west.blot.pdf

EUROIMMUN

Medizinische

Labordiagnostika

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

EUROIMMUN

Medizinische

Labordiagnostika

Features of conventional EUROIMMUN anti-Borrelia Westernblots

Monoclonal

reference ab

Specific

antigens

Monoclonal

reference ab

Specific

antigens

Monoclonal

reference ab

Specific

antigens

L83/100 22G3

p83

L83/100 22G3

p83

L83/100 22G3

p83

L75 3G10

p75

L60 E6

p60

MAB956

L39 B1

p41, Flag.

MAB956

L39 B1

p41, Flag.

p39, BmpA

L41 1C11

L39 B5

p41, Flag.

p39, BmpA

L34 1G8

L32 1F11

p34, OspB

p31, OspA

L30 1B10

L32 1F11

p30

L31 1F11

L30 1B10

p31, OspA

p29, OspA

p30

L22 1F8

p25, OspC

p21/22

L22 1F8

p25, OspC

L22 1F8

p25, OspC

MAB 962

p21/22

MAB 962

p21

L19 A11

p19

L19 A11

p19

Borrelia burgdorferi

Borrelia garinii

Borrelia afzelii

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

to read and have led some clinicians to abandon whole antigen

Westernblots. But recombinant Westernblots have a 23% lower

sensitivity (Wilske et al., Med. Microbiol. Immunol. 188: 139-144, 1999),

particularly with fresh infections, and therefore are not recommended

for reliable borreliosis diagnostics. Individual recombinant antigens

can, however, supplement whole antigen Westernblots. Additional use

of the newly identified major Borrelia antigen VlsE places the whole

antigen Westernblot once again at the forefront, including with regard

to convenient evaluation.

(

1 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: +49 4509 87430 · Fax 874334 · E-mail: euroimmun@euroimmun.de

L34 1G8

EUROIMMUN

Medizinische

Labordiagnostika

The newly identified VlsE – major antigen for Borrelia serology

What is VlsE and what is its function?

VlsE ( v ariable major protein- l ike s equence, e xpressed) 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-

membrane domains and anchor VlsE in the bacterial membrane, as well as variable and

invariable regions. The variable regions of VlsE face outwards and are constantly changed by

recombination (see below), whereby the attacking immune system consistently encounters

new, altered antigen epitopes. The invariable regions are masked by the variable regions and,

in living Borrelia bacteria, are protected from direct attack by the immune system. When

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

invariable and conserved regions of VlsE . These antibodies cannot bind to the Borrelia bacteria

in vivo, since the specific epitopes are masked, but they are highly suitable for diagnosis of

borreliosis because of the high level of conservation of their target antigens. Using enzyme

immunoassays (ELISA, EUROLINE-WB) Lyme borreliosis can be diagnosed in 85% of cases,

regardless of the species, through the detection of antibodies against VlsE alone.

invariable

regions

variable

regions

outer

Borrelia

membrane

inner

conserved

regions

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 v ariable major protein- l ike s equence( 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 4

vls cassette 5

vls cassette 6

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

VlsE protein

IR IR

invariable regions (IR)

conserved regions

(transmembrane domains)

Recombination and expression of VlsE

EUROIMMUN AG · D-23560 Lübeck (Germany) · Seekamp 31 · Tel: +49 4509 87430 · Fax 874334 · E-mail: euroimmun@euroimmun.de

EUROIMMUN

Medizinische

Labordiagnostika

Anti-Borrelia EUROLINE-WB – optimal borreliosis diagnostics with

Borrelia whole antigen extract plus VlsE

IgM

Rapid evaluation

IgG

Anti-Borrelia EUROLINE-WB

L60 E6

p17

p19

p21

p25,

OspC

p30

p31,

OspA

p39,

BmpA

p41,

Flag.

p83

VlsE

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)

showed that by additionally determining antibodies against VlsE the serological

hit rate can be increased by 20% compared to whole extract Westernblots and by

30% compared to recombinant antigen Westernblots. Of all recombinant antigens

tested, VlsE possesses the highest sensitivity for the detection of a Borrelia infection

(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

to the 32 nd Congress of the German Society for Rheumatology, Frankfurt, 2003).

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)

Performance of test with

IgM conjugate

IgG conjugate

OspC band

stained

Neither VlsE

nor OspC

band stained

VlsE band

stained

(

anti-Borrelia

IgM positive

Evaluation of

other bands

anti-Borrelia

IgG positive

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: +49 4509 87430 · Fax 874334 · E-mail: euroimmun@euroimmun.de

DY_2131_I_GB_A01

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