The use of luminol-dependent chemiluminescence assay for monitoring the phagocytic and opsonic activity in bovine whole blood

Abstract

Luminol-dependent chemiluminescence (CL) assay using whole bovine blood was developed to evaluate its phagocytic function and opsonic activity. The optimal assay system was as follows. A whole blood sample (100 μl) was mixed with 400 μl of Hanks balanced salt solution containing 25 m m Hepes, luminol (1 × 10 −4 m: final concentration) and zymosan (925·9 μg/ml: final concentration) were added and the mixture was measured continuously in a chemiluminometer. Final concentration of plasma in a reaction vial was 18·5%. Results were expressed as peak CL (cpm), CL index and the time (peak time) taken to show the peak CL. There was a significant correlation between the peak CL and the number of neutrophils in blood ( r = 0·722, P < 0·01, n = 25). The peak CL and peak time were closely related to the plasma concentration. The addition of sodium azide completely abolished CL response and Superoxide dismutase significantly inhibited CL response by 70% of the control. In the presence of sodium benzoate, catalase and histidine, a slight decrease of CL response was observed. CL response of whole blood was decreased considerably depending on the storage conditions and the relative percentages of the CL response under three different conditions were as follows: 83·3% at 5·7 h for 10 °C, 71·0% at 5 h for 30 °C and 60·5% at 4·3 h for 0 °C, as compared with the value which obtained from freshly drawn blood (10 min after blood collection). Whole blood CL assay was simple, rapid and is a useful tool for monitoring the phagocytic function and opsonic activities. It is potentially applicable to other animal systems.