Lyme disease testing uses a two-step process, and a single positive or negative result on your lab report doesn’t tell the full story. Both steps must be completed, and the results depend on which type of antibody your body has produced and how long it’s been since you were potentially infected. Here’s how to make sense of what your results actually mean.
How the Two-Tier System Works
Your Lyme test results come from a two-step process. The first step is a screening test (an enzyme immunoassay, or EIA) that checks your blood for antibodies against the Lyme bacteria. This initial test is sensitive but not very specific, meaning it catches most true cases but also flags some people who don’t actually have Lyme.
If that first test comes back negative, no further testing is done and the result is considered negative. If it comes back positive or equivocal (borderline), the lab automatically runs a second, more specific test on the same blood sample. You don’t need a new blood draw. The second test is traditionally a Western blot, which identifies antibodies targeting specific proteins on the Lyme bacteria. Some newer labs use a second EIA instead of a Western blot, an approach called modified two-tier testing (MTTT).
The key point: a positive first-tier result alone does not mean you have Lyme disease. It only means your blood moves on to step two.
What “Equivocal” Means on Your Report
An equivocal (or “indeterminate”) result on the first-tier screening test means your antibody level fell in a gray zone, not clearly positive and not clearly negative. Labs treat equivocal the same as positive for the purpose of moving to the second step. So if your report shows “equivocal” on the EIA, you should see a Western blot or second immunoassay result alongside it. If only the equivocal result appears with no second-tier test, the testing wasn’t completed.
Reading the Western Blot
The Western blot separates proteins from the Lyme bacteria and checks which ones your antibodies react to. Each reactive protein shows up as a “band” on the test strip, identified by its molecular weight in kilodaltons (kDa). Your lab report will list these bands and indicate which ones are present. Two separate blots are run: one for IgM antibodies (the early responders) and one for IgG antibodies (the longer-lasting ones).
IgM Western Blot
The IgM blot looks at three specific bands: 23, 39, and 41 kDa. For a positive IgM result, at least two of these three bands must be present. If only one band lights up, the IgM blot is considered negative. The IgM result is only clinically meaningful if you’ve had symptoms for 30 days or less, because IgM antibodies peak early in infection. A positive IgM blot in someone who has been sick for months is more likely a false positive than evidence of new infection.
IgG Western Blot
The IgG blot examines ten bands: 18, 23, 28, 30, 39, 41, 45, 58, 66, and 93 kDa. For a positive IgG result, at least five of these ten bands must be present. This is a high bar, which is intentional. It makes the test very specific, so a positive IgG blot is strong evidence of Lyme infection at some point.
If your report shows four IgG bands, that’s technically negative by standard criteria, even though it may feel close. The five-band threshold exists because many of these proteins overlap with proteins found in other bacteria, and requiring five reduces the chance of a false match.
IgM vs. IgG: What the Timing Tells You
Your body produces IgM antibodies first, typically within one to two weeks of infection. IgG antibodies follow in two to four weeks and persist much longer. This timeline matters for interpreting your results.
If only IgM is positive and your symptoms started recently, the result is consistent with early or acute Lyme disease. If only IgG is positive, it suggests either a later-stage infection or a past infection your immune system already responded to. If both are positive, it could reflect an infection that started weeks ago, with your immune response transitioning from early to sustained.
One important wrinkle: IgG antibodies can remain detectable for 10 to 20 years after successful treatment. In a study of patients treated for Lyme arthritis, 62% still had detectable IgG antibodies a decade or two later. These lingering antibodies do not indicate active infection. So if you were treated for Lyme in the past, a positive IgG result on a new test doesn’t necessarily mean you’re infected again. Lyme testing cannot distinguish between active infection and immune memory from a previous one.
Why Early Testing Often Misses Lyme
The biggest limitation of Lyme testing is timing. During the first few weeks of infection, when many people develop the characteristic bull’s-eye rash, the two-tier test catches only about 30% to 40% of true cases. Your immune system simply hasn’t produced enough antibodies yet for the test to detect them.
This means a negative result during the first few weeks after a tick bite does not rule out Lyme disease. If you have a bull’s-eye rash or other early symptoms and a negative test, your doctor may still diagnose and treat Lyme based on clinical judgment alone. Sensitivity improves significantly in later stages of infection, once the antibody response has had time to develop.
If your initial test was negative but symptoms persist, retesting a few weeks later can sometimes capture antibodies that weren’t detectable the first time around.
What Can Cause a False Positive
Several conditions can trigger a positive first-tier screening test in people who don’t have Lyme disease. The most common culprits are syphilis, relapsing fever (caused by related bacteria), and viral infections like Epstein-Barr virus (the cause of mono) and cytomegalovirus. People with rheumatoid factor, a protein found in many autoimmune conditions, are also prone to false positives on the initial screen.
This is exactly why the second-tier test exists. The Western blot separates out the individual bacterial proteins, which helps distinguish true Lyme antibodies from look-alikes produced during other infections. A false positive on the screening test will usually fail to meet the strict band criteria on the Western blot. Still, cross-reactivity between syphilis and Lyme can occasionally carry through to the second step, particularly on the 41 kDa band, which corresponds to a flagellar protein shared by several spiral-shaped bacteria.
Modified Two-Tier Testing
Some labs now use a newer approach called modified two-tier testing (MTTT), which replaces the Western blot with a second, different EIA. Instead of looking at individual bands, both tests measure overall antibody levels but target different sets of bacterial proteins. The CDC recognizes this as an acceptable alternative.
With MTTT, your report won’t show individual band results. Instead, you’ll see two immunoassay results. If both are positive or equivocal, Lyme antibodies are considered detected. One limitation of MTTT using combined (total) immunoassays is that it can’t always tell you whether the antibodies are IgM or IgG, so it may not distinguish early from late infection as clearly as the traditional approach. When separate IgM and IgG second-tier assays are used, a positive IgM with a negative IgG is interpreted as consistent with acute or recent infection.
Putting Your Results Together
Here’s how to read the most common result combinations on a standard two-tier report:
- First-tier negative: No evidence of Lyme antibodies. If you were tested within the first few weeks of a suspected bite, this doesn’t completely rule out infection.
- First-tier positive, Western blot negative (both IgM and IgG): The screening test was likely a false positive. No evidence of Lyme disease.
- First-tier positive, IgM positive, IgG negative: Consistent with early Lyme disease if symptoms began within the past 30 days. If symptoms have been present longer than 30 days, this pattern is unreliable and may be a false positive.
- First-tier positive, IgG positive: Evidence of Lyme antibodies, consistent with current or past infection. Does not confirm active disease on its own, especially in someone previously treated for Lyme.
- First-tier positive, both IgM and IgG positive: Strongest serologic evidence. Consistent with Lyme disease, though the clinical picture and symptom timeline still matter for interpretation.
No Lyme test result exists in isolation. Your symptoms, exposure history, how long you’ve been sick, and whether you’ve had Lyme before all factor into what the numbers on your report actually mean.