Enzymoimmunoassay (EIA) using sandwich method with insulin antibody bound beads and insulin antibody bound peroxidase was compared with radioimmunoassay (RIA) in the determination of insulin of human sera. The substrate solution comprised hydrogen peroxide and 5-aminosalicylic acid. The following results were observed: (1) The sensitivity of EIA was 5μU/ml, comparable to that of RIA. The linearity of the standard curve was obtained between 0 and 160μU/ml insulin. (2) The coefficient of variation of the intra-EIA was 14.2% at about 16μU/ml insulin and 7.5% at about 35μU/ml insulin, while that of inter-EIA 9.7% and 5.8%, respectively, which had not great difference from that of RIA. (3) The coefficient of correlation of insulin determined by EIA (Y) and RIA (X) in 170 human sera obtained at 50g oral glucose tolerarlce test was 0.918 (p<0.001) and the regression equation was Y=1.110x-4.278. (4) Proinsulin interfered with EIA, while C-peptide did not. (5) Hypoproteinemic sera revealed the discrepancy between EIA and RIA. The insulin ElA was suggested to be apPlicable clinically, although further study on its validity was needed.
In order to measure low levels of mitochondrial GOT (m-GOT) activity in eluates from DEAE-Sephadex A-50 minicolumn for the separate determination of serum GOT isoenzyme, a diazonium coupling procedure, modified from the method of Amador et al., and ultraviolet method for GOT determination were studied using System Olli 3000, a semiautomated computer-controled discrete type analyzer for end point and kinetic assay. To increase the sensitivity of the Fast Violet B dye method, low volume of the concentrated buffered substrate relative to that of sample is used, the incubation time is prolonged to E0 min., and the coloured product is measured at a fixed time after the addition of colour reagent without use of a diluent. By these means, GOT activity as low as 0.2 Karmen units can be precisely determined with a linearity of up to 6 Karmen units by the instrument. The photometer and the computational program developed for kinetic assay of the instrument exhibit several advantages for measuring GOT activity as low as 0.5 Karmen units by ultraviolet method with good precision.
Automatic analysis of acid phosphatase was made by the use of an autoanaeyzer system. This method automatizes the simultaneous determination of other acid phosphatases on one same sample, besides prostatic acid phosphatase. It may be considered of value in determining acid phosphatase activity which is very liabee to vary after blood sampling.
A simultaneous determination of urine VMA and HVA, as well as 5-HIAA was attempted by highspeed liquid chromatography. Urine was acidified with 6 N hydrochloric acid and extracted with ethyl acetate. The ethyl acetate layer was evaporated to dryness under reduced pressure. The residue was dissolved in 0.2 N acetic acid, and 40μl of the aliquot was injected on to the column. A highspeed liquid chromatograph was assembled from Waters ALC/GPC204 Type, the model 660 soovent programmer and packed with μBondapack C18. The mobile phase was consisted of water and methanol solution of PIC-B7. Elution was done gradiently by from 15 to 30% methanol in 10 minutes. The value of VMA was well correlated to that obtained colorimetrically. Moreover, the method proved to be very specific, reproducible with a good recovery. The technique was applied to the 10 urine specimens of normal subjects. The mean excretion of VMA was3.5±1.1 S. D. mg/day, 5-HIAA 3.3±1.3 S.D. mg/day and HVA 8.7±2.4 S.D. mg/day. In a case of phaeochromocytoma,they were 15.2mg/day, 6.2mg/day and 19.0mg/day respectively. Thus the method issimple and accurate enough for a routine simultaneous determination of urine VMA, HVA as well as 5-HIAA.
A method of quality control for the individual sample is presented by use of amultivariate statistical method. Multiphasic clinical chemistry data are processed by principal component analysis, and components with small variation are taken as indicators of the quality control. Abnormal values of these principal components are indicative of either unusual pathological states or mistakes of laboratory technics. The quality control with this method corresponds well to the data-check by the clinical pathologist.