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Antigen DetectionNOW AVAILABLE: A NEW GENERATION OF LYME DISEASE TESTS ANNOUNCING: THE LYME DOT BLOT ASSAY (LDA) for B. burgdorferi and THE REVERSE WESTERN BLOT Palo Alto, CA April 16, 2001 — IGeneX is offering two new tests — the Lyme DOT-BLOT Assay (LDA) for antigen and the Reverse Western Blot (RWB) for antigen. Both of these tests detect Lyme antigen shed in the urine. The Lyme DOT-BLOT is a screening test, and the RWB is confirmatory for the LDA positive samples. What is the Lyme DOT-BLOT Assay for Antigen (LDA)? The LDA is an immunoassay for the direct detection of Lyme antigen in urine that reacts specifically to rabbit anti-B. burgdorferi antibodies. The rabbit antibodies are specifically targeted to the following B. burgdorferi antigens: 23kDA–25kDA (Osp C), 31 kDA (Osp A), 34 kDA (Osp B), 39kDA and/or 93kDA. If the Borrelia specific antigen(s) are present in the urine sample, a dot with a bluish-purple precipitate forms on the membrane. The limit of detection in urine spiked with sonicated B. burgdorferi is 12.5 ng/ml of urine. In an in-house study performed on the “general population” (the age group was 3 years to 70 years) with no symptoms of Lyme, the assay specificity was greater than 96%. How Does the LDA Work? Urine is collected in a Becton Dickinson’s (BD) Vacutainer Brand Urine Collection kit and then sent to IGeneX. Urine can be stored in the refrigerator in the tube containing lyophilized maintenance formula (provided in the tube), and is stable for 7 days. The urine is processed following the IGeneX, Inc. proprietary sample processing protocol. The antigens present in the processed urine are immobilized onto a membrane in a DOT-BLOT format. The membrane is incubated with anti-B. burgdorferi specific antibodies. After washing the unbound antibodies, the bound anti-B. burgdorferi specific antibodies are reacted with horseradish peroxidase (HRP) conjugated anti-rabbit IgG. The membrane is washed to remove unbound conjugated antibody. Finally, the membrane is reacted with a precipitating color developing solution, which deposits a bluish-purple precipitate on dots that have antigens reacting to anti-B. burgdorferi specific antibodies: 23–25kDa (OspC), 31kDa (OspA), 34kDa (OspB), 39kDa and/or 93kDa. Interpretation of the LDA A positive result indicates that the urine reacted against rabbit anti-B. burgdorferi antibodies with specificity to the following B. burgdorferi antigens: 23-25kDa (OspC), 31kDa (OspA), 34kDa (OspB), 39kDa and/or 93kDa. Any positive sample is considered presumptive positive until confirmed by another method. We recommend the new Reverse Western Blot (RWB) and/or the Urine Multiplex PCR. A negative result indicates no reaction with the above listed antibodies. What is the Reverse Western Blot (RWB) for Antigen? The RWB can be performed as a confirmation test on any presumptive positive LDA sample. This test is strongly recommended on any presumptive positive LDA when a urine Multiplex PCR was not requested at the same time, or when the urine Multiplex PCR was negative. The RWB is an immunoassay for direct detection and identification of Lyme antigen in urine which specifically react with rabbit anti-B. burgdorferi antibodies. The rabbit antibodies are specifically targeted to the following B. burgdorferi antigens: 18kDA, 23kDA–25kDA (Osp C), 28kDa, 30kDA, 31kDA (Osp A), 34kDA (Osp B), 39kDA, 45kDA, 58kDA, 66kDA and 93kDA. If any of the Borrelia specific antigen(s) are present in the urine sample, bluish-purple bands are visualized on a membrane. How Does the RWB Work? The urine antigen(s) are separated in the presence of sodium dodecyl sulphate (SDS) by polyacrylamide gel electrophoresis. The resolved protein bands are then transferred by electrophoresis to a membrane. The membrane is incubated with anti-B. burgdorferi specific antibodies. After washing the unbound antibodies, the bound anti-B. burgdorferi specific antibodies are reacted with horseradish peroxidase (HRP) conjugated anti-rabbit IgG. The membrane is washed to remove unbound conjugated antibody. The membrane is finally reacted with a precipitating color developing solution, which deposits a bluish-purple precipitate on bands that have antigens reacting to anti-B. burgdorferi antibodies: 18kDA, 23kDA–25kDA, 28kDa, 30kDA, (Osp C), 31 kDA (Osp A), 34 kDA (Osp B), 39kDA, 45kDA, 58kDA, 66kDA and 93kDA. Interpretation of the RWB If one or more bluish-purple antigen band(s) are present in the urine sample at 18kDA, 23–25kDA (Osp C), 28kDa, 30kDA, 31kDA (Osp A), 34kDA (Osp B), 39kDA, 45kDA, 58kDA, 66kDA or/and 93kDA, the bands will be reported on the result report form. Any patient sample that does not have a bluish-purple band at for the above listed antigens will be reported as negative. Preliminary In-House Study In a preliminary study, the LDA, RWB, and Lyme Multiplex PCR were performed on 162 individuals’ urine samples. Urine samples (1 to 3 per patient) from the following were tested: 55 patients with Lyme Disease like symptoms; 95 from the general population; 7 normal urines spiked with autoimmune antigens, and 5 normal urines spiked with gamma globulins. Several of the patients had undergone an antibiotic challenge prior to urine collection.
All 7 autoimmune antigen spiked samples and all 5 gamma globulin spiked samples were negative by LDA, PCR and RWB. Specificity 141 individuals were negative by LDA. 4 patients suspected of Lyme Disease and 1 normal control were positive by LDA, but negative by either PCR or RWB. Therefore, these were considered false positives. Based on this data, the specificity of LDA is >96%. Sensitivity Conclusion
This demonstrates that in order to obtain optimum sensitivity, without sacrificing specificity, the LDA and Lyme Multiplex PCR should be performed on all urine samples, and the RWB should be used for confirmation of LDA positive samples that are negative by PCR. In addition to our new tests, IGeneX now offers a range of cost-effective panels. IGeneX,
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