Immunoblot interpretation criteria for serodiagnosis of early Lyme disease

Abstract

We monitored the antibody responses of 55 treated patients with early Lyme disease and physiciandocumented erythema migrans. Six sequential serum samples were obtained from patients before, during, and until one year after antibiotic therapy and analyzed by in-house enzyme-linked immunosorbent (ELISA) and immunoblot assays. An immunoblot procedure utilizing a gradient gel and an image analysis system was developed. A relational database management system was used to analyze the results and provide criteria for early disease immunoblot interpretation. Recommended criteria for the immunoglobulin M (IgM) immunoblot are the recognition of two of three proteins (24, 39, and 41 kDa). The recommended criteria for a positive IgG immunoblot are the recognition of two of five proteins (20, 24 [>19 intensity units], 35, 39, and 88 kDa). Alternatively, if band intensity cannot be measured, the 22-kDa protein can be substituted for the 24-kDa protein with only a small decrease in sensitivity. Monoclonal antibodies were used to identify all these proteins except the 35-kDa protein. With the proposed immunoblot interpretations, the sequential serum samples were examined. At visit 1, the day of diagnosis and initiation of treatment, 54.5% of the serum samples were either IgM or IgG positive. The peak antibody response, with 80% of the serum samples positive, occurred at visit 2, 8 to 12 days into treatment. The sensitivities of the IgM and IgG immunoblot for detecting patients that were seropositive into the study period were 58.5 and 54.6%, respectively, at visit 1 and 100% at visit 2. Twenty percent of the patients remained seronegative throughout the study. The specificities of the IgM and IgG immunoblots were 92 to 94% and 93 to 96%, respectively. The IgM immunoblot and ELISA were similar in sensitivities, whereas the IgG immunoblot had greater sensitivity than the IgG ELISA (P ‫؍‬ 0.006). Lyme disease, a multisystem disorder caused by infection with the spirochete Borrelia burgdorferi, is the most common vector-borne disease in the United States today. The diagnosis of early Lyme disease is usually based on the presence of an expanding erythematous lesion, erythema migrans (EM). However, this clinical marker may be absent in approximately 20 to 40% of patients. Although the diagnosis is primarily based on clinical findings, it may be assisted by the results of serological tests. The enzyme-linked immunosorbent assay (ELISA) has been widely used for detecting antibodies to B. burgdorferi. These assays are not standardized, resulting in tests with various levels of sensitivity and specificity. Some of these tests may result in false-positive reactions, especially when sera are from persons with other illnesses such as syphilis, sarcoidosis (18), or viral illnesses (21, 26). The Western immunoblot has also been used by investigators to study the antibody response to infection with B. burgdorferi, with variable results. This test has been reported to be more sensitive than ELISA for immunoglobulin M (IgM) detection (11, 14, 22)...

Serodiagnosis of Lyme Disease by Kinetic Enzyme‐Linked Immunosorbent Assay Using Recombinant VlsE1 or Peptide Antigens ofBorrelia burgdorferiCompared with 2‐Tiered Testing Using Whole‐Cell Lysates

“…The specificity of 2-tiered testing has generally been у95%, both in our unpublished studies and as reported by reference laboratories, including our own [13,14,42,43]. In the general practice of 2-tiered testing, standardization of the performance, scoring, and interpretation of immunoblots is sometimes difficult, and the quality of results provided to physicians may be lower than the best achievable [7,42,43]. Furthermore, our study used a single commercial ELISA and immunoblots from a single manufacturer for 2tiered testing; results with other products may vary.…”

Molecular characterization of a 6.6-kilodalton Borrelia burgdorferi outer membrane-associated lipoprotein (lp6.6) which appears to be downregulated during mammalian infection

“…Interestingly, like OspA and OspB, lp6.6 did not generate an antibody response in mice or rhesus monkeys following low-dose needle or tick inoculation, respectively, despite the fact that the native molecule is extremely immunogenic when administered in the context of killed borreliae. These findings appear to be relevant to human Lyme borreliosis in that antibodies directed against low-molecular-mass B. burgdorferi antigens typically are absent, particularly during early human infection (8,24,27,28,59). All of these findings lead us to postulate that, analogous to that of the OspA and OspB genes (25,54), the expression of lp6.6 occurs in the tick vector but is downregulated during mammalian infection.…”

“…In the present study, DNA sequence analysis allowed us to determine that the molecular mass of the 51-amino-acid mature, acylated polypeptide is about 6.6 kDa. Other studies have noted the existence in B. burgdorferi of low-molecular-mass polypeptides and lipoproteins (28,52,62), but their relationship to lp6.6 remains uncertain. As a cautionary note, investigators should be aware of the need to shorten resolution times during SDS-PAGE in order to visualize lp6.6 or other low-molecular-mass borrelial polypeptide constituents.…”

Learning and Mapping Lyme Disease Patient Trajectories from Electronic Medical Data for Stratification of Disease Risk and Therapeutic Response

“…Typically, laboratory testing of LD follows identification of cutaneous manifestations from visual inspection but these manifestations are not always present. Current guidelines recommend serologic testing, a twophase process consisting of an enzyme immunoassay within 30 days of symptom onset, followed by Western blot after 30 days from symptom occurs 7,8 if the early test is active. Even together, this diagnostic strategy has poor sensitivity, particularly during the acute phase, with false-negative rates of up to 50% 9 .…”