Development of a direct radioimmunoassay for serum progesterone

A direct radioimmunoassay for the measurement of progesterone in human serum is described. Progesterone 11-hemisuccinate was conjugated to tyrosine methyl ester (TME) by the mix anhydride method and then iodinated using chloramine-T. The radiochemical purity of different batches of¹²⁵I-progesterone was greater than 95% and showed 70–75% binding with excess antibody. Progesterone 11-hemisuccinate was coupled with BSA and injected to rabits. Antisera collected after three booster injections and having aK value of 1·10⁹1/M was selected for the assay. Significant reduction in binding with antibody was seen when hormone free serum was used in the assay system. Various blocking agents were tried to reduce the serum effects and none of them were found satisfactory. From a series of optimization experiments, an assay was developed without the use of blocking agents. This assay used a much higher concentration of antibody along with lower amount of serum sample (50 l). The optimized assay has a sensitivity of 0.5 nj/ml and a working range of 0.5 to 100 ng/ml. Serum samples were analyzed analysis showed good correlation between the results obtained from the present system and the DPC kit. (Y=0.93X+0.5,r=0.93, forn=25).     

Development of solid phase radioimmunoassay system using new polymeric magnetic micro-spheres

Magnetic particles were locally prepared by co-precipitation of Fe²⁺ and Fe³⁺ in an ammonia solution. The prepared microsphere were grafted with polyacrylamide acrylic acid by using gamma irradiation polymerization in presence of MBA as a cross linker. AFP antibody was immobilized on these beads and used as a solid phase in radioimmunoassay technique. The immunoreactivity of the developed assay was found to be influenced by different factors such as solid phase volume, incubation time, incubation temperature and storage period. A comparative study was performed between the developed assay system and others two ones. The maximum binding percent attained the value of 19.5% while the sensitivity was observed to be 1.3 IU/mL. The developed assay displayed acceptable precision estimated by repeated analysis of the quality control samples and the clinical samples analyzed by this assay showed a good correlation with that commercial kit (r = 0.998).     

Development of radioimmunoassay of intact laminin and its clinical evaluation as a tumor marker.

The concentration of laminin including laminin variants in serum samples was measured by a double-antibody radioimmunoassay using intact laminin. The mean level in 92 normal subjects (47 men and 45 women) was defined as 1 unit (U)/ml, and the cut-off value (2 S.D. above the mean) was 1.37 U/ml. Mean laminin level in 391 patients with various malignancies was 1.50 +/- 0.86 U/ml. Laminin levels were elevated in various cancer patients, and in 45.0% (176/391) the values exceeded the cut-off level; in patients with cancer of the stomach or pancreas, positivity rate exceeded 60%. Mean laminin concentration for 130 pregnant women (2.06 +/- 0.65 U/ml) was also significantly higher than that of normal controls, but concentrations for patients with various benign diseases were within a low range (0.55 +/- 0.29-1.10 +/- 0.29 U/ml). In the stomach or pancreas cancer patients, a positive correlation between laminin level and tumor progression or course of the disease was observed. These findings indicate that serum laminin level is potentially useful in the diagnosis and monitoring of certain cancers.     

Some problems of radioimmunoassay control

Problems and aims of RIA control are discussed and the survey of the major characteristics applied to the control is presented. An arrangement and organization of RIA control system which enable the objective and rational control on different levels are proposed.     

Recent developments in radioimmunoassay

Recent developments in the radioimmunoassay of steroid and peptide hormones are discussed. The modern requirement for the rigorous demonstration and maintainance of accuracy is emphasized. The future of immunoassay in clinical chemistry is discussed with special reference to quality control and automation.     

Clinical evaluation of immunoglobulin-E radioimmunoassay kit

An IgE RIA kit (Sandwich method; Dainabott), is used to obtain the following results. (1) Standard curve: Since the range of reproduction rate show 3.16-7.07% (C.V.), the curve become steep. (2) Incubations under controlled situation: Both of the incubations are controlled at 15-30 degrees C for 2 h. (3) Reproducibility test: Coefficients of variation (C.V.) of intra-assay and inter-assay variation are 2.32-3.94% and 2.92-3.92% respectively. (4) Recovery test: A result of the recovery test range between 100.1-101.7%. (5) Dilution test: Multiple dilution effects are observed. (6) Average counts of the serum IgE for the controlled and diseased groups: The average counts of the serum IgE for the controlled group, atopic diseased group, allergic rhinitis group and allergic bronchial asthma are 144.9 +/- 183.2 IU/ml, 1,099.0 +/- 2,782.4 IU/ml, 1,150.9 +/- 2,063.3 IU/ml and 600.7 +/- 686.4 IU/ml respectively. The value of the diseased groups have tendency to show higher averages than the controlled group. Since the controlled and diseased groups show wide distributions of the serum IgE level, there is no significant difference of two variations. However the diseased groups have tendency to show higher ratio of the serum IgE level in blood than the controlled groups. These basic researches are quite meaningful, because they are able to apply for a supplemental diagnosis of the atopic and parasitic disease.     

A radioimmunoassay for serum estradiol

A radioimmunoassay procedure to measure estradiol levels in human serum was developed using¹²⁵I labeled estradiol and antiestradiol antibody raised in-house using the estradiol-6-CMO-BSA as the immunogen. Controlled process serum was used to maintain the serum matrix and sample-standard identity. The assay was validated by analysing several (n=40) samples and comparing with established commercial assays. The assay had a sensitivity of 35 pg/mL and a range of 50 pg/mL to 5 ng/mL. The interassay C.V. were between 2 to 10% while the intraassay C.V. were between 2 and 8%. This assay is sensitive enough for monitoring ovulation and hypersecretions in ovarian tumours and was primarily developed to be used as an in-house assay in the local hospitals.     

A single reagent thyroxine radioimmunoassay

An homogenous radioimmunoassay of thyroxine using labelled antigen antibody complex as a single reagent is described. Variation of dose response curve slope with assay incubation time has been studied at two different antisera concentrations. The dissociation constant of the complex was found to be 3.5·10⁻⁴ s⁻¹. The assay involves only three pipettings and requires 45 minutes incubation at 37 °C and 10 μl of serum. Though the assay is essentially a nonequilibrium type, apparent increase in sample values, due to the drift of dose response curve for 45 minutes delay was less than 7.5%. Within and between assay variations were both less than 5% c. v. Assay has been validated by recovery experiments. Analysis of 40 serum samples by the proposed method and by an equilibrium assay gave similar values; r=0.957, Y=0.955X+0.478.     

Clinical usefulness of a trypsin radioimmunoassay kit

From the clinical use of RIA-gnost trypsin kit, the following results were obtained. 1. Standard curve showed a steep and good curve was shown. 2. Incubation: The condition for the first incubation was set at the room temperature for 10-24 hours and that for the second incubation at the room temperature for 3-5 hours. With these settings, satisfactory results were obtained. 3. Reproducibility and recovery: The C.V. of the reproducibility and the recovery were considered superior, and the values were below 10% and +/- 3%, respectively. 4. Correlation between trypsin and serum elestase-1: An excellent positive correlation (coefficient of correlation r = 0.889) was shown. 5. Serum trypsin concentration of normal and pancreatic diseases: The normal range was from 100 to 500 ng/ml. Acute pancreatitis rose obviously. Diabetes mellitus and chronic pancreatitis was below 500 ng/ml and the pancreatic cancer showed a tendency to scatter in the range of 50-1,250 ng/ml. The above results indicated that serum trypsin can be easily measured with high precision by using this method. Thus the method is considered useful for the diagnosis of pancreatic diseases.     

A rapid radioimmunoassay procedure for thyroxin

A sensitive thyroxin /T₄/ radioimmunoassay procedure with a short incubation time /10 min/ similar to a conventional stat test in clinical chemistry is described. The assay parameters such as concentration of antisera and¹²⁵I-T₄, and incubation temperature that affect the kinetics of antigen-antibody reaction have been studied. Within- and between-assay variations were less than 7% CV. Analysis of 45 serum samples by the proposed method and by a conventional assay gave similar results /Y=1.05X–0.187; r=0.990/.