Contents of Kit
Each kit contains the following components in sufficient quantities to perform the number of tests indicated on packaging label. Note: All reactive reagents contain sodium azide as a preservative at a concentration of 0.1% (w/v).
Plate. 96 wells configured in twelve 1x8-well strips coated with inactivated B. burgdorferi (B31 strain) antigen. The strips are packaged in a strip holder and sealed in an envelope with desiccant.
Conjugate. Conjugated (horseradish peroxidase) goat anti-human IgM (μ chain specific). Ready to use. One, 15 mL vial with a white cap.
Positive Control (Human Serum). One, 0.35 mL vial with a red cap.
Calibrator (Human Serum). One, 0.5 mL vial with a blue cap.
Negative Control (Human Serum). One, 0.35 mL vial with a green cap.
Absorbent Solution. One 12 mL bottle containing goat anti-human IgG (y chain specific) and phosphatebuffered-saline, (pH 7.2 ± 0.2). Ready to use.
Sample diluent. One 30 mL bottle (green cap) containing Tween-20, bovine serum albumin and phosphatebuffered-saline, (pH 7.2 ± 0.2). Green solution, ready to use. Note: Shake Well Before Use. Preservative added.
TMB. One 15 mL amber bottle (amber cap) containing 3,3',5,5' -tetramethylbenzidine(TMB). Ready to use. Contains DMSO < 15% (w).
Stop solution. One 15 mL bottle (red cap) containing 1M H2SO4. 0.7M HCl. Ready to use.
Wash buffer concentrate (10×): dilute 1 part concentrate + 9 parts deionized or distilled water. One 100 mL bottle (clear cap) containing a 10× concentrated phosphate-buffered-saline and Tween-20 solution (blue solution). NOTE: 1× solution will have a pH of 7.2 ± 0.2.
Note: Kit also contains:
1. Component list containing lot specific information is inside the kit box.
2. Package insert providing instructions for use.
Storage
1. Store the unopened kit between 2° and 8°C.
2. Coated microwell strips: Store between 2° and 8°C. Extra strips should be immediately resealed with desiccant and returned to proper storage. Strips are stable for 60 days after the envelope has been opened and properly resealed and the indicator strip on the desiccant pouch remains blue.
3. Conjugate: Store between 2° and 8°C. DO NOT FREEZE.
4. Calibrator, Positive Control and Negative Control: Store between 2° and 8°C.
5. Absorbent Solution: Store between 2° and 8°C
6. TMB: Store between 2° and 8°C.
7. Wash Buffer concentrate (10×): Store between 2° and 25°C. Diluted wash buffer (1×) is stable at room temperature (20° to 25° C) for up to 7 days or for 30 days between 2° and 8°C.
8. Sample Diluent: Store between 2° and 8°C.
9. Stop Solution: Store between 2° and 25°C.
General Description
Borrelia burgdorferi is a spirochete that causes Lyme disease. The organism is transmitted by ticks of the genus Ixodes. In endemic areas, these ticks are commonly found on vegetation and animals such as deer, mice, dogs, horses, and birds. B. burgdorferi infection shares features with other spirochetal infections (diseases caused by three genera in humans: Treponema, Borrelia, and Leptospira). Skin is the portal of entry for B. burgdorferi and the tick bite often causes a characteristic rash called erythema migrans (EM). EM develops around the tick bite in 60% to 80% of patients. Spirochetemia occurs early with wide spread dissemination through tissue and body fluids. Lyme disease occurs in stages, often with intervening latent periods and with different clinical manifestations.
In Lyme disease there are generally three stages of disease often with overlapping symptoms. Symptoms vary according to the sites affected by the infection such as joints, skin, central nervous system, heart, eye, bone, spleen, and kidney. Late disease is most often associated with arthritis or CNS syndromes. Asymptomatic subclinical infection is possible and infection may not become clinically evident until the later stages. Patients with early infection produce IgM antibodies during the first few weeks after onset of EM and produce IgG antibodies more slowly (1). Although IgM only may be detected during the first month after onset of illness, the majority of patients develop IgG antibodies within one month. Both IgG and IgM antibodies can remain detectable for years.
Isolation of B. burgdorferi from skin biopsy, blood, and spinal fluid has been reported (2). However, these direct culture detection methods may not be practical in the large scale diagnosis of Lyme borreliosis. Serological testing methods for antibodies to B. burgdorferi include indirect fluorescent antibody (IFA) staining, immunoblotting, and enzyme immunoassay (ElA).
B. burgdorferi is antigenically complex with strains that vary considerably. Early antibody responses often are to flagellum which has cross reactive components. Patients in early stages of infection may not produce detectable levels of antibody. Also, early antibiotic therapy after EM may diminish or abrogate good antibody response. Some patients may never generate detectable antibody levels. Thus, serological tests for antibodies to B. burgdorferi are known to have low sensitivity and specificity and because of such inaccuracy, these tests cannot be relied upon for establishing a diagnosis of Lyme disease (3,4).
In 1994, the Second National Conference on Serological diagnosis of Lyme disease recommended a two-step testing system toward standardizing laboratory serologic testing for B. burgdorferi. Because ElA and IFA methods were not sufficiently specific to support clinical diagnosis, it was recommended that positive or equivocal results from a sensitive ElA or IFA (first step) should be further tested, or supplemented, by using a standardized Western Blot method (second step) for detecting antibodies to B. burgdorferi (Western Blot assays for antibodies to B. burgdorferi are supplemental rather than confirmatory because their specificity is less than optimal, particularly for detecting IgM). Two-step positive results provide supportive evidence of exposure to B. burgdorferi, which could support a clinical diagnosis of Lyme disease but should not be used as a sole criterion for diagnosis.
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