Detection of anti-HBAg by solid phase radioimmunoassay using labeled human antibody]

A new solid-phase radioimmunoassay technique for the detection of anti-HBAg is described. The test is characterized by a neutralization step of guinea-pig anti-HBAg by means of HBAg-RIA positive pooled sera. The first results obtained using this technique gave 21 anti-HBAg positive sera (26.9%) among 78 institutionalized subjects. One of these sera was HBAg positive at the same time.     

A radioimmunoassay for human thyroglobulin: methodology and clinical applications

A specific double-antibody radioimmunoassay with a sensitivity of 2.5 ng/ml has been developed for measuring thyroglobulin (Tg) in human serum. As endogenous anti-Tg antibodies in serum interfere in the assay, only sera with a negative tanned red cell (TRC) test are suitable for analysis. Tg was detectable in 84.7% of the euthyroid subjects, with a mean value of 6.1 (values ranging from nondetectable to 43.0 ng/ml). Values were significantly higher in women than in men. Tg release by the thyroid appears to be under pituitary control, as suggested by TSH stimulation and T3 suppression tests. Elevated Tg levels were found in hyperthyroidism, simple goitre, and differentiated thyroid carcinoma. The significance of circulating Tg and the possible application of the Tg RIA are discussed.     

Direct solid-phase time-resolved immunofluorometric assay of cortisol in serum

A dissociation-enhanced lanthanide fluoroimmunoassay of serum cortisol based on time-resolved fluorescence is described. The assay is a direct assay, where cortisol immobilized on the wall of a microtiter-strip well competes with cortisol in the sample for the europium-labeled polyclonal antibody. The amount of bound europium-labeled antibody is inversely proportional to the amount of cortisol in the sample. Separation is accomplished by washing the strip well. The assay is carried out in 2 h, at room temperature; it is easy to perform and gives accurate and reliable results. A chaotropic agent, trichloracetic acid, was very effective in releasing cortisol from binding proteins. This finding will have practical importance in the immunoassay field.     

Detection of double-stranded RNA in Semliki Forest virus-infected cells by an indirect solid phase radioimmunoassay: an assay for interferon

An indirect solid phase radioimmunoassay using rabbit antibodies to the synthetic double-stranded RNA poly rI-rC and 125I-labelled sheep anti-rabbit IgG could detect 1 ng of poly rI-rC. This assay could also detect the presence of double-stranded RNA in Semliki Forest virus-infected chick fibroblasts and can be used as an assay for interferon activity.     

A single-step solid phase radioimmunoassay for quantifying human carbonic anhydrase I and II in cerebrospinal fluid

A single-step solid phase radioimmunoassay was developed to detect human carbonic anhydrase (CA) isoenzymes I (CA I) and II (CA II) in cerebrospinal fluid (CSF). The assay is capable of routinely detecting both isoenzymes at ng levels compared to the μg levels of the traditional catalytic methods, which failed to demonstrate any CA activity in CSF. When the values of immunoreactive CA II in CSF were corrected for blood contamination (the CA I/CA II ratio of blood was about 7.9), the amount of brain tissue originated CA II could be calculated. The CA II values in CSF samples from 13 patients with multiple sclerosis were higher than those in CSF samples from 11 patients with various peripheral neurological disorders. Since CA II has been specifically localized to oligodendrocytes and myelin, our preliminary results suggest the possibility of CA II leakage from oligodendrocytes and myelin into CSF in demyelinating diseases.     

Caffeine in plasma and saliva by a radioimmunoassay procedure.

Caffeine was analyzed in human plasma and saliva by a simple, rapid, and sensitive radioimmunoassay procedure. Immunization of rabbits with an antigen prepared by coupling 7-(5-carboxypentyl)-1,3-dimethylxanthine to bovine serum albumin resulted in the formation of antibodies selective for caffeine as opposed to various mono- and dimethylxanthines, mono-, di-, and trimethyluric acids and a variety of common drugs. The radioligand used for competitive binding studies was 7-(2,3-3H2-propyl)-1,3-dimethylxanthine. The procedure permits direct analysis of caffeine in plasma or saliva without extraction. Comparison with a high pressure liquid chromatography method for the analysis of caffeine gave satisfactory results and showed no evidence for interference by metabolites. A caffeine half-life of 4.0 hours determined by the radioimmunoassay was in agreement with previous work. Comparison of human plasma and saliva levels by the radioimmunoassay procedure indicated approximately equal concentrations in the two fluids.     

Stability of platelet activating factor (PAF) in human saliva. Quantitation by radioimmunoassay

Platelet activating factor (PAF) is thought to mediate many inflammatory processes and its involvement in health and disease may be clarified by examining PAF levels in human secretions. The known presence of PAF, the ease of obtaining samples and the relative stability of PAF in saliva, makes this fluid a preferred source for examination of PAF in health and disease. The activity of PAF-acetylhydrolase (the PAF degrading enzyme) in saliva was 1,000-fold lower than that found in human plasma. Extraction of saliva with chloroform/ methanol/water resulted in 70–90% recovery of PAF. Using the radioimmunoassay (RIA), PAF levels in the range 0.5–21 ng/ml were found in normal human salivas. These values were significantly higher than those reported from bioassay studies based on washed platelets. The validity of the RIA was checked by isolating and quantitating the PAF fraction from whole saliva extract, and by treatment of the extracts with the enzyme phospholipase A₂. Direct comparison of salivary PAF levels, determined by both platelet aggregation (PA) and RIA confirmed our original finding that values obtained were lower using the bioassay method. Furthermore, these bioassay values compared favourably with those in the literature. Investigations revealed the presence of a substance(s) in saliva which inhibited PAF-induced platelet aggregation but which did not affect the radioimmunoassay.     

Comparison of IgE values as determined by different solid phase radioimmunoassay methods.

Accurate measurement of low IgE concentrations if technically difficult. In this paper results obtained by a direct sandwich and three inhibition methods of radioimmunoassays are compared. For values above 50 U/ml good correlation was obtained with all methods. Below 50 U/ml, however, the inhibition methods tended to yield falsely high values. For very low concentrations, 1--10 U/ml the best correlation was obtained between the direct sandwich test (PRIST) and the inhibition test using a correction factor to allow for the non-specific effect of serum. The four methods were used to quantify IgE in cord serum samples from healthy individuals. The mean value obtained by PRIST was 0-4 U/ml and by the inhibition test, using a correction factor, 0-6 U/ml respectively. Because of its greater simplicity the direct sandwich test is recommended.     

Determination of cortisol in human plasma by radioimmunoassay. Use of the 125I-labelled radioligand

A radioimmunoassay for plasma cortisol featuring the gamma-emitting radioligand 125I-iodohistamine, coupled to cortisol-3-(O-carboxymethyl)-oxime, is described. The new procedure retains much of the specificity associated with the use of anti-cortisol-3-BSA sera with tritium-labelled radioligands, and has the further advantages that running costs are lower and there is a greater potential for automation. Cortisol values obtained by this procedure agree well with those obtained by a published specific radioimmunoassay using the tritiated cortisol radioligand. Specificity of the procedure was checked by comparing values obtained with and without thin-layer chromatography purication: correlation was excellent (r = 0.96). Satisfactory levels of sensitivity, precision, and accuracy were obtained.     

Serum cortisol and 11 deoxycortisol by liquid chromatography: clinical studies and comparison with radioimmunoassay.

We describe a liquid-chromatographic procedure for separating and measuring cortisol and 11-deoxycortisol in serum. We quantitated these steroids in patients who were undergoing various tests of pituitary and (or) adrenal function and compared the results with those obtained by two radioimmunoassays done in two different laboratories. Results of 48 tests done in 37 functionally normal humans are presented. Cortisol values for sera collected in the morning as determined by liquid chromatography were (mean +/- SD) 134 +/- 54 micrograms/L. Serum cortisol concentrations increased from 136 +/- 65 to 321 +/- 80 micrograms/L 60 min after injecting synthetic corticotropin and increased from 107 +/- 46 to 242 +/- 31 micrograms/L after insulin-induced hypoglycemia. Serum cortisol decreased from 142 +/- 49 to 26 +/- 20 micrograms/L after oral administration of metyrapone, while 11-deoxycortisol increased from less than 10 to 210 +/- 53 micrograms/L. Serum cortisol measured less than 10 micrograms/L the morning after oral ingestion of dexamethasone. Results of the dynamic tests of adrenal function correlated well with previously reported studies. However, the cortisol values obtained by our technique were generally lower than those obtained by radioimmunoassay, possibly owing to lack of specificity of the latter methods used here for comparison. In contrast, values for 11-deoxycortisol were the same by both methods. The present studies confirm the usefulness of liquid chromatography for measuring these two steroids in serum during tests of pituitary and adrenal function. Future refinements of the technique should continue to increase its clinical applications.     

Direct assay for testosterone in saliva: relationship with a direct serum free testosterone assay

A direct non-extraction radioimmunoassay for salivary testosterone is described using a modified commercial kit procedure that is in use for total serum testosterone (T). Serum free testosterone was also measured by direct radioimmunoassay. A significant correlation (r = 0.83, p less than 0.01, n = 194) was obtained between salivary and serum free testosterone in matched serum and saliva samples over a wide range of concentrations. Within- and between-batch precision for the salivary testosterone method was 11% and 18%, respectively at a concentration of 170 pmol/l. Recovery of added T was 89% +/- 15% (mean +/- 2 SD) dilution of high samples showed parallelism. Salivary testosterone measured by direct radioimmunoassay offers a simple cheaper alternative to serum free testosterone measurement with the additional advantages of a stress-free non-invasive sampling procedure.     

A quantitative assay of cortisol in human plasma by high performance liquid chromatography using a selective chemically bonded stationary phase

The extraction and subsequent liquid Chromatographie analysis of human plasma samples for cortisol is described.Extraction and chromatography are optimized, resulting in a recovery for cortisol of 96% and a detection limit of 1 μg cortisol in 100 ml plasma. The application of two chemically modified silicas has been evaluated.The specificity of the method was tested by field desorption — mass spectrometry experiments.     

Determination of human erythropoietin by radioimmunoassay. Method and clinical data

A sensitive competitive radioimmunoassay for the determination of circulating human erythropoietin (Epo) is presented. This RIA of human Epo is founded on the extensive application of a recombinant protein approach. Thus, highly purified recombinant human Epo is employed both as a tracer, immunogen and occasionally as standard in the RIA. The lower limit of detection is approximately 8 mU/ml and well below the mean value of Epo in normal individuals (25.6 +/- 6.6 mU/ml). The intra- and interassay variation are 6.3 and 9.2%, respectively. The documentation of consistent alterations in Epo plasma levels of selected categories of anemic patients establishes the clinical relevance of the immunoassay. The radioimmunoassay of human Epo fulfills reasonable requirements of a clinical routine analysis in terms of both specificity, sensitivity and practicability.     

Phenytoin and phenobarbital concentrations in saliva and plasma measured by radioimmunoassay.

Saliva and plasma levels of phenytoin (DPH) and phenobarbital (PB) in a series of epileptic patients were compared by means of a radioimmunoassat (RIA) that required only 10 mul of saliva or plasma. There was an excellent linear relation (r = 0.98) between the logarithms of the concentrations of DPH in the two fluids. The ratio saliva/plasma was remarkably constant at 0.10 and was unaffected by varying levels of PB. The ratio was close to the fraction of DPH reported unbound in plasma at 37 degrees. PB plasma and saliva levels were also closely related (r = 0.98 for logarithm of plasma and saliva levels). This relation was nonlinear [plasma ocncentration = 4.43 X (salivary concentration)0.86], but could be approximated by the ratio plasma/saliva = 3.4. The simplicity of sample collection and the sensitivity of the RIA procedure suggest that clinical monitoring of these anticonvulsant levels may be carried out by RIA on saliva samples.     

A new sensitive direct radioimmunoassay for human plasma renin and its clinical application

We developed new sensitive direct radioimmunoassay for human plasma renin. Renin was purified from Haas' preparation utilizing a pepstatin-C6-Sepharose affinity chromatography. Antiserum, prepared by immunizing rabbits with the purified renin, was used for the direct radioimmunoassay at a final dilution of 1:30,000. The antibody was specific for human renal and plasma renin, but did not cross-react with cathepsin D, trypsin, or renins of mouse, dog, and rat. Radioimmunoassay was performed by the double antibody technique using the delayed tracer addition method. In this method, a standard curve was obtained over a range from 0.2 to 8.0 ng/ml. The values from our assay correlated well with total renin activity measured as the generation rate of angiotensin I after trypsin activation (r = 0.78, p less than 0.01), but correlated weakly with active renin activity. This finding disclosed that both active and inactive renin were detected by this method. In normal participants, plasma renin concentration determined by direct radioimmunoassay was increased by standing and furosemide injection. The plasma renin concentration determined by direct radioimmunoassay of patients with essential hypertension (0.7 to 1.7 ng/ml) was not significantly different from values in normal controls (0.8 to 1.9 ng/ml). The values were higher in patients with renovascular hypertension (1.6 to 2.7 ng/ml), malignant hypertension (2.8 to 3.4 ng/ml) and Bartter's syndrome (1.8 to 2.5 ng/ml), but lower in patients with primary aldosteronism (0.4 to 0.8 ng/ml) than in normal controls. This newly developed radioimmunoassay for human renin was sensitive enough to estimate the levels of renin in plasma of patients with low renin hypertension. It provides a new tool for the understanding of the renin-angiotensin system under various clinical conditions.     

Molecular dissection of an hCG-beta epitope using single-step solid phase radioimmunoassay

BACKGROUND: Peptides and proteins have both sequence-specific (contiguous) and conformation-specific (discontiguous) epitopes. Sequence-specific epitopes are delineated by peptide approach and other robust methods like competition assays, gene expression assays, synthetic peptide library based assays etc. Available methods for delineation of conformation-specific epitopes are cumbersome (X-ray crystallography etc.), time-consuming and require costly sophisticated equipments. Hence, there is a need to develop a simple method for identification and mapping of conformation-specific epitopes. METHOD: In the single-step solid phase radioimmunoassay (SS-SPRIA), an immunochemical bridge of 'mouse IgG-anti-mouse IgG' was prepared in the polypropylene wells followed by adsorption with hCG specific monoclonal antibody (MAb) G(1)G(10).1. The extent of competitive inhibition in binding ability of (125)IhCG-beta with chemically or enzymatically modified hCG-beta to immobilized MAb G(1)G(10).1 in comparison to hCG-beta standards was utilized to identify the epitopic amino acid involved in epitope-paratope interaction. RESULTS: Data clearly suggest that the epitope under investigation consisted of Arg (94, 95) and Asp (99) at the core region with a Lys (104) and a His (106) in the proximity and absence of chymotrypsin susceptible Phe or Tyr in this region. CONCLUSION: The data of SS-SPRIA revealed the 93-100 loop of amino acid sequence, as the core region of conformation-specific epitope of hCG-beta at or near the receptor-binding region. Hence, SS-SPRIA seems to be a simple method for identification and mapping of conformation-specific epitopes.