Europium-labelled recombinant protein G. A fast and sensitive universal immunoreagent for time-resolved immunofluorometry.

Recombinant protein G was labelled with europium by conjugating the protein with Eu3+ chelate of a p-isothiocyanatobenzyl derivative of diethylenetriaminetetraacetic acid, a bifunctional chelating agent specifically optimized for labelling of immunoreagents with lanthanide ions. The labelling produced a universal reagent for time-resolved fluorometric immunoassays based on the principle of dissociative fluorescence enhancement (DELFIA). The optimum labelling level of about eight chelates per protein yielded a highly sensitive and stable reagent which retained its affinity for IgG and exhibited low non-specific binding to coated solid surfaces. The reagent was evaluated in an immunoassay of anti-tetanus antibodies in human serum samples and the results were compared to those obtained with Eu-labelled polyclonal and Eu-labelled monoclonal anti-human IgG antibodies. The detection limit of the assay was 0.003 mU/ml (0.3 microU per assay well). After a 100-fold dilution of the samples, the assay range extended from 0.3 mU/ml to 100,000 mU/ml with a linear range of five log orders. The incubation with Eu-labelled protein G reached equilibrium after a 15 min incubation. The rapid kinetics, the low non-specific background and the high specific binding suggest that Eu-protein G can serve as a universal label for immunoassays based on IgG binding to solid surfaces.

Affinity purification of bacterial luciferase and NAD(P)H:FMN oxidoreductases by FMN-sepharose for analytical applications.

A modified purification method for bacterial luciferases and NAD(P)H:FMN oxidoreductases is described which uses FMN-Sepharose alone or coupled to DEAE ion exchange chromatography for the simultaneous purification of luciferase and the various oxidoreductases from Vibrio harveyi, a bright mutant of Vibrio harveyi, Vibrio fischeri, and Photobacterium phosphoreum. This purification method is compared with DEAE-Sepharose Cl 6B fractionations from these organisms. Both methods allow the separation of oxidoreductases specific for either NADH or NADPH. The use of FMN-Sepharose coupled to DEAE-Sepharose fractionation allows the isolation of highly purified enzymes. Lacking interfering factors, these are very suitable for various analytical applications based on bacterial bioluminescence enzymes. The partially purified enzymes from the affinity column have higher specific activities than those obtained using DEAE-Sepharose.

Serologic evidence of Yersinia infection in patients with anterior uveitis.

Anterior uveitis is a common inflammatory eye disease associated with the HLA-B27 phenotype. Bacteriologic cofactors have been implicated in the pathogenesis of several diseases associated with HLA-B27. Using a sensitive enzyme-linked immunosorbent assay, we examined the incidence of previous Yersinia infection in a group of 28 consecutive patients with anterior uveitis. Twelve patients had a significantly increased antibody response to Yersinia, 8 of whom were HLA-B27 positive. Eight patients had IgM antibodies, possibly indicative of recent infection. There were no positive Yersinia serologic findings in our control group of 28 subjects, 13 of whom were HLA-B27 positive. A strong association was found between previous Yersinia infection and the development of anterior uveitis in HLA-B27-positive and HLA-B27-negative patients.

Rapid quantitation of DNA spot hybridization by flatbed scintillation counting.

The flatbed scintillation counting system (Betaplate) was used for quantitative measurement of the radioactive hybridization signal in detection of adenovirus and papillomavirus DNA in clinical specimens. In this method, 96 samples on a nylon membrane can be handled as a single entity throughout the hybridization and counting procedure. The technique is sensitive, rapid, and convenient in routine use when compared with conventionally applied methods for the numerical analysis of hybridization results. The assay principle allows simultaneous testing of large numbers of specimens.

Serological cross-reactions against Yersinia enterocolitica in patients infected with other arthritis-associated microbes.

In order to investigate arthritis-triggering, serologically cross-reactive antigens, sera from patients infected with arthritis-associated microbes, Salmonellae, Chlamydia trachomatis, and Sindbis-like alphavirus, were reacted against SDS-PAGE separated and immunoblotted Yersinia enterocolitica 0:3 antigens. These sera reacted with Yersinia to the same extent as did the control sera taken from patients with streptococcal, staphylococcal and Bordetella pertussis infections or from healthy blood donors. Moreover, the various sera produced different reactivity patterns, directed against several different antigens. Although sera from test subjects, as well as from controls including healthy individuals, recognized some Yersinia antigens, these patterns differed markedly from those recognized by sera taken from patients with Yersinia infection. Significantly, analysis of the reactivities against the different molecular weight antigen components of Yersinia revealed no dominant band or pattern which could thus have been defined as arthritis-associated.

Immunoblot analysis of IgM, IgG, and IgA responses to plasmid encoded released proteins of Yersinia enterocolitica in patients with or without yersinia triggered reactive arthritis.

The IgM, IgG, and IgA antibody responses of patients with Yersinia enterocolitica O:3 infection were studied by immunoblotting with plasmid encoded released proteins of Y enterocolitica as the antigens. The results indicate that antibodies of all three classes are most consistently directed against the proteins of molecular weights 25,000 and 36,000. Less than two months after the onset of infection 18 of the 19 patients with yersinia triggered reactive arthritis had IgA class antibodies against the released protein of mol. wt 36,000, whereas only eight of the 17 patients with non-arthritic yersiniosis had these antibodies. The same difference between the arthritic and non-arthritic patients was observed also 8-12 months after the onset of infection.

Comparison of bacteria with and without plasmid-encoded proteins as antigens for measurement of immunoglobulin M, G, and A antibodies to Yersinia enterocolitica by enzyme-linked immunosorbent assay.

Yersinia enterocolitica serovar O:3 bacteria with and without plasmid-encoded proteins were compared as antigens in an enzyme-linked immunosorbent assay. Good correlations between the two antigen preparations were obtained for immunoglobulin M (IgM), IgG, and IgA antibodies of patients with yersiniosis. For routine diagnostic purposes, these antigens are considered equal.

Factors associated with the development of reactive arthritis.

The role of the causative microorganism in the generation of reactive arthritis is decisive, but host factors are also of major importance. Patients who develop reactive arthritis after Yersinia enteritis show several interesting features in the immunological defence against Yersinia when compared to those who recover uneventfully. When all these peculiarities are taken together, they strongly indicate that in the patients developing reactive arthritis the causative microorganism enters host tissues to persist within the body for prolonged periods of time.

Immunoblot analysis of human IgM, IgG and IgA responses to plasmid-encoded antigens of Yersinia enterocolitica serovar O3.

Human IgM, IgG and IgA responses after infection with Yersinia enterocolitica serovar O3 were studied by immunoblotting sera against whole-cell homogenates of a plasmid-containing strain of Y. enterocolitica O3 and a plasmid-free strain derived from it; each strain was grown in conditions expressive for the plasmid. The antibodies observed were directed against several plasmid-encoded polypeptides. The response against different bacterial components decreased uniformly with time and the persisting antibody production was directed against several epitopes. Strong reactions to the prominent plasmid-specified antigens of mol. wts (10(3] 26, 34, 45 and 52.5 were found more often with IgG-class antibodies than with IgM or IgA; the latter immunoglobulins recognised, respectively, antigens of mol. wt (10(3] 26 and 45 (IgM) and 26, 34 and 52.5 (IgA). Immunoblotting of sera from patients with yersinia-triggered reactive arthritis did not reveal any antigens that were involved additionally or specifically. However, IgA-mediated recognition of certain antigens of mol. wts (10(3] 26, 34 and 52.5 tended to persist longer in the arthritic patients.

Virulence-plasmid is associated with the inhibition of opsonization in Yersinia enterocolitica and Yersinia pseudotuberculosis.

Plasmid-cured variants of virulent strains of Yersinia enterocolitica and Y. pseudotuberculosis were obtained by selection after growth in calcium-deficient medium. To obtain antigen preparations consisting of whole bacteria the original plasmid-containing strains and the plasmid-cured variants were grown in conditions favouring expression of the temperature-inducible outer membrane proteins of Yersinia (YOP) (37 degrees C, calcium-deficient culture medium). The presence or absence of the YOP on the bacteria was verified by immunoblotting. Opsonophagocytosis of YOP-negative Yersinia preparations (YOP-) was compared to that of YOP-containing ones (YOP+) in human polymorphonuclear leukocyte (PMN) chemiluminescence (CL) assay. The attachment of complement C3b on the surface of the bacteria after opsonization with normal human serum was determined by using a fluorescent anti-C3c-antibody and flow cytometry. YOP+ bacteria resisted opsonization in the absence of specific antibodies, as indicated by diminished C3b-fixation on bacteria and weaker CL response. This implies that virulence-plasmid-coded structures provide Y. enterocolitica and Y. pseudotuberculosis with an ability to avoid complement-mediated opsonization and phagocytosis.

Immunoblotting analysis of human IgM, IgG and IgA response to chromosomally coded antigens of Yersinia enterocolitica 0:3.

Human antibody response after Yersinia enterocolitica infection was studied by immunoblotting sequentially collected sera against a whole-cell homogenate of Y. enterocolitica serotype 0:3, grown under conditions restrictive for the plasmid. The antibodies observed were directed against a multitude of chromosomally coded antigens, and a considerable individual heterogeneity was found in the reactions of individual sera. The early (0-2 months) and late (greater than or equal to 11 months) responses were directed against the same antigenic determinants. Antibodies against different bacterial epitopes decreased evenly with time, indicating that several, if not all, antigenic epitopes of the bacteria are responsible for the prolonged antibody production. IgM responses by most patients declined within a few months but were surprisingly strong in some even one year after onset of the infection. IgG antibodies showed a strong reaction against a region corresponding to lipid A and core of the bacterial LPS, whereas IgM and IgA recognized this region less often. No other significant differences between IgM, IgG and IgA responses were observed. Immunoblotting of sera from patients with post-infection complications (arthritis, iritis, erythema nodosum) did not reveal any additional or specifically involved antigens. Altogether, these findings suggest that Yersiniae causing the original infection may hide in some of the patients for prolonged periods.

Monoclonal antibodies reacting selectively with core and O-polysaccharide of Yersinia enterocolitica O:3 lipopolysaccharide.

In order to obtain specific tools for studying the alterations of the immunochemical structure of Yersinia enterocolitica lipopolysaccharide in various conditions, we have produced monoclonal antibodies reacting with core and O-polysaccharide chains of Yersinia enterocolitica O:3 LPS. Immunizations were made with whole bacterial cells and outer membrane preparation, respectively. Monoclonal antibody 2B5 reacted in enzyme immunoassay with purified core-lipid A complex, and its binding was not inhibited by Polymyxin B, suggesting that the target determinant is in the outer core. 2B5 recognized 100% of all tested Y. enterocolitica O:3 strains (n = 152) and reacted to some extent also with many other gram-negative bacteria. In immunoblotting with 2B5, a band corresponding to core-lipid A complex was visualized both with Y. enterocolitica, Brucella abortus and Haemophilus influenzae. In immunofluorescence assay, the only positive reaction was seen with Y. enterocolitica. Monoclonal antibody A6 reacted in enzyme immunoassay with purified O-polysaccharide chains, recognized 100% of tested Y. enterocolitica O:3 strains, and showed no cross-reactions with other bacteria. A typical ladder pattern was not seen in the immunoblotting analysis with A6. This suggests that the O-chain of Y. enterocolitica O:3 may be different from those in other gram-negative bacteria. These two antibodies will make it possible to study the structural variations of Yersinia enterocolitica LPS more precisely than described before, because of their fine specificity against important immunogenic components of LPS. They will also be useful in serology measuring the immune response against the target determinants of these antibodies.

Amount and avidity of salivary and serum antibodies against Streptococcus mutans in two groups of human subjects with different dental caries susceptibility.

Immunoglobulin A (IgA) and IgG antibodies against Streptococcus mutans K1R and 10449 were measured in serum and in stimulated whole saliva from two groups of naval recruits, representing high or low caries susceptibility. The antibody assays were performed by using the enzyme-linked immunosorbent assay, and the results were expressed by a method able to estimate the amount of high-avidity and total specific antibodies. As a control, concentrations of salivary total immunoglobulins were related to the amounts of specific antibodies. Further, antibodies were assayed against three antigens, unrelated to the streptococci. No clear differences were observed in serum antibodies between the subjects with high or low caries susceptibility. However, in saliva, low caries susceptibility was associated with a high amount of total antigen-specific IgA, and possibly IgG, against S. mutans. This difference between the groups still existed when the amounts of specific antibodies were related to the amounts of salivary immunoglobulins. There were no differences in the amounts of total specific antibodies against the unrelated antigens. No differences were observed in the estimates of high-avidity anti-S. mutans antibodies between the groups, either in serum or saliva. Thus, within the limitations of the assays and crude antigen, lack of high-avidity antibodies is not responsible for caries susceptibility. Instead, the amount of anti-S. mutans antibodies seems to be linked with caries protection. The results of the present study indicate that salivary antibodies are linked with the control of human dental caries.