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EUROIMMUN Blot Systems for Borreliosis
Diagnostics: Refined and Unparalleled
" Certified according to EN ISO 9001 / EN 46001 / EN ISO 13485
" In line with all recommendations of the German Society for Hygiene
and Microbiology
" Borrelia whole antigen extract  up to 23% more sensitive than
mixes of recombinant antigens
" Newly identified major Borrelia antigen VlsE in addition:
a further 20% increase in sensitivity
" Evaluation at a glance: anti-Borrelia IgG and IgM
" EUROIMMUN: Experts in borreliosis serology!
Highly differential product spectrum,
including ELISA, IIFT, Westernblot and EUROLINE-WB,
antigen substrates for all relevant Borrelia species available
:
NEW
with
VlsE
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Features of conventional EUROIMMUN anti-Borrelia Westernblots
Monoclonal Specific Monoclonal Specific Monoclonal Specific
antigens
reference ab antigens reference ab reference ab antigens
L83/100 22G3 p83
p83
L83/100 22G3 L83/100 22G3 p83
L75 3G10 p75
L60 E6 p60
MAB956 p41, Flag.
MAB956 p41, Flag.
p41, Flag.
L41 1C11
L39 B1 p39, BmpA L39 B1 p39, BmpA
p39, BmpA
L39 B5
L34 1G8
L34 1G8 p34, OspB
p30 p31, OspA
L30 1B10 L31 1F11
L32 1F11 p31, OspA
L32 1F11 p29, OspA p30
L30 1B10
L22 1F8 p25, OspC L22 1F8 p25, OspC
L22 1F8 p25, OspC
MAB 962 p21/22
MAB 962 p21
MAB 962 p21/22
L19 A11 p19 L19 A11 p19
Borrelia garinii Borrelia afzelii
Borrelia burgdorferi
What role do Westernblots have in borreliosis diagnostics?
Westernblots allow the serological determination of antibodies against human pathogenic Borrelia species. These include B. burgdorferi,
B. garinii and B. afzelii. The German Society for Hygiene and Microbiology (Deutsche Gesellschaft für Hygiene und Mikrobiologie, DGHM),
the Robert Koch Institute (Berlin) and the Center for Disease Control (Atlanta, USA) recommend a two-step strategy for the serological
diagnosis of Lyme borreliosis: first a screening test  ELISA or indirect immunofluorescence (IIFT)  is employed, which must provide high
sensitivity. To check the specificity, positive and borderline reactions are then analysed by Westernblot. In fresh infections we recommend
performing ELISA/IIFT and Westernblot in parallel, since some weak reactions become detectable earlier in Westernblot than in the
screening tests.
Quality features of conventional EUROIMMUN anti-Borrelia Westernblots
EUROIMMUN Westernblots fulfil near to all requirements of the German Society for Hygiene and Microbiology (Wilske et al., Lyme Borreliosis:
Quality Standards for the Microbiological Diagnosis of Infectious Diseases (MiQ 12/2000):
" Borrelia whole antigen extract  prerequisite for early identification of fresh infections
" Clear separation of specific and unspecific antigen bands
" Characterisation of all diagnostically relevant bands with monoclonal reference antibodies
" Highly specific IgM detection with OspC  no false positive reactions with blood donors, as with some manufacturers products
" Specific evaluation templates, customized to each individual test kit
" Clear classification criteria for positive reactions  reaction of at least one (IgM) or at least two (IgG) specific bands
" Positive, borderline and negative control sera available on request
Whole antigen extract or recombinant antigens?
Westernblot systems with exclusively recombinant antigens are easier
Whole antigen Recombinant
Sensitivity n
to read and have led some clinicians to abandon whole antigen Westernblot Westernblot1
Westernblots. But recombinant Westernblots have a 23% lower
Erythema migrans 66 33.3% 10.6%
sensitivity (Wilske et al., Med. Microbiol. Immunol. 188: 139-144, 1999),
particularly with fresh infections, and therefore are not recommended
Neuroborreliosis (stage II) 42 57.1% 45.2%
for reliable borreliosis diagnostics. Individual recombinant antigens
Late-stage borreliosis 39 100% 97.4%
can, however, supplement whole antigen Westernblots. Additional use
of the newly identified major Borrelia antigen VlsE places the whole
Control group2 139 2.2% 2.2%
antigen Westernblot once again at the forefront, including with regard
1
to convenient evaluation.
Recombinant antigens used: p83/100, p39, p41i, OspC, Osp17, p58
2
118 blood donors, 11 syphilis patients, 10 patients seropositive for rheumatoid factors
EUROIMMUN AG · D-23560 Lübeck (Germany) · Seekamp 31 · Tel: [phone] · Fax 874334 · E-mail: [e-mail]
B.AFZELII
B.GARINII
B.BURGD
.
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The newly identified VlsE  major antigen for Borrelia serology
What is VlsE and what is its function?
VlsE (variable major protein-like sequence, expressed) is a newly characterized surface protein of Borrelia burgdorferi. Its significance for
the serological diagnosis of borreliosis was until recently overlooked. VlsE plays a key role in the survival strategy of Borrelia. After
penetration into the host organism, Borrelia bacteria constantly change their surface-expressed VlsE and, in this way, try to escape
recognition and elimination by the immune system.
The VlsE protein is divided into several sections: conserved regions, which serve as trans-
invariable
membrane domains and anchor VlsE in the bacterial membrane, as well as variable and
regions
invariable regions. The variable regions of VlsE face outwards and are constantly changed by
recombination (see below), whereby the attacking immune system consistently encounters
variable
new, altered antigen epitopes. The invariable regions are masked by the variable regions and,
regions
in living Borrelia bacteria, are protected from direct attack by the immune system. When
outer
deceased Borrelia bacteria are processed by antigen-presenting cells, the complete VlsE
protein is presented to the immune system and the host also forms antibodies against the
Borrelia
invariable and conserved regions of VlsE. These antibodies cannot bind to the Borrelia bacteria
membrane
in vivo, since the specific epitopes are masked, but they are highly suitable for diagnosis of
inner
borreliosis because of the high level of conservation of their target antigens. Using enzyme
conserved
immunoassays (ELISA, EUROLINE-WB) Lyme borreliosis can be diagnosed in 85% of cases,
regions
regardless of the species, through the detection of antibodies against VlsE alone.
VlsE on the surface of Borrelia
How is the variability of the VlsE variable regions generated?
For production of the VlsE protein, the DNA of Borrelia bacteria contains 15 to 20 so-called variable major protein-like sequence(vls)
cassettes, which act as a library of genetic information. They are each composed of 12 gene sections: six invariable regions (red) and six
variable regions (green). By combining different elements from these cassettes (so-called recombination), an almost unlimited number of
surface proteins differing in their variable regions can be produced. The DNA which is assembled from the individual cassettes and used
for synthesis of the protein is called vlsE (E = expressed). It also contains the transmembrane domains of the VlsE protein. VlsE is
expressed exclusively in vivo; cultured Borrelia do not contain this antigen.
Literature: Lawrenz et al., J. Clin. Microbiol. 37: 3997-4004 (1999); Eicken et al., J. Biol. Chem. 277: 21691-21696 (2002)
vls cassette 5 vls cassette 6
vls cassette 4 vls cassettes of
Borrelia DNA
(section)
Combination of individual
cassette elements
vlsE DNA
Expression of
VlsE protein
variable regions (VR)
VR VR VR VR VR VR
VlsE protein
IR IR IR IR IR IR
invariable regions (IR)
conserved regions
(transmembrane domains)
Recombination and expression of VlsE
EUROIMMUN AG · D-23560 Lübeck (Germany) · Seekamp 31 · Tel: [phone] · Fax 874334 · E-mail: [e-mail]
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Anti-Borrelia EUROLINE-WB  optimal borreliosis diagnostics with
Borrelia whole antigen extract plus VlsE
IgM Rapid evaluation IgG
L60 E6
Anti-Borrelia EUROLINE-WB
p17 p19 p21 p25, p30p31, p39, p41, p83 VlsE
OspC OspA BmpA Flag.
Anti-Borrelia EUROLINE-WB
EUROLINE-WB  a significant advance in antibody diagnostics
In the line blot system EUROLINE, purified, biochemically characterized antigens are printed as parallel lines at defined positions on the
membrane. Reactions can be evaluated effortlessly, but the spectrum of available antigens is often limited. Conventional native Western-
blot systems provide the complete antigen spectrum of cultured cells, tissue or infectious agents, whereby the individual proteins are
separated electrophoretically according to size. However, the multitude of antigens on the membrane strips makes the evaluation more
difficult, since the antigen bands often lie very close to one another. The EUROLINE-WB combines the advantages of both methods:
Ready prepared Westernblot strips are fitted with EUROLINE membrane chips, which are preprinted with either native, affinity
chromatographically purified antigens or recombinant antigens. For each antigen the most suitable membrane is selected and the
optimal coating procedure used.
Why is VlsE additionally employed as a recombinant antigen?
A study by the Max von Pettenkofer Institute (German Borrelia Reference Centre)
Positive reactions in neuro-
Antigen employed
showed that by additionally determining antibodies against VlsE the serological
borreliosis patients (n = 36)
hit rate can be increased by 20% compared to whole extract Westernblots and by
Recombinant VlsE 83.3%
30% compared to recombinant antigen Westernblots. Of all recombinant antigens
tested, VlsE possesses the highest sensitivity for the detection of a Borrelia infection
Whole antigen extract 63.8%
(Schulte-Spechtel et al., J. Clin. Microbiol. 41:1299-1303, 2003).These results could
be confirmed in an internal study (Meyer et al., scientific presentation submitted
Recombinant antigen mix:
52.7%
to the 32nd Congress of the German Society for Rheumatology, Frankfurt, 2003). p83/100, p39, p41i, OspC, Osp17, p58
Over 85% of IgG-positive sera could be identified at a glance by assessing the
VlsE band. VlsE allows detection of antibodies against all Borrelia species, and
the risk of a false negative reaction due to species differences is ten times lower.
Quality features of the EUROIMMUN anti-Borrelia EUROLINE-WB (whole antigen plus VlsE)
Easy evaluation without compromising sensitivity
Performance of test with
Borrelia whole antigen extract: high sensitivity, including for fresh infections
IgM conjugate IgG conjugate
VlsE antigen: high sensitivity for all species
OspC band VlsE band
Neither VlsE
Highly specific IgM reaction with OspC, healthy blood donors negative
stained stained
nor OspC
band stained
Identification of 85% IgG-positive sera at a glance (VlsE)
anti-Borrelia Evaluation of anti-Borrelia
Identification of atypical positive reactions (complete antigen spectrum)
IgM positive other bands IgG positive
Quality in line with DGHM recommendations
Evaluation of anti-Borrelia EUROLINE-WB
The following EUROIMMUN test systems contain the major Borrelia antigen VlsE: Anti-VlsE ELISA (Order no.: EI 2132-9601-1 G or M) and
Anti-Borrelia EUROLINE-WB (Order no.: DY 2131-1601-1 G or M). The evaluation system EUROLineScan, developed by EUROIMMUN,
provides fully automated evaluation of Westernblot strips and documentation of results.
EUROIMMUN AG · D-23560 Lübeck (Germany) · Seekamp 31 · Tel: [phone] · Fax 874334 · E-mail: [e-mail]
DY_2131_I_GB_A01