Brucella IgM ELISA

Enzyme immunoassay for the qualitative determination of IgM-class antibodies against Brucella in human serum or plasma (citrate, heparin). Brucella is a small Gram-negative bacterium (0.4-0.8 μm in diameter and 0.4-3.0 μm in length) which is nonflagellated, and non-spore-forming. They are named after the military doctor David Bruce, who on Malta in 1887 isolated the pathogens from the spleen of a soldier who died of undulating fever. Four species are pathogenic to human: Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis. All four species are exciters of Brucellosis, a disease characterized by undulating fever. Depending on exciter the disease is also called Morbus Bang (B. abortus) or Malta fever (B. melitensis). The pathogens are transmitted from animals, which are mainly affected. The infection is caused by contact with ill animals or their excrements as well as by non-pasteurized milk and milk products like fresh cheese from sheep or goat. Main entrances are skin wounds, conjunctives and digestive tract. The intact pathogens are transported by granulocytes into local lymph nodes, from where they spread haematogenous. All kind of organs can be infected. Symptoms depend up on the infected organ, where an inflammation takes place. Until now the pathogenic mechanism is not completely understood. Brucellosis appears worldwide. In non-pasteurized milk and milk products Brucella is viably and infectious for weeks. Bovine brucellosis caused by Brucella abortus is still the most widespread form, although reported incidence and prevalence of the disease vary widely from country to country (from <0.01 to >200 per 100,000 population). Brucella melitensis is endemic in areas where keeping of sheeps and goats is frequent. It causes serious human infections. Working with these bacteria in laboratories necessitates highest carefulness because of high contagiosity. Risk groups include abattoir workers, meat inspectors, animal handlers, veterinarians, and laboratorians. Brucellosis is a nationally notifiable disease and reportable to the local health authority. The qualitative immunoenzymatic determination of specific antibodies is based on the ELISA (Enzyme-linked Immunosorbent Assay) technique. Microtiterplates are coated with specific antigens to bind corresponding antibodies of the sample. After washing the wells to remove all unbound sample material a horseradish peroxidase (HRP) labelled conjugate is added. This conjugate binds to the captured antibodies. In a second washing step unbound conjugate is removed. The immune complex formed by the bound conjugate is visualized by adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product. The intensity of this product is proportional to the amount of specific antibodies in the sample. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorbance at 450/620 nm is read using an ELISA Microtiterplate reader.

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