and then increased and reached the maximum level by 1 to 2 years of age. After 3 years of age, the prevalence was almost stable.