Print version ISSN 0074-0276
On-line version ISSN 1678-8060
Mem. Inst. Oswaldo Cruz vol.89 no.2 Rio de Janeiro Apr./Jun. 1994
An inhibition ELISA to determine α macroglobulin levels in mouse plasma
Katholic University of Leuven, Dept. of Human Genetics, Leuven, Belgium
Instituto Oswaldo Cruz, Departamento de Ultraestrutura e Biologia Celular, Rio de Janeiro, Brasil
A sensitive method for quantifying mouse plasma α-macroglobulins (AM) using an inhibition ELISA is described. AM are important plasmaproteinase inhibitors that possibly act also as immunomodulatory molecules. The standard protocol develope in our experiments involves coating well with 10 µg/ml A2M in carbonate buffer, followed by incubation with a 1:1 (v/v) mixture of the plasma to be tested (diluted 1/1000) and goat anti-AM (diluted 1/1250). This is followed by further incubation, first with the enzyme-conjugated antibody and with the substrate prior to the reading of absorbance levels of the reaction products. Standard curve samples must be included in each plate, employing known amounts of the purified Murine Alpha-2-Macroglobulin (MuA2M) used for coating, with concentrations ranging from 0.001 to 10 µg/ml. Using test samples in triplicates and a 6-point standard curve in a single ELISA plate, 25 plasma samples can be tested accurately. The method offers an useful tool for establishing AM levelsin small samples of mouse plasma.
Key words: α-2-macroglobulin; A2M; ELISA; murinoglobulin