Cross-absorbed Antisera Research Articles

Comparative study of molecular and antigenic characterization for intratypic differentiation (ITD) of poliovirus strains

This study was designed to compare the sensitivity of a Sabin vaccine strain-specific PCR assay and an enzyme-linked immunosorbent assay with polyclonal cross-absorbed antisera (PAb-E) for intratypic differentiation (ITD) of polioviruses (PVs). These were used for the definitive characterization of the strains. Poliovirus strains isolated in L20B and RD cell lines were subjected to both PCR and ELISA. Both PCR and ELISA identified 3 (13.6%) out of 22 isolates, respectively as poliovirus Sabin 1. PCR identified 4 (18.2%) out of 22 isolates as poliovirus Sabin 2 and ELISA identified 2 (9.1%) out of 22 isolates as poliovirus Sabin 2. None of the two assay identified poliovirus Sabin 3. Both PCR and ELISA identified 12 (54.5%) out of 22 isolates, respectively as wild poliovirus (WPV) 1. None of the assays identified any of the isolates as WPV 2 and 3. Only PCR assay was able to identify the mixture of two poliovirus Sabin serotypes (a mixture of Sabin 1 and 2) and two mixtures of poliovirus Sabin 2 and 3. In this study, only ELISA was able to identified two invalid results. Invalid results observed in this study are of important practical implication to the emergence of vaccine-derived poliovirus. This may have epidemic potential. Hence, the two ITD assays are of paramount importance for identification of PVs. It is therefore recommended in line with WHO guideline that at least two methods be used for the ITD of poliovirus isolates, and each method should be based on a different principle (i.e., antigenic and genetic properties).

Polyclonal Antisera To Distinguish Strains and Form Variants of Photorhabdus (Xenorhabdus) luminescens

In this study antisera against Photorhabdus luminescens strains were prepared for the first time. P. luminescens is a bacterial symbiont of entomopathogenic nematodes belonging to the genus Heterorhabditis. To characterize P. luminescens strains and form variants, we produced polyclonal antisera against P. luminescens PE (obtained from nematode strain NLH-E87.3) and against the primary and secondary forms of P. luminescens PSH (obtained from nematode strain DH-SH1). In double-diffusion tests all form variants of strain PE reacted with the antiserum against the primary form, but each variant produced a different diffusion pattern. The primary and secondary forms of strain PSH were also serologically different. Antiserum 9226 reacted with almost all P. luminescens strains tested, but it reacted differently with each strain in the double-diffusion test, showing that the strains were serologically different. The specificity of the antisera was increased by cross-absorption. After cross-absorption the antiserum against the strain PSH primary or secondary form was specific for that form and did not react with the other form. Using the cross-absorbed antisera in immunofluorescence cell-staining tests, we could distinguish primary and secondary form cells in a mixed strain PSH culture.

Biochemical and serological characterization of Escherichia coli fimbrial antigen F165 2

Mannose-resistant hemagglutinating fimbrial antigen F165 is produced by Escherichia coli strains associated with septicemia in piglets and calves. A fimbrial component with an M r of 17 200 as determined by SDS-PAGE was purified to homogeneity from F165-positive E. coli strain 4787 of serogroup O115. This fimbrial component of F165 antigen was named F165 2. Separation procedures included fast protein liquid chromatography with a Superose 12 column followed by ultracentrifugation and 0.15 M ethanolamine buffer (pH 10.5) dissociation. Upon removal of ethanolamine, the fimbrial component reassociated into fimbriae. Amino acid composition analysis indicated that the fimbrial component molecule comprised 158 amino acid residues of which 37.3% were hydrophobic. The amino acid composition and the isoelectric point (9.5) were readily distinguishable from those of F1 fimbriae. The amino acid sequence was determined for approximately 40% of the molecule. For the first 33 residues, the F165 2 sequence was identical to that of F1B fimbriae and very similar to that of F1C. Fimbriae F165 2 could nevertheless be differentiated antigenically from F1C fimbriae as demonstrated by the immunodot technique using cross-absorbed antisera.

Comparison of four Mycoplasma arthritidis strains by enzyme immunoassay, metabolism inhibition, one- and two-dimensional electrophoresis, and immunoblotting

Four Mycoplasma arthritidis strains, two virulent (158p10p9 and 14124p10) and two avirulent (PG6 and H606), were examined for differences in their antigenic compositions. Rabbit antisera prepared against each strain were compared for their reactivities against both homologous and heterologous strains by enzyme-linked immunosorbent assay and metabolism inhibition assay. These tests confirmed a close serologic relationship among the four strains. Only by cross-absorbing each antiserum with intact cells from both homologous and heterologous strains could serologic differences be detected. Interestingly, antigenic variability was minimal among those antigens involved in the metabolism inhibition reaction, suggesting that they may be highly conserved within this species. No differences in protein composition could be detected by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, slight variation was apparent on two-dimensional gels, and antigens possessing strain-specific epitopes were detected by two-dimensional immunoblotting experiments performed with cross-absorbed antisera. The enzyme-linked immunosorbent assay, two-dimensional electrophoresis, and immunoblotting experiments showed that the two virulent strains were closely related to each other, while the avirulent strains were more distantly related to each other and to the other two strains. The relationship of M. arthritidis serologic diversity to virulence remains unclear: however, this study has demonstrated that it may be possible to subdivide M. arthritidis into serogroups on the basis of cross-absorption patterns and to identify those antigens bearing strain-specific epitopes by immunoblotting with cross-absorbed antisera.

Serological grouping ofTreponema hyodysenteriae

Two Australian isolates of Treponema hyodysenteriae which did not fit within the current serological grouping system for these bacteria were examined by agarose gel double immunodiffusion tests (AGDP). Isolate Vic1 was serologically unique, and we propose that it becomes the type organism for a new sixth serological group of T. hyodysenteriae (Group F). Isolate Q1 was unusual in that lipopolysaccharide (LPS) extracted from it reacted strongly in AGDP with serum raised against the type organism for serogroup D (A1), and also weakly with serum raised against the type organism for serogroup B (WA1). The nature of this cross-reactivity was examined by using cross-absorbed antisera in AGDP, and by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The pattern of serological cross-reactivity between Q1, A1 and WA1 was complex and was not fully defined, but the isolate Q1 apparently shared low molecular weight 'serogroup' LPS antigens with A1, and shared higher molecular weight LPS antigens with WA1. On this basis Q1 was designated as belonging to serogroup D, although it was recommended that this be qualified as D (B) to indicate the presence of weak cross-reactivity with serogroup B. Such serological cross-reactivity may have significance in relation to the development of immunity to T.hyodysenteriae. Isolate Q1 may be a potentially useful organism for vaccine development because of its ability to induce a good serological response to LPS of treponemes from both serogroups D and B.

Bacteroides vulgatus outer membrane antigens associated with carrageenan-induced colitis in guinea pigs.

Previous experiments with the carrageenan model for ulcerative colitis demonstrated that the inflammatory response in guinea pigs can be enhanced by immunization with Bacteroides vulgatus and subsequent feeding of this organism to experimental animals. The studies reported here show that antigens extractable from the bacterial outer membrane by EDTA are responsible for this effect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the outer membrane proteins from various strains as well as the lipopolysaccharides (LPS) extractable by the phenol-water method. Although the observed pattern of outer membrane proteins was complex, the strains could be divided into two electrophoretic types (phenons) on the basis of immunoblotting against a panel of antisera. Cross-absorbed antisera used in immunoblotting experiments identified four outer membrane proteins uniquely associated with the phenon capable of enhancing the colitis inflammatory response. These proteins had molecular weights of 100,000, 57,000, 34,000, and 21,000 when measured in 8% to 12% acrylamide gradient sodium dodecyl sulfate gels. Other antigens identified included at least one type of smooth LPS, three types of rough LPS, and a common antigen of 30,000 molecular weight among the strains of B. vulgatus tested. The outer membrane preparations were used in animal immunization and challenge experiments, and the severity of colitis was correlated with one electrophoresis type. The potential role of membrane proteins in the enhancement of colitis is discussed.

Identification of mycobacteria by specific precipitation of catalase with absorbed sera.

Cross-absorbed antisera have been prepared against catalase from reference strains of Mycobacterium asiaticum, Mycobacterium gordonae, Mycobacterium scrofulaceum, Mycobacterium simiae, and Mycobacterium szulgai. A total of 61 strains of mycobacteria were grown in small volumes of liquid medium and disrupted in sealed tubes in a cup horn sonicator, and the extracts were tested by a seroprecipitation technique against each of the reference antibody preparations. All 35 strains that belonged to one of the reference species reacted with the corresponding antibody, and none of the 61 extracts gave a significant cross-reaction with the absorbed antibody to a species to which it did not belong. This method appears to provide a rapid and accurate means of identifying mycobacterial cultures in the diagnostic laboratory.

Investigation of a waterborne outbreak of Campylobacter jejuni enteritis with a serotyping scheme based on thermostable antigens.

Serotyping of 11 human and 2 water isolates of Campylobacter jejuni associated with a waterborne outbreak revealed two serotypes among the human isolates. One of these (serotype 58) was a new serotype and was added to the serotyping scheme. Serotypes were defined by using extracted thermostable antigens and passive hemagglutination titrations of both unabsorbed and cross-absorbed antisera. Two water isolates of the same serotype as six human isolates provided evidence to link a contaminated water supply to the outbreak.

The surface antigens of orthopoxviruses detected by cross-neutralization tests on cross-absorbed antisera.

Cross-neutralization tests were done on accepted species and recently isolated members of the genus Orthopoxvirus using antisera which had been separately absorbed with the various viruses. The results provided evidence for the involvement of four neutralizing antigens, and their distribution among 13 virus strains was determined. Monkeypox (Congo-8-Lombe), camelpox (Gorgan), ectromelia (Mill Hill), 'Lenny' and elephant poxviruses had distinctive antigenic formulae. Lister and Wyeth vaccines were indistinguishable but different from Copenhagen and EM63 vaccines which were themselves distinct. Cowpox (Brighton), buffalopox (BP4), MK 10, and Moscow poxviruses were indistinguishable. Examples were found where viruses shared surface antigens but were not all neutralized by antibody to them. this reduced the practical value of the technique for virus identification. Evidence was also obtained for the existence in some viruses of a fifth antigen, antibody to which could block neutralization by antibody to one particular antigen.

Detection of strain-specific serological activity in antisera of tobacco mosaic virus, clover yellow mosaic virus, and potato virus X, by complement fixation

The strain-specific serological activity of 2 strains of tobacco mosaic virus (TMV) and 2 strains of clover yellow mosaic virus (CYMV) was confirmed by complement fixation tests. Eleven of 12 strains of potato virus X (PVX) proved to be serologically indistinguishable. One of the mottle strains was shown to differ from the other mottle and ringspot strains in tests with cross-absorbed antisera.