Early Lyme Disease Detection Improved With Single-Step Tests in New Study

FDA-cleared single-step immunoblot tests detected antibodies in a greater proportion of early Lyme disease samples than standard 2-tier testing (STTT), according to findings from a blinded analysis of more than 1100 serum samples. The results support the analytical approach underlying the iDart IgG and IgM Immunoblot Tests, which IGeneX reported have received US Food and Drug Administration 510(k) clearance for laboratory diagnosis of Lyme disease.¹,²

“Our FDA-cleared single-step immunoblot demonstrated improved early detection while simplifying the testing process, with the potential to significantly enhance patient care through earlier and more accurate diagnosis, more timely treatment, and ultimately better patient outcomes,” Jyotsna S. Shah, PhD, lead author and President of IGeneX, said in a press release.²

The study evaluated IgG and IgM immunoblot tests that incorporate recombinant protein targets from multiple Borrelia species and strains. Investigators compared the single-step tests with US Food and Drug Administration–cleared standard two-tier testing, the conventional approach that uses an initial enzyme immunoassay followed by confirmatory immunoblot testing when the first-tier result is positive or equivocal.¹

Why is early Lyme disease diagnosis challenging?

Serologic diagnosis of Lyme disease can be difficult early in infection because antibody levels may still be developing. This is clinically important because early diagnosis and treatment can help reduce the risk of disease progression. Aside from a characteristic erythema migrans lesion in an endemic area, laboratory diagnosis often relies on detecting serum antibodies to Lyme disease Borrelia antigens.

The new IgG and IgM immunoblot tests were designed to broaden antigenic coverage and simplify testing. The assays use a proprietary Lyme Screen Antigen derived from immunodominant regions of the VlsE protein across multiple Borrelia species, along with additional antigens including OspC, OspA, OspB, BmpA, FlaB, and P93.

How did the immunoblot tests perform?

Investigators evaluated more than 1100 serum samples, including a US Centers for Disease Control and Prevention premarketing panel, prospectively banked acute-phase samples from the Bay Area Lyme Foundation’s Lyme Disease Biobank, healthy control samples, and samples from individuals with potentially confounding infections or medical conditions.

In the CDC stage 1 Lyme disease serum panel, the IgG immunoblot test detected IgG antibodies in 58.3% of samples compared with 30.0% using the IgG standard two-tier test, a statistically significant difference. When IgG and/or IgM results were considered together in the same stage 1 CDC panel, the immunoblot tests detected antibodies in 81.7% of samples compared with 70.0% using STTT, although that difference did not reach statistical significance.

The immunoblot approach also outperformed STTT in the Lyme Disease Biobank cohort. Among 290 acute-phase sera, IgG immunoblot testing detected IgG antibodies in 19.0% of samples compared with 6.9% using IgG STTT. When IgG and/or IgM antibody detection was assessed, the positivity rate was 23.8% with the immunoblot tests compared with 16.9% with STTT.

In samples from patients with erythema migrans lesions, the combined IgG and IgM immunoblot tests detected antibodies in 30.2% of samples compared with 20.6% using STTT. Among patients with erythema migrans lesions larger than 5 cm, detection rates were 35.9% with immunoblot testing vs 24.6% with STTT.

What did discrepancy analysis show?

Among the 290 Lyme Disease Biobank samples, 22 sera were positive by IgG and/or IgM immunoblot but negative by IgG and IgM STTT. Discrepancy analysis confirmed that 21 of these 22 samples were true positives, including 17 from patients with erythema migrans lesions larger than 5 cm and 4 that tested positive on 2 different enzyme immunoassays.

The study authors noted that even with improved early detection, sensitivity remained limited in some early disease groups. For example, the combined IgG and IgM immunoblot tests detected antibodies in 35.9% of patients with acute early-stage Lyme disease and erythema migrans lesions larger than 5 cm, underscoring the ongoing challenge of serologic diagnosis early in infection.

How specific were the tests?

The immunoblot tests demonstrated high specificity across control samples. Among 387 sera from individuals with potentially cross-reactive conditions, including other tick-borne diseases, viral and bacterial infections, autoimmune disorders, fibromyalgia, and pregnancy, none were positive on the IgG immunoblot test. One parvovirus-19–positive serum sample was positive on the IgM immunoblot test, corresponding to an estimated IgM specificity of 99.7%.

Among healthy controls living in Lyme disease–endemic areas, estimated specificity was 99.4% for the IgG immunoblot and 100% for the IgM immunoblot. Among healthy controls from nonendemic areas, specificity was 100% for both tests.

“These findings represent a meaningful advancement in Lyme disease diagnostics, particularly given that commonly used FDA-cleared MTTT and STTT testing methods have been shown to miss 64–78% of early Lyme disease cases,” said Shah in the press release.²

References

1. Shah JS, Liu S, Calalo-Ramos C, et al. Single-step immunoblot tests with recombinant protein antigens for detecting IgG and IgM antibodies in Lyme disease. Microbiol Spectr. Published July 15, 2026. doi:10.1128/spectrum.00199-26
2. IGeneX Inc. New FDA-cleared single-step immunoblot IGeneX tests improve early detection of Lyme disease, study finds. Published July 15, 2026. Accessed July 22, 2026. https://igenex.com/press-release/new-fda-cleared-single-step-immunoblot-igenex-tests-improve-early-detection-of-lyme-disease-study-finds