Immunodiffusion test for diagnosing human pythiosis

An immunodiffusion test was developed for diagnosing subcutaneous and systemic pythiosis in humans. When culture filtrate antigen (CFA) from P. insidiosum was reacted against patient and rabbit antisera, 1–5 precipitin bands occured both in patient and rabbit antisera, and a line of identity also occured between patient and rabbit sera. When control P. insidiosum CFA was reacted with 30 apparently normal persons, 20 Thalassemia patients, 2 candidosis and 5 aspergillosis patients, no precipitin bands were found. P. insidiosum CFA also tested with rabbit antibodies to B. dermatitidis, C. immitis, H. capsulatum, P. brasiliensis, C. albicans, M. furfur and A. fumigatus revealed no cross reactions. This test is practical, sensitive and specific.     

Serological comparison of polyhedron protein and virions from four nuclear polyhedrosis viruses of plusiine larvae (Lepidoptera: Noctuidae)

Purified polyhedron proteins and purified, ultrasonicated virions of four nuclear polyhedrosis viruses (NPVs), separable into two morphologic groups of singly and multiply embedded virion types (SEVs and MEVs), were investigated by immunodiffusion and immunoelectrophoresis. The four viruses were Pseudoplusia includens SEV, Trichoplusia ni SEV, T. ni MEV, and Autographa californica MEV. In immunodiffusion, SEV polyhedron proteins formed two precipitin bands with antiserum to SEV polyhedron proteins, while MEV polyhedron proteins formed only one. All four proteins formed one precipitin band with antiserum to MEV polyhedron protein, with a spur between SEV and MEV proteins. In immunoelectrophoresis, mobilities of SEV proteins were significantly different from those of MEVs. Precipitin arc patterns were similar to those in immunodiffusion when electrophoresis was carried out at 4 C; at room temperature, a single arc of precipitation formed with all four proteins. SEV virions formed five possibly identical precipitin bands in immunodiffusion with antiserum to SEV virions. MEV virions formed three possibly identical precipitin bands when reacted with antiserum to MEV virions. Little or no cross-reactions were observed between SEV and MEV virions or between virions and polyhedron proteins. In immunoelectrophoresis, SEV virions formed three precipitin arcs in reactions with SEV antisera and none with MEV antisera; MEV virions formed two arcs with MEV antisera and none with SEV antisera. When antisera were subjected to electrophoresis, five arcs were formed by SEVs and three by MEVs in homologous systems, and none were formed in heterologous systems.     

Serological Comparison of the Three Morphological Phases of Coccidioides immitis by the Agar Gel Diffusion Method

Hyperimmune sera against spherules and against arthrospores of Coccidioides immitis were prepared by inoculation of rabbits. The antibody content of these sera was studied by the agar gel diffusion method. It was observed that antispherule pooled sera formed multiple precipitin bands with extracts of spherules and of arthrospores. The antiarthrospore pooled serum, however, failed to precipitate with the spherule extract, and formed a single band in the presence of an arthrospore solution. When the spherule and the arthrospore extracts were tested with a variety of different antisera, it was observed that the spherule preparation formed bands only in combination with anti-purified spherule pooled serum, whereas the arthrospore extract precipitated with anti-purified spherule, antiarthrospore, and anti-Histoplasma capsulatum pooled sera. It was also observed that a spherule culture supernatant solution formed five precipitin bands in combination with anti-spherule pooled sera, formed one band with pooled antiserum from rabbits with coccidioidomycosis, and did not precipitate in the presence of antiarthrospore pooled serum. Coccidioidin, however, formed two bands in the presence of any of these antisera. It was therefore concluded that extracts from the spherule phase of C. immitis differed from solutions obtained from the arthrospore and mycelial phases.     

Immunoelectrophoretic analysis of major component proteins in cystic fluid of Taenia solium metacestodes.

When cystic fluid of Taenia solium metacestodes (CF) was filtrated through Sephacryl S-300 Superfine, major proteins were in fractions III and IV. Major protein in fraction III was Band C protein of 150 kDa and that in fraction IV was Band N protein (Choi et al., 1990). When CF was electrophoresed in 0.9% agarose gel and reacted with anti-CF rabbit serum (RACF), two main bands, a long outer and a short inner band, were precipitated, together with 8 minor bands. RACF reacted with fraction III forming the long outer band whereas RACF formed the short inner band with fraction IV in immunoelectrophoresis (IEP). The long outer precipitin band of CF fraction III was similar to antigen B in hydatid fluid (HF) of Oriol et al. (1971), while the short inner band of CF fraction IV was similar to HF antigen 5 of Capron et al. (1967). When HF was reacted with RACF, the short inner band was immunoprecipitated without forming the long outer band. Common antigenicity between CF and HF seemed to exist in fraction IV rather than in fraction III of CF. Patient sera of neurocysticercosis reacted more frequently with fraction III than with fraction IV.     

Identification and genetic control of two rabbit high-density lipoprotein allotypes

Rabbits were immunized with high-density lipoprotein (HDL) isolated from the serum of other rabbits by ultracentrifugation and gel filtration. Two different precipitating antibodies were elicited which distinguished two antigenically different genetic variants, i.e., allotypes, of HDL. The allotypes were identified as HDL based on the observation that on immunoelectrophoresis the antigen-antibody precipitate formed by the reaction of each of the antiallotype antisera with electrophoresed rabbit serum appeared electrophoretically in the α₁ region and reacted with Sudan black and with β-naphthyl acetate. In addition, the precipitin bands formed by the reaction of each antiallotype antiserum with a normal rabbit serum coalesced with the precipitin band formed by the reaction of goat anti-HDL with the same normal rabbit serum. The inheritance of the two allotypes is controlled by a pair of allelic genes, as shown by genetic studies of 312 progeny from 83 rabbit families. This HDL locus, designatedLhj, was shown not to be linked to theLpq locus of low-density lipoprotein nor to any of five other loci controlling allotypic specificities of different rabbit serum proteins. The use of these allotypes for studying the structure and biosynthesis of HDL is discussed. This study was supported by Research Grant PHS AI-07043 (Dr. S. Dray) and PHS AI-09241 from the National Institutes of Allergy and Infectious Diseases. One of us (K. L. K.) is the recipient of National Institutes of Health Career Development Award (AI-28687).     

Common Antigen(s) in Cotton to Fusarium oxysporum f. sp. vasinfectum

Antigen-antibody reactions in agar gel, as demonstrated by the double diffusion technique, between cotton seed globulins and the antisera specific to each of the tested Fusarium oxysporum f. sp. vasinfectum isolates as well as the antiserum of F. moniliforme revealed that all the tested antisera of F. oxysporum f. sp. vasinfectum reacted with seed globulins except the Menoufi cultivar globulins. No precipitin lines were detected in the reaction between the antigenof the cotton cultivar Acala SJ2 versus the antiserum of P10 isolate. The 5 cultivars behaved differently with each fungal antiserum to the extent that they could be distinguished accordingly. When the seed globulins of the susceptible cultivars (Giza 74, and Bahtim 110) reacted with antiserum of the tested F. oxysporum f. sp. vasinfectum isolates, more precipitin lines were formed than the resistant cultivars. On the other hand, no obvious reaction was detected in case of F. moniliforme antiserum.     

A simple method for isolation of nuclei from <Emphasis Type="Italic">Basidiobolus ranarum</Emphasis> (Zygomycota)

A simple method for isolating the nuclei from Basidiobolus ranarum was established. To improve the yield and purity of nuclei, we investigated maceration methods, buffer composition, and centrifugation conditions to establish an optimal procedure. Basidiobolus ranarum cultured for 5 days was enzymatically macerated and then homogenized and filtrated through stainless steel sieves. The crude cell homogenate was loaded on a layer of buffer containing 50% glycerol and centrifuged at 1500 g. The resultant pellet contained pure nuclei.     

Serologic Changes Associated with the Encystment of Axenically Grown Hartmannella culbertsoni*

SYNOPSIS. Serologic reactions elicited by sonically ruptured trophic and cystic forms of Hartmannella culbertsoni were studied. The antigens of trophic amoebae reacted with their homologous rabbit antiserum showing multiple precipitin lines which could not be seen when the reacting antigens were treated with trypsin prior to application on the Ouchterlony plates. Antigens of trophic amoeba did not react with antiserum against cysts. Cyst antigens reacted with their homologous antiserum only after trypsin treatment. Antigens prepared from trophozoites excysting from cysts reacted positively with the antiserum against antigens of trophic amoebae. Antigens of trophic as well as cystic forms fixed guinea pig complement in presence of their homologous antisera. With the trophic form, this property was abolished after trypsin treatment. Non-specific complement fixation mediated by cyst antigens was abolished by treatment with cellulase. Antiserum against trophic amoebae immobilized trophozoites and, in the presence of guinea pig complement, led to their lysis.     

Immunological properties of gap junction protein from mouse liver

Hepatic gap junctions were purified as plaques from BALB/c mice and separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Antisera were raised in rabbits and rats against gap junction plaques as well as protein bands of the following apparent molecular weights: 44K to 49K ("dimer" proteins), 26K, and 21K. Using an enzyme immunoassay, we found that the reactivities of the different antisera towards gap junction plaques decreased in the following order: anti-plaque antisera, anti-26K antisera, anti-"dimer" protein antisera, and anti-21K antisera. The gap junction protein bands separated by SDS-polyacrylamide gel electrophoresis were transferred by blotting onto nitrocellulose paper and the immunological cross-reactivities were compared: the anti-26K antisera reated with the dimer protein bands and the 26K band but did not cross-react with the 21K protein band. The rabbit anti-21K antiserum reacted weakly with the 21K protein. The missing immunological cross-reaction of the 26K and the 21K protein band can be most easily explained if both proteins were independent of each other. No inhibition of metabolic cooperation between fibroblastoid mouse 3T6 cells was observed in the presence of Fab fragments prepared from rabbit antiplaque antiserum or from rabbit anti 26K antiserum. When the total proteins of plasma membranes from mouse liver were separated by SDS-polyacrylamide electrophoresis, only the 26K protein reacted with rabbit anti 26K antiserum. This result opens the possibility for direct quantitation of gap junction protein in tissues and cell fractions.     

Isolation of Fasciola hepatica genus-specific antigens

Santiago de Weil N., Hillyer G. V. and Pacheco E. 1984. Isolation of Fasciola hepatica genus-specific antigens. International Journal for Parasitology14: 197–206. The Fasciola hepatica antigens which induce antibody formation in acute fascioliasis were isolated by acid elution after reacting an F. hepatica tegument antigen extract with a CNBr-Sepharose 4B column coupled with IgG obtained from the serum of rabbits infected with fascioliasis for 6–10 weeks. These isolated antigens were further separated by gel filtration using a column packed with Sephacryl S-200. In this manner three major peaks were obtained. The best serologic antigens were found in peak 2 which had a mol. wt range of 14,000–43,000. This peak contains genus-specific F. hepatica antigens which are highly reactive with fascioliasis serum. These antigens do not cross-react with either Schistosoma mansoni or with bovine serum albumin by gel diffusion. Monitoring by ELISA and gel diffusion with heterologous and homologous antisera showed that as purification by antibody affinity chromatography proceeded, cross reactivity with S. mansoni was eliminated. The rabbit antiserum obtained against peak 2, when tested by immunoelectrophoresis with a crude F. hepatica extract shows one main band identical to the main band observed with serum from acutely infected rabbits. Up to two other minor bands can be detected using concentrated homologous antisera. Fractions obtained from preparative iso-electric focusing of the F. hepatica tegument extract were reacted with sera from rabbits with acute fascioliasis. Two main bands were observed in immunodiffusion with antigens eluting in a pH range of 7.4–8.7. When these fractions were monitored with anti peak 2 antisera, two precipitin bands appeared with antigens eluting in a pH range of 7.4–7.9. The F. hepatica genus-specific antigen pool was applied to ELISA to evaluate its ability to detect antibody in a primary F. hepatica infection in rabbits. A rise in absorbance values could be detected by 2 weeks of infection, reached high levels by 6 weeks and remained high through 28 weeks of infection.     

Serological identification of group D streptococci using commercial antisera

Three commercial group D streptococcal antisera were tested for the serological identification of 100 group D enterococci; 20 Streptococcus bovis; 5 isolated from each of the following streptococcal groups: A, B, C and G; and 3 isolates from serological group F. Antisera from Difco Laboratories, BBL, and Wellcome Reagents Limited were used in the classic capillary tube precipitin test on extracts prepared using the Rantz and Randall procedure. No false positive precipitin reactions were observed. Of the enterococcal isolates, all 100 reacted with the Wellcome, 99 reacted with the BBL, and 96 reacted with the Difco group D antisera. However, of the 20 S. bovis isolates, only 2 reacted with the BBL, and 1 reacted with both the Difco and the Wellcome antisera. Each antiserum was then used to prepare staphylococcal coagglutination (CoA) reagents and each isolate was subsequently tested. A simple extraction procedure was performed by suspending colonies of an isolate in a loopful of salin on a microscope slide and gently heating the slide directly in the flame of a Bunsen burner. All 100 enterococci and all 20 S. bovis gave positive results with the BBL and the Wellcome CoA reagents. Using the Difco reagent, 2 S. bovis isolates failed to produce postitive results. No false positive results were observed with the non-Group D isolates. Our results indicate that the CoA technique using commercial group D antisera may provide faster and sometimes more sensitive serological identification than the classic capillary tube precipitin test.     

Polyphosphate and orthophosphate content during the life cycle of Myxococcus coralloides D

Immunoglobulins, prepared from polyclonal rabbit antisera raised against Escherichia coli fimbrial antigens, colonization factor antigen (CFA)/I, and coli-surface-associated antigens (CS)1, CS2 and CS4, were used to assess antigenic cross-reactions between these four fimbrial types by Western immunoblotting. Antibodies in a serum, prepared against CS4, cross-reacted strongly with the fimbrial subunits of CFA/I, CS1 and CS2. Antibodies in sera prepared against CFA/I and CS1 gave weak reactions with CS1 or CFA/I respectively and also with CS2 and CS4, while the antiserum prepared against CS2 did not react. CS4 antiserum also reacted with the CS17 fimbrial subunit, but not with the subunits of fimbrial antigens: CFA/III, CS5, putative colonization factor (PCF) 0159:H4 or PCF0166.     

Detection of cross-reactive determinants shared by human monoclonal IgM reacting with myelin-associated glycoprotein

Monoclonal IgM from patients with peripheral neuropathy frequently have anti-myelin-associated glycoprotein (MAG) activity. We investigated the idiotypes of 10 monoclonal anti-MAG by using rabbit polyclonal antisera. Three groups of anti-idiotypic antisera could be distinguished. Four sera reacted only with the immunizing protein. Two sera reacted with a single other anti-MAG IgM in addition to the immunizing one. Immunoenzymatic studies showed that these two couples of anti-MAG IgM reacted identically with 100% cross-inhibition, indicating that the whole set of idiotypes identified by the rabbit antiserum was present on both IgM antibodies. The four other anti-idiotypic sera reacted with the homologous IgM, as well as with most of the other anti-MAG IgM. In contrast to the previous antisera the binding of these four sera to the homologous IgM could not be inhibited by other cross-reacting anti-MAG IgM. However, when a heterologous IgM was used for coating, these antisera with one exception showed complete cross-inhibition. The absence of inhibition by purified MAG of the patient of the anti-idiotypic antisera sera suggests that these antibodies are most likely to be directed against framework determinants rather than against combining site epitopes.     

Serological characterization of a ribonuclease from Hordeum vulgare

Specific rabbit antisera against purified Hordeum vulgare seedling RNase I from two winter barley cultivars each formed a single precipitin band when reacted with the homologous crude tissue extract. RNase antigen from either cultivar was equally reactive with both antisera when evaluated by immunodiffusion and immunoelectrophoresis. A small but consistent difference in anti-RNase specificity between cultivars was shown by passive hemagglutination inhibition, suggesting that molecular differences may exist between the two RNase antigens. Immunodiffusion and rocket immunoelectrophoresis were used to qualitatively test the cross-reactivity of protein preparations from various members of the genus Hordeum and species from other related grass genera. Neither antiserum showed cross-reactivity with soluble protein preparation from species outside the genus Hordeum. A few species within the genus Hordeum were cross-reactive. A modification of rocket immunoelectrophoresis was developed to determine the amount of RNase in unpurified tissue extracts. The technique involved a template-reservoir which allowed detection of 250 ng RNase in tissue extract volumes of 50 μl. The amount of RNase in unpurified protein extracts from the two cultivars of barley was similar.     

Gastrointestinal zygomycosis: a report of three cases

Three cases of gastrointestinal zygomycosis, probably caused by Basidiobolus ranarum, are described. The diagnosis was based on morphology of the fungal elements in infected tissues and histopathologic findings. All the three patients responded favorably to management strategy that included surgical resection of the infected portion of the bowel and institution of specific antifungal therapy.     

Analysis of the Antigenic Relationships Among Trichomonas,Histomonas, Dientamoeba,and Entamoeba.* II. Gel Diffusion Methods

SYNOPSIS. Antisera were developed in rabbits against Trichomonas gallinae, Histomonas meleagridis, Dientamoeba fragilis, Entamoeba invadens, and Entamoeba histolytica. In reactions between these antisera and antigens prepared from each of the 5 species the most numerous and strongest precipitin lines appeared on gel diffusion agarose plates between the homologous antigens and antisera. Anti-Trichomonas serum cross-reacted most strongly with Histomonas, somewhat less with Dientamoeba, but gave no lines with the 2 species of Entamoeba. Anti-Histomonas serum cross-reacted strongly with both Trichomonas and Dientamoeba, and weakly with E. invadens and E. histolytica. Dientamoeba antiserum gave many precipitin lines with Histomonas, fewer with Trichomonas, and fewest with the 2 species of Entamoeba. Stronger reactions were noted between anti-Dientamoeba serum and E. invadens than between this serum and E. histolytica. Immune sera prepared against the 2 species of Entamoeba gave the most numerous precipitin lines in intrageneric cross-reactions, but the reaction between either of these antisera and Histomonas was weak. Somewhat stronger reactions were observed between the 2 anti-Entamoeba sera and Dientamoeba. Trichomonas failed to react with either of the anti-Entamoeba sera.     

Immunologic Analysis by Gel Diffusion of Effects of Prolonged Cultivation on Histomonas meleagridis (Smith)*

SYNOPSIS. Antigens were prepared from each of 4 lines of Histomonas meleagridis: Hm-L1, a strain highly virulent for both turkeys and chickens; Hm-L1 /C12, Hm-L1 /C24, Hm-L1 /C52, 3 avirulent substrains derived from Hm-L1 after 12, 24, 52 weeks of in vitro cultivation, respectively. Hm-L1 strain and the 3 substrains were maintained in liquid nitrogen. Antisera were developed in rabbits against Hm-L1 and Hm-L1 /C24 parasites. Both antisera were reacted on gel diffusion plates with homologous and heterologous antigens. Two groups of precipitin lines and/or bands designated arbitrarily as A and B, were observed on the slides. Analysis of these bands revealed the common antigenic composition of the 4 histomonads with respect to some of the group A and group B antigens. The concentrations and numbers of precipitin lines in both groups increased, however, with the length of cultivation. These antigenic differences are discussed in the light of their possible relationship to pathogenicity.     

Purification of human alpha-fetoprotein

By employing complex and highly specialized immunochemical methods, several investigators have achieved purification of human α-fetoprotein (AFP) found in fetal serum and/or sera of patients with hepatoma. The present report describes a simpler method which results in the isolation of homogeneous preparation of AFP from human cord serum. AFP was purified by sequential use of Affi-Gel Blue affinity, DE-52 diethylaminoethyl cellulose ion-exchange, immunoadsorption with anti-albumin covalently coupled to Sepharose 4B, and Sephacryl S-200 molecular sieve chromatographic techniques. The homogeneity of the purified AFP was established by subjecting it to polyacrylamide gel electrophoresis, analytical isoelectric focusing, molecular sieve chromatography and immunological techniques. The purified AFP has a molecular weight of approximately 68,000 as determined by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and molecular sieve chromatography, and upon isoelectric focusing yielded a single band pI = 4.8. In addition, the purified AFP gives a single precipitin line when tested against rabbit antiserum to whole human hepatoma serum proteins, and no line(s) of precipitin when tested against rabbit antiserum to normal serum proteins.     

Histological and ultrastructural features of gastrointestinal basidiobolomycosis

Basidiobolus ranarum is a fungus found in the dung of amphibians, reptiles, and insectivorous bats. Its structural elements include both hyphae and zygospores. Patients with B. ranarum infection may present with subcutaneous, gastrointestinal, or systemic lesions. Here we report a case of gastrointesinal badidiomycosis in a 13-year-old male child who presented with acute abdomen. Exploration revealed a mass in the ascending colon. On histology, transmural granulomatous inflammation composed of abundant eosinophils, lymphocytes, histiocytes and giant cells was seen. Histochemical stains revealed broad, non-septate, hyphae-like structures surrounded by an eosinophilic sheath. On an ultrastructural level, fungal hyphae, spores, and macrophage-laden crystalloids were observed. The diagnosis of gastrointestinal basidiobolomycosis was established and the patient received antifungal treatment. This paper reviews the relevant literature regarding basidiomycosis, and discusses its diverse clinicopathological features, as well as distinguishing it from other diseases.     

Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid: Specificities and reactivities of the antibodies formed in rabbits to stearyl-isomaltosyl oligosaccharides

The specificities and the sizes and shapes of the antibody combining sites of the 15 antisera raised against various stearyl-isomaltosyl oligosaccharides were studied by quantitative precipitin and precipitin inhibition. The antibodies precipitated well with dextrans B512 and B1424 but less well with B1299S and B1355S. Only 3 of the 15 antisera reacted with linear dextrans; however, with about 50% of the added antibodies being precipitated, showing that most of the antibodies cannot bind to internal determinants along the dextran molecules and are similar to myeloma protein W3129 in having cavity-type sites which bind only to terminal nonreducing ends of α1 → 6 dextran. Antibodies differing in the sizes of their antibody combining sites were elicited in different rabbits by the same antigen. Of the 15 antisera studied, four have antibody combining sites as large as IM3, five as large as IM4, three as large as IM5 and three as large as IM6. The association constants for various isomaltose oligosaccharides of an antiserum (R-862) showing fewest bands in isoelectric focusing gel were determined by affinity electrophoresis and were comparable to W3129.