Western Blot vs Modified Two-Tier Testing in Lyme Disease
Modified Two-Tier Testing (MTTT) replaces Western blot with two enzyme immunoassays, but both approaches have important limitations—especially in early Lyme disease.
Newer Lyme disease tests were introduced to improve accuracy and simplify interpretation. However, the reality remains more complex.
Both traditional and newer testing methods may miss cases of Lyme disease.
The Traditional Two-Tier Lyme Disease Test
The traditional two-tier testing (TTT) algorithm combines:
- An initial ELISA or EIA screening test
- A confirmatory Western blot
Western blot testing evaluates antibody bands directed against specific Lyme disease proteins.
The test requires positive or equivocal ELISA findings followed by:
- 2 of 3 IgM bands, or
- 5 of 10 IgG bands
Western blot has helped provide serologic confirmation for some early Lyme disease cases since its adoption in 1994. The Western blot-based TTT has been considered sensitive for chronic manifestations by some doctors but not others. [1]
However, its performance in early Lyme disease has remained controversial.
The Proposal to Replace Western Blot
Researchers proposed replacing Western blot with newer testing strategies designed to simplify interpretation and improve standardization. [1]
The proposed Modified Two-Tier Testing (MTTT) approach evaluated combinations of:
- Whole-cell sonicate (WCS) EIA
- C6 peptide EIA
- VlsE chemiluminescence immunoassay (CLIA)
Given the lack of sensitivity in standard testing, the goal was to improve early detection while reducing subjectivity associated with Western blot interpretation. [1]
The Sensitivity Problem Remains
Despite enthusiasm for MTTT, important limitations remained.
The study found that traditional Western blot two-tier testing showed sensitivity ranging from 36% to 54% for early Lyme disease. [1]
The proposed MTTT protocols improved sensitivity modestly, ranging from 66% to 72% during convalescent Lyme disease. [1]
Neither approach reliably detected all early Lyme disease cases.
This reflects a fundamental biologic problem:
Both tests depend on antibody production, which may take time to develop after infection.
Why Some Clinicians Resisted Eliminating Western Blot
Western blot has recognized limitations, including subjective interpretation and lower sensitivity during early infection.
However, it also provides antigen-specific band information that many clinicians continue to find clinically useful.
The replacement protocols were also evaluated using relatively small datasets.
In the study, the proposed modified protocols were based on only 55 erythema migrans patients. [1]
Questions also remained regarding how well MTTT would perform in persistent or late-stage manifestations of Lyme disease.
Any recommendation to eliminate Western blot in favor of MTTT alone was considered premature because sensitivity remained low for both early and convalescent Lyme disease. [1]
What Happened After FDA Clearance?
Beginning in 2019, certain MTTT protocols received FDA clearance.
However, Western blot testing was not eliminated.
Both testing strategies remain in clinical use today.
Clinicians may choose one approach over another depending on testing availability, laboratory preference, and clinical context.
Why Lyme Disease Testing Still Has Limitations
Western blot and Modified Two-Tier Testing share several important limitations:
- Both detect antibodies rather than active infection
- Both may be negative early in disease
- Both depend on the timing of the immune response
- Both may miss some clinically significant cases
Changing the testing method does not change the biology of infection.
This diagnostic uncertainty is part of why Lyme disease tests the limits of medicine.
Why Clinical Judgment Still Matters
Laboratory testing supports diagnosis—but does not replace clinical evaluation.
Some patients present with symptoms before antibodies become detectable.
Others may remain seronegative despite ongoing symptoms.
Understanding symptom patterns and exposure history remains important. See the Lyme disease symptoms guide.
Frequently Asked Questions
Is Modified Two-Tier Testing better than Western blot?
MTTT offers greater standardization but still shares important limitations with Western blot testing, especially in early Lyme disease.
Can Western blot still be used for Lyme disease?
Yes. Western blot remains available and continues to be used in clinical practice.
Why are Lyme disease tests less accurate early on?
Most Lyme disease tests depend on antibody production, which may not yet be detectable during early infection.
Can Lyme disease tests miss active infection?
Yes. Both traditional and modified testing approaches may miss some cases, particularly early in illness.
Does a negative Lyme test rule out Lyme disease?
No. Clinical symptoms and exposure history remain important, especially when testing is performed early.
Clinical Takeaway
Western blot and Modified Two-Tier Testing both have important limitations.
While testing methods have evolved, neither approach reliably detects all cases—particularly early in disease.
Clinical judgment based on symptoms, exposure history, and disease patterns remains essential.
Related Articles
Lyme Disease Testing and Diagnosis
False Negative Lyme Disease Testing
Lyme Disease Misdiagnosis
Prevention of Lyme Disease
Delayed Lyme Disease Diagnosis
References
- Branda JA, Strle K, Nigrovic LE, et al. Evaluation of Modified 2-Tiered Serodiagnostic Testing Algorithms for Early Lyme Disease. Clin Infect Dis. 2017.
- Wormser GP, Dattwyler RJ, Shapiro ED, et al. The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines. Clin Infect Dis. 2006;43(9):1089-1134.
- Cameron DJ, Johnson LB, Maloney EL. Evidence assessments and guideline recommendations in Lyme disease. Expert Rev Anti Infect Ther. 2014.
Dr. Daniel Cameron, MD, MPH
Lyme disease clinician with over 30 years of experience and past president of ILADS.
Symptoms • Testing • Coinfections • Recovery • Pediatric • Prevention
I want to know….why can a tick be tested without problems, as where a human test is little better than 50%. In my case I had been tested 5-6 times over a span of 18 yrs. Never came back positive until my immune system shut down. I was bitten as a child at some point. Started getting symptoms of swollen muscles/joints at age 19. Finally had a positive test when I was 37. It’s insane to me that more studies aren’t done to better diagnose this disease.
The tick studies tests typically involve PCR or antigen tests. These tests are a problem in the body with the complexity of the DNA and proteins in the body. The two-tier depends on the human immune response.
So, if you have an immune disorder, the Two-tier test would not be effective at diagnosing someone infected with Lyme, Isn’t it?
I have not seen any data to understand how an immune disorder would affects tests.