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An animal model has been developed to study the evolution of intra-abdominal abscesses. Gelatin capsules containing pooled colonic contents and barium sulfate were prepared in an anaerobic chamber and implanted into the pelvic region of Wistar rats. The natural course of the ensuing disease was studied in various groups according to the source of the inoculum and sex of the recipient. Colonic contents derived from rats fed a grain diet produced a highly lethal disease with an 80% mortality rate for males and 100% for females. Most deaths occurred within 3 days of implantation, and autopsies showed generalized peritonitis. The addition of blood to the inoculum caused a rapidly fatal peritonitis in all animals. With an inoculum derived from meat-fed rats implanted in male recipients, there was a biphasic disease. Initially, there was peritonitis associated with 43% mortality. All animals that survived this acute period developed discrete intra-abdominal abscesses by the seventh postoperative day. The latter stage was characterized by an indolent course and progressive enlargement of abscesses.

An enzyme-linked immunosorbent assay has been developed by using IgM antibodies from the acute stage as a source to capture the antigen in stools of patients with epidemic non-A, non-B (NANB) viral hepatitis. 29/69 (42.3%) of the patients and 3/9 (33.3%) contacts were positive for a suspected NANB viral antigen. However, only 1/27 (3.7%) of the negative controls drawn from amongst the patients with amoebiasis, giardiasis, hepatitis due to virus A and healthy individuals was positive for NANB antigen in the stool. The suspected NANB viral antigen was more frequently detected in stools collected between the 14th and 18th day of icteric hepatitis. The study suggests that IgM antibodies from patients with acute viral NANB hepatitis react with an antigen present in the stools of a high proportion of patients with epidemic NANB viral hepatitis. This serological test may be useful to establish the etiological diagnosis of non-A, non-B (fecal-oral) viral hepatitis. ELISA-positive stools contained 27 nm viral particles.

A group of patients all regularly receiving blood products for a congenital bleeding disorder were investigated for evidence of past infection with hepatitis A and B, and compared with a control group. There was a significantly higher percentage of the test group who had markers of infection with these viruses, and 15 patients had symptoms of hepatitis during the study. The greater number of the test group showing evidence of past hepatitis A infection can only really be explained by acquiring antibody from blood products, especially since none of our patients with hepatitis had hepatitis A and there is evidence that this is not an infection associated with blood products. We conclude that the majority of patients in our study showing symptoms of hepatitis had non-A, non-B hepatitis.

Immunoglobulin E (IgE) was found by a double antibody radioimmunoassay technique (PRIST) in extracts of feces from 21 of 40 children with different kinds of allergy. 15 of the 22 children with gastrointestinal allergy and atopic dermatitis, but only 6 of the 17 patients with hay fever and/or bronchial asthma had detectable IgE in the extracts. The patients with gastrointestinal allergy also had the highest concentrations of fecal IgE, and the concentrations in feces did not correlate with the corresponding serum IgE levels. Furthermore, the presence of IgE in feces correlated with specific IgE antibodies in serum, measured as the sum of RAST classes to the food allergens wheat, fish, cow's milk and egg-white. IgE may therefore have been produced locally in the gut as a result of stimulation by food allergens. Since the concentrations of IgA in feces were also high in many children with allergy, and since some possibility of a positive correlation with high IgE concentration seemed to exist, the stimuli for local production of IgE and IgA may be interrelated.

Rabbits and guinea pigs were immunized with purified bovine rotavirus. Immunoglobulin G fractions of the resulting antisera were used in a standard four-layer solid-phase enzyme immunoassay (EIA) for rotavirus antigen in human faecal specimens. Samples negative for rotavirus in electron microscopy, when diluted in standard EIA buffers, regularly gave absorbance values lower than those obtained with buffer blank only. By further diluting of the samples the resulting absorbance values were found to increase to the blank levels. When all dilution buffers were supplemented with 1-5% of bovine serum, negative samples at any dilution gave absorbance values close to those of the buffer blanks. Similar results were obtained if the serum was replaced by 1-5 mM of phenylmethylsulphonyl fluoride, a synthetic broad spectrum serine-type protease inhibitor. Aprotinin, another protease inhibitor, was without effect. A similar inhibition pattern was obtained when faecal specimens were tested in a caseinolytic quantitative protease assay in the presence of the above inhibitors. These observations suggest that protease activity present in human faecal samples may cause false-negative results in solid-phase immunoassay for viral antigens, unless appropriate means are used to avoid this interference.

A simple method for the detection of human rotavirus in stools is described, using a double antibody sandwich enzyme-linked immunosorbent assay. Polysterene microtitre plates were used as solid phase. Four capture antibodies were tried, bovine, egg-derived, guinea pig and monoclonal antibody to rotavirus. Both bovine and egg-derived antirotavirus labelled with horseradish peroxidase were used as the detecting antibodies. The results obtained were compared with a commercially available ELISA, Rotazyme (Abbott Laboratories), and also with the direct detection of rotavirus by electron microscopy. Bovine antibody was found to be an unsuitable capture antibody due to non-specific false positive reactions.

Coproimmunoglobulins and specific coproantibodies were studied in 98 adult patients and 32 children in the course of infection caused by Sh. sonnei, and in 102 healthy individuals of various age. The concentrations of IgA, and, to a lesser extent, of the IgG and IgM proved to be significantly higher in the patients than in the healthy persons. Locally synthesized secretory IgA made up the main part of the total amount of the IgA in feces. The titres of specific coproantibodies were significantly higher in patients than in healthy persons; these antibodies, as identified by Coombs test with monospecific antiglobulin sera, mainly belonged to the secretory IgA. The local immune response to Sh. sonnei was more intensive in adults than in children. In adults the increase in the secretory IgA was revealed at the earlier stages of the disease, and specific antibodies were found more frequently and in higher titres than in children. The lesser intensity of local immune response in children apparently depends on a lesser maturity of the IgA system providing the local defence.

This paper describes the development of monoclonal antibodies generated against hepatitis A virus (HAV). Monoclonal antibodies (MCABs) from two murine hybridoma cell lines were found to bind to an epitope recognized in the sera of patients recovering from infection with HAV. Ascites fluids containing MCABs from one hybridoma (H1 C19) inhibited a maximum of 70% of the 125I-labeled polyclonal human anti-HAV from binding to HAV-antigen in a competitive radioimmunoassay, indicating that the MCAB recognizes a major epitope of HAV. Monoclonal anti-HAV that was coated onto polystyrene beads was as effective as polyclonal antibodies in capturing HAV antigen from extracts of human feces, marmoset liver, and cultured cells. Radiolabeled MCAB was used to screen sera for anti-HAV. A collection of 117 sera was tested for total anti-HAV by competitive radioimmunoassay utilizing either 125I-labeled human polyclonal or mouse monoclonal antibody. Thirty-four specimens were similarly reactive by both systems, while the remainder were negative. Likewise, 28 specimens were similarly positive for IgM anti-HAV, and 12 specimens were negative using each of the two labeled antibodies. The data show that anti-HAV induced during disease is directed against a common major epitope of HAV and that MC anti-HAV can be used effectively as a reagent to detect these antibodies.

An enzyme-immunoassay (EIA) using polystyrene beads as the solid phase, guinea pig anti-rotavirus or anti-adenovirus immunoglobulin as primary antibody, rabbit anti-rotavirus or anti-adenovirus immunoglobulin as secondary antibody, and horseradish peroxidase-conjugated swine anti-rabbit immunoglobulin as indicator antibody, has been developed for the detection of human rotavirus and adenovirus antigens from stool specimens. A comparison of the developed EIA and radioimmunoassay (RIA) used previously in our laboratory was made with 250 stool specimens from children with acute gastroenteritis. Two specimens were found negative by both rotavirus and adenovirus EIAs but not by RIAs, but in each of these cases confirmatory EIA tests showed them to be false negatives. The confirmatory EIA tests were also necessary in several cases to prove the specificity of the binding or to eliminate non-specific reactions with specimens giving low positive reactions in EIA. The developed EIA was found to be as specific, sensitive and reliable as RIA in the routine diagnosis of rotavirus and adenovirus gastroenteritis provided that appropriate confirmatory tests were included.

Volunteers were vaccinated subcutaneously and orally against typhoid fever; a determination was made of immunological reactions in the digestive tract secretions. Oral vaccination caused a much greater production of the secretory IgA than the subcutaneous one. Immunoglobulins of classes G and M were revealed in the intestinal contents only in a few volunteers. In studying the coprofiltrates obtained before the vaccination there was shown for the first time the presence of normal coproantibodies to typhoid fever, dysentery, tetanus, diphteria, botulism and gas gangrene; in the majority of the cases there was found a correlation between the presence of normal serum and secretory antibodies. Subcutaneous and oral vaccination caused an increase of coproantibodies specific for the vaccines used. Besides, the same vaccines stimulated the nonspecific response expressed in a rise of the level of other species of antibodies. Specificity of coproantibodies in the inhibitary passive hemagglutination reaction was demonstrated in the majority of the coprofiltrates examined.

The association between house dust allergy and asthma has long been recognized, and it has been demonstrated that a major allergen in house dust is related to the presence of mites of the genus Dermatophagoides. Using extracts of mite culture for skin testing, as many as 10% of the population and up to 90% of allergic asthmatics give positive immediate reactions. Although mites may occasionally become airborne during bed-making, it has also been demonstrated that they 'secrete or excrete' some allergen. Recently, we have shown that up to three-quarters of the serum IgE antibodies to mites are directed against a major allergen-antigen P1 (molecular weight 24,000). Using a radioimmunoassay it is possible to measure the concentration of this glycoprotein in both dust samples and mite cultures. These measurements, which are reported here, show that more than 95% of the allergen accumulating in mite cultures is associated with faecal particles.

Groups of 10 Mongolian gerbils, Meriones unguiculatus, were vaccinated with 1,500 gamma-irradiated Trichostrongylus colubriformis infective larvae (L3) or with non-irradiated larvae. 25 days later five gerbils from each group were necropsied and the remaining gerbils challenged with 1,500 non-irradiated T. colubriformis infective larvae. Systemic, local intestinal and coproantibody levels were compared in each group of gerbils 25 days after vaccination and 26 days after challenge. Strong local intestinal and faecal antibody responses were detected. Coproantibodies reflected antibody levels in the intestinal contents and in mucosal extracts. The results gave further support to the view that coproantibody measurements provide a sensitive index of immunity at mucosal surfaces to intestinal parasites.

Local antibody responses to challenge with 10,000 normal Haemonchus contortus larvae were evaluated in vaccinated and control Blackface sheep fed either a high or low protein diet. Anti-parasite antibody activity was found in faecal extracts of both vaccinated and control animals after infection. The coproantibody levels showed some correlation with abomasal mucosa antibody levels and faecal antibody activity was mostly associated with IgA. The low protein diet reduced local antibody responses in vaccinated and control sheep. However despite lower antibody levels the efficacy of vaccination was not diminished in sheep fed a low protein diet.

The development of microtiter solid-phase radioimmunoassays for the detection of Norwalk antigen and its antibody is described. The tests are simple to perform and are sensitive and specific. The test for antigen can be used on crude stool filtrates and suspensions. Both tests are at least as sensitive as immune electron microscopy and more sensitive than immune adherence assay.

A major allergen from an extract of the house dust mite, Dermatophagoides farinae, was shown to be extremely heterogenic with respect to charge. A slightly basic component of this allergen with a pI of 8, was purified by isoelectric focusing in two steps. The purified component, denoted antigen 19/20 IIa, seemed to be representative for the allergenic activity of the major allergen. The amino acid analysis suggested that antigen 19/20 IIa had a molecular weight of 9400 and contained one residue of galactosamine. SDS polyacrylamide gel electrophoresis did indicate a somewhat higher molecular weight of 14,500. Antibodies against the purified component cross-reacted with a crude extract of the mite Dermatophagoides pteronyssinus.

The structural characteristics of carcinoembryonic antigen (CEA) and CEA-like antigen in feces and meconium were examined using antibody raised against 2 antigens. When antibody to CEA was made, anti-alpha 1-acid glycoprotein (AG) (nonprecipitating antibody) besides the precipitating antibody for CEA was made. When antibodies against each CEA-like antigen (mol. wt. 180,000) purified from feces and meconium were tested, each antibody showed a fused precipitin line for 2 antigens. Some antibodies showed the non-precipitating antibody for AG. CEA and CEA-like antigen in feces and meconium may be composed of 2 portions (AG portion and non-AG portion). Antibody specific to these antigens may be the antibody directed to the non-AG portion.

Virus-like hepatitis A antigen (HA Ag) particles, presumably hepatitis A virus, were isolated from the liver, bile, and stool of three chimpanzees that had been infected with stool filtrates containing HA Ag particles. Specimens of serum, stool, liver biopsy material, and bile were obtained at selected intervals during the experiment. The animals developed mild hepatitis 19-21 days after inoculation, and antibody to HA Ag appeared de novo in their convalescent-phase serum. During acute illness, virus-like particles similar to the HA Ag particle were seen in liver cell cytoplasm by electron microscopy. HA Ag particles were detected by immune electron microscopy and a new radioimmunoassay in isopycnically banded samples of liver, bile, and stool. HA Ag particles were found at densities of 1.29-1.39 g/cm3, but the major peak density for antigen particles in samples of liver, bile, and stool was approximately 1.34 g/cm3. The fact that HA Ag particles can be recovered from chimpanzee liver, bile, and stool makes these potentially important sources of infectious and antigenic materials.

The authors evaluated the immunological results of enteral and subcutaneous injection of detergent Sonne dysentery vaccine to volunteers (changes in homo- and heterologous antibodies in the blood and saliva recorded by means of common indirect hemagglutination test and that modified according to Coombs' test, and IgG, IgM, and IgA concentration recording. Both immunization methods induced specific antibody formation: enteral vaccination caused chiefly the accumulation of secretory antibodies of IgA class, and subcutaneous -- of serum antibodies belonging to IgG class. The supposed mechanisms determining the capacity of enteral administration of the antigen to provoke an immune response different from that caused by its subcutaneous injection are discussed.

The specificity and sensitivity of the antibody neutralization test in the diagnosis of dysentery have been studied. This test has proved to be highly specific in experimental and clinical trials. The antibody neutralization test is more effective than the methods of the isolation of shigellae. The accuracy of this test can be increased by the elimination of different antierythrocyte agents from the secretions of persons under examination.

Levels of rotavirus-specific immunoglobulin G (IgG), IgA, IgM, and secretory immunoglobulin in maternal and cord serum, colostrum and milk, and infants' stools were measured by enzyme-linked immunosorbent assay in 92 mothers and their infants. Although antirotaviral IgG, IgA, and secretory immunoglobulin were present in most maternal sera, only IgG crossed the placenta. All samples of colostrum and milk tested contained antirotaviral secretory immunoglobulin and IgA except those of two women in whom IgA deficiency was subsequently described. Specific IgM and IgG were also detected in many colostral samples. Antirotaviral IgA was detected in many colostral samples. Antirotaviral IgA was detected in stools of breast-fed but not bottle-fed neonates. Apparently the human infant receives rotaviral antibodies both transplacentally and via maternal colostrum and milk.

A solid-phase, enzyme-linked immunospecific assay for measurement of different immunoglobulin classes of human rotavirus antibodies is described. The antigen, which was adsorbed directly to polyvinyl microtiter plates, consisted of a clarified cell culture stock of the simian rotavirus SA 11. The assay was sensitive and reproducible and could readily be calibrated to determine concentrations of each class of antibody. The assay was applied to measurements of rotavirus antibodies in serum, colostrum, milk, and fecal samples. It particularly facilitates investigations of the role of immunoglobulin A antibodies in immunity to rotavirus infections.

A direct fluorescent antibody technique for the detection of Treponema hyodysenteriae in faeces and bowel smears from pigs produced a better preparation than the indirect method and also had the advantages that it took less time, produced less non-specific fluorescnece and gave direct control over the availability of the reagents used.

Hepatitis B surface antigen (HBs Ag) was not detected in sewage by means of a sensitive affinity chromatography method combined with radioimmunoassay. The antigen was also absent from the feces and urine of 23 patients with HBs Ag antigenemia; this observation indicates that HBs Ag is rarely discharged into sewage. The absence of HBs Ag from feces is ascribed to antagonists of an enzymatic nature or to carboxypeptidase A, which destroys the antigen. Antagonists with similar effects were produced by three species of Pseudomonas but were not produced by various other bacteria. HBs Ag was also destroyed by two subtilisin enzymes. When hepatitis B sera were incubated with these enzymes or with the antagonists, small spherical particles, tubules, and the coats of Dane particles disappeared first, and Dane cores disappeared later. Although sewage or activated sludge did not affect the stability of HBs Ag, the results indicate that even Dane cores are not excreted in feces and that sewage plays a negligible role in the spread of HBs Ag, Dane cores, and viral hepatitis type B.

A human rotavirus complement-fixing (CF) antigen, prepared by purification of large volumes of fluid feces collected from children with winter diarrhea, was used to study the development and persistence of antibody in children with diarrhea and the prevalence of rotavirus antibody in Melbourne. In children with diarrhea, antibody rises were detectable within 4 to 6 weeks of the onset of illness, and the titers usually remained elevated for the next 1 to 2 years. CF antibody did not develop in two children with proven rotavirus infection aged less than 6 months, an age at which poor CF responses to other viruses have also been observed. A study of CF antibody levels in the general community showed that in Melbourne, most children have been infected with human rotavirus by the age of 3 years.

An ELISA was developed for the identification of rotavirus immune complexes in pig faeces. The relationship between rotavirus antigens, rotavirus immune complexes and rotavirus antibodies of IgA class was examined.

A collection of 104-fecal specimens from 45 patients with hepatitis A, 14 patients with hepatitis B, 10 patients with non-A, non-B hepatitis, 6 patients with diseases other than hepatitis, and 18 healthy adults were studied for the presence of secretory immunoglobulin A and immunoglobulin M to hepatitis A virus by solid-phase enzyme-linked immunosorbent assay and immune electron microsopy. Specific fecal antibody was found only in patients with hepatitis A. Of 54 specimens from patients with hepatitis A, only 10 (18.5%) possessed detectable levels of fecal antibody, and each of these was collected within 10 days from the onset of dark urine. All 10 fecal specimens contained hepatitis A-specific secretory immunoglobulin A, and 4 were also positive for hepatitis A-specific immunoblobulin M. Four of the 10 antibody-positive specimens also contained hepatitis A virus particles which could be shown by immune electron microscopy to be coated with specific secretory immunoglobulin A. Since specific fecal antibody was not detected in all the patients with hepatitis A that were studied, it would appear to have limited diagnostic value, although its detection is evidence of recent infection.

Specificity and sensitivity of the antibody neutralization test intended for detection of the O-antigen of enteropathogenic escherichia were checked under experimental conditions. Only 3 strains of the Klebsiella genus proved to neutralize the antibodies to the enteropathogenic escherichia of the serological group O20:K84. In the rest of the cases a positive result was obtained only in homologous combinations. In comparative study of the microbial cultures of the infected feces on hard nutrient media by means of bacteriological and serological methods the latter was found to be more sensitive, capable of detecting the homologous O-antigen with the bacterial concentration of not less than 5-10(5) microbial cells per 1 ml.

Carcinoembryonic antigen (CEA)-related antigen were isolated from meconium and compared with those in adult feces and tumor tissues. NCA-2 and NCA preparations were isolated from meconium. By conventional anti-CEA antisera, NCA-2 from meconium, NFA-2 from adult feces and CEA from tumor tissues were indistinguishable from each other. However, the CEA-distinctive determinant was absent in NCA-2-molecules. The NFA-2-distinctive determinant was detectable in 20% of NCA-2-molecules. The molecular weight of purified NCA-2 was estimated to be 150,000 to 170,000 determined by SDS-PAGE. NCA-2 revealed beta-region mobility electrophoretically. NCA-2 had amino acid and carbohydrate compositions similar to those of CEA and NFA-2.

An enzyme-linked immunosorbent assay was applied to evaluate the appearance of specific IgA in fecal eluates of 11 normal subjects given oral live Salmonella typhi Ty21a vaccine. All subjects after immunization showed a detectable rise in levels of specific IgA to both lipopolysaccharide and flagellar antigens of S. typhi.

A rapid, sensitive counterimmunoelectrophoresis assay was developed to detect adenovirus in stools of patients with gastroenteritis. The parameters of the assay were optimized, and its sensitivity and efficiency was examined. The assay promises to be a useful alternative to conventional techniques of cell culture isolation or electron microscopy for the detection of adenovirus in stool specimens.

A fecal filtrate of human origin containing the Norwalk agent of epidemic viral gastroenteritis was administered by stomach tube to chimpanzees in an attempt to induce diarrheal disease. Significant postchallenge serum antibody rises against Norwalk viral antigens were demonstrated in all animals using the techniques of immune electron microscopy and radioimmunoassay. In addition, viral antigens were detected in feces from five of nine animals using radioimmunoassay. Clinical illness characterized by diarrhea and/or vomiting did not occur. Infection was transmitted subsequently by feeding four additional chimpanzees a fecal filtrate prepared from one of the previously infected animals. Development of an antibody response in four animals and detection of viral antigen in two animals that received this passage filtrate indicated that viral replication had occurred in the absence of clinical illness. The availability of the chimpanzee as an experimental animal host susceptible to infection with the Norwalk agent should facilitate the study of epidemic viral gastroenteritis.

Two esterase activities were detected in pigeon dropping extracts (PDE). The first exhibited the ability to cleave p-tosyl-L-arginine methyl ester (TAMe), and the second utilized alpha-naphthyl acetate as its substrate. Both enzymes were stable at a temperature of 60 degrees C. Although the enzymes could be separated from each other through gel filtration chromatography, both were associated with PDE1 (a major antigen found in pigeon droppings) throughout the fractionation procedure. In addition, it was shown that the TAMe esterase exhibited protease activity by virtue of its ability to hydrolyze alpha-casein.

Isoelectric focusing (IEF) studies on pigeon dropping extracts (PDE) revealed that it is a complex mixture of components that are acidic in nature. Chromatographically purified antigens PDEB, PDE1, and PDE3 showed multiple bands in IEF, indicating a microheterogeneity of these components, with peak concentrations focusing at pH 6.1, 4.6, and 3.8, respectively. The isoelectric points are compatible with the chromatographic behavior of these antigens on DEAE-cellulose. Crossed immunoelectrofocusing (CRIF) resolved PDE into seven precipitin lines with rabbit antiserum in a pH gradient from 3.5 to 9.5, and into nine precipitable components in a pH gradient from 2.5 to 7.0. The complexity of this antigen source appears to reside in the heterogeneity of immunologically related antigens. A comparison of pigeon breeder's sera by CRIF of PDE revealed a qualitative difference in precipitation patterns obtained with symptomatic and asymptomatic individuals. Sera from all (eight out of eight) of the symptomatic breeders tested precipitated an unidentified component of PDE, whereas none (zero out of four) of the sera from asymptomatic breeders detected this antigen. These results suggest that CRIF of PDE is useful as a diagnostic tool and that some specific component of PDE may be involved in the pathogenesis of the disease.

We have developed an enzyme-linked immunosorbent assay for quantitating human immunoglobulin A in human secretions and serum. The procedure uses Immunobeads as the solid phase, has a sensitivity of 10 ng/ml, and can be completed in 1 day. Since there is no diffusion through agar, the assay does not require a correction factor when measuring secretory immunoglobulin A. This method should be useful in studies of local mucosal immunity in humans, especially when a very sensitive assay is needed.

An enzyme-linked immunosorbent assay (ELISA) has been developed for the identification of Coxsackie B antigens. This assay was capable of identifying and distinguishing all six Coxsackie B serotypes at concentrations one hundredfold to ten thousandfold less than could be detected by complement fixation (CF) systems. In addition, the Coxsackie B ELISA correctly identified the presence of Coxsackie B antigen in 19 of 21 tissue culture fluids and five of nine rectal swab specimens. Two additional rectal swab specimens reacted with Coxsackie B antisera but could not be conclusively serotyped. Tissue culture fluids and rectal swab specimens containing other viruses such as ECHO virus, Coxsackie virus A, rhinovirus, rotavirus and Norwalk virus were consistently negative in the assay. The Coxsackie B ELISA offers potential for the rapid identification of Coxsackie B antigens in clinical specimens and tissue culture systems.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of hepatitis A virus in human fecal specimens. Investigations with 88 fecal specimens from 77 patients with suspected viral hepatitis and 8 of their household contacts showed that ELISA was as specific and sensitive as radioimmunoassay and almost as sensitive as immune electron microscopy. The ELISA is quick and simple to perform, does not require sophisticated technical equipment, and can be read with the naked eye, making it suitable for field work and rapid diagnosis.