Pilot Study of Immunoblots with Recombinant Borrelia burgdorferi Antigens for Laboratory Diagnosis of Lyme Disease

Abstract

Accurate laboratory diagnosis of Lyme disease (Lyme borreliosis), caused by the spirochete Borrelia burgdorferi (BB), is difficult and yet important to prevent serious disease. The US Centers for Disease Control and Prevention (CDC) presently recommends a screening test for serum antibodies followed by confirmation with a more specific Western blot (WB) test to detect IgG and IgM antibodies against antigens in whole cell lysates of BB. Borrelia species related to BB cause tick-borne relapsing fever (TBRF). TBRF is increasingly recognized as a health problem in the US and occurs in areas where Lyme disease is prevalent. The two groups of Borrelia share related antigens. We have developed a modified WB procedure termed the Lyme immunoblots (IBs) using recombinant antigens from common strains and species of the BB sensu lato complex for serological diagnosis of Lyme disease. A reference collection of 178 sera from 26 with and 152 patients without Lyme disease were assessed by WB and IB in a blinded manner using either criteria for positive antibody reactions recommended by the CDC or criteria developed in-house. The sensitivity, specificity, positive and negative predictive values obtained with the reference sera suggest that the Lyme IB is superior to the Lyme WB for detection of specific antibodies in Lyme disease. The Lyme IB showed no significant reaction with rabbit antisera produced against two Borrelia species causing TBRF in the US, suggesting that the Lyme IB may be also useful for excluding TBRF.

Keywords: Borrelia burgdorferi; Lyme disease; immunoblot; laboratory diagnosis; tick-borne diseases; western blot.

Figure 1

Lyme IgM and IgG Immunoblots with Serum Samples. Ten representative serum samples 1–10 were tested by [I] Lyme IgM and [II] Lyme IgG IBs. P—positive control, C—calibrator and N—negative control. Control 1—conjugate control, and Control 2—serum control. The positions of target antigens used in the IB strips are shown. P39 EU—P39 from European BBsl species, P39 US—P39 from US BBsl species.

Figure 2

Comparison of Lyme IgM Western Blots and Lyme IgM Immunoblots. Results with serum samples (1–4) tested by Lyme IgM WB (I and III) and Lyme IgM IB (II and IV). P—positive control, C—calibrator and N—negative control. Control 1—conjugate control, and Control 2—serum control. The positions of target antigens in the IBs and WBs are shown. P39 EU—P39 from European BBsl species, P39 US—P39 from US BBsl species.

Figure 3

Lyme IgG Immunoblot Showing Reactions of Rabbit Antisera to Different BBsl and TBRF Borrelia species. Specific rabbit antisera produced against different BBsl (lanes 1–7) and TBRF (lanes 8 and 9). Borrelia species were tested individually on Lyme IgG IBs: Lane 1—B. burgdorferi ss B31, 2—B. burgdorferi ss 297, 3—B. afzelii, 4—B. garinii, 5—B. californiensis, 6—B. spielmanii, 7—B. valensiana, 8—B. hermsii, and 9—B. coriaceae. P—positive control human serum, N—negative control human serum, 1 Control—conjugate control, and 2 Control—serum control.