Development of a Multiantigen Panel for Improved Detection of Borrelia burgdorferi Infection in Early Lyme Disease.

The current standard for laboratory diagnosis of Lyme disease in the United States is serologic detection of antibodies against Borrelia burgdorferi. The Centers for Disease Control and Prevention recommends a two-tiered testing algorithm; however, this scheme has limited sensitivity for detecting early Lyme disease. Thus, there is a need to improve diagnostics for Lyme disease at the early stage, when antibiotic treatment is highly efficacious. We examined novel and established antigen markers to develop a multiplex panel that identifies early infection using the combined sensitivity of multiple markers while simultaneously maintaining high specificity by requiring positive results for two markers to designate a positive test. Ten markers were selected from our initial analysis of 62 B. burgdorferi surface proteins and synthetic peptides by assessing binding of IgG and IgM to each in a training set of Lyme disease patient samples and controls. In a validation set, this 10-antigen panel identified a higher proportion of early-Lyme-disease patients as positive at the baseline or posttreatment visit than two-tiered testing (87.5% and 67.5%, respectively; P < 0.05). Equivalent specificities of 100% were observed in 26 healthy controls. Upon further analysis, positivity on the novel 10-antigen panel was associated with longer illness duration and multiple erythema migrans. The improved sensitivity and comparable specificity of our 10-antigen panel compared to two-tiered testing in detecting early B. burgdorferi infection indicates that multiplex analysis, featuring the next generation of markers, could advance diagnostic technology to better aid clinicians in diagnosing and treating early Lyme disease.

Identification of anticitrullinated protein antibody reactivities in a subset of anti-CCP-negative rheumatoid arthritis: association with cigarette smoking and HLA-DRB1 'shared epitope' alleles.

INTRODUCTION: A hallmark of rheumatoid arthritis (RA) is the development of autoantibodies targeting proteins that contain citrulline. Anticitrullinated protein antibodies (ACPAs) are currently detected by the commercial cyclic citrullinated peptide (CCP) assay, which uses a mix of cyclised citrullinated peptides as an artificial mimic of the true antigen(s). To increase the sensitivity of ACPA detection and dissect ACPA specificities, we developed a multiplex assay that profiles ACPAs by measuring their reactivity to the citrullinated peptides and proteins derived from RA joint tissue. METHODS: We created a bead-based, citrullinated antigen array to profile ACPAs. This custom array contains 16 citrullinated peptides and proteins detected in RA synovial tissues. We used the array to profile ACPAs in sera from a cohort of patients with RA and other non-inflammatory arthritides, as well as sera from an independent cohort of RA patients for whom data were available on carriage of HLA-DRB1 'shared epitope' (SE) alleles and history of cigarette smoking. RESULTS: Our multiplex assay showed that at least 10% of RA patients who tested negative in the commercial CCP assay possessed ACPAs. Carriage of HLA-DRB1 SE alleles and a history of cigarette smoking were associated with an increase in ACPA reactivity-in anti-CCP(+) RA and in a subset of anti-CCP(-) RA. CONCLUSIONS: Our multiplex assay can identify ACPA-positive RA patients missed by the commercial CCP assay, thus enabling greater diagnostic sensitivity. Further, our findings suggest that cigarette smoking and possession of HLA-DRB1 SE alleles contribute to the development of ACPAs in anti-CCP(-) RA.

A novel algorithm to improve specificity in ovarian cancer detection.

BACKGROUND: Measurement of autoantibodies (AAbs) to tumor associated antigens has been proposed to aid in the early detection of ovarian cancer with high specificity. Here we describe a multiplex approach to evaluate selected peptide epitopes of p53 protein, and propose a novel approach to increase specificity and potentially sensitivity for discrimination between healthy women and women with cancerous masses. MATERIALS AND METHODS: 20-mer overlapping peptide epitopes of p53, generated by mapping the complete p53 sequence, were evaluated in a multiplex immunoassay for their detection of serum AAbs in patients with ovarian cancer, using Luminex technology. AAbs to the selected peptides and to p53 full length protein were then detected in a multiplex immunoassay evaluating 359 sera from healthy women and 285 sera from patients with early and late stage ovarian cancer. CA-125 levels were measured in all p53 AAb-positive sera. RESULTS: We considered the AAb results together to identify sera where both the full length protein and at least one selected peptide epitope were positive and chose cutoffs that reduced false positives from these AAbs to 1/359 samples, improving specificity. Using this combined approach, we could identify 7 AAb-positive patients that were negative for CA-125 (concentrations below 35 IU/mL); this represents 26% of the p53 positive patients in the total population. CONCLUSION: By detecting p53 AAbs in CA-125-negative sera, we demonstrated that combining measurement of AAbs to the full length p53 protein and one or more selected epitopes can potentially improve sensitivity and specificity for ovarian cancer detection.