What is Dr. Zubair Khalid's research focus?

Dr. Zubair Khalid specializes in molecular virology, mRNA vaccine development, and computational biology, with a focus on avian pathogens like IBDV and Avian Reovirus.

Where is Dr. Zubair Khalid currently working?

Dr. Zubair Khalid is a Postdoctoral Research Associate at the University of Maryland (UMD), specifically within the Department of Animal and Avian Sciences.

Lyme Western Blot: Principles, Interpretation, and Pitfalls

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-11

Introduction to Lyme Western Blot

The Lyme western blot is a confirmatory immunoblot assay used to detect antibodies against Borrelia burgdorferi, the spirochete bacterium that causes Lyme disease. Unlike a screening test, which prioritizes sensitivity to avoid missing any potential cases, the western blot is deployed as a second-tier test to increase diagnostic specificity. Its purpose is to distinguish true infection from false-positive results generated by less specific assays, particularly in patients with vague symptoms such as fatigue, arthralgia, or cognitive complaints.

The test detects the presence of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies directed against specific B. burgdorferi proteins. Because the bacterium expresses different antigens at different stages of infection, the pattern of antibody reactivity—the "bands" on the blot—carries diagnostic information about both the fact and the duration of infection. The Lyme western blot is not a standalone diagnostic tool; it is interpreted within a two-tier testing algorithm established by the Centers for Disease Control and Prevention (CDC) and requires correlation with clinical presentation.

This article explains the molecular basis of the test, the step-by-step mechanics of the procedure, the criteria for interpretation, and the common errors that lead to misdiagnosis. For a broader overview of the technique itself, see the Western Blot Test.

The Biology of Borrelia burgdorferi and Immune Response

Borrelia burgdorferi is a Gram-negative, motile spirochete transmitted to humans through the bite of infected Ixodes ticks. The bacterium has a highly unusual genome consisting of a linear chromosome approximately 910 kilobases in length plus more than 20 linear and circular plasmids. This genomic architecture allows extensive antigenic variation, which complicates both the host immune response and laboratory detection.

When the spirochete enters the host through a tick bite, it begins to replicate in the skin at the site of inoculation, producing the characteristic erythema migrans rash in approximately 70–80% of infected individuals. Over days to weeks, the bacterium disseminates hematogenously to joints, the heart, and the nervous system. The host immune system mounts a humoral response, producing IgM antibodies within 2–4 weeks of infection, followed by IgG antibodies that peak at 4–6 weeks and persist for years.

The proteins of B. burgdorferi that are most immunogenic and diagnostically useful are largely outer surface proteins and flagellar components. The flagellin protein, encoded by the flaB gene, is a 41 kDa protein that forms the axial filament of the spirochete's periplasmic flagella. It is highly immunogenic and produces an early and strong antibody response, but it is also cross-reactive with flagellins of other bacteria, including Treponema pallidum and oral spirochetes, which limits its specificity.

The outer surface proteins OspA (31 kDa) and OspC (23–25 kDa) are differentially expressed during the tick-to-mammal transmission cycle. OspA is expressed primarily in the tick midgut and is downregulated during feeding, while OspC is upregulated as the spirochete migrates to the tick salivary glands and enters the mammal. OspC is a major early antigen, and IgM antibodies against it appear within the first weeks of infection. OspA antibodies, in contrast, are rarely produced during natural infection but are prominent in individuals vaccinated with the now-discontinued LYMErix vaccine, a distinction with important interpretive consequences.

Other diagnostically significant antigens include the 39 kDa BmpA (basic membrane protein A), encoded by the bmpA gene, which is highly specific for B. burgdorferi; the 58 kDa antigen (GroEL, a heat shock protein); the 66 kDa protein (a flagellar hook protein); and the 93 kDa protein (a putative surface antigen). The 18 kDa and 21 kDa proteins are also used in some interpretive criteria, though their diagnostic value is debated.

Key Antigens in Lyme Disease

The table below summarizes the most diagnostically relevant B. burgdorferi antigens, their molecular weights, and their expression patterns.

Antigen	Molecular Weight (kDa)	Gene	Expression/Notes
OspC	23–25	ospC	Early infection; highly variable; strong IgM target
OspA	31	ospA	Tick midgut; vaccine antigen; rare in natural infection
Flagellin	41	flaB	Periplasmic flagella; early response; cross-reactive
BmpA	39	bmpA	Highly specific for B. burgdorferi
GroEL	58	groEL	Heat shock protein; cross-reactive with other bacteria
Flagellar hook	66	flgE	Structural protein; late IgG response
Unknown surface protein	93	—	Late infection; strong IgG response

How Western Blotting Works

The western blot is a technique for detecting a specific protein within a complex mixture. In the context of Lyme disease, the "mixture" is a lysate of B. burgdorferi that has been separated by electrophoresis and transferred to a membrane. The patient's serum is then applied, and any antibodies present bind to their cognate antigens. The procedure proceeds through four major phases: protein separation, membrane transfer, antibody probing, and detection.

Protein Separation

The first step is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). B. burgdorferi cells are cultured, harvested, and lysed in a buffer containing SDS, a denaturing detergent that binds to proteins at a ratio of approximately 1.4 g SDS per gram of protein. SDS imparts a uniform negative charge proportional to protein length, effectively eliminating differences in intrinsic charge. The sample is also heated to 95–100°C for 5 minutes in the presence of a reducing agent such as β-mercaptoethanol or dithiothreitol, which breaks disulfide bonds and ensures complete denaturation.

The denatured protein mixture is loaded into wells of a polyacrylamide gel composed of a stacking gel (typically 4% acrylamide, pH 6.8) and a resolving gel (typically 10–12% acrylamide, pH 8.8). An electric field is applied, and proteins migrate through the gel matrix. Smaller proteins migrate faster through the pores, so proteins are separated by molecular weight, with the smallest at the bottom. A molecular weight ladder is run in an adjacent lane to allow size calibration.

For Lyme western blots, commercial kits use a standardized lysate of B. burgdorferi strain B31, which has been fully sequenced. The lysate is electrophoresed, and the separated proteins are then transferred to a membrane.

Membrane Transfer

After SDS-PAGE, the separated proteins are transferred from the gel to a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. This is typically done using a "wet" transfer apparatus in which the gel and membrane are sandwiched between filter paper and placed in a transfer cassette. The cassette is submerged in transfer buffer (typically 25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) and an electric current is applied perpendicular to the gel surface. Proteins, which carry a net negative charge from SDS, migrate toward the anode and become immobilized on the membrane through hydrophobic interactions.

The transfer is usually performed at 100 V for 1 hour at 4°C, or at 30 V overnight. Methanol in the transfer buffer improves protein binding to the membrane but can cause shrinkage of the gel; some protocols use SDS (0.1%) in the transfer buffer to improve transfer efficiency of high-molecular-weight proteins. After transfer, the membrane is stained with a reversible dye such as Ponceau S to confirm that protein transfer occurred and to visualize the molecular weight markers.

Antibody Detection

The membrane is first incubated in a blocking solution—typically 5% non-fat dry milk or bovine serum albumin in Tris-buffered saline with Tween 20 (TBST: 20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.6)—to saturate nonspecific protein-binding sites. This step is critical; without it, antibodies will bind nonspecifically to the membrane surface, producing high background and false bands.

The patient's serum is then diluted (typically 1:100 to 1:200 for Lyme western blots) in blocking buffer and incubated with the membrane for 1–2 hours at room temperature or overnight at 4°C. During this incubation, antibodies in the serum bind to their specific B. burgdorferi antigens immobilized on the membrane. The membrane is then washed thoroughly with TBST to remove unbound antibodies.

Next, an enzyme-conjugated secondary antibody is applied. This secondary antibody is an anti-human immunoglobulin—either anti-human IgM or anti-human IgG, depending on which antibody class is being tested—conjugated to an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The secondary antibody binds to the Fc region of the patient's antibodies that are already bound to the membrane. After another series of washes, a chemiluminescent or chromogenic substrate is added.

For HRP, the substrate is typically luminol, which is oxidized in the presence of hydrogen peroxide to produce light. The light is captured on X-ray film or by a digital imager. For AP, substrates such as BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium) produce a purple precipitate directly on the membrane. In either case, the result is a series of dark bands at positions corresponding to the molecular weights of the B. burgdorferi antigens recognized by the patient's antibodies.

The entire procedure, from sample preparation to final image, is conceptually identical to that used in other applications, such as the Histone Western Blot, though the specific antibodies and lysates differ.

The Two-Tier Testing Protocol

The Lyme western blot is never performed as a first-line test. The standard diagnostic algorithm, recommended by the CDC and the Infectious Diseases Society of America (IDSA), is a two-tier protocol designed to balance sensitivity and specificity.

First-Tier Tests

The first tier consists of an enzyme-linked immunosorbent assay (ELISA) or an immunofluorescence assay (IFA). These tests are designed to be highly sensitive—they should catch nearly all true infections—but they are not highly specific. The ELISA works by coating a microtiter plate with a whole-cell lysate of B. burgdorferi or a recombinant antigen mixture, adding the patient's serum, and detecting bound antibodies with an enzyme-conjugated anti-human antibody. A colorimetric or chemiluminescent signal is generated, and the optical density is compared to a cutoff value.

The sensitivity of first-tier ELISA is approximately 70–80% in early localized disease (within 2 weeks of symptom onset) and greater than 95% in later stages. However, the specificity is only about 95%, meaning that 5% of healthy individuals will test positive. This is acceptable for a screening test because the purpose is to rule out infection, not to confirm it. A negative first-tier test effectively excludes Lyme disease in most clinical scenarios, and no further testing is performed.

Second-Tier Confirmation

If the first-tier test is positive or equivocal, the same serum sample is reflexively tested by western blot. The western blot is more specific because it detects antibodies against individual B. burgdorferi proteins rather than a pooled antigen mixture. A patient who is truly infected will typically produce antibodies against multiple specific antigens, producing a characteristic band pattern. A patient who has cross-reactive antibodies from another infection or an autoimmune condition will usually produce a weaker, less specific pattern.

The two-tier algorithm is deliberately conservative. It sacrifices some sensitivity in early disease—when the antibody response is still developing—to gain specificity and avoid false-positive diagnoses. This is particularly important because Lyme disease is overdiagnosed in clinical practice, and many patients with chronic fatigue, fibromyalgia, or other conditions are incorrectly labeled as having Lyme disease based on a single positive ELISA.

Interpreting Lyme Western Blot Bands

Interpretation of the Lyme western blot requires knowledge of which bands are diagnostically significant and how many must be present for a positive result. The CDC established standardized criteria in 1995, and these remain the reference standard for clinical diagnosis in the United States.

IgM Bands

IgM antibodies appear early in infection, typically within 2–4 weeks after the tick bite. The IgM western blot is therefore most useful in early localized or early disseminated disease. The CDC criteria require that at least 2 of the following 3 bands be present for a positive IgM result:

23 kDa (OspC)
39 kDa (BmpA)
41 kDa (Flagellin)

The IgM response is transient. It typically peaks at 3–6 weeks and then declines, even without treatment. By 8 weeks, the IgM response may be undetectable, so a negative IgM western blot does not exclude infection in a patient who has been symptomatic for more than 6–8 weeks.

IgG Bands

IgG antibodies appear later, typically 4–6 weeks after infection, and persist for years. The IgG western blot is therefore more useful in late or disseminated disease. The CDC criteria require that at least 5 of the following 10 bands be present for a positive IgG result:

18 kDa
23 kDa (OspC)
28 kDa
30 kDa
39 kDa (BmpA)
41 kDa (Flagellin)
45 kDa
58 kDa (GroEL)
66 kDa
93 kDa

The requirement for 5 of 10 bands is intentionally stringent. In a study of healthy blood donors, fewer than 1% had 5 or more IgG bands, whereas the vast majority of patients with culture-confirmed late Lyme disease met the criteria.

CDC Criteria

The table below summarizes the CDC interpretive criteria for both IgM and IgG western blots.

Antibody Class	Bands Evaluated	Minimum Bands for Positive	Typical Time to Positivity
IgM	23, 39, 41 kDa	2 of 3	2–4 weeks
IgG	18, 23, 28, 30, 39, 41, 45, 58, 66, 93 kDa	5 of 10	4–6 weeks

It is important to note that the presence of a single band—even the highly specific 39 kDa band—is not sufficient for a positive result. The criteria are deliberately based on the number of bands, not the identity of any single band, because no single antigen is both 100% sensitive and 100% specific.

Clinical Utility and Limitations

The Lyme western blot is a powerful confirmatory tool when used correctly, but it has well-defined limitations that must be understood to avoid misdiagnosis.

Early vs. Late Disease

The sensitivity of the western blot depends heavily on the stage of disease. In early localized disease (erythema migrans present, <2 weeks duration), the sensitivity of the IgM western blot is only approximately 40–60%. This is because the antibody response takes time to develop. A patient tested too early may have a negative western blot despite active infection. For this reason, the CDC recommends that patients with classic erythema migrans and a history of tick exposure be treated empirically without laboratory testing.

In contrast, the sensitivity of the IgG western blot in late disease (arthritis, neurologic manifestations) exceeds 95%. A negative IgG western blot in a patient with suspected late Lyme disease strongly argues against the diagnosis.

Cross-Reactivity

Cross-reactivity is a major source of false-positive results. Antibodies produced against other spirochetes—particularly Treponema pallidum (syphilis), Treponema denticola (oral spirochetes), and Leptospira species—can bind to B. burgdorferi antigens, especially the 41 kDa flagellin. Patients with other infections, including Epstein-Barr virus, cytomegalovirus, and rheumatoid arthritis, may also produce antibodies that cross-react with B. burgdorferi proteins. The two-tier algorithm mitigates this problem by requiring a positive or equivocal first-tier test before performing the western blot, but cross-reactivity can still produce false-positive western blots, particularly the IgM blot.

Common Pitfalls and Misinterpretations

Several recurring errors lead to incorrect interpretation of Lyme western blots. Understanding these pitfalls is essential for both clinicians and students.

Band Misidentification

The most common error is misidentifying bands on the membrane. Commercial western blot strips are calibrated with a molecular weight ladder, but the exact positions of bands can vary slightly between lots. A band at 22 kDa may be mistakenly called a 23 kDa OspC band, or a band at 40 kDa may be called a 41 kDa flagellin band. This is particularly problematic because the 23 kDa and 41 kDa bands are both part of the IgM criteria. Laboratories must use strict quality control measures, including running control sera with known band patterns, to ensure accurate band identification.

Timing of Test

Performing the western blot too early is a common error. A patient who presents within 1 week of a tick bite and symptom onset will almost certainly have a negative western blot because the antibody response has not yet developed. A negative result at this stage does not rule out Lyme disease and should not be used to withhold treatment if the clinical picture is consistent. Conversely, performing only an IgM western blot in a patient with months of symptoms is inappropriate; the IgM response has likely waned, and the IgG blot is the appropriate test.

Vaccination History

The LYMErix vaccine, which was available from 1998 to 2002, was based on recombinant OspA. Vaccinated individuals produce high-titer anti-OspA antibodies, which appear as a strong 31 kDa band on the western blot. Because OspA is not a criterion band for either IgM or IgG positivity, a vaccinated individual will not meet the CDC criteria based on the OspA band alone. However, the presence of an OspA band can confuse interpretation if the laboratory or clinician is not aware of the vaccination history. Moreover, some vaccinated individuals may have additional bands due to prior exposure or cross-reactivity, potentially leading to a false-positive interpretation. Clinicians should always inquire about vaccination history when interpreting a Lyme western blot.

Over-Reliance on the Test

The western blot is a laboratory test, not a diagnosis. A positive western blot in a patient with no clinical symptoms and no history of tick exposure is more likely a false positive than a true infection. The CDC criteria were developed to maximize specificity, but no test is perfect. The positive predictive value of the western blot depends on the pretest probability of Lyme disease. In a patient with a low pretest probability—for example, no tick exposure, no erythema migrans, and only vague fatigue—a positive western blot is more likely to be a false positive than a true positive. Conversely, in a patient with a classic erythema migrans rash and a history of tick exposure, a positive western blot merely confirms what is already clinically apparent.

Ignoring the Two-Tier Algorithm

Some clinicians order a western blot without first performing an ELISA, or they order both tests simultaneously. This violates the two-tier protocol and increases the risk of false-positive results. The western blot should only be interpreted in the context of a positive or equivocal first-tier test. If the ELISA is negative, the western blot should not be performed, and if it is performed, a positive result should be interpreted with caution.

Practical Summary: Key Takeaways

The Lyme western blot is a confirmatory immunoblot that detects IgM and IgG antibodies against B. burgdorferi proteins. It is used only after a positive or equivocal first-tier ELISA or IFA, as part of the CDC-recommended two-tier algorithm. The test separates B. burgdorferi lysate proteins by SDS-PAGE, transfers them to a membrane, and probes with patient serum followed by enzyme-conjugated anti-human IgM or IgG. A positive IgM result requires at least 2 of 3 bands (23, 39, 41 kDa); a positive IgG result requires at least 5 of 10 bands (18, 23, 28, 30, 39, 41, 45, 58, 66, 93 kDa). The test is most sensitive in late disease and least sensitive in early disease. Common pitfalls include misidentifying bands, testing too early, ignoring vaccination history, and over-relying on the test without clinical correlation.

Frequently Asked Questions

What is a western blot for Lyme disease?

A Lyme western blot is a laboratory test that detects antibodies against specific proteins of Borrelia burgdorferi, the bacterium that causes Lyme disease. It is used as a confirmatory test after a positive or equivocal first-tier screening test, such as an ELISA. The test produces a pattern of bands on a membrane, each corresponding to a specific B. burgdorferi antigen, and the pattern is interpreted according to standardized CDC criteria.

How does the Lyme western blot work?

The test begins with a lysate of B. burgdorferi that is separated by SDS-PAGE, which sorts proteins by molecular weight. The proteins are transferred to a nitrocellulose or PVDF membrane, and the patient's serum is applied. Any antibodies in the serum bind to their specific antigens on the membrane. After washing, an enzyme-conjugated anti-human IgM or IgG antibody is added, followed by a substrate that produces a visible or chemiluminescent signal. The result is a series of bands, each corresponding to a B. burgdorferi protein recognized by the patient's immune system.

Why is the western blot used for Lyme disease?

The western blot is used because it is more specific than the ELISA. The ELISA detects antibodies against a mixture of B. burgdorferi antigens and can produce false positives due to cross-reactivity with other bacteria or autoimmune conditions. The western blot resolves this by identifying antibodies against individual, specific B. burgdorferi proteins. The two-tier algorithm—ELISA followed by western blot—maximizes both sensitivity and specificity.

What do the bands on a Lyme western blot mean?

Each band corresponds to a specific B. burgdorferi protein, identified by its molecular weight. For example, a band at 23 kDa corresponds to OspC, a band at 39 kDa corresponds to BmpA, and a band at 41 kDa corresponds to flagellin. The presence of a band means the patient's immune system has produced antibodies against that protein. The pattern of bands—which ones are present and how many—determines whether the test is positive, negative, or equivocal.

What are the CDC criteria for a positive Lyme western blot?

For IgM, a positive result requires at least 2 of 3 bands: 23, 39, and 41 kDa. For IgG, a positive result requires at least 5 of 10 bands: 18, 23, 28, 30, 39, 41, 45, 58, 66, and 93 kDa. These criteria were established in 1995 and remain the standard for clinical diagnosis in the United States.

Can a Lyme western blot be false positive?

Yes. False positives can occur due to cross-reactivity with other spirochetes (such as Treponema pallidum), other infections (such as Epstein-Barr virus), or autoimmune conditions. The IgM western blot is particularly prone to false positives because the 41 kDa flagellin band is cross-reactive. False positives are more likely in patients with a low pretest probability of Lyme disease, which is why clinical correlation is essential.

When should a Lyme western blot be performed?

A Lyme western blot should be performed only after a positive or equivocal first-tier test (ELISA or IFA) in a patient with clinical symptoms consistent with Lyme disease. It should not be performed in patients with no symptoms or in those with a low pretest probability of infection. For patients with classic erythema migrans and tick exposure, treatment should be initiated without laboratory testing, as the test may be negative in early disease.

Key Takeaways

The Lyme western blot is a confirmatory second-tier test used only after a positive or equivocal ELISA or IFA, per the CDC two-tier algorithm.
The test detects IgM and IgG antibodies against specific B. burgdorferi proteins separated by SDS-PAGE and transferred to a membrane.
A positive IgM result requires 2 of 3 bands (23, 39, 41 kDa); a positive IgG result requires 5 of 10 bands (18, 23, 28, 30, 39, 41, 45, 58, 66, 93 kDa).
The test is highly sensitive in late disease (>95% for IgG) but poorly sensitive in early disease (<60% for IgM), so a negative result in early disease does not exclude infection.
False positives occur due to cross-reactivity with other spirochetes, viral infections, and autoimmune conditions; the IgM blot is especially prone to false positives.
Common pitfalls include misidentifying bands, testing too early, ignoring vaccination history (LYMErix produces an OspA band), and over-relying on the test without clinical correlation.
The western blot is a laboratory tool, not a diagnosis; interpretation always requires integration with clinical history, symptoms, and exposure risk.