Combination of antigens for Bb testing

Medical topics with questions, information and discussion related to Lyme disease and other tick-borne diseases.
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Joe Ham
Posts: 489
Joined: Fri 27 Jul 2007 6:15
Location: New Mexico, USA

Combination of antigens for Bb testing

Post by Joe Ham » Fri 5 Oct 2007 18:36

Recombinant flagellin A proteins from Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii in serodiagnosis of Lyme borreliosis.

J Clin Microbiol. 2001 Nov;39(11):4013-9.
Panelius J, Lahdenne P, Saxen H, Heikkilä T, Seppälä I.

Haartman Institute, Department of Bacteriology and Immunology, Helsinki, Finland. jaana.panelius@helsinki.fi

Genes for flagellin A (FlaA) proteins from European borrelial strains of Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii were cloned and sequenced.
An identity of 92 to 93% was observed in the flaA sequences of the different species.
Polyhistidine-tagged recombinant FlaA (rFlaA) proteins were produced in Escherichia coli and used as antigens in Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). In immunoglobulin G (IgG) WB, 71% (10 of 14) of the sera from neuroborreliosis and 86% (12 of 14) of those from Lyme arthritis patients reacted with one to three rFlaAs.
In IgG ELISA, 74% (14 of 19) and 79% (15 of 19) of patients with neuroborreliosis and arthritis, respectively, were positive.

The immunoreactivity in local European patient sera was stronger against rFlaA from B. garinii and B. afzelii than against rFlaA from B. burgdorferi sensu stricto.

Neither IgG nor IgM ELISA was sensitive in the serodiagnosis of erythema migrans. [Early acute stage?]

Serum samples from patients with syphilis and systemic lupus erythematosus showed mild cross-reactivity in IgG tests. Sera from Yersinia enterocolitica or beta-hemolytic Streptococcus infections showed only occasional responses.

With IgM ELISA, 58% (11 of 19) and 37% (7 of 19) of patients with neuroborreliosis and arthritis, respectively, were positive. Cross-reactive antibodies to FlaA, especially in serum samples from patients with rheumatoid factor positivity and Epstein-Barr virus infection, reduced the specificity of IgM serodiagnosis.

Therefore, rFlaA seems to have a limited role for IgM serodiagnosis, yet rFlaA might be useful in the IgG serodiagnosis of disseminated Lyme borreliosis.

PMID: 11682523 [PubMed - indexed for MEDLINE]
The full version is available:
http://www.ncbi.nlm.nih.gov/sites/entre ... stractPlus

Joe Ham
Posts: 489
Joined: Fri 27 Jul 2007 6:15
Location: New Mexico, USA

Re: Combination of antigens for Bb testing

Post by Joe Ham » Fri 5 Oct 2007 18:39

Species-specific serodiagnosis of Lyme arthritis and neuroborreliosis due to Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii by using decorin binding protein A.

J Clin Microbiol.2002 Feb;40(2):453-60

Heikkilä T, Seppälä I, Saxen H, Panelius J, Yrjänäinen H, Lahdenne P.

Hospital for Children and Adolescents, University of Helsinki, Finland. tero.heikkila@helsinki.fi

The antigenic potential of decorin binding protein A (DbpA) was evaluated in serodiagnosis of human Lyme borreliosis (LB).
The dbpA was cloned and sequenced from the three pathogenic Borrelia species common in Europe.

Sequence analysis revealed high interspecies heterogeneity.
The identity of the predicted amino acid sequences was 43 to 62% among Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii. The respective recombinant DbpAs (rDbpAs) were produced and tested as antigens by Western blotting and enzyme-linked immunosorbent assay (ELISA).

One hundred percent of patients with neuroborreliosis (NB) and 93% of patients with Lyme arthritis (LA) reacted positively.

Sera from the majority of patients reacted with one rDbpA only and had no or low cross-reactivity to other two variant proteins. In patients with culture-positive erythema migrans (EM), the sensitivity of rDbpA immunoglobulin G (IgG) or IgM ELISA was low.

The DbpA seems to be a sensitive and specific antigen for the serodiagnosis of LA or NB, but not of EM, provided that variants from all three pathogenic borrelial species are included in the combined set of antigens.

PMID: 11825956 [PubMed - indexed for MEDLINE]
The full text version is available:
http://www.ncbi.nlm.nih.gov/sites/entre ... stractPlus

Joe Ham
Posts: 489
Joined: Fri 27 Jul 2007 6:15
Location: New Mexico, USA

Re: Combination of antigens for Bb testing

Post by Joe Ham » Fri 5 Oct 2007 18:44

Significant improvement of the recombinant Borrelia-specific immunoglobulin G immunoblot test by addition of VlsE and a DbpA homologue derived from Borrelia garinii for diagnosis of early neuroborreliosis.

J Clin Microbiol. 2003 Mar;41(3):1299-303.

Schulte-Spechtel U, Lehnert G, Liegl G, Fingerle V, Heimerl C, Johnson BJ, Wilske B.

Max von Pettenkofer-Institut für Medizinische Mikrobiologie und Hygiene der Ludwig-Maximilians-Universität München, D-80336 Munich, Germany. Bettina.Wilske@mvp-bak.med.uni-muenchen.de

We investigated whether the recombinant Borrelia Western blot test previously described (B. Wilske, C. Habermann, V. Fingerle, B. Hillenbrand, S. Jauris-Heipke, G. Lehnert, I. Pradel, D. Rössler, and U. Schulte-Spechtel, Med. Microbiol. Immunol. 188:139-144, 1999) can be improved by the addition of VlsE and additional DbpA and OspC homologues.

By using a panel of sera from 36 neuroborreliosis patients and 67 control patients,

the diagnostic sensitivity of the recombinant immunoblot test was significantly increased (86.1% versus 52.7%) without loss of specificity

and was higher (86.1% versus 63.8%) than that of the conventional whole-cell lysate immunoblot test (U. Hauser, G. Lehnert, R. Lobentanzer, and B. Wilske, J. Clin. Microbiol. 35:1433-1444, 1997). Improvement was mainly due to the presence of VlsE and DbpA.

PMID: 12624072 [PubMed - indexed for MEDLINE]
The full text version is available:
http://www.ncbi.nlm.nih.gov/sites/entre ... stractPlus

Joe Ham
Posts: 489
Joined: Fri 27 Jul 2007 6:15
Location: New Mexico, USA

Re: Combination of antigens for Bb testing

Post by Joe Ham » Fri 5 Oct 2007 18:54

Improvement of Lyme borreliosis serodiagnosis by a newly developed recombinant immunoglobulin G (IgG) and IgM line immunoblot assay and addition of VlsE and DbpA homologues.

J Clin Microbiol. 2005 Aug;43(8):3602-9.
Goettner G, Schulte-Spechtel U, Hillermann R, Liegl G, Wilske B, Fingerle V.

Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität München, Pettenkoferstr. 9a, D-80336 Munich, Germany.

We developed and evaluated a recombinant Borrelia line immunoblot assay based on 18 homologues of seven different antigens, i.e., p100, p58, p41i, BmpA, VlsE, OspC, and DbpA.

Each recombinant antigen can be detected separately and is distinct even from homologues with identical molecular weights.

[Would this have implications for treatment?]

This blot was compared to the recently described recombinant Borrelia Western immunoblot assay (U. Schulte-Spechtel, G. Lehnert, G. Liegl, V. Fingerle, C. Heimerl, B. J. Johnson, and B. Wilske, J. Clin. Microbiol. 41:1299-1303, 2003).

To verify sensitivity and specificity, both blots were evaluated for reactivity with Borrelia-specific immunoglobulin G (IgG) and IgM antibodies with 85 sera from patients with different manifestations of Lyme borreliosis and 110 controls.

According to European interpretation criteria for Borrelia Western blots, which define a serum as positive when it recognizes at least two bands, sensitivity increased significantly from 70.6% (Western blot) to 84.7% (line blot) for IgG (P = 0.042) and from 40.0% (Western blot) to 73.8% (line blot) for IgM (P < 0.005).

The increased sensitivity for IgG detection is due to the new line blot technique, whereas the improvement in detection of IgM is mainly achieved through incorporation of the additional antigens.

Notably, the recombinant VlsE of Borrelia garinii strain PBi displayed the highest sensitivity of all antigens tested for IgG detection and is also one of the most useful antigens for IgM. Due to its excellent sensitivity and specificity combined with ease of evaluation, this line immunoblot assay offers a useful improvement in serodiagnosis of Lyme borreliosis.

PMID: 16081885 [PubMed - indexed for MEDLINE]
The full version is available:
http://www.ncbi.nlm.nih.gov/sites/entre ... stractPlus

Joe Ham
Posts: 489
Joined: Fri 27 Jul 2007 6:15
Location: New Mexico, USA

Re: Combination of antigens for Bb testing

Post by Joe Ham » Fri 5 Oct 2007 18:58

Laboratory diagnosis of infection caused by Borrelia burgdorferi

Ugeskr Laeger. 2006 Aug 21;168(34):2805-7.
[Article in Danish]

Dessau RB, Bangsborg JM, Jensen TP, Hansen K, Lebech AM, Andersen CØ.
Dansk Selskab for Klinisk Mikrobiologi, Dansk Selskab for Infektionsmedicin. ram.dessau@dadlnet.dk

The laboratory diagnosis of Lyme disease in Denmark is reviewed with recommendations for serological testing. In Denmark the laboratory testing is performed with an ELISA technique.

Most laboratories use an assay based on purified flagella antigen.
[Do I understand this correctly? that these labs are looking for WB band 41 or a part of that band?]

The two-tier approach with Western Blot as confirmatory testing is not recommended since the contribution to the diagnostic specificity is only marginal.

Predictive values of Lyme serology are presented, based on the estimated prevalence of the different stages of Lyme disease in Denmark.

PMID: 16942701 [PubMed - indexed for MEDLINE]

Joe Ham
Posts: 489
Joined: Fri 27 Jul 2007 6:15
Location: New Mexico, USA

Re: Combination of antigens for Bb testing

Post by Joe Ham » Fri 5 Oct 2007 19:04

[Western-blot with VLSE protein and "in vivo" antigens in Lyme borreliosis diagnosis]
Przegl Epidemiol. 2006;60 Suppl 1:177-85.
[Article in Polish]

Zajkowska J, Kondrusik M, Pancewicz S, Grygorczuk S, Swierzbińska R, Hermanowska-Szpakowicz T, Czeczuga A, Sienkiewicz I.
Klinika Chorób Zakainych i Neuroinfekcji AM w Białymstoku.

The aim of the study was the evaluation of the efficiency of Western blot (EcoLine) test detecting simultaneous presence of IgM and IgG antibodies against B. burgdorferi in diagnosis of early and late stage of Lyme borreliosis.
The comparison of results achieved by performing test Western-blot, ELISA (based on recombinant antigens of three genospecies of Borrelia) and EIA (based on antigens of one B. burgdorferi genospecies).
The tests Western blot: EcoLine (Virotech) with antigens "in vivo", ELISA Borrelia IgM, IgG recombinant (Biomedica), EIA: B. b. ss. IgG, EIA B. garinii IgG, EIA B. afzelii IgG (TestLine) were used.
Results showed efficacy of detecting IgM, IgG antibodies against VlsE simultaneously and IgG antibodies against "in vivo" antigens in diagnosis of early stages of Lyme disease when atypical picture skin lessions raise diagnostic doubts and in discerning early and late stage of disease.
The EIA tests based on one B. burgdoreferi genospecies seem less effective in comparison to ELISA tests based on 3 genospecies antigens.

PMID: 16909799 [PubMed - indexed for MEDLINE]

[It is believed that only Bb ss is present in North America but I wonder what would be the results of using combination antigens for other genotypes. Would the abysmally high rate of false negatives decline?

That question came to mind because Igenex argues that their higher positive detection rate is because they use local isolates of Bb ss. Most other labs use a a commercial kit based on a high passaged strain B-31 (an "East coast" strain).]

Nick
Posts: 299
Joined: Wed 19 Sep 2007 19:10
Location: Zeeland, Netherlands

Re: Combination of antigens for Bb testing

Post by Nick » Sat 6 Oct 2007 14:48

Joe Ham wrote:[It is believed that only Bb ss is present in North America but I wonder what would be the results of using combination antigens for other genotypes. Would the abysmally high rate of false negatives decline?

That question came to mind because Igenex argues that their higher positive detection rate is because they use local isolates of Bb ss. Most other labs use a a commercial kit based on a high passaged strain B-31 (an "East coast" strain).]
I think you are right, we see similar differences between the more sensitive/reliable Western Blots in Europe and the standard blots. It is a fact that Bb can easily change certain genes like those for OSPC and Vlse, both by intragene transfer (which can occur e.g. inside the host after infection) or plasmid exchange between separate Bb organisms (aquiring new gene from different Bb strain/species). Some of these highly variable genes are the specific ones that are looked for in the blot. With all the different version of these genes and their rapid evolution, I have no doubt that the standard antigene tests are missing some strains completely.

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