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Biomedical subjects

Thomas B Martins

Publications and source records attributed to Thomas B Martins.

18 recordsLinked to original sources
PubMed2006

Development of a multiplexed fluorescent immunoassay for the quantitation of antibody responses to group A streptococci.

The host immunologic response to group A streptococcal infections gives rise to numerous antibodies directed against cellular and extracellular bacterial antigens. For determining individual immune status, or studying the pathogenesis of group A streptococcal associated diseases, such as acute rheumatic fever (ARF), an assay capable of determining antibodies responses to multiple antigens would be of great advantage. We have developed a microsphere based, multiplexed immunoassay for the simultaneous quantitation of antibodies to nine different extracellular, ARF related tissue and group A streptococci specific antigens using only 5 microl of sample. Through the selection of microspheres and serum diluent, non-specific antibody binding was reduced by 17%. Different formulations of the coupling buffer were found to greatly influence the efficiency of coupling antigens to the carboxylated microspheres. Monoclonal antibodies against the different antigens demonstrated assay specificity as well as sensitivities of less than 1 ng/ml of antibody. This multiplexed assay should be a powerful research and clinical tool in determining antibody responses to group A streptococcal infections and in potentially determining the role of a variety of cross-reactive antigens in rheumatic fever and rheumatic heart disease.

Antibodies, Bacterial↗
PubMed2006

The flow cytometric analysis of cytokines using multi-analyte fluorescence microarray technology.

Cytokines, which are small peptides that act as hormones of the immune system, affect cells throughout the body in a variety of different ways. These cellular signaling molecules often have synergistic or opposing effects on various cell types and often different cytokines have overlapping activities. There is great advantage, therefore, to be able to assess a pattern of cytokine responses in certain inflammatory, autoimmune, transplant or immunodeficiency states. This is one of the major advantages of the new particle-based flow cytometric assays, which have become available. We have employed such assays to analyze up to 10 different cytokines in cultured supernatants of stimulated mononuclear cells and in as little as 75 microL of serum from patients with a variety of different disorders. In developing these assays and validating them for use in our esoteric reference laboratory (ARUP Laboratories), we have found that a variety of heterophile antibodies can lead to both false positive and false negative results. This review will describe the development of our multi-analyte cytokine assays and document the interference derived from heterophile antibodies. Lastly, we will point out various procedures that we have utilized to include internal controls directly in the assays, which allow one to routinely detect these interfering antibodies, as well as methods we have developed to circumvent the interference posed by these antibodies.

Animals↗
PubMed2006

Performance characteristics of six IMMULITE 2000 TORCH assays.

TORCH is an acronym for Toxoplasma gondii (Toxo), other microorganisms (eg, syphilis), rubella virus (RV), cytomegalovirus (CMV), and herpes simplex virus (HSV) that are associated with congenital abnormalities from maternal infection. We evaluated linearity, imprecision, and comparison with commercially available methods of the IMMULITE 2000 (Diagnostic Products, Los Angeles, CA) Toxo IgG, Toxo IgM, RV IgG, RV IgM, CMV IgG, and HSV IgG assays. Linearity and imprecision results were acceptable. The IMMULITE 2000 assays show good concordance with other commercially available methods except for Toxo IgM and RV IgM. Toxo IgM showed better concordance with a consensus of 3 of 4 (Access, Beckman Coulter, Fullerton, CA; IMMULITE 2000; Platelia, Bio-Rad Laboratories Diagnostics Group, Redmond, WA; and Vidas, bioMerieux, Hazelwood, MO) assays than with Access alone. The RV IgM assay showed better concordance with the Zeus method than with the Diamedix method (Diamedix, Miami, FL). The IMMULITE 2000 TORCH assays studied show acceptable performance and are suitable for routine clinical use. Some commercial assays for Toxo IgM and RV IgM show rather poor concordance.

Animals↗
PubMed2006

Risk factor analysis of plasma cytokines in patients with coronary artery disease by a multiplexed fluorescent immunoassay.

Coronary artery disease (CAD) is the leading cause of death in the United States. Increasing evidence suggests involvement of inflammation in the atherosclerotic process. We examined cytokines and other inflammatory markers in 865 patients with chest pain in whom coronary angiography revealed no evidence of CAD or CAD with or without concomitant myocardial infarction (MI). We developed a multiplexed immunoassay to simultaneously assess the plasma concentrations of 8 cytokines (interferon gamma, interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor alpha), IL-2r, and soluble CD40 ligand in the patient groups. Concentrations of C-reactive protein (CRP) and IL-18 also were determined. Significant differences (P < .05) between no CAD and combined CAD groups were found for IL-2, IL-4, IL-6, IL-12, and IL-18. When the no CAD group was compared with the group with CAD with subsequent MI, significant differences were found for proinflammatory markers IL-6 (P pound .001), IL-8 (P = .017), and CRP (P pound .001). Cytokine profiles may have a role in differentiating patients with CAD with MI from those with chest pain due to other disorders and in deciphering the role of inflammation in the pathogenesis of CAD.

Angina Pectoris↗
PubMed2006

Comparison of complement fixation with two enzyme-linked immunosorbent assays for the detection of antibodies to respiratory viral antigens.

We compared complement fixation (CF) for the measurement of antibodies against influenza A, influenza B, respiratory syncytial virus (RSV), human adenovirus, and parainfluenza viruses 1, 2, and 3 (para-1, para-2, and para-3) with 2 enzyme-linked immunosorbent assays (ELISA kits, A and B). The IgG ELISA kits compared very well with each other except for the influenza A and B IgG ELISAs. The IgG ELISAs, in general, did not agree with CF In contrast, the IgM ELISAs compared well with CF and each other except for the consensus parainfluenza panel from ELISA B. The poor agreement of the IgG ELISAs with the CF test can be explained by the increased sensitivity of the ELISAs and differences between CF antigens and the ELISA antigens. The influenza A and influenza B ELISA antigens differed between both kits, which may explain their poor agreement. The ELISA is a suitable replacement for CF, providing greater sensitivity, isotype specificity, and ease of use.

Antigens, Viral↗
PubMed2004

The Bartonella henselae sucB gene encodes a dihydrolipoamide succinyltransferase protein reactive with sera from patients with cat-scratch disease.

Bartonella henselae is a recently recognized pathogenic bacterium associated with cat-scratch disease, bacillary angiomatosis and bacillary peliosis. A recombinant clone expressing an immunoreactive antigen of B. henselae was isolated by screening a genomic DNA cosmid library by Western blotting with sera pooled from patients positive for B. henselae IgG antibodies by indirect immunofluorescence (IFA). The deduced amino acid sequence of the 43.7 kDa encoded protein was found to be 76.3 % identical to the dihydrolipoamide succinyltransferase enzyme (SucB) of Brucella melitensis. SucB has been shown to be an immunogenic protein during infections by Brucella melitensis, Coxiella burnetii and Bartonella vinsonii. The agreement between reactivity with a recombinant SucB fusion protein on immunoblot analysis and the results obtained by IFA was 55 % for IFA-positive sera and 88 % for IFA-negative sera. Cross-reactivity was observed with sera from patients with antibodies against Brucella melitensis, Mycoplasma pneumoniae, Francisella tularensis, Coxiella burnetii and Rickettsia typhi.

Acyltransferases↗
PubMed2004

Heterophile antibody interference in a multiplexed fluorescent microsphere immunoassay for quantitation of cytokines in human serum.

While modern immunoassays provide sensitive and specific means for the quantitation of cytokines in biological fluids, heterophile antibodies are still a well-recognized cause of interference in the measurement of cytokines in these assays. We have developed a multiplexed fluorescent microsphere immunoassay for the simultaneous quantification of 10 cytokines in only 75 microl of serum. During the development of this multiplexed assay, the amount of assay interference due to heterophile antibodies was also determined, and methods for detecting heterophile interference and minimizing its effect were incorporated into the assay. Heterophile antibodies resulted in significantly elevated cytokine values compared to those of normal blood bank samples. These falsely elevated values, and thus the components of the assay the heterophile antibodies were binding to, were identified through the use of internal controls. This information was then used to design assay-specific blockers and absorbents that were shown to significantly reduce falsely elevated cytokine values while not affecting the standard and control values. The fluorescent multiplexed microsphere-based immunoassay can be used to quantitate multiple cytokines from a single sample and should be a useful tool in furthering our understanding of the role of cytokines in disease processes.

Antibodies, Heterophile↗
PubMed2004

Evaluation of multiplexed fluorescent microsphere immunoassay for detection of autoantibodies to nuclear antigens.

Antibodies to extractable nuclear antigens (ENA) are found in a variety of collagen vascular diseases. Determining the individual specificities of these antibodies is extremely useful in establishing the disease diagnosis and in some cases the prognosis. With a multiplexed fluorescent microsphere immunoassay, reactivity to five of the most diagnostically useful ENA was measured in 249 serum samples, including samples from 56 patients previously documented to have systemic lupus erythematosus (SLE). Results of the multiplexed assay were compared to results from established ENA enzyme-linked immunosorbent assays (ELISAs), and the agreement, sensitivity, and specificity, respectively, for the five ENA evaluated were as follows: SSA, 99.1, 100.0, and 98.8%; SSB, 98.6, 88.9, and 99.5%; Sm, 97.6, 95.8, and 97.9%; RNP, 97.2, 92.7, and 98.8%; Scl-70, 93.6, 50.0, and 99.0%. In the 56 confirmed SLE patients, the frequency of significant concentrations of autoantibodies with the multiplexed assay was 21.4% for SSA, 7.1% for SSB, 10.7% for Sm, 32.1% for RNP, and 0% for Scl-70. The new flow cytometric bead-based multiplexed assay showed excellent correlation with the well-established single-analyte ELISA methods for four of five the ENA markers investigated in this study. The most notable discrepancies between the two assays were for the Scl-70 antigen, which was most often resolved in favor of the multiplexed assay. Our studies show that the multiplexed microsphere-based immunoassay is a sensitive and specific method for the detection and semiquantitation of ENA antibodies in human sera.

Antibodies, Antinuclear↗
PubMed2004

Evaluation of an in vitro assay for gamma interferon production in response to Mycobacterium tuberculosis infections.

The tuberculin skin test (TST) is the "gold standard" for detecting infection with Mycobacterium tuberculosis. We compared the TST using purified protein derivative to the QuantiFERON-TB test (QFT). Two groups were examined. Group 1 individuals (n = 66) (low risk) were at low risk for exposure to M. tuberculosis and were not Mycobacterium bovis BCG vaccinated. Group 2 (n = 29) include individuals who were likely to have been exposed to a high prevalence of M. tuberculosis infections and were BCG vaccinated. Group 1 individuals were given a TST. Group 2 individuals were not given a TST because of possible adverse reactions. A 10- to 15-mm indurated area 48 h after TST was considered positive. A positive QFT result was defined as a significant gamma interferon response to M. tuberculosis antigen, Mycobacterium avium antigen, and a nonspecific mitogen stimulus and no response in the negative control. In group 1, 60 of 66 individuals (90.9%) were negative by both methods, and 1 person was positive by both methods. There was one QFT-negative, TST-positive case, one QFT-positive, TST-negative case, and three conditional QFT-positive, TST-negative cases. In group 2, 12 of 29 (41.4%) were positive by QFT and considered likely to be TST positive because of prior BCG vaccination. QFT testing in our low-risk group resulted in an agreement of 96.8%, a sensitivity of 50%, and a specificity of 98.4% compared with TST results. QFT testing with TST in low-risk groups can aid in the detection of latent M. tuberculosis infections.

Adult↗
PubMed2004

Evaluations of commercial West Nile virus immunoglobulin G (IgG) and IgM enzyme immunoassays show the value of continuous validation.

West Nile virus was introduced into the United States in 1999 and in only four seasons has become endemic east of the Rocky Mountains. Recently, immunoglobulin M (IgM)-capture enzyme immunoassays for the detection of West Nile virus-specific IgM and indirect IgG enzyme immunoassays for the detection of IgG antibodies against West Nile virus were made available from Focus Technologies and PANBIO, Inc. We evaluated these commercial IgG and IgM test systems and determined agreement, sensitivity, and specificity for the assays, compared to immunofluorescence assay and the Centers for Disease Control and Prevention's IgM-capture enzyme-linked immunosorbent assay (ELISA). Initially, the Focus and PANBIO IgM enzyme immunoassays had at least 95% agreement, sensitivity, and specificity, and, based on the 95% confidence intervals, both IgM-capture assays performed similarly. The IgG assays also performed well, although the Focus IgG assay demonstrated greater specificity (98.8%) and the PANBIO IgG assay demonstrated greater sensitivity (99.3%). However, for 400 samples consecutively submitted for West Nile virus antibody testing during 2 days of the 2003 West Nile virus season, agreement, clinical sensitivity, and clinical specificity were 93.1, 98.0, and 92.4%, respectively, for the PANBIO IgM assay and were 97.4, 100.0, and 97.1%, respectively, for the Focus IgM assay. The specificities observed in this second evaluation equates to an overall false-positivity rate of 6.3% in the PANBIO West Nile virus IgM-capture ELISA versus 2.5% with the Focus West Nile virus IgM-capture ELISA. This experience demonstrates the importance of continuously evaluating the performance of an assay in order to detect any changes in assay performance as the test population evolves.

Adult↗
PubMed2003

Comparison of two commercial enzyme-linked immunosorbent assays with an immunofluorescence assay for detection of Legionella pneumophila types 1 to 6.

Members of the genus Legionella are characterized as gram-negative, motile, freshwater-dwelling bacteria that were responsible for a pneumonia outbreak among American Legion members in 1976. Because clinicians routinely order serologic testing for Legionella pneumophila serogroups 1 to 6 as a screen for possible L. pneumophila infections, we evaluated the Wampole Laboratories L. pneumophila type 1 to 6 immunoglobulin G (IgG) and IgM combined enzyme-linked immunosorbent assay (ELISA) and the Zeus Scientific L. pneumophila type 1 to 6 IgG-IgM-IgA multispecific combined ELISA systems and compared them to an IgG-specific immunofluorescence assay (IFA) for L. pneumophila serogroups 1 to 6. The Centers for Disease Control and Prevention recommends that the positive titer cutoff for an IFA be 1:256. Regardless of where the positive IFA cutoff titer is placed, however, the sensitivity of both commercial assays was below what would be acceptable for a screening assay. With a 1:256 IFA titer as the positive cutoff, the agreement, sensitivity, and specificity of the Wampole ELISA were 74.6, 21.4, and 98.4%, respectively. The agreement, sensitivity, and specificity of the Zeus ELISA were 72.6, 10.5, and 100.0%, respectively. We recommend that any laboratories attempting to replace an IFA type 1 to 6 screen with an alternative ELISA carefully investigate the sensitivity of the replacement assay.

Antibodies, Bacterial↗
PubMed2003

Detection of IgG and IgM to West Nile virus. Development of an immunofluorescence assay.

West Nile virus (WNV) had its first recorded appearance in the western hemisphere in 1999 and has continued to spread across the United States, necessitating the development of serologic procedures to diagnose infection. We developed an immunofluorescence assay (IFA) protocol for the detection of WNV-specific IgG and IgM antibodies in serum and cerebrospinal fluid (CSF) specimens. We tested 82 serum and 16 CSF samples and compared the results with WNV IgG enzyme-linked immunosorbent assay (ELISA) and IgM antibody-capture (MAC) ELISA results. Agreement, clinical sensitivity, and clinical specificity for the IgG IFA were 92%, 100%, and 90%, respectively, and 98%, 96%, and 100% for the IgM IFA, respectively. Extensive arbovirus cross-reactions occurred in the IgG assays, but only minimal cross-reactions were observed in the IgM assays. The IFA protocol described herein is a cost-effective and sensitive alternative to ELISA and MAC-ELISA for the serologic diagnosis of WNV infection.

Antibodies, Viral↗
PubMed2002

Comparison of four enzyme immunoassays for the detection of immunoglobulin G antibody against glomerular basement membrane.

Goodpasture syndrome is a life-threatening autoimmune kidney and pulmonary disease that is characterized by pulmonary hemorrhage, renal failure, and the presence of autoantibodies against glomerular basement membrane (GBM) by indirect fluorescent antibody (IFA) techniques. In 1988, these antibodies were found to be specific for the noncollagen region of the alpha3 collagen IV chain. The antigen is characterized by a restricted tissue distribution occurring mainly in the kidneys and lungs. Specific enzyme immunoassays (EIAs) for anti-GBM have now been developed for in vitro diagnostic use in the laboratory. Our objective in this study was to compare the results obtained using four different EIAs for detecting anti-GBM IgG antibody with those using the standard IFA method using tissue from human kidney. Thirty-two patients with suspected Goodpasture syndrome, and 10 control sera were included in the study. GBM EIAs were purchased from or donated by the following vendors: Scimedx Corporation (Denville, NJ), INOVA Diagnostics (San Diego, CA), The Binding Site (Birmingham, England), and Wieslab (distributed by DiaSorin, Inc., Stillwater, MN). Percent agreement, sensitivity, and specificity were calculated for each EIA as compared to IFA. The results were as follows: Binding Site: 83.3, 100.0, and 72.0%; Wieslab: 95.2, 94.1, and 96.0%; INOVA: 96.2, 100.0, and 92.3%; and Scimedx: 81.0, 100.0, and 68.0%. We conclude that the INOVA and Wieslab GBM IgG EIAs compared well with the standard GBM IFA method using tissue from human kidney. The Scimedx and Binding Site EIAs had eight and seven false-positive results, respectively, when compared to GBM IFA using human kidney. The authors recommend conducting thorough evaluations of EIAs that screen for anti-GBM antibody before their implementation in the clinical laboratory. We also recommend confirming GBM EIA-positive sera by the standard IFA method using tissue from human kidney.

Anti-Glomerular Basement Membrane Disease↗
PubMed2002

Development of internal controls for the Luminex instrument as part of a multiplex seven-analyte viral respiratory antibody profile.

The ability of the Luminex system to simultaneously quantitate multiple analytes from a single sample source has proven to be a feasible and cost-effective technology for assay development. In previous studies, my colleagues and I introduced two multiplex profiles consisting of 20 individual assays into the clinical laboratory. With the Luminex instrument's ability to classify up to 100 distinct microspheres, however, we have only begun to realize the enormous potential of this technology. By utilizing additional microspheres, it is now possible to add true internal controls to each individual sample. During the development of a seven-analyte serologic viral respiratory antibody profile, internal controls for detecting sample addition and interfering rheumatoid factor (RF) were investigated. To determine if the correct sample was added, distinct microspheres were developed for measuring the presence of sufficient quantities of immunoglobulin G (IgG) or IgM in the diluted patient sample. In a multiplex assay of 82 samples, the IgM verification control correctly identified 23 out of 23 samples with low levels (<20 mg/dl) of this antibody isotype. An internal control microsphere for RF detected 30 out of 30 samples with significant levels (>10 IU/ml) of IgM RF. Additionally, RF-positive samples causing false-positive adenovirus and influenza A virus IgM results were correctly identified. By exploiting the Luminex instrument's multiplexing capabilities, I have developed true internal controls to ensure correct sample addition and identify interfering RF as part of a respiratory viral serologic profile that includes influenza A and B viruses, adenovirus, parainfluenza viruses 1, 2, and 3, and respiratory syncytial virus. Since these controls are not assay specific, they can be incorporated into any serologic multiplex assay.

Adenoviridae↗
PubMed2002

Comparison of a multiplex flow cytometric assay with enzyme-linked immunosorbent assay for auantitation of antibodies to tetanus, diphtheria, and Haemophilus influenzae Type b.

We developed a multiplexed indirect immunofluorescence assay for antibodies to Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays (ELISAs) for the same analytes. By both methods, 75 (92.6%) of 81 of random serum samples had protective levels of antibody to Tet (> or = 0.1 IU/ml). For Dip, 81.5% of the samples had protective antibody levels (> or = 0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex. Protective levels (> or = 1.0 microg/ml) of antibody to Hib were found in 45.0% of the samples tested by ELISA and in 39.0% of the samples tested by Luminex. The correlations (R(2)) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively. There was also similar agreement between Luminex and ELISA for sera collected before and 1 month after Tet, Dip, and Hib vaccine administration. Both methods detected strong postvaccination responses. The Luminex method is an attractive alternative to ELISA since it reduces labor and reagent costs, as well as assay time.

Antibodies↗
PubMed2002

A multiplexed fluorescent microsphere immunoassay for antibodies to pneumococcal capsular polysaccharides.

We developed a multiplexed indirect immunofluorescent assay for antibodies to pneumococcal polysaccharides (PnPs) based on the Luminex multiple analyte profiling system (Luminex, Austin, TX). The assay simultaneously determines serum IgG concentrations to 14 PnPs serotypes: 1, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 12F; 14, 18C, 19F, and 23F. To assess the specificity of the multiplexed assay for each individual serotype, inhibition-of-binding studies were conducted using adult serum samples obtained after pneumococcal vaccination. Except for the closely related serotypes 9V and 9N, we demonstrated inhibition by homologous serotypes of more than 95% and inhibition by heterologous serotypes of less than 15% for all 14 PnPs serotypes. There was, however, high heterologous inhibition of 50% or greater with some serotypes. These cross-reacting antibodies could not be removed by preabsorption with pneumococcal C-polysaccharide but were removed by additional preabsorption with serotype 22F polysaccharide. The multiplexed Luminex assay showed good overall agreement with a well-established enzyme-linked immunosorbent assay that is currently recommended for evaluation of pneumococcal vaccine immunogenicity.

Antibodies, Bacterial↗
PubMed2002

Determination of cytokine responses using a multiplexed fluorescent microsphere immunoassay.

We used a multiplexedfluorescent microsphere immunoassay to develop a sandwich capture assay to assess simultaneously the production of thymus helper (TH) 1- and TH2-type cytokines in tissue culture supernatant obtained from stimulated peripheral blood mononuclear cells. The assay then was used to assess the cytokine production of patients with hyperimmunoglobulinemia E syndrome and in cord blood from neonates. The multiplexed assay has a reportable range of less than 10 to 50,000 pg/mL. For linearity and recovery studies, R2 values for the 6 cytokines ranged from 0.988 to 0.999 for samples spiked with known concentrations of recombinant cytokine standards and for patient samples. The assay showed good specificity, with little cross-reactivity between cytokines. Results from supernatants of Staphylococcus aureus-stimulated peripheral blood mononuclear cells obtainedfrom 6 patients with hyperimmunoglobulinemia E syndrome showed significantly less interferon (IFN)-gamma production than cells from healthy control subjects. Cord blood cells from neonates produced significantly less interleukin 12 and IFN-gamma than cells from adults in group B streptococci-stimulated mononuclear cells. The fluorescent multiplexed microsphere immunoassay can be used to quantitate multiple cytokines from 1 sample and should be useful for further understanding of the cytokine role in disease.

Cells, Cultured↗