A sensitive method for quantifying mouse plasma alpha-
macroglobulins (AM) using an inhibition ELISA is described. AM are
important plasma proteinase inhibitors that possibly act also as
immunomodulatory molecules. The standard protocol developed in our
experiments involves coating well with 10ug/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 ug/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 levels in small
samples of mouse plasma.
