Christine M. Litwin

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.

Cloning and Characterization of vuuA, a Gene Encoding the Vibrio vulnificus Ferric Vulnibactin Receptor

ABSTRACT The ability of Vibrio vulnificus to acquire iron from the host has been shown to correlate with virulence. Many iron transport genes are regulated by iron, and in V. vulnificus, transcriptional regulation by iron depends on thefur gene. The N-terminal amino acid sequence of a 72-kDa iron-regulated outer membrane protein purified from a V. vulnificus fur mutant had 53% homology with the first 15 amino acids of the mature protein of the Vibrio choleraevibriobactin receptor, ViuA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for VuuA, the vulnibactin receptor of V. vulnificus. Analysis of the DNA sequence of the vuuApromoter region demonstrated a sequence identical to the upstream Fur box of V. cholerae viuA. Northern blot analysis showed that the transcript was strongly regulated by iron. The amino acid sequence of VuuA was 74% identical to the sequence of V. choleraeViuA and was homologous to those of several TonB-dependent outer membrane receptors. An internal deletion of the V. vulnificus vuuA gene resulted in the loss of expression of the 72-kDa protein and the loss of the ability to use transferrin or vulnibactin as a source of iron. This mutant showed reduced virulence in an infant mouse model. Introduction of a plasmid containing the completeviuA coding sequence and 342 bp of upstream DNA into the mutant restored ferric vulnibactin and ferric transferrin utilization to the mutant.

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.

Heterophile Antibody Interference in a Multiplexed Fluorescent Microsphere Immunoassay for Quantitation of Cytokines in Human Serum

ABSTRACT 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 μl 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.

Evaluation of Multiplexed Fluorescent Microsphere Immunoassay for Detection of Autoantibodies to Nuclear Antigens

ABSTRACT 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.

Multiplex analysis of heterophil antibodies in patients with indeterminate HIV immunoassay results

We hypothesized that heterophil antibodies reactive with animal proteins used in blot preparation caused nonspecific staining (NSS) on HIV Western blot (WB) studies, causing indeterminate results. We analyzed samples showing NSS on HIV WB using a multiplexed immunoassay to simultaneously measure IgG antibodies to animal IgG (bovine, goat, sheep, mouse) and bovine serum albumin. Heterophil antibodies reactive with IgG from several animal species were detected in 23 (49%) of 47 samples showing NSS on HIV WB; 15 positive samples demonstrated antibodies to all 5 antigens. Similar IgG heterophil antibodies were detected in only 2 (8%) of 24 control samples. Of the HIV WB samples with a positive HIV-1 enzyme-linked immunosorbent assay (ELISA) result at the time of WB testing (11/47), heterophil antibodies were found in 8 (73%) of 11. Preabsorption with bovine, goat, and sheep IgG removed heterophil antibodies detected by the multiplexed assay and, in some cases, eliminated reactivity in ELISA and WB testing. Heterophil antibodies are associated with indeterminate HIV immunoassay results and are an important cause of false-positive HIV ELISA results. Multiplexed immunoassays provide a powerful tool for screening patients for heterophil antibodies and resolving possible false-positive results.

Comparison of Four Enzyme Immunoassays With a Western Blot Assay for the Determination of Type-Specific Antibodies to Herpes Simplex Virus

Most current enzyme immunoassays (EIAs) differentiate inadequately between types 1 and 2 herpes simplex virus (HSV) antibodies since significant cross-reactivity exists. We compared 4 IgG type-specific EIAs using a Western blot assay for resolution of discrepant results. The Diamedix had sensitivities of 100% for types 1 and 2 but specificities of only 71% and 61%, respectively. The cross-reactivity rate was 82% in positive samples tested. For HSV types 1 and 2, the Zeus sensitivities were 92% and 98%, respectively; specificities were 72% and 79%, respectively; the cross-reactivity rate was 54%. For HSV types 1 and 2, the Wampole sensitivities were 98% and 95%, respectively; specificities were 68% and 85%, respectively; the cross-reactivity rate was 47%. For HSV types 1 and 2, the Meridian sensitivities were 98% and 90%, respectively; specificities were 96% and 100%, respectively; no cross-reactivity was found between positive samples tested. While the Diamedix, Zeus, and Wampole assays showed good sensitivity, they lacked type specificity. The Meridian EIA offers the highest specificity along with no observed cross-reactivity. This EIA may be an easier, reliable alternative to Western blot for the determination of HSV type-specific antibodies.

Evaluations of Commercial West Nile Virus Immunoglobulin G (IgG) and IgM Enzyme Immunoassays Show the Value of Continuous Validation

ABSTRACT 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 Preventions 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.

Limited Utility of Culture for Mycoplasma pneumoniae and Chlamydophila pneumoniae for Diagnosis of Respiratory Tract Infections

ABSTRACT We assessed the utility of culture for Mycoplasma pneumoniae and Chlamydophila pneumoniae to diagnose respiratory tract infections. Compared to PCR and IgM serology, culture was less sensitive and had extremely low yield. Culture is not recommended for these pathogens, and this method should be eliminated from routine practice.

Evaluation of Helicobacter pylori immunoglobulin G (IgG), IgA, and IgM serologic testing compared to stool antigen testing.

ABSTRACT The utility of Helicobacter pylori serology was evaluated in 4,722 specimens and compared to stool antigen detection. Immunoglobulin M (IgM) sensitivity (6.8%) was unacceptably low. Key performance differences were observed in IgG specificity, IgA sensitivity, and specificity between adults and children that may warrant differentiating optimal serologic cutoff values by age.