C6 Peptide-Based Multiplex Phosphorescence Analysis (PHOSPHAN) for Serologic Confirmation of Lyme Borreliosis

Abstract

BackgroundA single-tier immunoassay using the C6 peptide of VlsE (C6) from Borrelia burgdorferi sensu stricto (Bb) has been proposed as a potential alternative to conventional two-tier testing for the serologic diagnosis of Lyme disease in the United States and Europe.ObjectiveTo evaluate the performance of C6 peptide based multiplex Phosphorescence Analysis (PHOSPHAN) for the serologic confirmation of Lyme borreliosis (LB) in Russian patients.MethodsSerum samples (n = 351) were collected from 146 patients with erythema migrans (EM); samples from 131 of these patients were taken several times prior to treatment and at different stages of recovery. The control group consisted of 197 healthy blood donors and 31 patients with other diseases, all from the same highly endemic region of Russia. All samples were analyzed by PHOSPHAN for IgM and IgG to Bb C6, recombinant OspC and VlsE proteins, and C6 peptides from B. garinii and B. afzelii.ResultsIgM and IgG to Bb C6 were identified in 43 and 95 out of 131 patients (32.8 and 72.5%, respectively); seroconversion of IgM antibodies was observed in about half of the patients (51.2%), and of IgG antibodies, in almost all of them (88.4%). Additional detection of OspC-IgM and VlsE-IgM or IgG to C6 from B. garinii or B. afzelii did not contribute significantly to the overall sensitivity of the multiplex immunoassay.ConclusionsThe multiplex phosphorescence immunoassay is a promising method for simultaneously revealing the spectrum of antibodies to several Borrelia antigens. Detection of IgM and IgG to Bb C6 in the sera of EM patients provides effective serologic confirmation of LB and, with high probability, indicates an active infection process.

Lyme Disease: Beyond Erythema Migrans

“…Detection of IgM and IgG antibodies of C6 peptides using ELISA has a sensitivity of >70% for detecting Lyme disease. Pomelova et al showed that of their 146 Lyme disease patients, 51% had seroconversion of IgM antibodies, and 88% had seroconversion of IgG antibodies [28]. Liange et al showed that in acute infected patients, including early localized and early disemminated infection) sensitivity of the C6 ELISA was 74%, In late phase disease, sensitivity was 100%.…”

Multiplex immunoassay for detection of immunoglobulin G to herpes simplex virus types 1, 2 and cytomegalovirus based on PHOSPHAN technology

“…Для создания мультиплексного теста была применена биочип-технология ФОСФАН ТМ [16], эффективность которой для серологической диагностики ряда инфекций была продемонстрирована ранее [17]. Для решения поставленных задач в состав иммуночипа, помимо рекомбинантных белков gG1 и gG2, были включены лизатные антигены ВПГ и рекомбинантный мозаичный антиген ЦМВ.…”

“…Выполнение мультиплексного теста. ФОСФАН проводили по методикам, аналогичным описанным ранее [17]. Исследуемые образцы сыворотки крови, предварительно разведенные 1:100 в 0,01 М фосфатно-солевого буфера, рН 7,4, вносили по 100 мкл в лунки микропланшета с напечатанными антигенами и инкубировали 2 ч на шейкере при комнатной температуре.…”

“…Значения К поз. рассчитывали относительно порогового уровня (ПУ) интенсивности фосфоресценции, определенного предварительно в оптимизационных экспериментах, как описано ранее [17]. Значения ПУ составили 50, 30, 150 или 400 импульсов для определения IgG к ЦМВ, ВПГ-1/2, ВПГ-1 и ВПГ-2 соответственно.…”

Autoimmune Diseases of the Innate and Adaptive Immune System including Atopic Dermatitis, Psoriasis, Chronic Arthritis, Lyme Disease, and Alzheimers Disease

“…With the Borrelia spirochetes being found in the brains in Alzheimer's disease, [24][25][26] and with Montauk knee being a representation of Lyme arthritis, and with biofilms present in both joints and brains, all that is missing is proof of the innate immune system activity. However, increased activity of the adaptive immune system with IgG has been noted [31].…”