Single-tier C6 peptide ELISA kit vs 2-tier testing

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
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Single-tier C6 peptide ELISA kit vs 2-tier testing

Post by RitaA » Tue 23 Oct 2012 9:35
Diagn Microbiol Infect Dis. 2012 Oct 10. pii: S0732-8893(12)00360-4. doi: 10.1016/j.diagmicrobio.2012.09.003. [Epub ahead of print]

Single-tier testing with the C6 peptide ELISA kit compared with two-tier testing for Lyme disease.

Wormser GP, Schriefer M, Aguero-Rosenfeld ME, Levin A, Steere AC, Nadelman RB, Nowakowski J, Marques A, Johnson BJ, Dumler JS.


Division of Infectious Diseases, New York Medical College, Valhalla, NY 10595, USA.



The 2-tier serologic testing protocol for Lyme disease has a number of shortcomings including low sensitivity in early disease; increased cost, time, and labor; and subjectivity in the interpretation of immunoblots.


The diagnostic accuracy of a single-tier commercial C6 ELISA kit was compared with 2-tier testing.


The C6 ELISA was significantly more sensitive than 2-tier testing with sensitivities of 66.5% (95% confidence interval [CI] 61.7-71.1) and 35.2% (95% CI 30.6-40.1), respectively (P < 0.001) in 403 sera from patients with erythema migrans. The C6 ELISA had sensitivity statistically comparable to 2-tier testing in sera from Lyme disease patients with early neurologic manifestations (88.6% versus 77.3%, P = 0.13) or arthritis (98.3% versus 95.6%, P = 0.38). The specificities of C6 ELISA and 2-tier testing in over 2200 blood donors, patients with other conditions, and Lyme disease vaccine recipients were found to be 98.9% and 99.5%, respectively (P < 0.05, 95% CI surrounding the 0.6 percentage point difference of 0.04 to 1.15).


Using a reference standard of 2-tier testing, the C6 ELISA as a single-step serodiagnostic test provided increased sensitivity in early Lyme disease with comparable sensitivity in later manifestations of Lyme disease. The C6 ELISA had slightly decreased specificity. Future studies should evaluate the performance of the C6 ELISA compared with 2-tier testing in routine clinical practice.

Copyright © 2012 Elsevier Inc. All rights reserved.

[PubMed - as supplied by publisher]
Some additional information from the blog Relative Risk, with my blue highlighting: ... stics.html
16 OCTOBER 2012

C6 Diagnostics

Notes from:
Wormser GP, Schriefer M, Aguero-Rosenfeld ME, Levin A, Steere AC, Nadelman RB, Nowakowski J, Marques A, Johnson BJ, Dumler JS. Single-tier testing with the C6 peptide ELISA kit compared with two-tier testing for Lyme disease. Diagn Microbiol Infect Dis. 2012 Oct 10.

The public health service recommendations in support of 2-tier testing (CDC, 1995) provide for the development of alternatives to 1 or both steps provided that equal or better performance is demonstrated by such alternative methods (Proceedings of the Second National Conference on Serologic Diagnosis of Lyme Disease, 1995). In principle, an ELISA of sufficiently high specificity and sensitivity would provide diagnostic information similar to existing 2-tier testing. A variety of recombinant and synthetic antigens have been evaluated in previous studies for use in serodiagnosis of Lyme disease. In particular, the VlsE protein and the highly conserved 25-amino acid peptide (“C6 peptide”) derived from the sixth invariant region of this protein have been shown to be both sensitive and specific antigens in the ELISA and immunoblot formats. The aim of the present study was to determine whether the C6 ELISA by itself is a suitable alternative to 2-tier testing by evaluating the comparative diagnostic accuracy in over 550 sera from well-characterized Lyme disease patients and in more than 2200 control sera.

The C6 Lyme ELISA kit (Immunetics, Boston, MA, USA), a test kit approved by the FDA for use as a first-tier assay, was modified for this study. Kits provided by the manufacturer for this study incorporated a simplified and comparable cut-off formula that was based on a negative calibrator serum. This modification yielded sensitivity and specificity statistically equivalent to what had been demonstrated with the original cut-off formula. Reproducibility testing of the C6 ELISA kit using the simplified cut-off yielded intra-assay and interassay coefficients of variation (CV) of 10% and 11.6%, respectively. Intra-assay and interassay CVs for the kit with the original cut-off were 9.5% and 14.3%, respectively. In all other respects, the modified and original kits were the same. The C6 peptide used as antigen in the kit is derived from the B. burgdorferi B31 strain sequence, which differs from the originally described IP90 sequence by 4 amino acids. The kit is formatted as an indirect ELISA in which both IgG and IgM antibodies to C6 peptide are detected by an enzyme conjugate. The C6 ELISA testing was performed at New York Medical College, the CDC, and at Immunetics.

The patient sera selected for this study were chosen to represent a broad range of clinical manifestations of Lyme disease from well-characterized patients. The sera comprised multiple reference panels from different sources that had been previously collected and frozen at −80 °C. The 569 Lyme disease sera studied were obtained from 528 patients. Erythema migrans was defined based on clinical diagnosis alone for 172 sera and on clinical diagnosis in conjunction with microbiologic confirmation of B. burgdorferi infection by either culture or polymerase chain reaction (PCR) for 231 sera.

In this evaluation of the performance of serologic methods for detection of antibodies to B. burgdorferi, the C6 ELISA as a standalone test had significantly greater sensitivity than either the WCS ELISA-based 2-tier testing method (66.5% versus 35.2%, P < 0.001) or the C6 ELISA-based 2-tier testing method (66.5% versus 34.5%, P < 0.001) for patients with erythema migrans.

In contrast, on sera from the groups of patients with neurologic or rheumatologic manifestations of Lyme disease, each of the 3 testing methods had high sensitivity without a statistically significant difference (P < 0.05). The specificity of either of the 2-tier testing methods on more than 2200 control sera, however, exceeded that of the C6 ELISA alone by 0.6 percentage points (99.5% versus 98.9%, 95% CI surrounding the difference of 0.04 to 1.15), and this difference was significant (P < 0.05). The 0.4–0.7 percentage point lower specificity values observed for these serologic methods in the endemic compared with the nonendemic blood donor group might be explained by the inclusion of individuals with prior exposure to B. burgdorferi in the endemic group, although this hypothesis cannot be evaluated due to the lack of information on donor histories. Our overall results for specificity are quite comparable to that for the C6 ELISA in another published study that involved a sizeable but smaller number of control serum samples (present study 98.9% [2216/2241] versus 98.4% [1279/1300], P = 0.22). The authors of that report (Branda et al., 2011) observed that a novel 2-tier strategy using a WCS ELISA followed by the C6 ELISA would have increased the specificity to 99.5%, a figure that was identical to the conventional WCS ELISA–immunoblot-based 2-tier testing. A reanalysis of our study data with such an algorithm yielded the same 99.5% specificity value.

Interestingly, however, in the group of individuals with other disease conditions as well as in the group of LymeRx® vaccine recipients, which collectively might be more representative of the target population for testing than blood donors, the specificity of this 2-tier ELISA algorithm was identical to that of the C6 ELISA alone. Serial 2-step algorithms generically offer improved specificity at the cost of some loss of sensitivity; in the present study, the serial ELISA algorithm would result in a loss of about 6% in sensitivity for erythema migrans patients, and a similar decrease was observed by Branda et al. (2011). A cost–benefit analysis of this tradeoff may indicate its utility relative to other algorithms.

Overall, our results indicate that the C6 ELISA should be preferred over 2-tier testing in patients with early infection, for example, in patients with a skin lesion(s) for whom a clinical diagnosis is uncertain.

Given the ease of performance, objectivity and greater simplicity of single-tier testing using an ELISA, the C6 ELISA may be preferred over 2-tier testing in patients with early neurologic Lyme disease and other manifestations of early disseminated Lyme disease.

An advantage of 2-tier testing over the C6 ELISA is its higher specificity. The high specificity of 2-tier testing found in this study, however, is not consistent with observations in clinical practice where overreading of weak bands on the IgM immunoblot in particular has served to reduce specificity and stimulate interest in alternative testing strategies to avoid the IgM immunoblot entirely. Another potential advantage of 2-tier testing over C6 ELISA is that the former provides information on the presence of an expanded IgG response specifically, a serologic prerequisite for the diagnosis of late Lyme disease. Thus, for the diagnosis of late Lyme disease, it may be advantageous to perform an IgG immunoblot to supplement the C6 or another ELISA.
Just for reference purposes, here's an earlier article by Wormser et al about the C6 test:
Clin Infect Dis. 2008 Oct 1;47(7):910-4.

Effect of Borrelia burgdorferi genotype on the sensitivity of C6 and 2-tier testing in North American patients with culture-confirmed Lyme disease.

Wormser GP, Liveris D, Hanincová K, Brisson D, Ludin S, Stracuzzi VJ, Embers ME, Philipp MT, Levin A, Aguero-Rosenfeld M, Schwartz I.


Division of Infectious Diseases, Department of Medicine, New York Medical College, Valhalla, NY 10595, USA.



A potential concern with any serologic test to detect antibodies to Borrelia burgdorferi is whether the epitopes incorporated in the test provide sufficient cross-reactivity to detect infection with all of the pathogenic strains of the species. This is a particular concern for the C6 test, which is based on reactivity to a single peptide.


C6 testing and 2-tier testing were performed on acute-phase serum samples obtained from >158 patients with erythema migrans for whom the genotype of the borrelial isolate was defined on the basis of an analysis of the 16S-23S ribosomal DNA spacer region and/or on the genetic variation of the outer surface protein C gene (ospC). The sonicated whole cell-based enzyme-linked immunosorbent assay, the immunoblots used in the 2-tier testing, and the C6 assay all used antigens from B. burgdorferi sensu stricto strain B31.


The sensitivity of C6 testing (69.5%) was greater than that of 2-tier testing (38.9%) (P<.001); the difference in sensitivity, however, was statistically significant only for patients infected with 2 of the 3 ribosomal spacer type-defined genotypes. The lower sensitivity of 2-tier testing was attributable to the low sensitivity of the immunoblot tests, rather than the first-tier enzyme-linked immunosorbent assay. There was also a trend for the sensitivity of 2-tier testing to vary according to the ospC genotype for the 14 genotypes represented in the study (P=.07); this relationship was not observed with C6 testing.


Lack of sensitivity of the C6 test because of strain diversity seems less likely to be a limitation of this serologic test, compared with 2-tier testing in North American patients with early Lyme disease.

[PubMed - indexed for MEDLINE]
Free PMC Article
The full article is here:


Serum specimens from patients with Lyme disease

Serum specimens that had been stored at −80°C were tested using the C6 and 2-tier methods as part of a performance comparison. The serum samples were originally obtained from patients with erythema migrans whose clinical diagnosis was confirmed by the growth of B. burgdorferi from culture of a skin biopsy or blood sample, with use of methods reported elsewhere [6]. Serum specimens were obtained before antibiotic treatment was initiated. The patients who provided the serum samples were subjects in approved research studies at New York Medical College (Valhalla). The patients resided in Westchester County, New York, or in the immediately surrounding geographic areas. Information on duration of illness and number of erythema migrans skin lesions was obtained from study records.


A limitation of our study is that the patients were infected in a single location in New York State or in the immediately surrounding geographic areas; thus, the results may not be generalizable to infections acquired in other locations. Another limitation may be related to the choice of the MarDx/Trinity Biotech immunoblot kits as the second-tier test. This may have contributed to the reduced sensitivity observed in this study, because 1 comparative study has shown that the IgM kit is less sensitive than some other immunoblot assays [27].

The IR6 peptide in some VlsE proteins of strains of other species of B. burgdorferi sensu lato, such as Borrelia afzelii and B. garinii, may vary by up to 5 amino acids, compared with the IR6 region of the VlsE protein of B. burgdorferi B31 [3]. In North America, only strains of B. burgdorferi are known to cause Lyme disease, and variations in the IR6 region among North American isolates of B. burgdorferi have not been directly examined with a large number of isolates. Lack of sensitivity of the commercially available C6 test attributable to strain diversity, although a theoretical concern, has not yet been demonstrated and, on the basis of our findings in New York State, seems unlikely to be a limitation of this serologic test.
Here's the link for the Immunetics C6 peptide test kit:
C6 (B. burgdorferi) Lyme ELISA

For in vitro diagnostic use
Catalog # DK-E352-096


Lyme disease can be difficult to diagnose. Traditional Lyme ELISA tests are short on specificity and can generate over 20 percent false positive results. This uncertain performance can result in time-consuming, expensive Western Blot confirmatory tests. The C6 Lyme ELISA from Immunetics sets a new standard in Lyme test accuracy, and delivers more reliable results sooner and more economically than any other ELISA. It demonstrates 99 percent specificity in an average population – equivalent to the specificity of Western Blot testing and a 10-fold improvement over Whole Cell Sonicate ELISAs. Additionally, the C6 Lyme ELISA is a far more sensitive assay – showing twice the sensitivity of the standard Western Blot in patients with symptoms of acute or early Lyme disease. These features mean that more patients are correctly detected as positive for the disease. From early onset to late stage disseminated infection, C6 Lyme ELISA demonstrates superior results.

Accurate: Western Blot accuracy in an ELISA
Sensitive: More sensitive detection of antibodies in early and late Lyme disease than competing kits
Specific: Eliminates vast majority of other ELISA's false positives at the screening step
Broadly applicable: Detects infection with all strains of Borrelia which cause Lyme disease, including European strains B. garinii and B. afzelii.
Convenient: One hour turnaround time, breakaway strips for any number of samples, ready-to-use reagents and controls included
Cost-effective: Greatly reduces the number of samples sent for expensive and lengthy confirmatory testing


Immunetics has pioneered the use of the C6 peptide, a 26-amino acid sequence within the Borrelia membrane protein VlsE. This is the first truly specific and sensitive marker for infection with the Borrelia burgdorferi spirochete. This peptide has been shown to represent the immunodominant portion of VlsE, containing substantially all of the antigenic reactivity of the whole protein. The C6 peptide sequence is highly antigenic, generating an immune response in nearly all human cases soon after infection, and is specific to Borrelia strains causing Lyme disease. It is not found in other infectious organisms, eliminating cross-reactivity at the source.

Immunetics’ C6 ELISA uses a synthetic version of the C6 peptide antigen*. This precisely defined antigen makes possible a highly sensitive, specific and reproducible ELISA for infection with the B. burgdorferi spirochete. Current ELISA tests do not match the specificity or sensitivity of Immunetics’ C6 ELISA because they are based on Whole Cell Sonicates (WCS) containing multiple antigens, leading to cross-reactivity and inaccurate or ambiguous results.

Along with superior performance, Immunetics’ C6 Lyme ELISA offers a rapid and easy-to-use protocol. Turnaround time is approximately one hour, and tests can be run on automated ELISA systems or manually. The C6 Lyme ELISA kit is approved for in vitro diagnostic use.

U.S. Patents 6,475,492, 6,719,983, and 6,740,744 and International Patents
FDA approved and CE marked
C6 test kits (Cat # DK-E352-096) include:

Coated microwell plate with 96 breakaway wells
Ready to use reagents – everything needed to perform the assay
Positive and negative controls
Record sheet
One-hour protocol instructions

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Re: Single-tier C6 peptide ELISA kit vs 2-tier testing

Post by RitaA » Fri 26 Oct 2012 8:14

Is the C6-peptide ELISA test really the best available option at this time?

Would it be too expensive/impractical to create a test that checks for both VlsE and BBK32 -- and possibly even more antigens -- to cover all possible strains of Bb? Saying that we'll pick up "nearly all" cases of Lyme disease by using the C6-peptide ELISA just doesn't seem good enough to me. Besides, the C6-peptide ELISA still can't distinguish between an active and a past infection.

I've been wondering about this because I seem to recall reading about a possible lack of VlsE when it comes to strain N40.

Now I've just read that a BBK32-based test might be a more appropriate choice: ... poster.pdf
Lyme Disease: Developing an ELISA Based Diagnostic Assay


Lyme disease (LD) is the most common vector-borne disease in temperate climates. The CDC reports a >350% increase in U.S. incidence over the last 15 years. LD is caused by the transmitted bacterium Borrelia burgdorferi (Bb), and is readily treated by antibiotics if infection is diagnosed early. CDC-recommended methods for serological testing for LD have several limitations, including: 1) low sensitivity of the assay in early stages of Bbinfection; 2) poor diagnostic range, resulting in the inability to distinguish between active and prior infections; 3) most current diagnostic tests use Bb lysates or peptides from two infection-associated antigens (VlsE and OspC) to detect serum antibodies against Bb; however, the expression patterns and functions of these antigens during infection are not well understood. Recently, we determined that the Bb early antigen BBK32 mediates bacterial adhesion to vascular surfaces during bloodborne pathogen dissemination. We are producing a library of purified BBK32 sequence variants, domains and peptides for investigation of the BBK32-mediated vascular adhesion mechanism and epitope-mapping of dissemination-blocking BBK32 antibodies. We will use these BBK32 variants to develop an ELISA assay for identification of BBK32 peptides which are most highly immunogenic early in infection (IgM antibodies) and which exhibit a diagnostic range sensitive enough to distinguish between current and previous Bb infections. Development and validation of ELISA assays will be carried out using serum samples from control patients and clinically- and laboratory- diagnosed LD patients obtained from the OAHPP Public Health Laboratory. The sensitivity and specificity of the BBK32-based ELISA assay will be compared to the sensitivity and specificity of commercial kits used for diagnosis of LD. Earlier diagnosis of LD using BBK32-based ELISA assays could lead to more rapid introduction of antibiotic therapy, and more sensitive methods of determining the efficacy of antibiotic treatment.

•Most current diagnostic tests use whole Borrelia lysates or selected antigens to detect serum antibodies against B. burgdorferi.
•ELISA with C6 peptide (26 mer synthetic peptide analogue of the invariable region 6 (IR6) of the VIsE variable major protein like sequences has been shown to be highly sensitive and specific for the detection of B. burgdorferi infection. The C6 assay has a limited diagnostic range.
•More recent assay developments: Luciferase immuno-precipitation system (LIPS) developed for diagnosing B. burgdorferi, infections uses a synthetic antigen called VOVO (consists of repeated antigenic peptide sequences, VIsE-OspC-VIsE-OspC).
•Problem: Genetic variation in antigens in different bacterial strains

BBK32 is an early antigen, conserved, less variation between strains, critical for blood-borne dissemination stage of disease and likely a target of host antibody response
If the following statements are accurate, even BBK32 may not be present in all strains and/or all stages. I'm more than a bit confused at the moment, and would appreciate anyone who can help me understand this: ... f=5&t=4264
Differentiating Borrelia burgdorferi strains

Furthermore, we failed to detect bbk32, which encodes a fibronectin-binding adhesin, in one "N40" strain. ... 46&p=30975
Comparing Bb Strains 31 and N40D10/E9

Based upon the studies here, it can be predicted that N40D10/E9 disseminated infection at lower doses may be enhanced by its lower binding to epithelial cells at the site of inoculation due to the absence of BBK32. ... 32&p=30894
Lyme neuroborreliosis test improvements

Three recombinant antigens, decorin binding protein A (DbpA), BBK32, and outer surface protein C (OspC), and IR(6) peptide of borrelial VlsE protein, were evaluated for the diagnosis of neuroborreliosis (NB), using cerebrospinal fluid (CSF) and serum samples from 89 patients.

Antibodies to BBK32 were detectable mainly in early disease. Antibodies to DbpA and IR(6) were observed in early and late NB.

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Re: Single-tier C6 peptide ELISA kit vs 2-tier testing

Post by RitaA » Fri 26 Oct 2012 8:51

By the way, I'm not the only one who has noticed there is contradictory information available when it comes to BBK32: ... 47532-g004
Citation: Lin T, Gao L, Zhang C, Odeh E, Jacobs MB, et al. (2012) Analysis of an Ordered, Comprehensive STM Mutant Library in Infectious Borrelia burgdorferi: Insights into the Genes Required for Mouse Infectivity. PLoS ONE 7(10): e47532. doi:10.1371/journal.pone.0047532


In contrast, mutation of bbk32, encoding a well-characterized fibronectin-binding protein, had little apparent effect on infectivity; prior studies had yielded contradictory information regarding the requirement of BBK32 for infectivity [60]–[62].
References 60 to 62 (as noted above) are:
60. Li X, Liu X, Beck DS, Kantor FS, Fikrig E (2006) Borrelia burgdorferi lacking BBK32, a fibronectin-binding protein, retains full pathogenicity. Infect Immun 74: 3305–3313.

61. Seshu J, Esteve-Gassent MD, Labandeira-Rey M, Kim JH, Trzeciakowski JP, et al. (2006) Inactivation of the fibronectin-binding adhesin gene bbk32 significantly attenuates the infectivity potential of Borrelia burgdorferi. Mol Microbiol 59: 1591–1601.

62. Norman MU, Moriarty TJ, Dresser AR, Millen B, Kubes P, et al. (2008) Molecular mechanisms involved in vascular interactions of the Lyme disease pathogen in a living host. PLoS Pathog 4: e1000169.

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Re: Single-tier C6 peptide ELISA kit vs 2-tier testing

Post by radicale » Sat 27 Oct 2012 19:08

Oh Rita, why are you wasting your time learning about Lyme related diagnostics. After all, didn't you hear the tests we use now are super sensitive and specific.....

On a serious note, It's really disheartening to come across the facts which show the opposite and which impact people's lives for no good reason. Good enough has become perfect, allowing people to be taken advantage of due to uncertainty (real). Instead of striving for the perfect diagnostic test, the IDSA has sunk incredible amounts of time and money trying to discredit a dozen or so MD's (their words), instead of using those resources to improve diagnostics. Idiots.

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Re: Single-tier C6 peptide ELISA kit vs 2-tier testing

Post by RitaA » Sat 27 Oct 2012 20:57

radicale wrote:Oh Rita, why are you wasting your time learning about Lyme related diagnostics. After all, didn't you hear the tests we use now are super sensitive and specific.....
Yes, I'm pretty sure I've read that once or twice. ;)

Saying that there's still lots of room for improvement when it comes to testing for Lyme disease is definitely an understatement. I can only hope that some researchers are equally unsatisfied with tests that pick up "nearly all" instead of all cases of Lyme disease. I realize that few things in life are perfect, but a missed diagnosis of Lyme disease can so easily change the course of a person's life.

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Re: Single-tier C6 peptide ELISA kit vs 2-tier testing

Post by k10 » Sun 9 Feb 2020 13:47

This post has been very informative. I am in the beginning stages of my diagnosis. I just received my lab results and was hoping you could explain why the results are contradictory and frankly, I find them confusing. My C6 came back positive with high levels and score of 4.5. My blot test came back negative with all bands showing absent. How can one test show such high numbers while the other shows nothing. Which one is the false negative?

I am trying to be an informed patient at this point, since 9 years ago a doctor dismissed my circular ring rash as just an infected insect bite.

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