An anti-insulin antibody IgG-coated polystyrene ball of 3.2 mm in diameter was incubated with insulin or sample in a total volume of 50 μl in a small glass tube (3.5 × 20 mm) and subsequently with affinity-purified (anti-insulin) Fab'-peroxidase conjugate in a 50 μl in the same size glass tube. The peroxidase activity bound to the ball was assayed by using fluorimetry, in which the terminal volume for fluorimetric measurement was reduced to 0.5 ml. As a result, detection limit of insulin was as low as 0.3 nU (12.5 fg)/tube. Human insulin in normal saliva was determined by using this highly sensitive sandwich enzyme immunoassay. Gel filtration of concentrated human saliva sample showed that the major peak with insulin immunoreactivity eluted at the same position as monocompone porcine insulin. The level of insulin in normal human saliva was 2.8 + 2.47 (SD) μ/ml (n=8), and a response of saliva insulin secretion by glucose loading was evidenced.