The 2017 Proposal to Replace Western Blot Testing

In 2017, researchers from Massachusetts General Hospital and Harvard Medical School proposed eliminating the Western blot from Lyme disease diagnosis.

Their argument was straightforward: replace the traditional two-tier testing algorithm (ELISA followed by Western blot) with a combination of enzyme immunoassays—specifically, a whole-cell sonicate (WCS) EIA, C6 EIA, and VlsE chemiluminescence immunoassay (CLIA).

This approach, called Modified Two-Tier Testing (MTTT), promised to improve early detection while eliminating the complexity and variability of Western blot interpretation.

The proposal gained attention because Western blot testing had documented limitations. It requires visual band interpretation, shows laboratory-level variability, and often remains negative in early infection when treatment is most effective.

But eliminating Western blot entirely raised concerns—particularly about whether the proposed replacement was actually better.

Why There Was Resistance to Replacing Western Blot

The Western blot-based two-tier test had allowed doctors serologic confirmation for some early Lyme disease cases. The test requires that positive or equivocal ELISA results be confirmed with 2 of 3 IgM bands or 5 of 10 IgG bands.

While some doctors considered the Western blot-based two-tier test sensitive for chronic manifestations, others disagreed. Regardless, it had been the standard since 1994 and provided serologic confirmation for patients who might otherwise have been dismissed.

My concern in 2017 was that the proposed Modified Two-Tier Testing protocols weren’t demonstrably better—they were just different.

The Sensitivity Problem With Both Approaches

When researchers evaluated the proposed MTTT protocols, the sensitivity numbers were telling:

Western Blot Two-Tier Testing:
– 36-54% sensitivity for early Lyme disease
– Higher sensitivity for chronic manifestations (though debated)

Modified Two-Tier Testing:
– 66-72% sensitivity on convalescence
– Based on only 55 erythema migrans patients

The sensitivity improvement was modest at best. More concerning, MTTT sensitivity for chronic manifestations of Lyme disease had not been determined.

Branda and colleagues argued that MTTT protocols “provide comparable or greater sensitivity in acute EM, and similar specificity compared with conventional 2-tiered testing, obviating the need for Western blots.”

But eliminating Western blot in favor of MTTT seemed premature. A sensitivity of 36-54% for early Lyme disease and 66-72% for convalescent Lyme disease was still low. Both approaches missed substantial numbers of genuine cases.

Moreover, the proposed MTTT sensitivity was based on a small sample size and had not been validated for the chronic presentations that many clinicians encounter.

What Actually Happened: FDA Clearance and Clinical Adoption

Despite initial resistance, Modified Two-Tier Testing moved forward. Beginning in 2019, the FDA cleared certain MTTT algorithms for clinical use.

These protocols use two enzyme immunoassays with different antigen targets—typically a first-tier EIA followed by a second-tier EIA with different Borrelia antigens—rather than ELISA followed by Western blot.

The goals were standardization, automation, faster results, and elimination of subjective Western blot band interpretation.

However, the FDA clearance did not eliminate Western blot testing. Both approaches remain available, and clinicians can choose which testing strategy to use based on clinical context.

Current State: Two Options, Same Fundamental Limitations

Today, physicians have two serologic testing pathways:

Traditional Two-Tier Testing:
– ELISA or IFA screening
– Western blot confirmation if positive/equivocal
– Shows specific antibody band patterns
– Familiar to clinicians

Modified Two-Tier Testing:
– First-tier EIA
– Second-tier EIA with different antigens
– Automated and standardized
– Faster results

Both approaches have advantages. Western blot provides specific antigen reactivity information. MTTT offers standardization and reduces subjective interpretation.

But both share the same fundamental constraint: they detect antibodies, not active infection.

Neither solves early seronegativity (antibodies take weeks to develop). Neither addresses immune suppression, antibiotic-altered responses, or interpretation of persistent symptoms after treatment.

Changing testing methodology changes laboratory workflow—it does not change the biology of Borrelia infection or immune response timing.

Why Clinical Judgment Still Matters More Than Testing Method

The debate over Western blot versus Modified Two-Tier Testing highlights a larger truth: laboratory methodology matters less than clinical judgment.

Whether using traditional two-tier testing or MTTT, serologic testing remains most reliable several weeks after infection begins—precisely when early treatment windows have already passed.

Up to 30% of patients never develop the classic erythema migrans rash. Many present with nonspecific symptoms that overlap with other conditions. Some test negative despite active infection.

This is why the CDC emphasizes that Lyme disease is a clinical diagnosis supported by laboratory testing—not defined by it.

When symptoms and exposure history suggest Lyme disease, treatment should not be delayed pending serologic confirmation. Early intervention prevents progression to chronic manifestations.

Both Western blot and MTTT provide useful information when positive. But negative results—regardless of methodology—do not rule out infection when clinical suspicion remains high.

Frequently Asked Questions

Is Modified Two-Tier Testing better than Western blot?
MTTT offers standardization and automation but does not fundamentally solve early seronegativity. Both approaches have similar limitations in detecting early infection when treatment is most effective.

Can I still get Western blot testing?
Yes. Western blot remains available and is still used by many clinicians. The introduction of MTTT provided an alternative, not a replacement.

Which testing method should I use?
This is a clinical decision best made with your physician based on your symptoms, exposure history, and timing. Both methods have limitations, and clinical judgment often matters more than test selection.

Why doesn’t either test work well in early infection?
Both Western blot and MTTT detect antibodies, which take weeks to develop. Early Lyme disease occurs before the immune system has mounted a detectable antibody response.

What if my test is negative but I have symptoms?
Negative serologic testing does not rule out Lyme disease, especially in early infection. Clinical diagnosis based on symptoms and exposure history is appropriate when testing is unreliable.

Clinical Takeaway

The proposal to eliminate Western blot testing in 2017 sparked important debate about Lyme disease diagnostics. The concern was not whether standardization and automation had value—they do. The concern was whether replacing one imperfect test with another imperfect test would actually help patients. The answer, years later, is nuanced. Modified Two-Tier Testing now exists alongside Western blot as an alternative approach. Both methods improve diagnostic certainty when positive. But neither solves the fundamental challenge: early Lyme disease, when treatment matters most, is precisely when serologic testing is least reliable. This is why “saving” the Western blot in 2017 was important—not because Western blot is perfect, but because eliminating a familiar, albeit flawed, tool before proving its replacement was substantially better would have served laboratory standardization at the expense of clinical flexibility. Today, clinicians have both options. But the real lesson remains unchanged: clinical judgment based on symptoms and exposure history matters more than which laboratory algorithm processes the blood sample.