Immunoblot for the detection of IgG/IgM-antibodies against Borrelia burgdorferi

SUMMARY AND EXPLANATION OF THE TEST

The Lyme disease was named after the city of Lyme in Connecticut, where this disease was found for the first time (1975). The transmission results  from bites by infected ticks (USA; lxodes damminii, Europe: lxodes ricinus). Therefore, the disease is endemic where these ticks can be found (north and middle Europe and some parts of the south, USA, Canada and Japan). In these areas 2-20% of all ticks are infected by Borrelia burgdorferi, the causing agent of Lyme disease, a Spirochaeta bacteria, first discovered by Willy Burgdorfer in 1982.

Similar to the clinical picture caused by its Spirochaeta relative Treponema pallidum, Lyme disease shows three distinct stages of disease:

1. Early stage - Erythema chronicum migrans (ECM) a changing skin reddening at the site of the tick bite)
2. Generalization - Lymphocytoma, carditis, lymphocytic meningoradiculitis (change to a multisystemic disease with skin, heart, nerves etc. affected)
3. Late disease - Arthritis, Acrodermatitis chronica atrophicans (ACA), progressive Borrelia-encephalomyelitis (neuroborreliosis) (chronic disease with skin, synovia, and brain affected)

The diagnosis of Lyme disease is very difficult, because of the symptoms described above often only one, rarely two and very seldom three can be observed. For example the typical ECM can be found in only 50 % of all cases at the maximum. Growing Borrelia in culture is very difficult and time consuming, therefore serology is the only laboratory test for the diagnosis of Lyme disease.

The prevalence of significantly increased antibody titers is dependent on the stage of disease and on its duration. Therefore in stage I, only in 20 - 50 % of all cases can increased antibody titers be expected (mostly IgM). In stage II the prevalence increases to 70 – 90 % (IgG and IgM). Only in stage III can increased titers be found in nearly all cases, but exceptions have been reported.

Persistent IgM-antibodies, unspecifities, variations dependent on methods and the used antigens make the interpretation of the results more difficult. The diagnosis should therefore be validated by more than one test. For example all borderline and weak positive results should be confirmed by a westernblot (recommendation of the Center of Disease Control (CDC) in Atlanta, USA).

Westernblotting is highly specific, because an antibody response against a range of special Borrelia specific antigens can be observed and, therefore, cross-reactivities can mostly be eliminated, whereas in an Elisa well all antigens are coated, the specific and the unspecific.

REFERENCES

1. Benach, J.L., Bosler, E.M., Burgdorfer, W., Hanrahan, J.P., Colemann, J.L. et al. Spirochetes isolated from the blood of two patients with Lyme disease. New England J. of Med. 1983; 308: 740.
2. Bergström, S., Sjöstedt, A., Dotevall, L., Kaijser, B., Ekstrand, B., Wallberg, C., et al. Diagnosis of Lyme Borreliosis by an enzyme immunoassay detecting immunglobulin G reactive to purified  Borrelia burgdorferi cell components. Eur. J. Clin. Mikrobiol. Infect. Dis. 1991; 5: 422-427.
3. BGA Merkblatt Nr. 56 Bundesgesundheitsblatt 4/91 Lyme-Borreliose - Erkennung und Verhütung.
4. Burgdorfer, W. und Barbour, A. C. Lyme disease - a tick-borne spirochetosis? Science 1982; 216: 312.
5. Barbour, A. G., Heiland, R. A., Howe, T. R. Heterogenity of major proteins in Lyme disease Borrliae: a molecular analysis of North American and European isolates. J. Infect. Dis. 1985; 152: 478-484.
6. Blenk, H. Serodiagnostik der Lyme-Borreliose. MTA 1993; 8: 575-580.
7. Craft, J. E., Fisher, D. K., Shimato G. T., Steere, A. C. Antigens of Borrelia burgdorferi recognized during Lyme disease. J. Clin. Invest. 1988; 78: 934-993.
8. Magnarelli, L. A., und Anderson, J. F. Early detection and persistence of antibodies to Borrelia burgdorferi in persons with Lyme disease. Zbl. Bakt. Hyg. 1986; A 263; 392.
9. Magnarelli, L. A., Miller, J. N., Anderson, J. F., Riviere, G. R. Cross-reactivity of   nonspecific treponemal antibody in serologic tests for Lyme disease. J. Clin. Mikrobiol.  1990; 6: 1276-1279.
10. Putzker, M., Mertes, T., Sobe, D. Beeinflussung der serologischen Diagnostik von Infektionen mit Borrelia burgdorferi durch Kreuzreaktionen. Doppelinfektionen mit FSME-Virus. Lab. med. 1991; 15: 223-227.
11. Putzker, M., Schmell, K. W., Sauer, H. Die Lyme-Borreliose - eine klinisch und labordiagnostisch schwer faßbare Multisystemerkrankung. GIT Labormedizin 1991; 11: 461-469.
12. Raoult, D., Hechemy; K. E., Baranton, G. Cross-reaction with Borrelia burgdorferi antigen of sera from patients with HIV-infection, Syphilis, and Leptospirosis. J. Clin. Mikrobiol. 1989; 10: 2152-2155.
13. Schmidt-Wolf, J. und Sticht-Groh, V. Zur Lyme-Krankheit. Humorale Antikörperbestimmung gegen Borrelia burgdorferi in drei verschiedenen Patientengruppen. Ärztl. Lab. 1989; 35: 153.
14. Wilske, B. Serodiagnostik der Lyme-Borreliose. Z. Hautkr. 1988; 63: 511.
15. Wilske, B., Preac-Mursic, V., Fuchs, R., Schierz, G. Diagnostik der Lyme-Borreliose. Diagnose und Labor 1990; 1: 24-36.
16. Zöller, L., Haude, M., Sonntag, H.-G. Validität der Standardverfahren in der Serodiagnostik der Lyme-Borreliose und Einfluß methodischer Varianten. Lab. med. 1990; 14: 404-411.

 Borrelia burgdorferi IgG-Immunoblot

The following antigens can be identified by this immunoblot:

93 kD - Protein from the membrane vesicles at the outer surface of Borrelia. The immunoresponse against this protein is highly specific. In most cases only IgG-antibodies against this antigen can be observed. The p93 is a typical late or chronic phase marker, because antibodies against the protein can very often be detected in the sera of patients suffering from Lyme arthritis. In some cases IgM antibodies against p93 can be found. This could be a hint that the disease will develop into a chronic stage, but this is only an assumption so far.

62-72 kD - Heat-shock proteins. These proteins are not specific to Borrelia; cross-reactions to a lot of other bacteria are reported. These bands are of diagnostic value when antibodies against these proteins can be observed together with antibodies against p93. This is a further support for the diagnosis of a chronic stage of disease. In conclusion: a broad band pattern between 60 and 93 kDa is a strong support for the diagnosis of chronic Lyme disease with consequences for the therapy.

60 kD - Common antigen. Not specific for Borrelia-infections. A lot of cross-reactivities are known.

48 kD - Specificity is controversial. Can support the diagnosis of Lyme disease.

41 kD - Flaggelin-protein. Not specific for Borrelia. Cross-reactivities to a lot of other bacteria with endoflagellae are well documented. In spite of this it is a valuable marker, because antibodies  against this protein can be observed in every stage of the disease. For example, in early Lyme disease in many cases only IgM-antibodies against p41 and the highly specific Osp C protein can be observed. When both proteins are labeled in an IgM-blot this is are very strong support for diagnosis of early Lyme disease. But if no other specific bands are labeled, the finding remains unclear, especially in an IgG-blot.

39 kD - p39-protein. Highly specific for Borrelia. Marker of an early infection. IgM antibodies will be observed. If there is a strong marked reaction against the flagellin, it may happen that the p39-band cannot be surely differentiated from p41.

37 kD - Specificity not sure, but supports a positive finding.

34 kD - OspB (Outer surface protein B) is highly specific for B. burgdorferi.

31 kD - OspA (Outer surface protein A) is highly specific for B. burgdorferi according to literature. In this isolation the protein is over-expressed. Therefore cross-reactivities and unspecific binding can be observed in several cases. Therefore it is in this case not highly specific.

30 kD - Highly specific for B. burgdorferi. Function not known.

29 kD - OspD (Outer surface protein D) is highly specific for B. burgdorferi.

28 kD - Highly specific for B. burgdorferi. Function not known.

25 kD - OspC (Outer surface protein C) is highly specific for B. burgdorferi. There will often be IgM antibodies observed, not so often IgM antibodies. Because of that it is a very good marker of the acute phase.

21 kD - Belongs probably to the OspC-Complex. Therefore highly specific for B. burgdorferi. Also, early marker.

18 kD - Specificity is controversial. Not proved for B. burgdorferi when marked alone.

Many other bands may be found on Lyme-blots, which cannot be assigned to the above discussed proteins. These may be labeled Borrelia antigens, but because nothing is known about their function and specificity they must be omitted from the interpretation.

Additional help in interpretation (from manufacturer)

IgG:

IgG-antibodies can be detected against all antigens discussed above. For a positive interpretation at least two of the bands described as specific should be labeled, and in addition at least two of the other bands should be identified. One specific band alone is not sufficient for diagnosis of Lyme disease, but supported by at least four other characterized bands Lyme disease is highly probable. A broad band spectrum above 60 kD together with at least one specific band supports the diagnosis of late or chronic Lyme disease.

IgG positive:

IgG borderline:

CDC criteria (not validated for this kits)

IgG:

LIMITATIONS OF USE