IGeneX Innovations
	January 2002 Published by IGeneX, Inc. 795 San Antonio Rd, Palo Alto, CA 94303 800/832-3200
Questions and Answers Selected Antigen References
LYME (BORRELIA) ANTIGEN ASSAYS: QUESTIONS AND ANSWERS Nick S. Harris, Ph.D., ABMLI IGeneX is the only laboratory that performs a urine antigen test for Lyme Disease. Following are some of the most frequently asked questions and answers. What is the Lyme Dot-Blot Antigen Assay (LDA)? The LDA is a third generation antigen assay, based on the proprietary technology of US patent 4,888,276. It replaces the LUAT, the Lyme Urine Antigen Test, which was a second generation test. In the LDA, the patient’s urine is reacted with a special nitrocellulose membrane to bind any Borrelia antigen, which may be present in the urine. The membrane is incubated with anti-B. burgdorferi antibodies with a specific affinity to the following Borrelia antigens: 23-25 kDa (Osp C), 31 kDa (Osp A), 34 kDa (Osp B), 39 kDa, and 83-93 kDa. This step allows the anti-B. burgdorferi antibodies to bind to the Borrelia antigens on the membrane. After removing excess antibodies, the bound anti-B. burgdorferi antibody is detected using a horseradish-peroxidase conjugated second antibody. A positive reaction is reflected as a blue dot on the membrane, hence the name Lyme Dot-Blot Antigen. What is the difference between the LDA and the LUAT? The concept of detecting antigen in urine is the same for both methods, but the methods are considerably different. The LDA is a simpler assay to perform and has fewer repeats. The LUAT used a conjugated antibody, which did not include an antibody to Osp C, and the results had to be read with a fluorescent ELISA reader. The LDA uses a non-conjugated specific anti-B. burgdorferi antibody and has a visible endpoint. Urine samples collected for the LDA have longer stability, meaning fewer rejected specimens. Return to Top Why is IGeneX the only laboratory performing an antigen test on urine? IGeneX started as part of 3M Corp., the company that invented the patented Lyme Urine Antigen Test, and was instrumental in the assay’s development. Many of the same research team members that developed the assay moved to IGeneX. The NIH (National Institutes of Health) developed another patented assay for Lyme Antigen in Urine. This assay requires an Electron Microscope and gold-conjugated antibody, and has never successfully been commercialized for routine patient use by any clinical reference laboratory. What does the LDA detect? The LDA detects pieces of the Lyme bacteria. Why do you look for antigen in the urine? Studies (see references) in animal models of Lyme disease utilizing mice, rats, rabbits, guinea pigs and dogs have found Lyme antigen in the urine. In addition, one of the best sources to culture the Lyme bacteria in experimental animals has been the bladder epithelium. This source has been better than blood, spleen, kidney and comparable to or better than heart and brain. It may be that the Lyme bacteria rinds bladder epithelium a privileged site and that antigen is shed into the urine from the bladder. Why do you recommend three samples from three separate days? We have observed that Lyme patients with active disease may have a positive antigen. While some patients are positive most of the time they are tested, the majority of patients are only sporadically positive. Initial clinical studies with physician volunteers who collected multiple times per day for a week, demonstrated that three morning samples provided the optimum sensitivity versus cost basis for the average patient. A current analysis of more than 500 positive LDA patients who had submitted three urines, collected on three different days, indicates that the chance of finding a positive LDA with two only days of urine samples was 80–85 % and with one day of urine was 50–55%. Do you recommend the LDA as the initial or screening test after a tick bite? Our published studies show that only 8% of patients positive for Lyme urine antigen have anti-B. burgdorferi antibody in their serum at the same time. Based on these studies, our hypothesis is that “high antibody levels of anti-B. burgdorferi would interfere with antigen detection.” Therefore, we recommend that patient sera should first be screened with a good quality Lyme Western Blot IgM and IgG. If those assays are not conclusive, then an antigen and/or PCR test would be the next logical step. Return to Top How should the urine samples be stored? The Becton Dickinson Grey top tubes that we provide should be REFRIGERATED until shipment. PLEASE DO NOT FREEZE THE SAMPLES. Please contact the office for a test kit which includes all the collection and shipping materials. Why do physicians prescribe antibiotics before doing the LDA? Many physicians have observed that an antibiotic challenge for a few days before the assay seems to increase the chances of finding antigen in urine, or at least is more predictable. Antibiotics kill spirochetes which may be present in the body, and the antigenic parts are shed through the urinary tract. We can not perform clinical studies along this line because it is prohibited for laboratories. We provide the protocols as a courtesy from our physician clients who have offered them for use by other physicians. Do you have to take antibiotics to get a positive LDA? No, antibiotics are probably not mandatory. In the previously mentioned publication about the Lyme Urine Antigen Test, approximately 30% of the patients testing positive by the LUAT were not specifically challenged prior to the assay. The challenge does seem to make the test more predictable, since antibiotics would kill the Lyme bacteria and may make multiple testing events over several weeks unnecessary. What other conditions may affect the LDA? Based upon studies with Marylynn Barkley, M.D., Ph.D. at UC Davis it seems that menses is a very sensitive time to look for antigen in the urine of women with Lyme Disease. Initial studies examined women who had been clinically treated for persistent Lyme Disease, but who had never had a positive laboratory marker for the disease. Urine samples collected at the start of menses for three to five days were positive for Lyme antigen, thus supporting the clinical diagnosis of Lyme Disease. What other Lyme assays are useful with urine samples? The PCR assay for Borrelia in urine is helpful and probably should be done at the same time as the LDA. In fact, IGeneX offers a panel that consists of three LDA’s and a pooled urine sample from the three days, which is assayed by PCR. The benefit of this panel is twofold: the PCR detects some patients missed by the LDA and also acts as a confirmation in 30–40% of the positive LDA’s. In addition, we offer a unique assay called the Reverse Western Blot (RWB). This test is only available as a confirmation of positive LDA’s. Return to Top When, in the disease process, can the test be per formed? Lyme antigen has been detected in urine as early as three days after a tick bite. Antigen has also been detected in patients with active disease many years after the purported exposure. What is the Reverse Western Blot (RWB) and how is it different from the regular Western Blot? The regular Western Blot detects anti-B. burgdorferi antibody in patient serum. Patient serum is reacted to commercial B. burgdorferi antigen bound to a membrane. Specific Lyme antibodies, if present, are detected. The Lyme Reverse Western Blot (RWB) detects Lyme antigens in patient urine. Antigens in urine are first immobilized to a membrane. Membrane bound antigens are then detected with defined commercial anti-B. burgdorferi antibodies. This is a difficult and expensive procedure, and is performed only if the LDA is positive and a PCR on the urine is negative. Is Lyme the only disease in which an antigen test is performed? No. In fact, with laboratory analysis, the test of choice for most infectious disease detection is an antigen or DNA test. The reason is that antigen or PCR is direct detection. If you find antigen in a patient, they have disease or at least have the bacteria, parasite, virus, fungus, etc. in their body. An antibody test, on the other hand, is an indirect assay and only implies that a person has been exposed and not necessarily that the disease organism is still present in their body. Can the LDA have false positive results? Any test can have false positive results, but laboratory assays are designed with a balance between sensitivity and specificity. If you had a test with 100% sensitivity, it would have many false positives. For example, the screening test for anthrax has a sensitivity approaching 100%, but probably has 30–40% false positives. That is good, because if you miss a patient they can die. Laboratories try to achieve 90–95% sensitivity and 90–95% specificity. Based upon our studies, the LDA has a sensitivity of 99+% if there is 12.5 ng/ml of antigen in the urine. The test has a measured specificity of 93%. This means that there is a 7% chance of a false positive. We do recommend confirmation of positive LDA samples by PCR and/or RWB. We have found no cross-reactions with common urine bacteria or with Babesia or Ehrlichia. We have seen some non-specific false positives with urine that contains very high levels of red blood cells and very high levels of nitrites. For this reason, all urines are tested for RBC’s and nitrites. If positive for nitrites or if 3+ for RBC’s, the sample(s) is rejected. Those rejected urines can be tested by PCR. Return to Top Does a positive LDA indicate active disease? A positive LDA supports or indicates the presence of the Lyme bacteria in the body. Physicians can use the LDA results in conjunction with the patient’s clinical history and symptoms to make a diagnosis of Lyme Disease. A reference laboratory performs tests to support a physician’s suspicion about disease. The reference laboratory never makes a diagnosis. If I test negative for Lyme antigen does it mean I do not have Lyme disease? No. A negative LDA only suggests that the Lyme antigens were below the limit of detection or were absent in the test sample. In Lyme Disease, a single episode of negative testing by either PCR or LDA should not be taken as indicating the absence of disease. First of all, that is part of a medical diagnosis made by a physician. Often times multiple testing events need to be performed in order to have a high level of confidence that the organism is not present. Is the LDA test approved by the FDA? No. The LDA is not FDA approved. Getting approval from the FDA is a laborious process and usually reserved for test kits manufactured for resale to other laboratories and hospitals. Thus, we are looking for a diagnostic manufacturing partner to do this. Many tests run in clinical laboratories are not FDA approved. For example, all PCR assays for Lyme, Babesia and Ehrlichia, as well as many Western Blot assays, are not FDA approved nor are many assays for cancer. Why did IGeneX discontinue the LUAT? We discontinued the LUAT because we ran out of one of the critical reagents used in the test. Two years prior, we had started a development program for the new LDA. We are constantly working to improve all of our testing. The LDA is more sensitive than the LUAT and cost effective as well. It is easier to manufacture than the LUAT and does not require conjugation of the highly specific anti-Borrelia antibodies. Why don’t we find Lyme antigen studies published in the mainstream scientific journals? Lyme is a political “hot potato” and editors must unfortunately make tough decisions that usually follow the academic mainstream. We have tried unsuccessfully and will continue to submit studies. Currently we have a seven-center study underway regarding Lyme symptoms and the detection of antigen in urine. This study will be submitted for publication. Return to Top Does insurance pay for antigen testing and if so why does IGeneX not bill insurance? Many insurance companies, including Medicare, do pay for antigen testing. We do bill Medicare, but have elected not to bill insurance companies because we would rather invest revenue in researchers than billing clerks. Even though IGeneX is a small laboratory, 30% of the annual budget is for research on new tests and improvements of existing ones. Why is the LDA not available to New York State Residents? New York State currently requires that we provide all information about the LDA test, including proprietary information. Because of the proprietary issues, we have not submitted any LDA documents to the New York Department of Health. We hope to reach some type of agreement in the future about the confidential nature of our intellectual property. New York State physicians, however, can use the LDA on their patients residing outside of New York, just not on residents of New York State.
Selected Antigen References Bosler and Schulze. 1986. The prevalence and significance of Borrelia burgdorferi in the urine of feral reservoir hosts. Zentralbl Bakteriol Microbiol Hyg [A] 263:40–44. Schwann, et al. 1988. The urinary bladder, a consistent source of Borrelia burgdorferi in experimentally infected white-footed mice (Peromyscus leucopus). J Clin Microbiol 26: 893–805. Hyde FW, et al. 1989. Detection of antigens in urine of mice and humans infected with Borrelia burgdorferi, etiologic agent of Lyme disease. J Clin Microbiol 27:58–61. Shelburne CE. 1989. Method and composition for the diagnosis of Lyme Disease. US Patent 4,888,276. Goodman JL, et al. 1991. Molecular detection of persistent Borrelia burgdorferi in the urine of patients with active Lyme disease. Infect Immun 59: 269–278. Dorward DW, et al. 1991. Immune capture and detection of Borrelia burgdorferi antigens in urine, blood or tissues from infected ticks, mice, dogs and humans. J Clin Microbiol 29: 1162–1170. Drulle J, and E Eiras. Persistence of Borrelia burgdorferi antigen in patients receiving long-term antibiotic therapy. Presented at V Int Conf Lyme Borreliosis. Abstract 70E. Washington, DC. 1992. Harris N, Drulle J, Eiras E and BG Stephens. Detection of B. burgdorferi antigen and antibody in patients presenting with an erythema migrans. Presented at 6th annual Lyme disease scientific conference. May 5–6, 1993. Atlantic City, NJ. Magnarelli LA, et al. 1994. Detection of Borrelia burgdorferi in urine of Peromyscus leucopus by inhibition enzyme-linked immunosorbent assay. J Clin Microbiol 32: 777–782. Harris NS and BG Stephens. 1995. Detection of Borrelia burgdorferi antigen in urine from patients with Lyme Borreliosis. JSTBD 2: 37–41. Bayer et al. 1996. Borrelia burgdorferi DNA in the urine of treated patients with chronic Lyme disease symptoms. A PCR study of 97 cases. Infection 24: 347–353. Harris NS, Harris SJ, Joseph JJ and BG Stephens. Borrelia burgdorferi antigen levels in urine and other fluids during the course of treatment for Lyme Disease: A case study. Presented at the VII International Congress of Lyme Borreliosis meeting. June 16–21, 1996. San Francisco, CA. Barkley M, Harris N and B Szantyr. The Lyme Urine Antigen Test (LUAT) during antibiotic therapy in women with recurrent menstrual cycles. Presented at the 10th Annual Scientific Conference on Lyme Borreliosis, NIH, Bethesda, MD. April 28–30,1997. Harris, NS. Antigen Detection of Borrelia burgdorferi in Urine. Presented at the 11th Scientific Conference on Lyme Borreliosis, New York, NY. April 25–26,1998.
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IGeneX, Inc. 795 San Antonio Rd., Palo Alto, CA 94303 USA Tel. 650.424.1191 / 800.832.3200 Fax. 650.424.1196