Chemistry: molecular biology and microbiology – Kit
Reexamination Certificate
2002-11-07
2004-06-22
Navarro, Mark (Department: 1645)
Chemistry: molecular biology and microbiology
Kit
C530S300000, C530S350000
Reexamination Certificate
active
06753183
ABSTRACT:
Lyme borreliosis is the commonest infectious disease of humans transmitted by ticks in the Federal Republic of Germany. In contrast to Russian spring-summer encephalitis (RSSE) which is likewise transmitted by ticks, Lyme borreliosis is not confined to a few endemic areas but occurs in all the states of the FRG. Infestation of the main vector in Europe, Ixodes ricinus, with the pathogen of Lyme borreliosis, the spirochete
Borrelia burgdorferi
, in Southern Germany is about 20% of adults, about 10% of nymphs and about 1% of larvae. The main vector in the USA, Ixodes dammini, may be up to 100% infected by Borrelia in highly endemic areas.
B. burgdorferi
belongs to the family of spirochetes. Spirochetes are spiral bacteria 8-30 &mgr;m long. They consist of an outer coat, the endoflagella in the periplasm and the protoplasmic cylinder. The protoplasmic cylinder is a complex of cytoplasm, internal cell membrane and peptidoglycan. Representatives of the spirochetes which are pathogenic for humans include, beside
B. burgdorferi
, the Borrelia of relapsing fever (for example
B. recurrentis
), the pathogen of syphilis (
Treponema
(
T
.)
pallidum
) and the Leptospira. As a result of the close immunological relationship of the pathogens, cross-reactions are a problem in the serological detection of antibodies in cases of syphilis and Lyme borreliosis with assays currently available.
Infection with
B. burgdorferi
results in a complex clinical picture which can, similarly to syphilis, be divided into three different stages. The principal manifestations are:
Early phase:
Stage I
Erythema migrans
Bannwarth's lymphocytic
meningoradiculitis (LMR)
Borrelia lymphocytoma
Late phase:
Stage III
Lyme arthritis
Acrodermatitis chronica
atrophicans (ACA)
Chronic Borrelia
encephalomyelitis
Less common clinical manifestations are: carditis, myositis, iritis and panophthalmitis. Transmission by the pathogen crossing the placenta is possible but to date only a few cases of congenital Lyme borreliosis have been recorded. The various stages may occur singly or in combination.
B. burgdorferi
infection may also have a subclinical course. Epidemiological studies on 375 clinically confirmed cases show some peculiarities in the age and sex distribution of the various clinical manifestations. Thus, patients with Erythema migrans were commonest in the 30 to 60 year age group. Neuro-logical manifestations showed two peaks with age: the first in children and young people up to 20 years of age, and the second in 40 to 70 year-olds. Lyme arthritis was observed to be commonest in 30 to 60 year-olds. Patients with ACA were never below 30 years of age. ACA affects women distinctly more often than men. Serological testing showed predominantly positive IgM findings in patients with Erythema migrans, and predominantly positive IgG findings when there were neurological manifestations, in an immunofluorescence assay. With the late manifestations of ACA and Lyme arthritis, the IgG titers were regularly elevated, and IgM antibodies were now detectable only in exceptional cases.
Available for diagnosis are both pathogen detection and antibody detection. Pathogen detection in material from patients (skin biopsies, CSF, puncture fluids) is recommended especially in the early stage (Erythema migrans) when antibody detection is frequently negative. However, a complex nutrient medium is required for culturing
B. burgdorferi
(Preac-Mursic, V.; Wilske, B.; Schierz, G. (1986): European
Borreliae burgdorferi
isolated from humans and ticks—culture conditions and antibiotic susceptibility. Zbl. Bakt. Hyg. A 163, 112-118) and cultivation is therefore restricted to special laboratories. In addition, a time of up to 5 weeks is required to isolate the pathogen.
B. burgdorferi
is isolated from skin samples in 50-70% of cases with cutaneous manifestations and in 3-5% of cases with neuroborreliosis (Preac-Mursic, V.; unpublished results).
Antibody detection (IgM, IqG) is carried out on serum and, when there are neurological manifestations, also from CSF. The serological finding depends on the stage of the disease, the duration of the symptoms and any antibiotic therapy which has already been applied. Thus, antibody detection with assays available to date is successful only in 20-50% of cases with Erythema migrans, in 50-90% of cases with neurological manifestations and in 90-100% in cases with ACA and arthritis.
Therapy of Lyme borreliosis is predominantly carried out with penicillin G, tetracyclines, erythromycin or cephalosporins. Although Lyme borreliosis frequently resolves spontaneously in the early stages, even then late manifestations are not ruled out. This is why therapy in the early stage is indispensable. In addition, clinical resolution after antibiotic therapy can be achieved when there are late manifestations only in some of the cases (for example only about 50% of cases with Lyme arthritis).
This is why Lyme borreliosis should be diagnosed as early as possible. Since (as already explained) pathogen isolation is costly, time-consuming and, moreover, not always successful, better serodiagnostic assays ought to be developed. The methods used to date (immunofluorescence assay (IFA), indirect hemagglutination assay (IHA), enzyme-linked immunosorbent assay (ELISA)) frequently fail in the early stages. The antigens employed for these assays are all
B. burgdorferi
cells or whole-cell ultrasonicates. The use of different
B. burgdorferi
strains as antigen in the ultrasonicate ELISA leads to differing test results. Immobilization of cells on slides or ultrasonicate antigen on microtiter plates is followed by incubation with serum or CSF and detection of the Borrelia—specific antibodies with a second fluorescence- or peroxidase-labeled antibody of the appropriate immunoglobulin class. The reaction is then quantified either in a fluorescence microscope (IFA) or after a color reaction in a photometer (ELISA).
Broad cross-reactions of the pathogen
B. burgdorferi
with other bacterial pathogens, especially with
T. pallidum
, the syphilis pathogen, is a problem for the specificity of the assays. Since the assay antigens generally consist of lysates of the whole pathogen there is also detection of antibodies against so-called common antigens (Hansen, K.; Hindersson, P.; Pedersen, N. S. (1988): Measurement of antibodies to the
Borrelia burgdorferi
flagellum improves serodiagnosis in Lyme disease. J. Clin. Microbiol., 26, 338-346). Common antigens are widely distributed proteins with highly conserved sequences, that is to say the common antigens of Borrelia, Treponema as well as many other bacteria have common epitopes. Besides this, false-positive reactions may occur in the IgM-IFA or IgM-ELISA when the sera have rheumatoid factor activity. Therefore, in order to make the assays more specific, in the detection of IgG and IgM antibodies a preabsorption of the sera with a Treponema ultrasonicate, and additionally for the detection of IgM antibodies also absorption with rheumatoid factor absorbent, is carried out.
An object of the present invention is therefore to provide immunologically active proteins from
Borrelia burgdorferi
which are used in an assay kit which does not have the abovementioned disadvantages. An additional aim is that this assay kit makes it possible rapidly and reliably to detect antibodies directed against
Borrelia burgdorferi.
Another object of the present invention is to provide monoclonal antibodies which are directed against particular immunologically active proteins from
Borrelia burgdorferi
. A further aim is to provide immunologically active proteins which are suitable as vaccines against infections caused by Borrelia strains.
Testing of patients' sera from different stages of the disease of Lyme borreliosis in a Western blot, and testing of non-Lyme borreliosis patients (especially syphilis patients) for cross-reactivity with
B. burgdorferi
resulted in the finding of immunologically active proteins (
B. burgdorferi
antigens) which, on the one hand, elicit a good antibody response af
Fuchs Renate
Motz Manfred
Preac-Mursic Vera
Soutscheck Erwin
Wilske Bettina
Mikrogen Molekularbiologische Entwicklungs-GmbH
Navarro Mark
Schwegman Lundberg Woessner & Kluth P.A.
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