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Interferon (IFN)-γ release assays (IGRAs) are probably the most accurate tests for the detection of latent Mycobacterium tuberculosis infection, but IGRAs are labour intensive and the transport of samples over longer distances is difficult. IFN-γ-induced protein (IP)-10 is expressed at 100-fold higher levels than IFN-γ, and IP-10 release assays have comparable performance to IGRAs. The aim of this study was to explore the diagnostic potential of a novel IP-10 release assay based on dried plasma spots (DPS). The presence of IP-10 and IFN-γ was determined in plasma and in DPS by ELISA. Diagnostic algorithms for plasma and DPS tests for IP-10 were developed on a training cohort comprising 60 tuberculosis (TB) patients and 59 healthy controls. Diagnostic accuracy was assessed in a validation cohort comprising 78 TB patients and 98 healthy controls. Plasma was measured in Spain and DPS samples were sent to Denmark using the conventional postal service for analysis. IP-10 was readily detectable in both plasma and DPS, and correlation was excellent (r2 = 0.95). QuantiFERON-TB Gold In-Tube (QFT-TB) and IP-10 in DPS and plasma rendered comparable sensitivity (78%, 82% and 84%, respectively), specificity (100%, 97% and 97%, respectively) and indeterminate rates (p>0.55). The DPS-based IP-10 test has comparable diagnostic accuracy to the QFT-TB and samples can be sent via conventional mail over long distances for analysis without affecting the results.

Background: Adult opsoclonus-myoclonus (OM), a disorder of eye movements accompanied by myoclonus affecting the trunk, limbs, or head, is commonly associated with an underlying malignancy or precipitated by viral infection. Methods: We present the first two reports of post-streptococcal OM associated with antibodies against a 56 kDa protein. Two young girls presented with opsoclonus and myoclonus following a febrile illness and pharyngitis. Protein purification techniques were employed. Amino acid sequences of human neuroleukin (NLK) and streptococcal proteins were compared using the protein-protein BLAST application. Results: The antigen was identified as NLK (glucose-6-phosphate isomerase, GPI). GPI is present on the cell surface of streptococcus making the protein a candidate target for molecular mimicry. Conclusions: We have identified NLK as an antigenic target in two patients with post-streptococcal OM. The pathogenicity of the antibodies is uncertain. The potential role of anti-neuroleukin antibodies in the pathogenesis of OM is discussed. We propose that OM may represent a further syndrome in the growing spectrum of post-streptococcal neurological disorders. The role of streptococcus in OM and the frequency with which anti-NLK responses occur in both post-infectious and paraneoplastic OM should be investigated further.

Background: In vitro stimulation of whole blood or isolated blood cells with specific antigens is used for several purposes. Immediately following incubation with antigens, samples have to be centrifuged to stop the reactions by remaining cells and the supernatant refrigerated or analysed directly to preserve the analytes of interest, which makes samples difficult to prepare outside laboratories. We have tested whether spotting whole blood on filter paper after activation can be used in one of the tests for Mycobacterium tuberculosis infection (MTI), the QuantiFERON®-TB Gold In Tube test (QFT), where the spotting technique can make it suitable for use in locations without facilities like a centrifuge and a refrigerator. Materials and methods: Samples from 22 individuals undergoing screening for MTI and 10 healthy controls were incubated, centrifuged and IFN-γ measured by Enzyme-linked immunosorbent assay (ELISA), as described in the kit insert. In parallel, activated blood was spotted on filter paper (Schleicher & Schuell) and dried. The dried blood spot samples were analysed for 21 inflammatory markers with an in-house assay based on Luminex technology. Results: Our multiplex measurements of inflammatory markers in samples from suspected MTI patients confirmed the IFN-γ findings in the QFT. IL-2, GM-CSF, IL-5, and IL-1β were also found as useful markers for MTI. We were not able to distinguish between active tuberculosis and latent MTI. Conclusion: Applying blood on filter paper after incubation makes in vitro stimulation tests feasible in locations where heat and electricity is unavailable.

Inflammatory periodontal diseases are provoked by bacteria which adhere to teeth at the gingival margin and form plaques containing toxins detectable by their effect on mammalian cells in culture. The aim of this study was to make toxin-neutralizing monoclonal antibodies and determine whether they detect antigen in specific oral bacteria. Bacterial plaque was collected from teeth and homogenized, and the fluid phase (plaque extract) was boiled or first fractionated over Sephacryl S-300. Hybridomas from immunized mice secreted immunoglobulin M (IgM) antibodies which reacted to plaque antigens. Neutralization was detected by an increase in the growth of HL60 cells which were exposed to plaque toxins in the presence of IgM from hybridoma culture or ascitic fluids. However, the neutralization was obvious only when the plaque toxins reduced growth by 50% or less. Plaque toxin preparations were found to contain proteases which hydrolyzed all of the IgM in ascitic fluids within 24 h. Replenishing the IgM daily preserved protection compared with protection from IgM from other hybridomas or saline only. The decrease in the specific activity of plaque proteins caused by replenishing one such antibody (3hE5) was 2.5-fold compared with activity with unreplenished 3hE5, 3.8-fold compared with activity with saline only, and 10.7-fold compared with activity with replenished, unrelated antibody. The neutralizing IgM detected an array of 14,000- to 22,000-molecular-weight antigens. The native toxins may be aggregates of these antigens, or the array may indicate fragments of an undetected, larger antigen or a common, nonpeptide adduct. Only 0.5 to 0.8% of the bacteria from sites with periodontitis and grown on blood agar contained antigen. One group of reactive bacteria was identified as Actinomyces odontolyticus serotype I. Other isolates were identified as Staphylococcus epidermidis, but antigen disappeared from the these isolates within 6 weeks of subculture. Epitope-containing antigens were also found in streptococcal and Eikenella isolates, and it is likely that the antigens from only some of these bacteria are toxic.

We have developed a simple, rapid, and low-cost "paper-bridged" method for Salmonella phase reversal. More than 3500 isolates were tested in our laboratory, and the results indicated that this paper-bridged method is a useful alternative for phase reversal.

We have experimentally immunized rats intratracheally with sonicated Pseudomonas aeruginosa to develop a high antibody response systemically and locally. The systemic antibodies were measured by a standard enzyme-linked immunosorbent assay (ELISA) on serum samples, whereas the local antibodies were measured on eluates from paper discs containing saliva. High levels of IgA antibodies were elicited in saliva whereas only traces of IgG antibodies were detected; the opposite results were found in serum. The saliva disc method permits serial measurements from the same animal and therefore offers the possibility to follow post-immunization antibody responses.

Given the technical difficulties of the ELISA method, a gelatin particle agglutination test (MLPA) has been developed recently for the detection of anti-PGLI antibodies. The purpose of this study was to compare these 2 tests. MLPA was found to be less specific than ELISA (91% versus 98%, chi 2 = 66.8, p less than 0.001). The sensitivity of both tests was of 95% for the diagnosis of multibacillary patients. In the case of paucibacillary patients. MLPA was found to be less sensitive than ELISA (21% versus 35%, chi 2 = 6.98, p greater than 0.01). The agreement between the 2 tests for a positive or a negative result was satisfying (85% to 100%), except for the weakly seropositive individuals (71%). The correlation between OD obtained with ELISA and antibody titre obtained with MLPA was statistically significant (r = 0.70, p less than 0.001). Conversely to ELISA, MLPA was not applicable on blood samples absorbed on filter paper without a serious loss of sensitivity. In conclusion, this study demonstrated that the MLPA test can only reliably detect anti-PGLI antibodies in multibacillary cases.

Antigenic competition is known to be a widespread phenomenon when using crude extracts of antigens (e.g., Escherichia coli cytoplasmic proteins) for immunization. This non-specific form of immune suppression can be partially overcome by passive immunization with antibodies against dominant antigens (which are the suppressive molecules) before injection of the antigenic mixture. Blocking these immunodominant antigens or antigenic determinants by a passively administered antibody permits antibody responses against hitherto weakly or non-immunogenic molecules.

An ammonium sulfate precipitate derived from a culture filtrate of Nocardia asteroides contained 20-30 protein subunits, as identified by gel electrophoresis. Western blot analysis of these proteins (obtained from serum samples from patients with systemic nocardiosis) revealed that all of the 17 samples reacted with a subunit of a protein with an apparent molecular weight of 55,000 and that 11 of the 17 samples reacted with a subunit with a molecular weight of 31,000. In contrast, only two of 25 serum samples obtained from healthy controls or patients hospitalized without nocardial or mycobacterial disease reacted with these subunits. Sera from 21 patients infected with Mycobacterium tuberculosis, a historically troublesome cross-reactive group, failed to react with either the 55,000 or the 31,000 molecular weight subunit. The presence of only two protein subunits that reacted with sera from humans with nocardiosis suggests that these molecules may have use as diagnostic reagents or probes of the immunologic reaction to Nocardia. Infection with Nocardia asteroides has become

When pilus+ Gc were introduced into a male subject's urethra, they gave rise to pilus+ variants whose pilin mRNAs differed from that of input Gc. The differences stemmed from the Gc genome's single complete pilin gene having undergone gene conversion by different partial pilin genes' sequences and by different length stretches of a single partial pilin gene. In some instances, the variant's pilin mRNA appeared to reflect two independent gene-conversion events that used sequences from two different partial pilin genes. The resulting variants' pilins exhibited antigenic differences compared with the pilin polypeptide of input Gc; these differences were discernible by immunoblotting with mAbs. Amino acid and antigenic changes occurred in a segment of the variants' pilin polypeptides that previously was thought to be conserved or constant in sequence.

Filter paper discs have been used in the enzyme immunoassay (EIA) as solid phase instead of polystyrene plates. The use of paper discs has made it possible to achieve a multiple increase in the sensitivity of sandwich EIA, thus permitting the detection of Yersinia pestis capsular antigen at a concentration of 0.4 ng/ml. Paper discs can be used not only for the sorption of antigen and antibodies, but also for the affinity purification of preparations.

The outer membranes of gram-negative bacteria are considered to be of importance in host-bacteria interaction, in protective immunity, and occasionally in subclassification within a species. In this study, the outer membranes of several strains of Yersinia enterocolitica and Y. pseudotuberculosis were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the appearance of the major proteins depended on the temperature at which they were solubilized in SDS. A protein was identified with the use of two-dimensional gels and preparative SDS-PAGE, which was equivalent to the "heat-modifiable protein" (protein II) of other Enterobacteriaceae species. A monoclonal antibody, 4G1, was generated against an isolated preparation of this Y. enterocolitica protein. This antibody was tested with whole cell bacterial antigens of 46 individual bacterial strains. The reactive strains included only Y. enterocolitica and Y. pseudotuberculosis. In addition, the reactivity of the 4G1 monoclonal antibody preparation could be absorbed only with Y. enterocolitica and Y. pseudotuberculosis, and not with other strains of bacteria. The reactivity of this 4G1 monoclonal antibody was also tested by the Western Blot technique. Six individual strains were tested: a Y. enterocolitica serotype 0:3, a Y. enterocolitica serotype 0:9, an Escherichia coli, a Salmonella typhimurium, a Shigella flexneri, and a Klebsiella pneumoniae. The 4G1 antibody reacted with only the proteins of the two Y. enterocolitica strains. In conclusion, the equivalent of the heat-modifiable protein was present in Y. enterocolitica and Y. pseudotuberculosis. Moreover, this protein also carried a species-specific antigenic determinant.

We studied the antigenic cross-reactivity of surface proteins among various strains of Legionella pneumophila and other Legionella species by using a novel method of antibody purification. Anti-bacterial antibodies in hyperimmune sera were adsorbed to and eluted from the surface of recombinant E. coli cells that express individual L. pneumophila antigens on their surface. These affinity-purified antibodies were then used to probe protein immunoblots prepared from the test strains to detect cross-reactive domains. We found that antigenic proteins are generally conserved in all L. pneumophila serogroups. Although some of these antigenic domains are shared with members of other Legionella species, they are associated with proteins of different molecular mass. Our approach to the study of antigenic cross-reactivity has potential advantages over similar studies that use either monoclonal antibodies or monospecific antibodies prepared by immunization with purified antigens.

Purpura hemorrhagica is an acute disease of the horse characterized by edema of the head and limbs, leucocytoclastic vasculitis, petechial hemorrhages in mucosae, musculature and viscera, and sometimes glomerulonephritis. It is usually associated with strangles, an upper respiratory tract disease of the horse caused by Streptococcus equi. We have detected and characterized immune complexes in the sera of horses with poststrangles purpura hemorrhagica by using PEG precipitation and Western blot analysis. The immune complexes contained IgA and S. equi-specific antigens similar to those found in acid extracts. We propose that purpura hemorrhagica is an immune complex-mediated disease.

Protein G is an immunoglobulin (IgG)-binding bacterial cell wall protein recently isolated from group G streptococci. We have investigated the avidity of protein G for various monoclonal and polyclonal Ig of the IgG class, and compared it with the binding properties of protein A, the staphylococcal Fc-binding protein. Radiolabeled Ig were mixed with Sepharose-coupled protein G or protein A, and the amounts of radioactivity bound to the matrix-coupled bacterial proteins were determined. The avidity was found to be greater for protein G than for protein A for all examined Ig. Protein G bound all tested monoclonal IgG from mouse IgG1, IgG2a, and IgG3, and rat IgG2a, IgG2b, and IgG2c. In addition, polyclonal IgG from man, cow, rabbit, goat, rat, and mouse bound to protein G, whereas chicken IgG did not. The binding property of protein G was additionally exploited in the Western blot assay, in which iodine-labeled protein G was used successfully for the detection of a rat monoclonal antibody against ovalbumin, and for the detection of rabbit and goat polyclonal whole antisera against human urinary proteins. In these experimental situations, protein G was found to be a powerful reagent for the detection of IgG, and consequently the antigen against which these antibodies are directed.

A patient with cutaneous vasculitis during infective endocarditis due to Lactobacillus casei was studied. Immune complexes (IC) were isolated from serum at the time of diagnosis and after 4 wk of therapy. Purification of IC used differential polyethylene glycol precipitation and competitive binding to staphylococcal protein A. In situ radioiodination of IC was performed, followed by SDS-polyacrylamide gel electrophoresis (PAGE). Anti-IC antisera were raised in rabbits by immunization with purified IC. IC were characterized by SDS-PAGE followed by electrophoretic transfer to nitrocellulose, incubation with antiserum and then with 125I protein A, and autoradiography. Although early and late IC differed quantitatively, there were no differentiating immunochemical features. Both IC contained a 60,000 dalton component that did not react with preimmune serum nor with anti-normal human serum. This component reacted with antiserum rendered specific for L. casei by affinity chromatography. The restricted antigen-antibody representation in IC contrasted with a wider panel of antibody activity in patient serum. The Western blot analysis proves to be an ideal method for the characterization of IC because of its sensitivity, dissociative capability, and preservation of immunoreactivity. IC isolated at a time removed from the original antigenic challenge may provide insight into the nature of the inciting antigen.