Minimum Incubation Time Research Articles

Methodological and clinical observation of chromogenic substrate for plasma prekallikrein and prekallikrein assay

On the assay method by the use of chromogenic peptide substrate (Chromozym PK) consisting of Bz-Pro-Phe-Arg-pNA the quantitative observations on plasma kallikrein are performed methodologically and clinically, and the following results are obtained.1) The minimum incubation time to activate plasma prekallikrein with kaolin requires about 20 minutes at 0°C. However, the incubation time is shorten to 3 minutes at 25°C or 37°C following a rapid decrease of kallikrein activity in the reaction mixture.2) The standard curve of the PK assay using chromogenic substrate with 0.94mmol/l at the optimal pH (7.9) demonstrates a linear manner within 8 minutes incubation at 37°C.3) For the purpose of anticoagulant sodium citrate is better than other remedies including sodium oxalate, EDTA and heparin, and it is observed that heparin can not be used for this chromogenic assay because of producing turbid precipitate.4) The plasma stored at 4°C or -20°C are stable on the amidase assay for one week.5) When the pooled normal plasma is taken as 100 per cent, the lenear relationship between diluted plasma and ΔOD is obtained in the range to 6.25 per cent.6) Coefficients of variation is low, normal and high activities are calculated as 13.7%, 2.6%, and 0.95%, respectively.7) Normal values of plasma prekallikrein and kllikrein in Japanese adults are in the range of 38.5-73.3mU/ml and 0-3.55mU/ml, respectively.8) Although the levels of plasma prekallikrein and kallikrein in the majority of tested patients are in the normal range, one case with DIC shows extremely low concentration of plasma prekallikrein, and six cases with liver cirrhosis are distributed in the subnormal range.9) The plasma samples of 11 cases from 14 pregnant women show high levels (42-63mU/ml) of plasma kallikrein accompanying with lower levels of plasma prekallikrein.10) Furthermore, all plasma samples from 6 cases taking estrogen derivatives as oral contraceptive indicate high levels of plasma prekallikrein (84-110mU/ml), and this fact seems to be worthy to explain the tendency to thrombosis in such conditions created by the pills.

Polarisation fluoroimmunoassay of gentamicin

A polarisation fluoroimmunoassay has been developed for the routine determination of gentamicin levels in serum. The method employs fluorescein-labelled gentamicin which is easily, reproducibly and economically prepared and has excellent shelf-life. The assay is fast (minimum incubation time 2 min) and requires 1.25 μl of serum. There is no separation step prior to readout of results. Recovery experiments reveal no serum effects. Intra- and inter-assay coefficients of variation are of the order of 10% or less. Estimations of gentamicin levels in patient serum samples correlate closely with those obtained by bioassay ( r = 0.93) and radioimmunoassay ( r = 0.97).

Clinical use of a solid-phase radioimmunoassay specific for human chorionic gonadotropin

A rapid radioimmunoassay specific for human chorionic gonadotropin (hCG) has been developed. Elimination of cross-reactivity with luteinizing hormone (hLH) was obtained by utilizing an antiserum with a specificity for the beta subunit of hCG. A solid-phase system, in which the antibody was bound to CNBR-activated sepharose in a preliminary step, reduced the minimum incubation time of the assay to two hours. The assay has been found to be of particular value in those conditions where in the concentration of hCG as measured by previously described assays would be affected by circulating levels of hLH: i.e., early implantation, unruptured ectopic pregnancy, threatened abortion, and trophoblastic disease under chemotherapy.

The metabolism of phytohaemagglutinin during incubation with human peripheral lymphocytes.

The mechanism of action of PHA in in vitro leucocyte cultures was investigated with respect to the fate of the mitogen during the 3‐day blastogenic response. It was demonstrated that incubation of the leucocytes with PHA for only 5 minutes followed by incubation for 72 hours in medium devoid of PHA was sufficient to initiate mitosis and blastogenesis. However, the minimum incubation time with PHA required to induce maximum blastogenesis was 6 hours. Furthermore, it was demonstrated that the mitogen is bound to cells in culture but the experimental findings suggest that this cell‐bound mitogen is not degraded during the 3‐day incubation period.

SCAPHYTOPIUS ACUTUS (SAY), A NEWLY DISCOVERED VECTOR OF CELERY-INFECTING ASTER-YELLOWS VIRUS

Scaphytopius acutus (Say) transmitted a celery-infecting strain of aster-yellows virus from infected periwinkle (Vinca rosea L.) and aster (Callistephus chinensis Nees) to periwinkle, aster, and ladino clover (Trifolium repens L.). It acquired the virus during feeds as short as 4 hours, and transmitted it during a 2-hour feeding period. The minimum incubation time for the virus in the insect was between 21 and 26 days. After an acquisition feed of 7 days and an incubation period of at least 30 days, 11 of 21 insects transmitted the virus to separate periwinkle test plants.