An evaluation of the conditions necessary for optimal protein A-gold labelling of capsular antigen in ultrathin methacrylate sections of the bacteriumPasteurella haemolytica

Summary The protein A-gold (PAG) probe is a particulate immunocytochemical probe that is eminently suitable for quantification. In order to obtain critical results from the technique, a specific and reproducible probe is needed. To this end, the concentration of probe, the variation of labelling different sections within a single grid, the effect of washing procedures, the variation of labelling with time and temperature and the effect of different storage conditions on the probe have been investigated using PAG labelling of capsular antigen on ultrathin methacrylate sections of the bacteriumPasteurella haemolytica.The results indicate that in this antigen-antibody system, and using a 20nm probe, optimal results are achieved with 2×10¹² particles/ml, a labelling time of 60min at room temperature and the PAG probe, which will have been stored at 4°C, should be between 1-and 5-weeks-old. The efficiency of the probe is tested by evaluating different primary antibody concentrations, by evaluating cross reactions of the primary antibody and by evaluating the relative amounts of antibody against internal components of the bacterium present in different antisera.