Detection of adenovirus by enzyme-linked immunosorbent assay.

A solid-phase direct enzyme-linked immunosorbent assay (ELISA) was developed for the detection of adenovirus antigen in extracts of infected cells by using antihexon serum. Results with simulated clinical specimens consisting of normal nasal wash specimens seeded with varying concentrations of adenovirus type 5 showed that antigen could be detected in extracts of HEp-2 cell cultures inoculated with 10(2.5) 50% tissue culture infective doses (TCID50) and 10(1.5) TCID50 after 2 and 4 days of incubation, respectively. Fifty-three clinical nasal wash specimens containing adenovirus type 5 (stored for 5 years at -70 degrees C) were used to evaluate antigen detection by ELISA in HEp-2 cell extracts and by manifestation of cytopathic effect in human embryonic kidney cells. After 2 days of incubation, 62% were positive by ELISA, whereas none was positive for cytopathic effect. After 4 days of incubation, 76% were ELISA positive and 47% were positive for cytopathic effect. The results according to infectivity titers indicated that clinical specimens containing 10(3.0) TCID50 or greater were all positive by ELISA after 2 days of incubation in HEp-2 cells, and by 4 days all but one specimen containing 10(2.0) TCID50 or greater were ELISA positive. ELISA and immunofluorescent methods for antigen detection were compared using 24 of the 53 clinical specimens containing adenovirus type 5. Nearly equivalent sensitivities were demonstrated. These results suggest that ELISA may provide an alternative method of detecting and identifying adenoviral infections in humans.     

Investigation of sheep-associated malignant catarrhal fever virus infection in ruminants by PCR and competitive inhibition enzyme-linked immunosorbent assay.

Development of control measures for the gammaherpesviral disease of cattle known as sheep-associated malignant catarrhal fever (SA-MCF) has been hampered by a lack of accurate diagnostic tests either for the causative virus or for antibody against that virus. A recently developed competitive-inhibition enzyme-linked immunosorbent assay (CI-ELISA) for the detection of antibody to malignant catarrhal fever (MCF) virus (MCFV) in ruminants based on a monoclonal antibody to a widely conserved epitope of MCFV (H. Li, D. T. Shen, D. P. Knowles, J. R. Gorham, and T. B. Crawford, J. Clin. Microbiol. 32:1674-1679, 1994) and a PCR assay based on previously reported primers (S. I. F. Baxter, I. Pow, A. Bridgen, and H. W. Reid, Arch. Virol. 132:145-159, 1993) were used to detect anti-MCFV antibody and SA-MCFV DNA in sheep and other ruminants. The PCR amplified a specific 238-bp SA-MCFV genomic DNA fragment from peripheral blood lymphocytes of adult sheep and other ruminants with clinical MCF. Of 144 samples from randomly selected healthy adult sheep, 143 (99%) were positive by PCR and 136 (94%) were positive by CI-ELISA. The agreement between the two assays exceeded 95%. Of nine samples collected from cattle and deer with clinical MCF of apparent sheep origin, seven were CI-ELISA positive and all 9 were PCR positive. Among 59 serum samples from presuckling lambs, none contained antibody detectable by CI-ELISA. After suckling, maternal anti-MCFV antibody was detectable for about 10 +/- 3 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of airborne reindeer epithelial antigen by enzyme-linked immunosorbent assay inhibition

Hypersensitivity reactions to reindeer epithelial (RE) allergens have been recently demonstrated among reindeer herders. To determine the concentration of airborne RE antigens a method based on inhibition of the enzyme-linked immunosorbent assay (ELISA) was developed. Dust samples were collected in workshops where reindeer leather was processed and the workers had inhaled dry epithelial dust during their working shifts. Specific IgE to RE allergen could be detected in one of 5 workers. RE antigen concentrations varied from 0.01 microgram to 3.9 micrograms/m3 in the air of the workshop. All workers except one claimed work-associated respiratory symptoms.     

Detection of equine antibody to Babesia equi merozoite proteins by a monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay.

A competitive inhibition enzyme-linked immunosorbent assay (CI ELISA) was developed to detect antibody to Babesia equi. One hundred fifty-four equine serum samples from 19 countries were tested for antibody to B. equi by the complement fixation test and by CI ELISA. The CI ELISA and complement fixation test results agreed in 94% (144) of the serum samples tested. The 10 discrepant serum samples were retested and analyzed for ability to immunoprecipitate in vitro translation products from B. equi merozoite mRNA. Five discrepant results were clearly resolved in favor of the CI ELISA, and the remaining five discrepancies were not definitively resolved.     

Serological diagnosis of bovine neosporosis by Neospora caninum monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay.

Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, causes abortion and congenital infection in cattle. To investigate specific methods of antemortem diagnosis, the antibody responses of infected cows were evaluated by immunoblot assay and competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) by using a monoclonal antibody (MAb), MAb 4A4-2, against N. caninum tachyzoites. MAb 4A4-2 bound diffusely to the exterior surface of N. caninum tachyzoites and recognized a single 65-kDa band in immunoblots. MAb 4A4-2 was unreactive to antigens of two closely related apicomplexan protozoa, Toxoplasma gondii and Sarcocystis cruzi. Binding of MAb 4A4-2 was inhibited by mild periodate treatment of N. caninum antigen, demonstrating the carbohydrate nature of the epitope. Immunoblot analysis of N. caninum tachyzoite antigens with sera from cows with confirmed Neospora-induced abortion revealed at minimum 14 major antigens ranging from 11 to 175 kDa. Although the recognized antigens varied from cow to cow, antigens of 116, 65, and 25 kDa were detected in all cows with abortion confirmed to be caused by N. caninum. The binding of MAb 4A4-2 to N. caninum tachyzoite antigen was consistently inhibited by sera from Neospora-infected cows in a CI-ELISA format and was not inhibited by sera from Neospora antibody-negative cows. Furthermore, sera from cattle experimentally infected with T. gondii, S. cruzi, Sarcocystis hominis, or Sarcocystis hirsuta, which had cross-reactive antibodies recognizing multiple N. caninum antigens by immunoblot assay, did not inhibit binding of MAb 4A4-2 in the CI-ELISA. Thus, MAb 4A4-2 binds a carbohydrate epitope on a single N. caninum tachyzoite surface antigen that is recognized consistently and specifically by Neospora-infected cattle.     

Detection of Borrelia burgdorferi in urine of Peromyscus leucopus by inhibition enzyme-linked immunosorbent assay.

An inhibition enzyme-linked immunosorbent assay was developed to detect Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, in urine from white-footed mice (Peromyscus leucopus). Of the 87 urine specimens tested from 87 mice collected in widely separated tick-infested sites in Connecticut, 57 (65.5%) contained detectable concentrations of spirochetal antigens. Forty-seven (62.7%) of 75 serum samples analyzed contained antibodies to B. burgdorferi. In culture work with tissues from bladders, kidneys, spleens, or ears, 50 of 87 mice (57.5%) were infected with B. burgdorferi. Thirty-eight (76%) of 50 infected mice had antigens of this spirochete in urine, while 36 (72%) individuals had infected bladders. Of those with infected bladders, 24 (66.7%) mice excreted subunits or whole cells of B. burgdorferi into urine. Successful culturing of B. burgdorferi from mouse tissues, the presence of serum antibodies to this bacterium, and detection of antigens to this spirochete in urine provide further evidence that multiple assays can be performed to verify the presence of B. burgdorferi in P. leucopus.     

Detection of respiratory syncytial virus in nasopharyngeal secretions by inhibition of enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay developed for the demonstration of respiratory syncytial (RS) virus immunoglobulin G antibodies was used for the detection of RS virus in specimens of nasopharyngeal secretions (NPS) obtained from children with acute respiratory disease. Samples of NPS were incubated with a fixed amount of standard serum (human serum antibodies to RS virus) before being added to the enzyme-linked immunosorbent assay test plate. A decrease in the optical density value determined for this standard serum was seen with majority of NPS specimens from which RS virus had been isolated in tissue culture. The reliability and the specificity of this inhibiton test were supported by experiments with purified RS virus and by tests with NPS specimens containing other respiratory viruses.     

Detection of antibodies to Anaplasma marginale by an improved enzyme-linked immunosorbent assay with sodium dodecyl sulfate-disrupted antigen.

Sensitivities of two enzyme-linked immunosorbent assays (ELISAs) with particulate and sodium dodecyl sulfate (SDS)-disrupted Anaplasma marginale antigen were compared. The quotient of positive reference sera divided by the absorbance quotient of a negative reference serum at identical dilution was termed the signal-to-noise ratio. Optimal signal-to-noise ratios were dependent on both pretreatment of antigen and antigen concentration. SDS disruption of anaplasmal antigen resulted in a markedly improved signal-to-noise ratio of ELISA compared with ELISA with untreated antigen at identical antigen and serum dilutions. This represented higher sensitivity and lower background absorbance of the ELISA with disrupted antigen. SDS-disrupted A. marginale antigen was standardized by protein determination, and antigen, as well as precoated microtiter wells, was stored frozen without apparent loss of antigenic properties. ELISA results were in agreement with results of positive and negative control sera tested by the complement fixation test or by light microscopy Anaplasma diagnosis in Giemsa-stained blood films.     

Detection of antibodies to alphaviruses by enzyme-linked immunosorbent assay.

Cell culture-derived antigens detected antibodies to alphaviruses in human sera with the enzyme-linked immunosorbent assay technique. Results correlated with those from hemagglutination inhibition and neutralization tests.     

Detection of antibodies to influenza virus M protein by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay test system was developed in which purified influenza virus M protein was used for the detection of M antibody in human sera. Antibody levels to influenza A virus M protein were monitored in sera from a vaccine study population by using an enzyme-linked immunosorbent assay technique with purified M protein as the adsorbent antigen. A 10-fold variation in titers of preexisting M antibody was observed in this population of young adults. Increases of anti-M titer of 7- to 24-fold were observed upon immunization with Formalin-inactivated vaccine or after natural infection. The antibody response to M protein was dissociated from the response to the hemagglutinin or neuraminidase antigens. The M antibody response preceded or was coincident with the antibody response to H1 hemagglutinin upon natural exposure to circulating virus.     

Detection of Fusarium sp. contamination in corn by enzyme-linked immunosorbent assay

Fusarium verticillioides is a primary corn pathogen and one of the main producers of fumonisins, a group of mycotoxins that cause several diseases in animals and probably also affect humans. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on polyclonal antibodies was developed to detect the exoantigen of this fungus in corn and its correlation with traditional methods for mould detection was evaluated. Forty freshly harvested corn samples were analysed for F. verticillioides exoantigens, as well as for total mould count, ergosterol and fumonisin levels in order to evaluate the relationship between these parameters. In addition, F. verticillioides was grown in brain heart infusion (BHI) broth in order to evaluate the correlation between biomass and exoantigen concentration. There was no significant correlation between exoantigen concentration and total mould count, Fusarium sp. count and fumonisin levels. The correlation coefficient between exoantigens and ergosterol content was 0.52 and between biomass and F. verticillioides exoantigens in BHI broth was 0.84. These results suggest that this ic-ELISA has potential for Fusarium sp. detection in corn samples.     

Detection of eosinophil cationic protein (ECP) by an enzyme-linked immunosorbent assay

Eosinophil cationic protein (ECP) is a highly basic and potent cytotoxic single-chain zinc-containing protein present in the granules of the eosinophilic granulocytes. ECP appears to be involved in defence against parasites and in the tissue damage seen in subjects with allergic and inflammatory disease. To investigate ECP release from in vitro activated human eosinophils and to study the involvement of eosinophils in health and disease, we have developed a sensitive and specific enzyme immunoassay. ECP was purified from normal human peripheral blood eosinophils and polyclonal antibodies to ECP were subsequently raised in rabbits. The ELISA utilizes the biotin/avidin method and measures ECP within the range 15-1000 ng/l. The intra- and interassay coefficients of variation were 6% and 10%, respectively, and the recoveries of 12 and 25 pg of purified ECP added to diluted serum samples were 108 +/- 14.5% (mean +/- SD, n = 12) and 107 +/- 7.5%, respectively. The high sensitivity, reproducibility and specificity of this ELISA makes it suitable for the determination of minute amounts of ECP in in vitro systems as well as in various biological fluids.     

Detection of Anaplasma marginale rickettsemia prior to onset of clinical signs by using an antigen capture enzyme-linked immunosorbent assay.

An antigen capture enzyme-linked immunosorbent assay was developed by using monoclonal antibodies to conserved epitopes on the Anaplasma marginale MSP1a surface protein. The assay sensitivity was 1.1 (+/- 0.5)% parasitized erythrocytes, and all infected cattle were detected prior to development of 2.0%-parasitized erythrocytes. Positive tests preceded the onset of anemia by a mean of 2 days. The assay was specific for anaplasmosis, as demonstrated by nonreactivity with another common hemoparasitic pathogens.     

Detection of infectious pancreatic necrosis virus using an inhibition enzyme-linked immunosorbent assay

An inhibition enzyme-linked immunosorbent assay was used to detect infectious pancreatic necrosis (IPN) virus. In this assay the presence of virus was determined by measuring the decrease in titer of a known antiserum after incubation with a sample suspected to contain virus. The titer of the antiserum was measured with an indirect enzyme-linked assay. Compared to the double antibody sandwich method this assay required fewer reagents (only one anti-IPN serum was required). This assay was also sensitive enough to detect virus at levels of 1 X 10(2) TCID 50/ml. of purified virus and was able to detect virus in samples obtained in the field.     

Detection of cytomegalovirus in urine samples by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytomegalovirus (CMV) in urine using monoclonal antibodies directed against CMV as a capture for viral antigen. The assay was capable of detecting virus at 10^2.3TCID₅₀/ml as determined by titration of stock virus, strain Ad169. The assay was found to have a sensitivity of 65% and a specificity of 100% when 73 coded stored urine specimens were examined. Assuming that the poor sensitivity was due to loss of antigen following storage, we proceeded to analyse fresh urine specimens. Surprisingly, the assay gave negative results with 46 fresh urines known to contain CMV; however, following storage at +4°C for two weeks, 35 (76%) of these samples gave ELISA results in the positive range. This detection of CMV, after storage at +4°C, could be due to degradation of virus particles leading to release of soluble glycoproteins into the medium or to the presence of an inhibitory substance in fresh urine that is destroyed during storage.     

Detection of rubella antibodies by hemagglutination inhibition, indirect fluorescent-antibody test, and enzyme-linked immunosorbent assay.

Using hemagglutination inhibition (HAI) as a reference method, 292 (40 nonimmune, 252 immune) human serum samples were tested by indirect fluorescent antibody (IFA) and enzyme-linked immunosorbent assay (ELISA) for immune status and quantitation of rubella antibodies. The overall agreement with HAI for immune status was 99.7% (291/292) with IFA and 98.6% (288/292) with ELISA. Two specimens (0.7%, 2/292), negative by HAI, were equivocal by ELISA. Initially a 6.5% (19/292) overall disagreement was obtained for immune status evaluation between HAI and IFA, which was reduced to 0.3% (1/292) upon repeat testing. All of these samples were near the immune/nonimmune cutoff point (95 samples), reflecting an initial disagreement of 20% (19/95) in this category (HAI titers less than 1:20). Likewise, an initial overall disagreement of 4.5% (13/292) was obtained between HAI and ELISA which was reduced to 0.7% (2/292) upon repeated testing. Eleven of the 13 samples were near the immune/nonimmune cutoff point, reflecting an initial disagreement of 11.6% (11/95) with sera having an HAI antibody titer of less than 1:20. Quantitation of rubella antibodies by IFA showed an overall correlation with HAI of 86.6% within less than twofold titer and 99.3% within less than fourfold titers. In testing the ability of ELISA to quantitate antibody, a correlation coefficient (r) of 0.996 was obtained by plotting the measured average optical density (405 nm) of ELISA against the corresponding log of HAI titer. Both IFA and ELISA showed good correlation with HAI for immune status evaluation and for quantitation of rubella antibodies. Technically the HAI was the most cumbersome to perform, whereas IFA was the least technically demanding. Originally, 308 samples were tested; 16 samples (5.2%) could not be evaluated by IFA because of a high level of nonspecific fluorescence. The strict requirement of controlling the temperature range (23 to 24 degrees C) during substrate hydrolysis proved to be a problem with the ELISA test in our laboratory.     

Detection of Rift Valley fever virus antigen by enzyme-linked immunosorbent assay.

A double-antibody (sandwich) enzyme-linked immunosorbent assay (ELISA) was adapted to detect Rift Valley fever virus antigen. Antibodies were purified from hyperimmune mouse and rabbit sera by affinity chromatography, using CNBr-activated Sepharose 4B coupled to a beta-propiolactone-inactivated sucrose-acetone-extracted suckling mouse liver antigen. In the assay, antigen was captured by mouse antibody adsorbed to polystyrene plates and then detected by reacting sequentially with rabbit anti-Rift Valley fever virus antibody and swine anti-rabbit immunoglobulin G conjugated to alkaline phosphatase. ELISA proved to be useful in measuring viral antigen in different animal systems. However, great variation was found in the amount of antigen per PFU encountered in different circumstances. The ELISA system was optimized using supernatant fluids from infected Vero cell cultures and had a sensitivity of 10(5) PFU/ml. Hamsters develop progressive viremia, much as seen in susceptible domestic animals, such as lambs; ELISA could reliably detect 10(6) PFU/ml of viremic hamster serum. Rhesus monkeys with Rift Valley fever infection were positive by ELISA even when viremias were only 5 X 10(3) PFU/ml. ELISA also proved to be useful in measuring viral antigen in infected mosquitoes.     

Detection of antibodies to caprine arthritis-encephalitis virus by protein G enzyme-linked immunosorbent assay and immunoblotting.

Sera from goats suffering from caprine arthritis-encephalitis contained antibodies to virus proteins of 15, 17, 28, 40, and 130 kilodaltons in immunoblots of maedi-visna virus. We propose to use immunoblotting as a validation test for enzyme-linked immunosorbent assay and demonstrate that the specificity of indirect enzyme-linked immunosorbent assay can be improved by replacing second antibody by a protein G-avidin-biotin conjugate.