Reference Antigen Research Articles

Assessment of viral replication in eggs and HA protein yield of pre-pandemic H5N1 candidate vaccine viruses

H5N1 infection and the potential for spread from human to human continue to pose a severe public health concern. Since vaccination remains the most effective way to prevent a potential H5N1 pandemic, the World Health Organization (WHO) Collaborating Centers (CCs) and Essential Regulatory Laboratories (ERLs) engineered and developed a panel of H5N1 pre-pandemic vaccine viruses for pandemic vaccine preparedness as well as production of antigen potency testing reagents (reference antigen and reference anti-serum) for vaccine standardization. To develop a strategy utilizing a number of biochemical methods for the characterization of the viral growth properties and protein yield in eggs, we have selected eight H5N1 pre-pandemic viruses and determined the viral Egg Infectious Dose 50 (EID50), total protein yield, hemagglutinin (HA) to nucleoprotein (NP) ratios (HA:NP), and HA1 content of each virus. Our results showed that all the tested H5N1 vaccine viruses grew to high titers in eggs. The total viral protein yield varies within a narrow range, whereas there were greater differences in the HA:NP protein ratios among the eight viruses. The RP-HPLC based HA1 content analysis demonstrated that the viruses A/Anhui/1/2010, A/Hubei/1/2005, and A/goose/Guiyang/337/2006 contained higher HA contents than other five viruses including A/Vietnam/1203/2003. Our approach for analyzing virus growth and protein yield will allow us identify optimal vaccine virus in a timely manner. In addition, we successfully purified the HA proteins of H5N1 vaccine viruses by optimizing bromelain cleavage conditions. Our studies on the HA protein purification may improve the quality control of the production of influenza vaccine test reagent.

Simultaneous quantification of the viral antigens hemagglutinin and neuraminidase in influenza vaccines by LC–MSE

Current methods for quality control of inactivated influenza vaccines prior to regulatory approval include determining the hemagglutinin (HA) content by single radial immunodiffusion (SRID), verifying neuraminidase (NA) enzymatic activity, and demonstrating that the levels of the contaminant protein ovalbumin are below a set threshold of 1μg/dose. The SRID assays require the availability of strain-specific reference HA antigens and antibodies, the production of which is a potential rate-limiting step in vaccine development and release, particularly during a pandemic. Immune responses induced by neuraminidase also contribute to protection from infection; however, the amounts of NA antigen in influenza vaccines are currently not quantified or standardized. Here, we report a method for vaccine analysis that yields simultaneous quantification of HA and NA levels much more rapidly than conventional HA quantification techniques, while providing additional valuable information on the total protein content. Enzymatically digested vaccine proteins were analyzed by LC–MSE, a mass spectrometric technology that allows absolute quantification of analytes, including the HA and NA antigens, other structural influenza proteins and chicken egg proteins associated with the manufacturing process. This method has potential application for increasing the accuracy of reference antigen standards and for validating label claims for HA content in formulated vaccines. It can also be used to monitor NA and chicken egg protein content in order to monitor manufacturing consistency. While this is a useful methodology with potential for broad application, we also discuss herein some of the inherent limitations of this approach and the care and caution that must be taken in its use as a tool for absolute protein quantification. The variations in HA, NA and chicken egg protein concentrations in the vaccines analyzed in this study are indicative of the challenges associated with the current manufacturing and quality control testing procedures.

Application of deglycosylation to SDS PAGE analysis improves calibration of influenza antigen standards

Each year the production of seasonal influenza vaccines requires antigen standards to be available for the potency assessment of vaccine batches. These are calibrated and assigned a value for haemagglutinin (HA) content. The calibration of an antigen standard is carried out in a collaborative study amongst a small number of national regulatory laboratories which are designated by WHO as Essential Regulatory Laboratories (ERLs) for the purposes of influenza vaccine standardisation. The calibration involves two steps; first the determination of HA protein in a primary liquid standard by measurement of total protein in a purified influenza virus preparation followed by determination of the proportion of HA as determined by PAGE analysis of the sample; and second, the calibration of the freeze-dried reference antigen against the primary standard by single radial immunodiffusion (SRD) assay. Here we describe a collaborative study to assess the effect of adding a deglycosylation step prior to the SDS-PAGE analysis for the assessment of relative HA content. We found that while the final agreed HA value of the samples tested was not significantly different with or without deglycosylation, the deglycosylation step greatly improved between-laboratory agreement.

Establishing China's national standards of antigen content and neutralizing antibody responses for evaluation of enterovirus 71 (EV71) vaccines

Enterovirus 71 (EV71) is a highly infectious agent that causes hand-foot-mouth disease (HFMD) in humans. Effective vaccination against EV71 infection is critically important, given the recent outbreak of HFMD in the Asia-Pacific region, where it has shown significant mortality and morbidity. There is currently no approved anti-viral therapy available to treat the disease. While several vaccine manufacturers are actively developing EV71 vaccines, there are no international reference standards available to conduct quality control on EV71 vaccines or to assess the effectiveness of EV71 vaccines in immunized populations. In the current report, antigen reference standard based on the C4 subtype of the EV71 vaccine strain was developed. In addition, neutralizing antibody (NTAb) reference panels were analyzed and standards with various neutralizing titers were selected. These reference antigens were used to calibrate vaccine samples from several producers and found that five EV71 antigens and the national reference standards showed good linearity and parallelism. Moreover, mice immunized with various vaccines at doses standardized by these national references showed comparable NTAb responses. Finally, the national NTAb reference panels were found to effectively reduce assay discrepancy between different labs. Taken together, these national reference standards are highly valuable for the standardization and evaluation of EV71 vaccines.

Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza vaccines

Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009–2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified.

Emprego de estirpes Leptospira spp. isoladas no Brasil na microtécnica de soroaglutinação microscópica aplicada ao diagnóstico da leptospirose em rebanhos bovinos de oito estados brasileiros

O objetivo do presente trabalho foi investigar a conveniência do emprego de estirpes de leptospiras autóctones isoladas no Brasil, na coleção de antígenos da microtécnica de soroaglutinação microscópica (SAM) aplicada a leptospirose. Foram amostradas por conveniência 109 propriedades e 9820 bovinos, fêmeas em idade reprodutiva, distribuídos em 85 municípios, dos Estados de Goiás, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Paraná, Rio Grande do Sul, Santa Catarina e São Paulo. Dos 9820 animais examinados, 5806 (59,12%) foram reagentes na SAM para pelo menos um sorovar com a coleção de 23 sorovares de referência. Com a coleção de antígenos de referência e dez estirpes autóctones houve 6400 (65,17%) reagentes, com diferença significativa entre as proporções (p=0,001). Os sorovares mais prováveis identificados com a coleção de antígenos de referência foram Hardjo (43,03%), Shermani (20 %), Wolffi (9,96%), Grippothyphosa (5,42%) e Pomona (4,28%). Com a coleção ampliada por dez estirpes isoladas no Brasil, os sorovares mais prováveis foram Hardjo (31,00%), Guaricura-M4/84 (22,50%), Shermani (15,43%), Wolffi (4,76%), Grippothyphosa (3,71%) e Autumnalis (3,24%). O sorovar Guaricura, estirpe M4/84, isolada de bovinos e búfalos no Estado de São Paulo, despontou como um dos três sorovares mais freqüentes nos Estados de Goiás, Mato Grosso, Mato Grosso do Sul, Minas Gerais e São Paulo. A introdução de estirpes autóctones na coleção de antígenos da SAM propiciou a confirmação do diagnóstico de leptospirose em 594 animais (6,00%) classificados como não reagentes pela coleção de referência (p=0,001).

Impact of antigenic and genetic drift on the serologic surveillance of H5N2 avian influenza viruses

BackgroundSerologic surveillance of Avian Influenza (AI) viruses is carried out by the hemagglutination inhibition (HI) test using reference reagents. This method is recommended by animal health organizations as a standard test to detect antigenic differences (subtypes) between circulating influenza virus, vaccine- and/or reference- strains. However, significant discrepancies between reference antisera and field isolates have been observed during serosurveillance of influenza A viruses in pig and poultry farms. The objective of this study was to examine the effects of influenza virus genetic and antigenic drift on serologic testing using standard HI assays and reference reagents. Low pathogenic AI H5N2 viruses isolated in Mexico between 1994 and 2008 were used for phylogenetic analysis of AI hemagglutinin genes and for serologic testing using antisera produced with year-specific AI virus isolates.ResultsPhylogenetic analysis revealed significant divergence between early LPAI H5N2 viruses (1994 - 1998) and more recent virus field isolates (2002 - 2008). Results of the HI test were markedly influenced by the selection of the AI H5N2 virus (year of isolation) used as reference antigen for the assay. These analyses indicate that LPAI H5N2 viruses in Mexico are constantly undergoing genetic drift and that serosurveillance of AI viruses is significantly influenced by the antigen or antisera used for the HI test.ConclusionsReference viral antigens and/or antisera need to be replaced constantly during surveillance of AI viruses to keep pace with the AI antigenic drift. This strategy should improve the estimation of antigenic differences between circulating AI viruses and the selection of suitable vaccine strains.

Immunogenicity and Reactivity of Novel Mycobacterium avium subsp. paratuberculosis PPE MAP1152 and Conserved MAP1156 Proteins with Sera from Experimentally and Naturally Infected Animals

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Development of genetic tools and completion of the M. avium subsp. paratuberculosis genome sequencing project have expanded the opportunities for antigen discovery. In this study, we determined the seroreactivities of two proteins encoded at the 5' and 3' regions of the MAP1152-MAP1156 gene cluster. MAP1152 encodes a PPE protein, and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Recombinant MAP proteins were overproduced and purified from Escherichia coli as maltose-binding protein (MBP) fusions. Immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of mice and rabbits immunized with live M. avium subsp. paratuberculosis cells and against samples from naturally infected cattle. In immunoblot assays, MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme-linked immunosorbent assay for the recombinant proteins was developed and used to test preclassified positive and negative serum samples from naturally infected and noninfected cattle. Samples, with one exception, displayed no seroreactivity against the MBP-LacZ fusion protein (P > 0.05), the negative-control antigen. MAP1152 displayed seroreactivity against all positive sera but no seroreactivity to the negative sera (P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between noninfected and infected cattle (P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, both proteins are immunogenic in mice and rabbits, and M. avium subsp. paratuberculosis-infected cattle mount a humoral response to both MAP1152 and MAP1156 cross-reactive epitopes. These findings have potential applications to diagnostics, vaccine production, and elucidation of the immunopathogenesis of JD.

An alternative method for preparation of pandemic influenza strain-specific antibody for vaccine potency determination

The traditional assay used to measure potency of inactivated influenza vaccines is a single-radial immunodiffusion (SRID) assay that utilizes an influenza strain-specific antibody to measure the content of virus hemagglutinin (HA) in the vaccine in comparison to a homologous HA reference antigen. Since timely preparation of potency reagents by regulatory authorities is challenging and always a potential bottleneck in influenza vaccine production, it is extremely important that additional approaches for reagent development be available, particularly in the event of an emerging pandemic influenza virus. An alternative method for preparation of strain-specific antibody that can be used for SRID potency assay is described. The approach does not require the presence or purification of influenza virus, and furthermore, is not limited by the success of the traditional technique of bromelain digestion and purification of virus HA. Multiple mammalian expression vectors, including plasmid and modified vaccinia virus Ankara (MVA) vectors expressing the HAs of two H5N1 influenza viruses and the HA of the recently emerging pandemic H1N1 (2009) virus, were developed. An immunization scheme was designed for the sequential immunization of animals by direct vector injection followed by protein booster immunization using influenza HA produced in vitro from MVA vector infection of cells in culture. Each HA antibody was highly specific as shown by hemagglutination inhibition assay and the ability to serve as a capture antibody in ELISA. Importantly, each H5N1 antibody and the pandemic H1N1 (2009) antibody preparation were suitable for use in SRID assays for determining the potency of pandemic influenza virus vaccines. The results demonstrate a feasible approach for addressing one of the potential bottlenecks in inactivated pandemic influenza vaccine production and are particularly important in light of the difficulties in preparation of potency reagent antibody for pandemic H1N1 (2009) virus vaccines.

Development and Application of an Electrochemical Plate Coupled with Immunomagnetic Beads (ELIME) Array for Salmonella enterica Detection in Meat Samples

Salmonella is one of the main organisms causing outbreaks of foodborne illness, and meat is one of the major vehicles of salmonellosis throughout the world. A novel analytical immunosensor array, based on a 96-well electrochemical plate coupled with immunomagnetic beads (ELIME array), is proposed for the detection of Salmonella in meat samples. After an optimization study, using Salmonella enterica serotype Enteritidis as reference antigen, the ability of the method to interact with a large number of Salmonella serovars commonly present in food was evaluated. The assay was then used to analyze samples of pork, chicken, beef, and turkey experimentally inoculated with Salmonella as well as real samples. The results were compared with those from the International Standard of Organization (ISO) culture method. The comparison showed that the ELIME array is able to detect a low number of Salmonella cells (1-10 CFU per 25 g) after only 6 h of incubation in a pre-enrichment broth. The investigation revealed a very good agreement between culture and ELIME array methods for meat samples, reducing the time for performing the analysis and obtaining the results quickly.

Local Borrelia burgdorferi sensu stricto and Borrelia afzelii strains in a single mixed antigen improves western blot sensitivity

Aims:This study evaluates the use of local Borrelia burgdorferi sensu stricto and Borrelia afzelii strains in a single mixed antigen for in-house IgG western blots in the routine diagnostic setting...

Prevalence of Leptospirosis in Dairy Cattle from Small Rural Production Units in Toluca Valley, State of Mexico

In order to know the seroprevalence of Leptospira spp. in stabled dairy cattle, a study was conducted from 2004 to 2006 in which 416 sera were tested using a microscopic agglutination test conducted on microplates. A collection of culture reference antigens, each representing a serogroup, was used for these tests. Results showed that 10.33% (43) of the animals had antibody titers ranging from 1:100 to 1:1600. The main serovars detected in these tests were L. interrogans serovar hardjo and L. interrogans serovar canicola. It is important to note that these serovars represent a high risk for transmission to other susceptible animal species, between individuals, and to human health. This serological survey provides useful information establishing the presence or absence of these serovars in this type of herd. The range of antigens used in this study included serovars representative of all common serogroups.

Allergen concentration in natural rubber latex

Hevea brasiliensis latex serum is commonly used as the in vivo and in vitro reference antigen for latex allergy diagnosis as it contains the full complement of latex allergens. This study quantifies the concentrations of the significant allergens in latex serum and examines its suitability as an antigen source in latex allergy diagnosis and immunotherapy. The serum phase was extracted from centrifuged latex that was repeatedly freeze-thawed or glycerinated. Quantitation of latex allergens was performed by two-site immunoenzymetric assays. The abundance of RNA transcripts of the latex allergens was estimated from the number of their clones in an Expressed Sequence Tags library. The latex allergens, Hev b 1, 2, 3, 4, 5, 6, 7 and 13, were detected in freeze-thawed and glycerinated latex serum at levels ranging from 75 (Hev b 6) to 0.06 nmol/mg total proteins (Hev b 4). Hev b 6 content in the latex was up to a thousand times higher than the other seven latex allergens, depending on source and/or preparation procedure. Allergen concentration was reflected in the abundance of mRNA transcripts. When used as the antigen, latex serum may bias the outcome of latex allergy diagnostic tests towards sensitization to Hev b 6. Tests that make use of latex serum may fail to detect latex-specific IgE reactivity in subjects who are sensitized only to allergens that are present at low concentrations. Latex allergy diagnostics and immunotherapy that use whole latex serum as the antigen source may not be optimal because of the marked imbalance of its constituent allergens.

Allergen Concentration in Natural Rubber Latex

RATIONALE: The serum from Hevea latex is commonly used as the in vivo and in vitro reference antigen for latex allergy diagnosis since it contains the entire complement of latex allergens. This study determines the concentrations of the significant allergens in latex serum and examines its suitability as an antigen in latex allergy diagnostics and immunotherapy.

Fully human IgG and IgM antibodies directed against the carcinoembryonic antigen (CEA) Gold 4 epitope and designed for radioimmunotherapy (RIT) of colorectal cancers

BackgroundHuman monoclonal antibodies (MAbs) are needed for colon cancer radioimmunotherapy (RIT) to allow for repeated injections. Carcinoembryonic antigen (CEA) being the reference antigen for immunotargeting of these tumors, we developed human anti-CEA MAbs.MethodsXenoMouse®-G2 animals were immunized with CEA. Among all the antibodies produced, two of them, VG-IgG2κ and VG-IgM, were selected for characterization in vitro in comparison with the human-mouse chimeric anti-CEA MAb X4 using flow cytometry, surface plasmon resonance, and binding to radiolabeled soluble CEA and in vivo in human colon carcinoma LS174T bearing nude mice.ResultsFlow cytometry analysis demonstrated binding of MAbs on CEA-expressing cells without any binding on NCA-expressing human granulocytes. In a competitive binding assay using five reference MAbs, directed against the five Gold CEA epitopes, VG-IgG2κ and VG-IgM were shown to be directed against the Gold 4 epitope. The affinities of purified VG-IgG2κ and VG-IgM were determined to be 0.19 ± 0.06 × 108 M-1 and 1.30 ± 0.06 × 108 M-1, respectively, as compared with 0.61 ± 0.05 × 108 M-1 for the reference MAb X4. In a soluble phase assay, the binding capacities of VG-IgG2κ and VG-IgM to soluble CEA were clearly lower than that of the control chimeric MAb X4. A human MAb concentration of about 10-7 M was needed to precipitate approximatively 1 ng 125I-rhCEA as compared with 10-9 M for MAb X4, suggesting a preferential binding of the human MAbs to solid phase CEA. In vivo, 24 h post-injection, 125I-VG-IgG2κ demonstrated a high tumor uptake (25.4 ± 7.3%ID/g), close to that of 131I-X4 (21.7 ± 7.2%ID/g). At 72 h post-injection, 125I-VG-IgG2κ was still concentrated in the tumor (28.4 ± 11.0%ID/g) whereas the tumor concentration of 131I-X4 was significantly reduced (12.5 ± 4.8%ID/g). At no time after injection was there any accumulation of the radiolabeled MAbs in normal tissues. A pertinent analysis of VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver.ConclusionOur human anti-CEA IgG2κ is a promising candidate for radioimmunotherapy in intact form, as F(ab')2 fragments, or as a bispecific antibody.

Immunodiagnosis of ABPA.

For the diagnosis of allergic aspergillosis demonstration of specific immune responses to allergens has been accepted as a significant paradigm. Elevated levels of total IgE and Aspergillus fumigatus specific IgE and IgG antibodies are important criteria for diagnosis of ABPA. Although reference antigens or standardized methods are not available, there are a number of relevant recombinant antigens, which have been isolated in recent years. Several techniques have been employed in the demonstration of specific antibodies against antigens of Aspergillus fumigatus in the sera of patients. Of these methods, the widely followed ones are ELISA, radioimmunoassay, Western blot, and agar gel double diffusion. Recently, semi-automated methods have been developed using recombinant allergens to detect circulating antibody against Aspergillus. However, these methods have not been evaluated widely. Here we review the immunodiagnostic methods currently in use for allergic bronchopulmonary aspergillosis.

IDENTIFICATION OF ANTIGENIC AND ALLERGENIC NATURAL RUBBER LATEX PROTEINS BY IMMUNOBLOTTING1*

ABSTRACT Quantitation of proteins in finished natural rubber latex (NRL) products is essential in predicting their allergenic potential. The ASTM standard Modified Lowry method for measuring total protein content has been used for several years. Most recently, ASTM published a standard for more sensitive and more specific enzyme immunoassay for quantitation of antigenic NRL proteins. It is an ELISA inhibition assay, using rabbit anti NRL sera. Since the measurement of proteins in this method depends on recognition capacity of rabbit antibodies, the selection of an appropriate protein source for rabbit immunization is crucial for the accuracy of such test. In this study, we evaluated the composition of NRL proteins from ammoniated (AL) and nonammoniated (NAL) raw latex and from finished NRL products, and compared the effectiveness of sera from rabbits immunized with NRL proteins, to react with those extracts. Immune rabbit sera were analyzed by immunoblotting against extracts of several samples of AL, NAL, and glove proteins. In the NAL extracts, we identified 26–28 protein bands by SDS-PAGE. AL samples had between 6 and 9 bands with a great variation in the band positions among the samples. The Western blot analysis showed that anti-AL rabbit serum reacted with 4–9 protein bands in various AL extracts. The highest intensity of reaction was observed with the extract used to immunize the rabbits. Similar reaction was observed with anti-NAL serum. However, when the antisera were blotted against NAL extracts, anti-NAL serum reacted more strongly and with a larger number of proteins than anti-AL serum. In summary, anti-NAL serum recognized an equal number of proteins in AL extract as anti-AL serum. However, anti-AL serum recognized fewer protein molecules in NAL extract than anti-NAL serum. Our findings suggest that NAL extract contains more individual proteins than other extracts, and sera from rabbits immunized with this antigen have a greater capacity to react with a wide spectrum of NRL proteins. This finding may be helpful in selecting the representative reference antigen and antiserum for further efforts in NRL protein quantitation. *The submitted manuscript has been authored by a contractor of the U.S. Government. Accordingly, the U.S. Government retains a non-exclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for Government purposes.

Detection of antibodies to human immunodeficiency virus (HIV) that recognize conformational epitopes of glycoproteins 160 and 41 often allows for early diagnosis of HIV infection.

On the basis of human immunodeficiency virus (HIV) needlestick studies, the time to seroconversion for anti-HIV antibodies is 1-9 months (mean, approximately 2-3 months). However, an earlier marker of an immune response to HIV often occurs-serum anti-HIV antibodies reactive with live HIV-infected cells, termed "early HIV antibodies." The specificities of these antibodies are characterized by the recognition of type-specific conformational epitopes of the HIV envelope glycoprotein (gp) 160 and gp41. By use of a third-generation native HIV(IIIB) gp160 enzyme immunoassay (EIA), detection of HIV antibodies occurred, on average, 33 days earlier than did detection by commercial EIA and 25 days earlier than did detection by the reference antigen and reverse-transcription polymerase chain reaction (RT-PCR) assays in 3 of 5 HIV seroconversion panels. A fourth panel possessed early HIV antibodies that reacted with HIV(213) but not with HIV(IIIB), allowing for detection of HIV antibodies approximately 3 weeks earlier than by RT-PCR or other current tests.

High diversity in Curtobacterium flaccumfaciens pv. flaccumfaciens characterized by serology and rep-PCR genomic fingerprinting

The diversity found in culture collection strains of Curtobacterium flaccumfaciens pv. flaccumfaciens was characterized by serology and repetitive-sequence-based polymerase chain reaction (rep-PCR). Using strain NCPPB 559 as the reference antigen, immunofluorescence tests with polyclonal and monoclonal antibodies confirmed that no epitope appeared to be common to all strains of this pathovar. Screening of hybridoma cell lines againsl strains of this pathovar, as well as against other members of this genus and species, identified two distinct epitopes. Phylogenetic analysis of the rep-PCR genomic fingerprints of Curtobacter, Clavibacter, and Rathayibacter species, grouped all the C. flaccumfaciens strains and pathovars together, but the C. flaccumfaciens pv. flaccumfaciens strains did not form a single cluster. Rather, they dispersed into two clusters, along with strains of other pathovars of this species. Members of one cluster reacted with one of the monoclonal antibodies, while members of the other did not.

Interferon-gamma production in response to in vitro stimulation with collagen type II in rheumatoid arthritis is associated with HLA-DRB1(*)0401 and HLA-DQ8.

IFN-γ was measured in supernatants after in vitro stimulation of peripheral blood mononuclear cells with collagen type II (CII), purified protein derivative or influenza virus. IFN-γ production in response to CII was similar in rheumatoid arthritis (RA) patients and healthy control individuals. The IFN-γ response to purified protein derivative and influenza virus was lower in RA patients, reflecting a general T-cell hyporesponsiveness in RA. After recalculating the response to CII taking this hyporesponsiveness into account the CII response was higher in RA patients, and was associated with human leucocyte antigen (HLA)-DRB1*0401 and HLA-DQA1*0301-DQB1*0302 (HLA-DQ8). Rheumatoid arthritis patients with elevated serum levels of immunoglobulin (Ig)G anti-CII antibodies had lower CII-induced IFN-γ production than patients with low anti-CII levels. The relative increase in CII-reactivity in RA patients as compared with healthy control individuals, and the association of a higher response with RA-associated HLA haplotypes, suggest the existence of a potentially pathogenic cellular reactivity against CII in RA.