B-066 VeraBIND™ Borrelia Antibody Test: Multiplex Capture, Enrichment, and Detection of Borrelia Burgdorferi Antigens-Specific Immunoglobulins From Saline Oral Rinse Samples
Authors: Shamsundar K , Ansari K I , Bergmann S , Kleven K , Soldo J 
Published: October 2024

Abstract

Background
Lyme disease is the most prevalent tick-borne infection in the United States and ranks among the most frequently diagnosed globally. While most patients respond well to prompt antibiotic treatment, there is a sizeable subset of patients that go undetected and are subjected to enduring prolonged and debilitating symptoms such as pain, fatigue, and cognitive dysfunction. The lack of reliable early diagnostic tests makes managing the disease easier, as conventional methods often yield unreliable results. To address this unmet need, Veravas utilized the VeraBIND™ Antibody Detection Kit to develop an ultra-sensitive and specific RUO test to multiplex capture and detect low-abundant pathogen-specific IgA, IgG, and IgM immunoglobulins from serum and saline oral rinse (SOR).

Methods
Preanalytical sample cleaning: VeraBIND Clean Beads are designed to target and efficiently remove a broad spectrum of endogenous human immunoglobulin interferences, thereby mitigating false assay signal response. 60 µL of serum, or 1,300 µL of SOR, were pre-analytically cleaned using 100 µL of VeraBIND Clean Beads. Multiplex immunoglobulin capture and enrichment. The cleaned samples were subsequently incubated with a pool of B. burgdorferi OspC, DpbA, and VlsE antigen-coated VeraBIND Capture Beads to capture, purify, and enrich total IgA, IgG, and IgM. Multiplex Detection: The captured and enriched IgA, IgG, and IgM immunoglobulins were multiplex detected, and their levels were measured using a triplex fluorescent conjugate (iFluor™488, 546, 597 labeled anti-hIgA, IgG, and IgM antibodies).

Results
Validation of the VeraPATH Borrelia burgdorferi antibody test (RUO): Using the CDC Research I sera panel (N=32) and Bay Area Lyme Foundation sera panel (N=19), and an immunoglobulin-based multiplex algorithm, the VeraPATH Borrelia burgdorferi antibody test correctly detected antibodies against Borrelia burgdorferi with 100% sensitivity and 100% specificity (N= 51, PPV 95%CI 82-100%; NPV 95%CI 85-100%). Notably, this accuracy is not contingent on detection of singular immunoglobulin class but rather based on an algorithm defining “positive” when samples test positive for at least 2 of 3 immunoglobulins (IgA & IgG, or IgA & IgM, or IgG & IgM, or all three detected) and “negative” when samples test negative for at least 2 of 3 immunoglobulins (IgA & IgG, or IgA & IgM, or IgG & IgM, or all three not detected). SOR-based Borrelia burgdorferi antibody test: We collected SOR samples from an individual clinically diagnosed with Lyme disease within two weeks after a tick bite (Stage I LD), and five SOR samples from presumably healthy Veravas employees. Using the VeraPATH Borrelia burgdorferi antibody test, we successfully detected both IgA and IgM immunoglobulins in the SOR of the patient with Lyme disease. No immunoglobulin classes were detected in the SOR of four out of the five Veravas employees. Surprisingly, the fifth employee tested positive for all three immunoglobulin classes (IgA, IgG, and IgM) against Borrelia burgdorferi. This employee’s serum was subsequently confirmed positive using 2-tier ELISA and Western blot.

Conclusions
The VeraPATH Borrelia burgdorferi antibody test eliminates sample interferences and matrix effects and multiplex-captures, enriches (over 6.5-fold), and detects immunoglobulins against Borrelia burgdorferi from both SOR and serum sample types.