|Budget Amount *help
¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1991 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1990 : ¥700,000 (Direct Cost : ¥700,000)
The present study was undertaken to establish clinical usefulness of urinary hemoglobin under pathlogic conditions. Hemoglobin (Hb) was purified from erythrocytes and its rabbit antibody was thus prepared. Using purified Hb as standard and rabbit antibody, a manual latex agglutination test and a radioimmunoassay of a double antibody system were established. Based on previous experiences, Hb was obtained by isoelectric focusing : hemolysate was at first obtained by hemolyzing with distilled water containing a equal volume of toluene. Hb rich fractions were thus separated on isoelectricfocusing under pH 6-8 and sucrose density gradient from 5 t6 50%. A highest peak from PH 7.02-7.08 was collected and used as standard and immunogen. Rabbit antibody was prepared according to established methods previously reported.
A latex agglutination test was thus established in the same manner as previously reported. There was no positive reaction in 14 normal urine. In evaluating pathologic urine, the
result by the test showed a good coincidence with that by a dipstick test. Provided urine osmolarity was low, the present test could more sensitively detect minute amounts of Hb. The merit of the test based on antigen-antibody reaction was that it can detect Hb without interference of ascorbic acid and myoglobin.
Since the variation of microhemoglobulinuria may be of clinical significance, a double antibody radioimmunoassay was establised. The assay was quite sensitive to detect about 10 ng/ml of Hb and intra-assay precision in purified Hb was satisfactory. however, precision, recovery and a dilution test were all poor in evaluating urinary Hb. This was explained by generation of methohemoglobin upon exposure to air. A further study is needed to prevent Hb from being degenerated.