Infantile gastroenteritis associated with excretion of pestivirus antigens.

Faeces from children under 2 years old who had gastroenteritis that could not be attributed to recognised enteric pathogens were examined with a monoclonal-antibody-based immunoassay for Pestivirus antigens. Such antigens were detected in 30 of 128 episodes of gastroenteritis. Children without diarrhoeal disease and children infected with rotaviruses had little evidence of Pestivirus infection (faeces positive in 1 of 28 and 1 of 31, respectively). The diarrhoeal disease in children excreting Pestivirus antigens resembled that in other children except that it was more commonly associated with signs and symptoms of respiratory inflammation.

Enzyme immunoassay for detection of human immunodeficiency virus antigens in cell cultures.

A sensitive and specific avidin-biotin enzyme immunoassay that uses immunoreagents from naturally infected individuals was formulated for the detection of human immunodeficiency virus (HIV) antigens. A total of 500 cell culture samples from 111 cultures were tested in this assay and in a reverse transcriptase (RT) assay. Of 353 samples that were positive in the immunoassay, 174 were positive and 179 were negative in the RT assay. The specificity of the immunoassay results was supported by the failure of samples to react with nonimmune serum, by the ability of an anti-HIV type 1 monoclonal antibody to block the reactivity of selected samples, and by the appearance of RT activity in samples drawn from some cultures after a longer period of cultivation. HIV antigens were detected in 174 of 176 RT-positive samples (sensitivity, 98.9%). A comparison of the kinetics of antigen production and RT activity revealed that detectable antigen levels frequently preceded the appearance of RT activity. Thus, 50% of virus-containing cultures were identified within 9 days by immunoassay compared with 14 days by RT assay. In addition, RT activity was often detected intermittently in cultures sampled on several days, whereas antigen levels did not decline after initial appearance. Enzyme immunoassays for HIV antigen detection that use easily obtained reagents and simple technologies could facilitate laboratory and clinical research which requires cultivation of viruses.

Antibodies to rotaviruses in chickens' eggs: a potential source of antiviral immunoglobulins suitable for human consumption.

The prevalence of antibodies to human rotaviruses in commercially available eggs and egg products that are suitable for human consumption was investigated. The yolks of virtually all of the individual eggs and pasteurized pooled egg preparations contain antirotavirus antibodies detectable by means of enzyme immunoassay systems. Also, the eggs and egg preparations are capable of inhibiting the growth of two strains of rotaviruses in tissue culture. Chromatographic studies indicated that the antigen-binding activity is limited largely to the immunoglobulin fractions of the egg yolks. The antibody levels in eggs can be increased by the immunization of hens with purified rotavirus preparations, and the immunoglobulins isolated from the eggs of immunized hens can prevent the development of rotavirus gastroenteritis in experimentally infected animals. Egg preparations might serve as a practical source of antiviral antibodies suitable for consumption by infants and young children.

Self-contained enzymic membrane immunoassay for detection of rotavirus antigen in clinical samples.

A self-contained enzymic membrane immunoassay (SCEMIA) system has been developed for the detection of viral antigens in clinical samples. The assay system makes use of antiviral antibodies bound to a nylon membrane, a flow-through washing procedure, and a clearly visible endpoint of the enzymic reaction. A SCEMIA system with antibodies against rotavirus detected rotavirus antigen, within 15 min, in all faecal samples from children with gastroenteritis that were positive for antigen in a standard microplate enzyme immunoassay, which took 4 h to complete. In addition, the SCEMIA could detect rotavirus in faecal samples collected from infected individuals both before and after antigen could be detected by a standard immunoassay system. Rotavirus antigen was not detectable in control children who did not have evidence of rotavirus infection. SCEMIA systems are an accurate, rapid, and inexpensive means for the practical diagnosis of viral infections in human beings.

Antibody to human rotavirus in cow's milk.

Rotavirus infection is an important cause of gastroenteritis in infants and young children. Since the virus replicates in the intestinal lumen, we investigated the presence and effectiveness of rotavirus antibody in three forms of milk: raw milk, pasteurized milk, and commercially available infant formulas. Both raw and pasteurized milk contained detectable levels of IgG1 antibody directed at rotavirus. On the other hand, little or no anti-rotavirus antibody was detected in commercially available infant formulas or other sterile milk preparations. The milk samples with rotavirus antibody were capable of inhibiting the replication of simian, bovine, and human rotaviruses in tissue culture. In addition, they were capable of protecting mice from infection and disease in a murine model of rotavirus infection. On the other hand, the formula preparations were incapable of modifying the in vitro replication of rotavirus strains in tissue culture and did not prevent symptomatic gastroenteritis in the mouse model. We conclude that the alteration of milk-processing procedures or the addition of effective antibodies to milk preparations commonly used in the nutrition of young children may alter the clinical course of rotavirus infection or decrease the transmission of rotavirus throughout susceptible populations.

Use of an enzyme-linked immunosorbent assay to measure antigenaemia during acute plague.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure concentrations of the specific F1 antigen of the plague bacillus in biological fluids. The assay employed a monoclonal antibody to capture the antigen. Sensitivity of the assay was 0.4 ng of F1 antigen. ELISA-inhibition was used to confirm the specificity of the reactions.This assay detected F1 antigen in two of ten sera from patients with acute bubonic plague and indicated that antigenaemia in man during plague may reach levels of 4-8 mug of F1 antigen per ml of serum.The probability for a correct serodiagnosis of plague was improved when the patients' sera were tested for both antibody and antigen. Two patients with antigenaemia did not have antibody, while two patients with antibody lacked antigenaemia.

Enzyme immunoassays for the detection of bacterial antigens utilizing biotin-labeled antibody and peroxidase biotin--avidin complex.

This report describes the use of biotin-labeled antibodies in sensitive enzyme immunoassay systems for the measurement of bacterial antigens. Biotinylated immunoglobulins could be reproducibly formulated using N-hydroxysuccinimide biotin ester to link biotin to the immunoglobulin. Binding of the biotinylated antibody to solid-phase antigen was efficiently measured by reaction with a complex consisting of biotinylated antibody and unlabeled avidin. This immunoassay system was at least as sensitive as ones which utilized enzyme-labeled or fluorescein-labeled antibodies for the detection of antigens from Streptococcus pneumoniae and Haemophilus influenzae type b. In the case of Streptococcus pneumoniae the assay system could detect antigen in the supernatants from broth cultures containing as few as 10(3) organisms/ml. Assay systems utilizing biotinylated antibody and avidin-biotin complex can provide for sensitive, specific assays for the measurement of microbial antigens.

Rapid multiple-determinant enzyme immunoassay for the detection of human rotavirus.

Enzyme immunoassays (EIAs) can be used to detect rotavirus antigens accurately in human stool specimens. Conventional EIA systems require prolonged incubations and multiple washing steps, thus making them impractical for truly rapid diagnosis. However, more rapid solid-phase EIA systems can be devised which make use of simultaneous binding of enzyme-labeled and solid-phase antibodies at immunologically distinct antigenic sites. A multiple-determinant EIA system was devised that was capable of detecting small quantities of rotavirus antigen in less than 40 min. This assay system accurately detected rotavirus antigen in 45 stool specimens positive for rotavirus. No false-positive reactions were noted provided that appropriate control reactions were performed. The rapid double-determinant EIA system provides an accurate, objective means for the rapid diagnosis of human viral infections.

Gastroenteritis associated with enteric type adenovirus in hospitalized infants.

Enteric types of adenovirus have recently been identified as a causative agent of infantile gastroenteritis. We utilized enzyme immunoassay and tissue culture techniques to evaluate prospectively the role of ET Ad in diarrhea occurring in hospitalized infants. We found that ET Ad was associated with 14 of 27 cases of diarrhea occurring during a 12-week study period in the late autumn and early winter months; ET Ad was found in the stool of only one of 72 children without diarrhea (P less than 0.001). Although adenoviruses other than ET Ad were found in the stools of two of the 27 children with diarrhea, such viruses were also found in the stools of five of 72 children without diarrhea and thus could not be statistically correlated with acute gastroenteritis. Children infected with ET Ad had diarrhea for a mean of 8.0 days, compared to a mean duration of 4.2 days for the children with gastroenteritis not associated with ET Ad. Thirteen of the 14 children with ET Ad gastroenteritis had respiratory symptoms such as cough, rhinorrhea, or wheezing, six had roentgenographic evidence of pneumonia, and three children had bilateral conjunctivitis. This study documents that ET Ad can be an important cause of acute gastrointestinal disease in hospitalized infants and young children and that gastrointestinal infections with ET Ad can be associated with a high rate of respiratory disease.

Comparison of fluorescent and colorigenic substrates for enzyme immunoassays.

A variety of substrates can be employed in enzyme immunoassays (EIAs) for the measurement of enzyme-labeled immunoreactants. We compared the sensitivities of a fluorescent and a colorigenic substrate in an EIA system for the measurement of Haemophilus influenza purified polyribose phosphate. After a 10-min substrate incubation, the EIA in which the fluorescent substrate was used could detect 10 pg of polyribose phosphate per ml, whereas the EIA in which the colorigenic substrate was used required the addition of 640 pg of polyribose phosphate per ml to generate a positive reading. However, the use of longer substrate incubation periods led to an increase in sensitivity of the colorigenic EIA. After an incubation period of 240 min, the sensitivity was equal to that of the EIA in which the fluorescent substrate was used. These results suggest that the ultimate limit of sensitivity of EIA systems is determined by the nature of the antigen-antibody reactions. However, the use of high-energy substrates in EIA systems can allow for the attainment of maximal sensitivity after short enzyme-substrate incubation periods.

Enzyme immunoassays for measurement of cytomegalovirus immunoglobulin M antibody.

The diagnosis of congenital cytomegalovirus (CMV) infection is often accomplished by the detection of circulating antibody directed against CMV. We devised a method for measuring CMV-specific immunoglobulin M (IgM) based on the isolation of IgM antibody by reaction with a solid phase coated with antihuman IgM. The determination of IgM antibody specific for CMV was accomplished by the subsequent addition of CMV or control antigen and enzyme-labeled CMV antibody (solid phase-IgM method). We compared the sensitivity and specificity of this method with those of a conventional form of solid-phase enzyme immunoassay in which CMV antigen is bound to the solid phase (solid phase-antigen method). Both assay systems were capable of detecting CMV-specific IgM antibody in the sera of 10 babies with documented CMV infection and in those of the mothers of 4 of these babies. The solid phase-IgM method yielded negative results in all 66 sera available from babies who did not have congenital CMV infection. On the other hand, the solid phase-antigen system yielded false-positive results in 12 (18%) of these sera. In addition, the solid phase-antigen system yielded false-positive results in 8 of 12 sera obtained from patients with demonstrable rheumatoid factor. However, the solid phase-IgM system yielded negative results for the rheumatoid sera, provided that appropriate control reactions were performed. The solid phase-IgM system is thus a specific and sensitive method for the determination of CMV IgM antibody.

Investigation of enzyme immunoassay time courses: development of rapid assay systems.

An enzyme immunoassay (EIA) consists of a series of antigen-antibody reactions which result in the binding of an enzyme-labeled antibody to a solid phase. The performance time of an EIA determination is thus largely dependent upon the time required for the antigen-antibody reactions. In an attempt to develop a rapid EIA system, we investigated the time course of an EIA system for the measurement of Haemophilus influenzae type b polysaccharide. We found that, although the use of short incubations led to a decrease in sensitivity, an assay system utilizing 10-min incubation periods was still capable of detecting antigen at a concentration of 1 ng/ml. Important factors in the sensitivity of EIAs with short incubation times were the performance of the reaction at 37 degrees C and the incubation of the solid phase with constant agitation. Utilizing these techniques, we developed an EIA system for the measurement of H. influenzae type b polysaccharide which could be completed in less than 30 min. This system was sufficiently sensitive to detect H. influenzae polysaccharide in the cerebrospinal fluids of nine patients with proven H. influenzae meningitis. Thus, EIA systems utilizing short incubation times might be useful for the rapid detection of infectious antigens in body fluids.

Staphylococcal protein A-enzyme immunoglobulin conjugates: versatile tools for enzyme immunoassays.

This report describes the use of peroxidase-labeled staphylococcal protein A to prepare conjugates suitable for direct enzyme immunoassays (EIAs). Such conjugates were used to develop direct EIA systems for the measurement of two antigens associated with human infections, human rotavirus and Haemophilus influenzae type b polysaccharide. Both systems were as sensitive or more sensitive than currently available EIAs for the measurement of standard antigens. In addition, both systems could be utilized to correctly identify the antigens in clinical specimens obtained from sick patients. Efficient enzyme conjugates prepared with enzyme-labeled staphylococcal protein A were simple to formulate providing that immunoglobulin of the appropriate animal species was available as the source of antibody. The use of such conjugates might increase the availability of practical EIA systems for the measurement of a wide range of medically important antigens.

Chemical selection of mutants that affect alcohol dehydrogenase in Drosophila. II. Use of 1-pentyne-3-ol.

We describe a procedure for the selection of alcohol dehyrogenase negative mutants in Drosophila. The method consists of exposing eggs and larvae to low concentrations of 1-pentyne-3-ol dissolved in the culture medium. Only those flies with greatly reduced levels of alcohol dehydrogenase activity survive. In addition, genotypically negative flies die if their mothers are alcohol dehydrogenase positive. Using this procedure and formaldehyde to generate mutants, we were able to detect seven alcohol dehydrogenase negative mutants out of 350,000 individuals subjected to selection. At least five of the mutants contain small deletions that include the alcohol dehydrogenase locus.