Determination of rat β2‐microglobulin in urine and in serum. I. development of an immunoassay based on latex particles agglutination

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Abstract
Rat β2‐microglobulin has been isolated from the urine of rats pretreated with sodium chromate. The purified protein was used to raise antisera in two rabbits. Treatment of the antisera with ammonium sulphate followed by gel chromatography led to an immunoglobulin fraction which was used to coat latex particles by physical adsorption. The latex suspension was used in an automated system including an optical particle counter to analyse the protein in serum, urine and cerebrospinal fluid. Serum contains 5.8 mg of β2‐microglobulin/l, of which 33% is found as a 55000 dalton complex while 66% is present as the ‘free’ protein. The daily urinary excretion of β2‐microglobulin in females is about 2 μg of which 7% is found to be a 65000‐dalton complex while 92% is the free protein. From this, it can be calculated that the fractional urinary excretion of β2‐microglobulin is about 0.03%. The cerebrospinal fluid contains about 1 mg of β2‐microglobulin/l. Preliminary tests also suggest that the method can be adapted for non‐automated turbidimetric detection. In the automated assay, the within‐ and between‐assay coefficients of variation are less than 10% for the three biological fluids tested. The analytical recovery in the urine is 93%. In urine, β2‐microglobulin undergoes proteolytic degradation at pH below 6. This does not represent a serious drawback to its use as a sensitive index of tubular function since in most experimental circumstances, rats excrete urine with a pH above this value.