MICROPARTICLE-ENHANCED NEPHELOMETRIC IMMUNOASSAY FOR CASEINOMACROPEPTIDE IN MILK

A microparticle-enhanced nephelometric immunoassay has been developed for the determination of caseinomacropeptide (CMP) in bovine milk. It is based on the nephelometric quantification of the competitive immuno agglutination of a microparticle-CMP conjugate with an anti-kappa-case in (kappa-CN) antiserum. This one step immunoassay was sensitive (dete ction limit in reaction mixture, 16 mu g/l), accurate (linear recovery of CMP in dilution overloading) and reproducible (CV 7-14% for within and between run precision). Because of the specificity of the polyclo nal antiserum used, it was necessary to separate CMP from kappa-CN by ultrafiltration before the quantification of bovine milk CMP. Under th e conditions of milk ultrafiltration used, kappa-CN was entirely retai ned (> 99.5%) but the concentration of CMP measured in milk ultrafiltr ates was underestimated (by similar to 25%) compared with its concentr ation in whole milk. Microparticle-enhanced nephelometric immunoassay of CMP, with a calibration range from 0.32 to 20 mg/l for 20-fold dilu ted milk ultrafiltrate, allowed contamination of bovine milk by rennet whey as low as 5 ml/l to be detected. Applied to ultrafiltrates from milk stored at 4 degrees C, this immunoassay also detected proteolysis of kappa-CN not revealed by measurement of kappa-CN concentration in milk. A statistical lower limit of 3.21 mg/l was determined as the inc rease in CMP concentration in milk ultrafiltrates that indicated proba ble kappa-CN proteolysis in the milli sample. Previously demonstrated to be an easy to perform method for assaying the main proteins of bovi ne milk, microparticle-enhanced nephelometric immunoassay thus also ap peared to be appropriate to quantify CMP so as to detect slight contam ination of milk by whey and to indicate the proteolysis of kappa-CN du ring milk storage at low temperature.