Two radioimmunoassay (RIA) systems for genistein have been established, based on polyclonal antibodies against genistein-4'-O-(carboxymethyl)ether-bovine serum albumin and genistein-7-O-(carboxymethyl)ether-bovine serum albumin conjugates. The sensitivities of assays were 4.44 and 10.4 fmol (1.2 and 2.8 pg)/tube, respectively, the intraassay coefficients of variation ranged from 3.54 to 9.30%, the interassay C.V. varied from 6.72 to 19.7%, depending on the type of method and on genistein concentration. The cross-reactivities with other chemically related compounds (with exception of genistein derivatives at the position used for construction of the immunogen) were 5.5 and 6.1% for daidzein and 3.9 and 0.04% for formononetin in RIAs using reagents prepared through positions 4'- and 7- of genistein, respectively. The method was used for measurement of genistein levels in 26 omnivore subjects and in three volunteers after consumption of a meal prepared from 125 g of cooked whole soybeans. The values obtained in ether extracts from human sera were almost identical for both RIA systems, indicating that both RIAs measure the same entity.

Competitive radioimmunoassay standard curves for progesterone were established with tritiated progesterone and six different anti-progesterone monoclonal antibodies ranging in affinity from 1.23 x 10(8) to 2.82 x 10(10) l/mol. The detection limits for these curves ranged from 133 to 6,670 pmol/l final assay concentration (16.7 to 839.1 pg/tube). Separately, a mass-action mathematical model was used to predict lower detection limits for assays run under equivalent conditions with the six antibodies. When compared to the experimental data the predictions were reliable for the higher affinity antibodies but became less accurate as the affinity decreased.

The localization of FMRFamide-related peptide (FaRP) immunoreactivity was determined during different stages of development of the rat tapeworm Hymenolepis diminuta. In the adult worm (14 days old), FaRP immunostaining was most intense in the scolex and concentrated in the central nervous system (cerebral ganglia and transverse commissures) and around the lips of the suckers. In the strobila, medial and lateral longitudinal nerve cords (LNCs) and ladder-like connecting commissures were the only tissue stained. Immunoreactivity in the medial LNCs of the adult tapeworms extended only to and included proglottides containing developing testis and seminal receptacle but disappeared in proglottides in which primordial ovaries were first detected. Radioimmunoassay confirmed that FaRPs were concentrated in the scolex/neck region of the adult worm (3.9 +/- 1.5 pmol mg protein-1), whereas the lowest concentrations (0.2 +/- 0.19 pmol mg protein-1) were recovered from the regions of the strobila containing shelled eggs. The pattern of FaRP immunoreactivity observed in 5- and 7-day-old worms was similar to that seen in adult worms, but in 2- and 3-day-old worms the pattern of immunoreactivity observed in the cerebral ganglia, transverse commissures, and LNCs differed significantly as compared with that seen in older worms. These results indicate differential utilization and/or roles for FaRPs during development and suggest both central and sensory roles in this tapeworm.

Kisspeptin is a hypothalamic peptide hormone that plays a pivotal role in pubertal onset and reproductive function. Previous studies have examined hypothalamic kisspeptin mRNA expression, either through in situ hybridisation or real-time RT-PCR, as a means quantifying kisspeptin gene expression. However, mRNA expression levels are not always reflected in levels of the translated protein. Kisspeptin-immunoreactivity (IR) has been extensively examined using immunohistochemistry, enabling detection and localisation of kisspeptin perikaya in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). However, quantification of kisspeptin-IR remains challenging. We developed a specific rodent radioimmunoassay assay (RIA) capable of detecting and quantifying kisspeptin-IR in rodent tissues. The RIA uses kisspeptin-10 as a standard and radioactive tracer, combined with a commercially available antibody raised to the kisspeptin-10 fragment. Adult female wistar rat brain samples were sectioned at 300 µm and the ARC and AVPV punch micro-dissected. Brain punches were homogenised in extraction buffer and assayed with rodent kisspeptin-RIA. In accord with the pattern of kisspeptin mRNA expression, kisspeptin-IR was detected in both the ARC (47.1±6.2 fmol/punch, mean±SEM n = 15) and AVPV (7.6±1.3 fmol/punch, mean±SEM n = 15). Kisspeptin-IR was also detectable in rat placenta (1.26±0.15 fmol/mg). Reverse phase high pressure liquid chromatography analysis showed that hypothalamic kisspeptin-IR had the same elution profile as a synthetic rodent kisspeptin standard. A specific rodent kisspeptin-RIA will allow accurate quantification of kisspeptin peptide levels within specific tissues in rodent experimental models.

Plasma immunoreactive renin was measured by sandwich radioimmunoassay, under various physiological and pathological conditions. Enzymatic activities of active renin and trypsin-activatable inactive renin were also measured. There was a significant correlation between plasma immunoreactive renin concentration (IRRC) and total (active plus inactive) renin concentration, as estimated by enzymatic activity. In plasma from normotensive volunteers and hypertensive patients, the IRRC were 279 +/- 37 pg/ml and 268 +/- 29 pg/ml, respectively. After the intravenous injection of furosemide, the plasma IRRC in normotensive volunteers increased significantly. IRRC was significantly higher in plasma from juvenile diabetics than in plasma from age-matched disease-free children. Thus, renin secretion in children with diabetes mellitus is increased.

An immunoassay employing (125)I labeled enterotoxins A and B and polystyrene tubes coated with specific antibodies was used for detection and quantitation of enterotoxin in food. Ham salad, cheddar cheese, custard, condensed milk, and salami were studied. Enterotoxin was successfully determined in all the foods by simple extraction procedures. The assay was sensitive to 1 to 10 ng of toxin per g of food; nonspecific inhibitions were 15% or less.

An improved method for the purification of 125I-labelled PTH is presented. Following iodination of the hormone by the chloramine-T method, the reaction mixture is transferred directly to a 0.9 x 9 cm column of carboxymethyl-cellulose. Two major peaks emerge with the ammonium acetate gradient. Peak I represents labelled intact bPTH, Mr 9600, of good quality, i.e. a high B0 and a low NSB in the radioimmunoassay. Peak II, Mr approximately 12000-18000, gives a high B0, but also a high NSB and is less suitable for RIA. Peak II is not the result of iodination damage, as it is also present in the unlabelled PTH (Inolex); it is suggested that it represents either a pre-prohormone or an aggregate of PTH. The label produced is usable for up to 3 months. Trasylol and benzamidine protect the label against proteolytic degradation equally well; the latter, however, is considerably cheaper.

Antibodies against thyroxine (T4) were detected in a patient of systemic lupus erythematosus associated with chronic thyroiditis and a patient with primary myxedema. Both patients were clincally hypothyroid with elevated serum TSH. Serum T4 values measured with solid phase radioimmunoassay were very high (over 25ug/dl), but were undetected with polyethylene glycol separation method. On the extraction of the sera with ethanol, the concordant low values of T4 were obtained with both radioimmunoassay methods and these values were compatible with clinical findings and serum TSH levels. Major portion of 125I-T4 binding of sera was found in 7S fraction on Sephadex G-200 column chromatography and T4-binding protein migrated to the gamma-globulin region on cellulose acetate electrophoresis. The 125I-T4 binding of IgGs were competitively inhibited by the addition of unlabelled T4 in both patients. In Case 1, the association constant (Ka) for binding to T4 was 6.1 ×108 l/mol and the binding capacity was 4.8ng T4/mg IgG. The anti-T4 antibody of Case 1 cross reacted with T3 and resulted in falsely high or low T3 values with radioimmunoassay. Ka and the binding capacity of case 2 were 9.2×109 l/mol and 0.11ng T4/mg IgG respectively. The clinical significance of these antibodies was discussed.

The dodecapeptide angiotensin-(1-12) [Ang-(1-12)] functions as an intracrine/paracrine substrate for local production of angiotensin II. We developed a reliable and specific radioimmunoassay (RIA) method for the measurement of Ang-(1-12) in human plasma and urine using an affinity purified antibody fraction directed towards the C-terminus of the human Ang-(1-12) sequence. The RIA method was applied to quantify the Ang-(1-12) in plasma and urine collected from thirty-four human subjects (29 treated with antihypertensive medicines and 5 untreated patients). Plasma Ang-(1-12) level was significantly higher (P < 0.05) in patients with systolic blood pressure ≥140 mm Hg (n = 10) compared to the group with systolic blood pressure <140 mm Hg (n = 24). No significant difference (P = 0.22) was found in spot urine between the groups. Our study also shows that the polyclonal antibody neutralizes the cleavage sites of the human Ang-(1-12) from recombinant human chymase (rhChymase) and serum angiotensin converting enzyme (ACE) mediated Ang II generating hydrolysis. Overall, this newly developed RIA method is reliable and applicable to accurately quantify the Ang-(1-12) level in clinical samples (plasma and urine). Further, our in vitro neutralization study suggests that the anti-Ang-(1-12)-antibody might be used as an in vivo therapeutic agent for preventing Ang-(1-12)/Ang II-mediated hypertension and organ damage.

Salivary steroid measurement is a popular way to assess endocrine hormone levels, but efficient sample collection can be challenging because the use of stimulants can interfere with valid measurement. The aim of this study was therefore to identify a stimulant that can be used in assessment of the steroid hormones cortisol (C), testosterone (T), progesterone (P) and estradiol (E2) without impairing their quantification by radioimmunoassay. Study 1 and 2 explored the suitability of potential stimulants in comparison to unstimulated saliva collection. Study 3 tested stimulants under standardized conditions in water. Across all three studies, Parafilm® wax foil performed best and was therefore tested once more and validated as a saliva stimulant in Study 4. No significant differences between unstimulated saliva and Parafilm®-stimulated saliva could be found for any of the four hormones assayed. Therefore, Parafilm® appears to be a suitable saliva flow stimulant for assaying the salivary steroid hormones C, T, P and E2 by radioimmunoassay.

We studied the effect of various sample preparation procedures on rat brain met-enkephalin content, measured by radioimmunoassay. Whole brain met-enkephalin content of rats killed by decapitation followed by immediate tissue freezing was similar to that of rats killed by microwave irradiation and to those of rats anesthetized with pentobarbital or halothane before killing, whether previously perfused with paraformaldehyde or not. In contrast, a decrease (up to 80%) in met-enkephalin concentrations was observed when brain samples were frozen and thawed to mimic the procedure utilized in the "punch" technique for analysis of discrete brain nuclei. This decrease was totally prevented by paraformaldehyde perfusion of the brain prior to sacrifice. Brain perfusion did not alter the amount of immunoassayable met-enkephalin extracted from tissue or its profile after Sephadex chromatography. Paraformaldehyde perfusion results in better morphological tissue preservation and facilitates the "punch" dissecting technique. Paraformaldehyde perfusion may be the procedure of choice for the measurement of neuropeptides in specific brain nuclei dissected by the "punch" technique.

The pregnancy rate during in-vitro fertilization (IVF) following progesterone supplement still remains very low at around 20%.

 TSH-receptor (TSHR)-autoantibody (TRAb) is the serological hallmark of Graves' disease (GD). Recently, 3rd-generation radioimmunoassays (RIA) employing monoclonal TRAb such as M22 or T7 instead of TSH for the inhibition of human TRAb binding with solid-phase TSHR (coated tubes) have been introduced into laboratory routine.

Most thyroid hormone determinations in animals are based on immunoassays adapted from those used to test human samples, which may not reflect the actual values of thyroid hormone in horses because of the presence of binding proteins. The aims of the present study were i) to establish a novel radioimmunoassay (RIA) using a more simple and convenient method to separate binding proteins for the measurement of total thyroxine (T4) in horses and ii) to validate the assay by comparing total T4 concentrations in yearling horses raised in different climates. Blood samples were collected from trained yearlings in Hokkaido (temperate climate) and Miyazaki (subtropical climate) in Japan and from adult horses in estrus and diestrus. T4 was extracted from both serum and plasma using modified acid ethanol cryo-precipitation and sodium acetate ethanol methods. Circulating total T4 concentrations were determined by RIA. T4 concentration by sodium acetate ethanol was appropriately detectable rather than sodium salicylate method and was the same as for acid ethanol method. Furthermore, this sodium acetate ethanol method required fewer extraction steps than the other methods. Circulating T4 concentrations in yearlings were 225.98 ± 20.89 ng/ml, which was higher than the previous reference values. With respect to climate, T4 levels in Hokkaido yearlings tended to be higher than those in Miyazaki yearlings throughout the study period. These results indicated that this RIA protocol using a modified sodium acetate ethanol separation technique might be an appropriate tool for specific measurement of total T4 in horses.

A relaxin-like gonad-stimulating peptide (RGP) from starfish Patiria (=Asterina) pectinifera is the first identified invertebrate gonadotropin for final gamete maturation. An antiserum against P. pectinifera RGP (PpeRGP) was produced by immunizing rabbits with a PpeRGP sulfanyl-polyethylene glycol derivative conjugated with keyhole limpet hemocyanin (KLH) as the antigen. The antiserum was used for the development of a specific and sensitive radioimmunoassay (RIA) for the measurement of RGP. In binding experiments using radioiodinated PpeRGP and antiserum against PpeRGP, a displacement curve was obtained using radioinert PpeRGP. The sensitivity of the RIA, defined as the amount of PpeRGP that significantly decreased the counts by 2 SD from the 100% bound point, averaged 0.040±0.002pmol PpeRGP per 100μl assay buffer (0.40±0.02nM) in 10 assays. Intra-assay and inter-assay coefficients of variation were 6.1% and 2.7%, respectively. Serial dilution of whole homogenates from the radial nerve cords and circumoral nerve-rings of P. pectinifera produced displacement curves parallel to the PpeRGP standard. Thus, the amounts of PpeRGP were determined as 1.54±0.09pmol/mg wet weight of radial nerves and 0.87±0.04pmol/mg wet weight of nerve-rings, respectively. On contrary, pyloric stomach, pyloric caeca, tube-feet, ovaries, testes, and ovarian follicle cells did not react in the RIA system. Furthermore, the A- and B-chains of PpeRGP, Asterias amurensis RGP, bovine insulin, and human relaxin did not show cross-reactivity in the RIA. These results strongly suggest that the RIA system is a highly specific and sensitive with respect to PpeRGP.

Ultrasensitive enzymatic radioimmunoassay (USERIA) was compared to radioimmunoassay and enzyme-linked immunosorbent assay in determining the amount of benzo(a)pyrene [B(a)P] metabolite covalently bound to guanine in DNA. In the USERIA approach, DNA either with or without B(a)P metabolite modification was adsorbed on the wells of microtiter plates, and rabbit antiserum to B(a)P metabolite-modified DNA [B(a)P-DNA] was then added. Antibodies reacted specifically with the B(a)P-DNA attached to the surface of the plate. After reaction between goat anti-rabbit IgG conjugated to alkaline phosphatase and rabbit IgG bound to the solid phase, this specific antigen-antibody reaction was enzymatically amplified by alkaline phosphatase conversion of [3H]adenosine 5'-monophosphate to [3H]adenosine. Following chromatographic separation from [3H]adenosine5'-monophosphate, [3H]adenosine was measured by liquid scintillation counting. The amount of [3H]-adenosine formed was linearly related to the amount of B(a)P-DNA in 10 ng DNA attached to the solid phase. As little as 3 fmol of bound B(a)P metabolite can be detected by noncompetitive USERIA, while 10 fmol of the adducts in 25 microgram DNA [1 B(a)P-DNA adduct per 7 X 10(6) nucleotides] can be measured by the competitive USERIA approach. Under our standard competitive procedure with 1 microgram DNA in the antigen-antibody reaction mixture, USERIA is approximately 500-fold more sensitive than radioimmunoassay and 5-fold more sensitive than enzyme-linked immunosorbent assay for the detection of B(a)P-DNA adducts. These highly sensitive assays should be extremely useful for studies in DNA damage and repair by B(a)P metabolites as well as in studies on environmental exposure to this ubiquitous carcinogen.

In vitro oxytocin (OXT) release from isolated posterior pituitary lobes (PPL) of adult male Wistar rats was measured under basal and K+- stimulated conditions using a specific, sensitive radioimmunoassay. A basal release of 0.95 +/- 0.20 ng OXT/lobe/10 min was estimated in standard Locke's bathing solution. An excess of K+ (56 mmol/l) augmented the OXT secretion to 18.1 +/- 2.24 ng OXT/lobe/10 min in the presence of 2.2 mmol/Ca++. A stimulatory effect of K+ excess was also determined in Ca++-free medium and in Ca++ free medium enriched with 0.7 mmol/l EGTA. An inhibitory effect on K+-stimulated OXT release was achieved by raising the Mg++ concentration from 1.0-8.0 mmol/l of bathing fluid. During prolonged K+ stimulation the rate of OXT release declined exponentially. Estimation of the OXT content of PPLs after prolonged stimulation with K+ excess revealed that the lobes still contained 80% of their original OXT content.

Radioimmunoassay (RIA) is the "gold standard" method for evaluation of serum cortisol concentration. The VIDAS cortisol test is an enzyme-linked fluorescent assay designed for the MiniVidas system. The aim of this study was to compare the VIDAS method with RIA for measurement of bovine serum cortisol concentration. Cortisol concentrations were evaluated in 40 cows using both VIDAS and RIA methods, the latter as the reference method. A paired Student's t-test, Pearson's correlation analysis, Bland-Altman plot, and Deming regression analysis were used to compare the two methods. There was no statistically significant difference between mean serum cortisol concentrations measured by VIDAS or RIA methods (P = 0.6570). Both methods were able to detect significant differences in mean low and high cortisol concentrations (P < 0.00014 RIA and P < 0.0016 VIDAS). The correlation coefficient was low, but a Bland-Altman plot and Deming regression analysis show neither constant nor proportional error. The VIDAS method produced slightly higher values than RIA, but the difference was small and in no case did the mean value move the normal range. Results suggest that VIDAS method is suitable for the determination of bovine serum cortisol concentration in studies of large numbers of animals.

The most sensitive and specific investigative method for the diagnosis of narcolepsy type 1 (NT1) is the determination of hypocretin-1 (orexin-A) deficiency (≤110 pg/mL) in cerebrospinal fluid using a radioimmunoassay (RIA). We aimed to assess the reliability of the Phoenix Pharmaceuticals hypocretin-1 RIA, by determining the lower limit of quantification (LLOQ), the variability around the cutoff of 110 pg/mL, and the inter- and intra-assay variability.

Sensitivity and specificity of passive hemagglutination (RCA), solid phase radioimmunoassay (RIA), and immunoelectroosmophoresis (IEOP) were compared under experimental and clinical conditions. In dilution experiments with sera containing hepatitis B antigen (HB Ag) of known subtypes, the sensitivity for an ad subtype serum was RIA (1), RCA (1/2), IEOP (1/256) and for an ay subtype serum RCA (1), RIA (1/8), IEOP (1/128). An evaluation of the National Institutes of Health, Division of Biologics Standards test panel number 2 demonstrated HB Ag in 34 of 60 samples by RIA, in 33 by RCA, and in 25 by IEOP. HB Ag was detected in 57.5% of 200 outpatients with a tentative diagnosis of hepatitis by RIA, in 54% by RCA, and in 42.5% by IEOP. In 1,661 volunteer blood donors, 13 (0.78%) were "positive" for HB Ag by RIA, 11 (0.66%) by RCA, and 3 (0.18%) by IEOP. However, absorption experiments indicated that at least six of the above RIA positive and five of the RCA positive sera exhibited nonspecific positive reactions.