Human Lyme Disease antibody ELISA Kit

Sample

serum, plasma

Species Reactivity

Human

Detection Method

iELISA

Intended Use

The Borrelia burgdorferi (B. burgdorferi) IgG/IgM Enzyme-Linked Immunosorbent Assay (ELISA) is intended for the qualitative presumptive (first-step) detection of total (IgG/IgM) antibodies to Borrelia burgdorferi in human serum. This ELISA should only be used for patients with signs and symptoms that are consistent with Lyme disease. Equivocal or positive results must be supplemented by testing with a standardized Western-blot(second-step) procedure. Positive supplemental (second- step) results are supportive evidence of exposure to B. burgdorferi and can be used to support a clinical diagnosis of Lyme disease. The diagnosis of Lyme disease must be made based on history, signs (such as erythema migrans), symptoms, and other laboratory data, in addition to the presence of antibodies to B. burgdorferi. Negative results (either first- or second step) should not be used to exclude Lyme disease.

Contents of Kit

Each kit contains the following components in sufficient quantities to perform the number of tests indicated on the package label.
1. Purified B. burgdorferi antigen, strain B-31 (passed less than 15 times, washed, concentrated and detergent treated in glycine buffer) coated microassay plate: 96 wells, configured in twelve 1×8 strips, stored in a foil pouch with desiccant. (96T: one plate)
2. B. burgdorferi Serum Diluent Type V: Ready for use. Contains gentamycin and sodium azide (< 0.1%) as preservatives. (96T: one bottle, 30 mL)
3. Cutoff Calibrator (Calibrator): human serum or defibrinated plasma. Sodium azide (< 0.1%) and pen/strep (0.01%) added as preservatives, with kit specific factor printed on vial label. The Cutoff Calibrator is used to calibrate the assay to account for day-to-day fluctuations in temperature and other testing conditions. (96T: one vial, 0.4 mL) *
4. High Positive Control: human serum or defibrinated plasma. Sodium azide (< 0.1%) and pen/strep (0.01%) added as preservatives, with established range printed on vial label. The High Positive Control is utilized to indicate the upper limit of dynamic range of the assay. (96T: one vial, 0.4 mL) *
5. Low Positive Control: human serum or defibrinated plasma. Sodium azide (< 0.1%) and pen/strep (0.01%) added as preservatives, with established range printed on vial label. The Low Positive Control is utilized to control the assay near the cutoff of the assay. (96T: one vial, 0.4 mL) *
6. Negative Control: human serum or defibrinated plasma. Sodium azide (< 0.1%) and pen/strep (0.01%) added as preservatives, with established range printed on vial label. The Negative Control is utilized to control the negative range of the assay.(96T: one vial, 0.4 mL) *
7. Horseradish-peroxidase (HRP) Conjugate: Ready for use. Goat antihuman IgG/IgM, containing Proclin 300® (0.1%) as a preservative. (96T: one bottle, 16 mL)
8. Chromogen/Substrate Solution Type I: Tetramethylbenzidine (TMB), ready to use. (96T: one bottle, 16 mL)
9. Wash Buffer Type I(20× concentrate): dilute 1 part concentrate + 19 parts deionized or distilled water. Contains TBS, Tween-20 and Proclin 300® (0.1%) as a preservative. (96T: one bottle, 50 mL)
10. Stop Solution: Contains a H₂SO₄ solution, ready to use. (96T: one bottle, 15 mL)
Note: serum vials may contain excess volume.

Storage

1. Store unopened kit between 2°C and 8°C. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date.
2. Unopened microassay plates must be stored between 2°C and 8°C. Unused strips must be immediately resealed in a sealable bag with desiccant, and returned to storage at 2°C and 8°C. If the bag is resealed with tape, the wells are stable for 30 days. If the bag is resealed with a heat sealer, the wells are stable until their labeled expiration date.
3. Store HRP Conjugate between 2°C and 8°C.
4. Store the Cutoff Calibrator, High Positive Control, Low Positive Control and Negative Control between 2°C and 8°C.
5. Store Serum Diluent and 20X Wash Buffer between 2°C and 8°C.
6. Store the Chromogen/Substrate Solution between 2°C and 8°C
7. Store 1× (diluted) Wash Buffer at room temperature (21°C to 25°C) for up to 5 days, or up to 1 week between 2°C and 8°C.
Note: If constant storage temperature is maintained, reagents and substrate will be stable for the dating period of the kit. Refer to package label for expiration date. Precautions were taken in the manufacture of this product to protect the reagents from contamination and bacteriostatic agents have been added to the liquid reagents. Care should be exercised to protect the reagents in this kit from contamination.

Precision

The precision of the Borrelia burgdorferi IgG/IgM ELISA was determined by testing six different sera ten times each on three separate days. The mean coefficients of variation from the intra- and inter-assays are presented in Table. With appropriate technique the user should obtain precision of < 15% CV.

X = Mean Thyroglobulin Value
S.D. = Standard Deviation
C.V. = Coefficient of Variation
The methods in NCCLS EP5 were utilized for precision parameters.

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 a characteristic rash called erythema migrans (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. 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. However, these direct culture detection methods may not bepractical 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 (EIA). B. burgdorferi is antigenically complex with strains that vary considerably. Early antibody responses often are to flagellin, 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. Thus, serological tests for antibodies to B. burgdorferi are known to have low sensitivity and specificity, and because of such inaccuracy, these test cannot be relied upon for establishing a diagnosis of Lyme disease.
In 1994, the Second National Conference on Serological Diagnosis of Lyme disease recommended a two-steptesting system toward standardizing laboratory serologic testing for B. burgdorferi.Because ELISA and IFA methods were not sufficiently specific to support clinical diagnosis, it was recommended that positive or equivocal results from a sensitive ELISA 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 criterion for diagnosis. Antibody detection methods do not provide definitive results for establishing or ruling out a diagnosis of Lyme disease in that the predictive value of positive results will be low when specimens are tested from patients with diagnoses that are not supported by history of exposure, symptoms, signs, and laboratory data.