Competitive Protein Binding Method Research Articles

25-Hydroxyvitamin D: A Difficult Analyte

In their article in the current issue of Clinical Chemistry , Farrell et al. (1) describe the latest in a number of studies (2, 3) highlighting the method-related variability in serum 25-hydroxyvitamin D (25-OHD)2 results. There is common agreement that 25-OHD is a “difficult” analyte. That quality is generally ascribed to its hydrophobic nature, its existence in several different molecular forms, its tight binding to vitamin D– binding protein (VDBP) and, until recently, the absence of reference materials or a reference measurement procedure (RMP) against which assays could be standardized. This last problem was mitigated to some extent with the introduction in 2009 of the NIST Standard Reference Materials (SRM 972 and SRM 2972) and the acceptance of the NIST and University of Ghent liquid chromatography–tandem mass spectrometry (LC-MS/MS) assays as RMPs (4). Unfortunately, 3 of the 4 SRM 972 reference materials are either spiked with exogenous metabolites or diluted with equine serum, characteristics that render these reference materials unsuitable for many immunoassays (5, 6). The history of 25-OHD methodology could serve as a case study of the consequences of transferring a rigorous but labor-intensive method from the unhurried atmosphere of the research laboratory to the bustle of a routine clinical laboratory having to meet tight deadlines. The pioneering competitive protein-binding (CPB) method of Haddad and Chyu (7) involved solvent extraction and chromatography, with the results of every sample corrected for procedural losses. Subsequent attempts at simplifying a CPB method by omitting the chromatography stage proved unsuccessful (8), and an automated version (Nichols Advantage) introduced in 2004 was withdrawn in 2006. The successive abandonment of sample extraction, chromatography, and correction for procedural losses in immunoassays has undoubtedly contributed to the inconsistencies reported by Farrell et al., as have differences in assay standardization. Nevertheless, results submitted to …

A modified competitive protein binding method for measuring plasma progesterone levels.

A modified competitive protein binding method for measuring plasma progesterone levels.

The effect of somatostatin and SSTR3 on proliferation and apoptosis of gastric cancer cells

In the present study, we detected the expression of SSTR3 protein in 40 patients with gastric adenocarcinoma and 40 cases of normal gastric mucosa by immunoperoxidased staining. SSTR3 mRNA and protein were also examined in gastric cancer cell lines and eternal gastric epithelial cell line by RT-PCR, immunofluorescence and Western blot. The effect of octreotide on the growth of gastric cancer cells was examined by MTT test, and the apoptosis by flow cytometry. Competitive protein binding method was also used to evaluate the role of SSTR3. The results were: (1) SSTR3 protein existed in the membrane of gastric cancer cells. In normal gastric mucosa, SSTR3 protein distributed to the cellular membrane and cytoplasm or interstitial tissue in submucosa. The expression of SSTR3 protein was significantly lower in gastric cancer compared with normal mucosa. Moreover, the poor-differentiated adenocarcinoma was lower than the well-differentiated adenocarcinoma, and the similar result in cell lines. (2) Octreotide could inhibit the growth and induce the apoptosis of gastric cancer and normal epithelial cells that expressed SSTR3, but didn’t affect the cells with no or weakly expression of SSTR3. (3) When the cells were administrated octreotide in combination of SSTR3 antibody, the effect of octreotide decreased dramatically. The preliminary study suggested that SSTR3 might play a role in the growth and apoptosis of gastric cancer. In those gastric cancers that expressed SSTR3, octreotide could be effective in inhibiting cell growth and inducing cell apoptosis through mediation of SSTR3.

How Much “UFC” Is Really Cortisol?

When urinary free cortisol (UFC) determinations became readily available for clinical use in 1968 (1), chromatographic methods were cumbersome. The first competitive protein-binding method (radiotransinassay) (1) used human corticosteroid-binding globulin (transcortin) as the binding protein. The specificity of the assay was enhanced by the use of Fuller’s earth as the agent to adsorb the unbound fraction because it takes up some of the competing steroids differentially (2). The reference interval was ∼10–100 μg/day (30–300 nmol/day). When radioimmunoassays, based on the same competitive binding principle but using antibodies raised to cortisol linked to albumin, became popular during the 1970s, it was assumed that these would be more specific for cortisol, but this assumption was not warranted. As pointed out recently (3), most of the articles published over the past 20 years have quoted even higher values, reflecting a significant lack of specificity. In 1976, Chattoraj et al. (4) found values for UFC after combined thin-layer and column chromatography that were approximately one-half those of the original method (1), as did Schoneshofer et …

The Evolution of Immunoassay as Seen Through the Journal Clinical Chemistry

A brief historical review shows that the first reference in the Journal to an immunologically based method was in 1962 [in a method for lipoproteins using an immunoprecipitation technique (1)]. In 1962 and 1963, there were two references to enzyme-mediated methods (again the first in the Journal) for glucose and oxalate! In 1965 came the first reference to a competitive protein binding method for thyroxine (2), which was also automated (!). In contrast, in 1966 there was a reference to the effect of ultrasound on isoenzymes! In 1968 came the second reference to an immunologically based assay, this time for growth hormone (3), as well as, in contrast, the use of nuclear magnetic resonance for studying low molecular weight constituents! I mention these other landmark papers because enzyme-mediated and protein-binding assays reflect other examples of biorecognition systems that have also matured with great success (particularly in the case of enzymes) as well as other analytical concepts that in the 1960s may have been considered ahead of their time, in relation to what has been achieved in laboratory medicine. In 1969, papers on immunoassay began to blossom, with more publications on the assessment of thyroid status, the issues of sensitivity in relation to quantification of low concentration proteins in cerebrospinal fluid, and reagent quality control. These early papers should be placed chronologically in the context of the publication on the radioimmunoassay of insulin by Yalow and Berson (4) in 1959 and the measurement of thyroxine by Ekins about that time. Thus, in the first 15 years of the Journal, there were approximately 10 papers on immunologically based assays or their utilization; in 1997 there were in excess of 100 papers in which immunoassay methods were utilized (>35% of the total number of papers published). Looking at the Editor’s perspective on the …

Modification of a Competitive Protein Binding Assay for Determination of Inositol 1,4,5-Trisphosphate

A modification of a competitive protein-binding method for the determination of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), facilitated by the use of a cell harvester, is described. The method is based on the competition of [3H]Ins(1,4,5)P3 with Ins(1,4,5)P3 in the sample for binding to a binding protein prepared from bovine adrenal cortex. The assay is carried out in 96-well microtiter plates in a final volume of 100 μl and free [3H]Ins(1,4,5)P3 is separated from bound by filtration using a cell harvester. This allows the rapid measurement of large numbers of samples, with high reproducibility. Ins(1,4,5)P3 bound to a single class of high-affinity receptors with a KD of 2.3 ± 0.2 nM and a Bmax of 289 ± 7 fmol/mg of protein. The binding was rapid, reaching equilibrium after 20 min, and reversible. The sensitivity of the assay allows the determination of amounts of Ins(1,4,5)P3 as small as 0.1 pmol, which corresponds to a concentration of 5 nM in the sample. The application of the method to measure the formation of Ins(1,4,5)P3 in DDT1 MF-2 smooth muscle cells activated with ATP or bradykinin is described. The modification is a rapid, sensitive, convenient, and relatively inexpensive method for the determination of Ins(1,4,5)P3 in large numbers of samples.

Relationship between plasma adrenocorticotrophic hormone and cortisol concentrations in chronic alcoholic patients with depression

In 20 hospitalized male chronic alcoholic patients, plasma adrenocorticotrophic hormone (ACTH) levels were estimated by radioimmunoassay and cortisol levels by the competitive protein binding (CPB) method with radioactive selenium-75 on admission and during abstinence along with rating of the degree of depression immediately after the acute phase of the ethanol withdrawal syndrome was over. Duration of drinking ranged from 5 to 25 years and average daily ethanol intake was between 100 and 150 g. Plasma ACTH levels were found to be raised in drinking chronic alcoholics. There was a positive and statistically significant correlation between depression ratings and plasma ACTH concentrations ( r = + 0.379; P < 0.05). In chronic alcoholism the negative feedback mechanism seems to be disturbed between plasma ACTH and cortisol levels which are not normalised after 1 week of total abstinence. Chronic ethanol ingestion might have a direct stimulatory effect on the adrenal cortex leading to dysregulation of the hypothalamo-pituitary-adrenocortical axis.

Improvements in the simultaneous determination of calcidiol and calcitriol in human serum or plasma.

Technical improvements have been applied to reduce the time required for the determination of calcidiol and calcitriol in serum or plasma. The modifications include the use of Sep-Pak C18 cartridges for the extraction of calciol metabolites from serum instead of a classical liquid/liquid extraction, a considerable shortening of the HPLC purification time compared with our previously described method, and the application of HPLC with UV detection at 254 nm of calcidiol as an alternative to the usual competitive protein binding methods. In none of the 199 samples where calcidiol was determined by HPLC did we observe a detectable peak of ercalcidiol. Quantitation of calcidiol by HPLC and competitive protein binding was compared in 5 series of assays. The correlation between the two methods was 0.99. The average slope of the linear regression line when the HPLC values were plotted versus the competitive protein binding values was 1.14. The average intercept was 0.19 nmol/l. The mean within-run coefficient of variation for calcidiol in these series was 5% for the competitive protein binding method, and 4% for HPLC method. Between-run coefficients of variation were 6% and 12% for the competitive protein binding and for the HPLC method, respectively. Within-run and between-run coefficients of variation for calcitriol were 6% and 15%, respectively.

Fundamental aspects of radioreceptor assays

Significant advances in understanding the actions of hormones, neurotransmitters and drugs resulted from the development of new analytical procedures. Among these advances the discovery that, for a given drug (or hormone or neurotransmitter), there exists a receptor site on membranes to which the substance must attach in order to evoke a biological response, led to the setting up of the radioreceptor assays. Thus ligand-binding techniques can be used as assays for measuring drug levels in biological tissues and fluids. Radioreceptor assays are based on the principle of competitive protein-binding methods; when a radioligand and an unlabelled ligand are present together with a specific receptor preparation, the amount of radioactive ligand bound to the receptor is a quantitative function of the amount of unlabelled ligand present in the incubation medium. The technology of the radioreceptor assays is simple and rapid to perform when optimal conditions for binding are determined. Radioreceptor assays are highly sensitive, reliable, precise and accurate. Their chief limitation relates to specificity as any substance having an appreciable affinity for the receptor displaces the specifically bound radioligand. Paradoxically this lack of specificity may be in some cases advantageous in that it allows for the detection of the parent compound and active metabolites in proportion to their affinity for the specific receptor. During recent years, radioreceptor assays have been applied to quantitative determination of hormones, neurotransmitters and drugs. Among these, the radioreceptor assay of benzodiazepines is a typical example. In addition to drug analysis and drug monitoring, radioreceptor assays have been found to be versatile tools for basic studies on the receptor itself, on the endogenous ligand(s) for the receptor and on the discovery and the identification of biologically active chemical entities (pharmacological screening). The future of radioreceptor assays in the pharmacological sciences appears to be promising.

Relationship between plasma cortisol concentrations and depression in chronic alcoholic patients

Plama cortisol levels were estimated by the competitive protein binding (CPB) method with radioactive selenium-75 in 32 male chronic alcoholics with depression. The degree of depression was rated by the Hamilton Depression Rating Scale (HDRS). Duration of drinking ranged from 5–25 years with an average daily amount of ethanol intake of 100–150 g (equivalent to half a bottle of spirits). Plasma cortisol levels were often raised in drinking chronic alcoholics and there was a strongly positive and statistically highly significant correlation between them and depression ratings ( r = + 0.56; P = 0.001). It is possible that the diurnal rhythm of cortisol secretion is disturbed or adreno-cortical activity is stimulated in these patients due to chronic ethanol ingestion. It is further suggested that brain serotonin deficiency known to occur in chronic alcoholics might lead to raised plasma cortisol levels in ethanol-induced depression.

Vitamin D and femoral neck fractures in elderly South Australian women.

The vitamin D status of women with femoral neck fractures and of male and female nursing-home residents in South Australia was assessed and compared with that of ambulant normal elderly women and young male and female controls. Serum 25-hydroxyvitamin D (25OHD) was assayed with a competitive protein-binding method including a chromatography step. The mean serum 25OHD levels in the patients with femoral neck fractures (39.2 [SE 2.6] nmol/L) and in the nursing-home residents (28.9 [SE 1.3] nmol/L) were significantly lower than those in the ambulant elderly women (67.6 [SE 3.9] nmol/L) and young controls (76.9 [SE 3.7] nmol/L) (P less than 0.001 in each case). The serum 25OHD levels were unrelated to age in the normal ambulant subjects. There was a significant seasonal variation in the results of routine 25OHD assays performed in our laboratory, but no such variation was seen in the nursing-home residents. We conclude that vitamin D deficiency is common in housebound subjects, and that femoral neck fractures occur mainly in subjects who were housebound before the fracture.

Effect of melatonin alone or in combination with human chorionic gonadotropin or ovine luteinizing hormone on the in vitro secretion of estrogens or progesterone by granulosa cells of rats.

This work was undertaken to learn what effect, if any, melatonin might have on estrogen and progesterone production in vitro. Granulosa cells (10(6)/tube) were harvested from immature Sprague-Dawley rats, previously primed with diethylstilbestrol. Such cells were incubated for 90 min in medium only or in medium to which hCG (10 or 100 IU) or melatonin (3, 30, or 300 pg) had been added separately and in combination. Melatonin alone had no significant effect on the amount of estrogen produced, but in combination with hCG, a significant increase in estrogen followed (P less than 0.05). Estrogen was measured by RIA. More mature granulosa cells from rats primed with both diethylstilbestrol and porcine FSH were similarly collected and incubated in medium alone or in medium containing ovine LH (oLH; 0.3 or 3 ng) and/or melatonin (23 or 23 X 10(2) pg). The progesterone produced was measured by a competitive protein-binding method. A significant increase (P less than 0.05) in progesterone followed the addition of either melatonin or oLH alone, but a substantially greater increase was seen when oLH and melatonin were combined. These results show that, at least over a short period (90 min), melatonin is progonadal, in that it augments gonadotropic stimulation of granulosa cells and leads to increased synthesis of estrogen or progesterone. Melatonin may also independently stimulate granulosa cell production of progesterone.

Effect of saikosaponins on dexamethasone suppression of the pituitary-adrenocortical system.

The effect of saikosaponins d and a on corticosterone secretion was investigated in dexamethasone-treated rats (250μg/kg, 0.64×10-6mol/kg, i.p. 3h before saponin injection). Plasma corticosterone level 30min after saponin injection was determined by the competitive protein binding method. Saikosaponins d and a both had no effect at low doses, but elevated plasma corticosterone levels at higher doses. ED50 values for corticosterone response to saikosaponins d and a were 1.4mg/kg (1.8×10-6mol/kg) and 3.8mg/kg (4.9×10-6mol/kg) in the dexamethasone-treated rats, and these values were 4.2 and 4.3 times greater than the ED50 values determined in rats without dexamethasone treatment, respectively.

Production, characterization and clinical application of antisera to cortisol.

In the present paper the production of highly specific and sensitive antisera to cortisol is described. The antisera were generated in rabbits using both cortisol-3-monooxime and cortisol-3,20-dioxime derivatives. Bovine thyroglobulin was used as a carrier protein. Antibody characteristics were determined by radioimmunoassay procedure. Antibody titers observed after immunization with the monooxime conjugate were significantly higher (p less than 0.001) than those obtained with the dioxime conjugate. In addition, cross reactivity for various naturally occurring steroids was markedly lower and sensitivity was increased as compared to dioxime antisera (15 vs. 40-60 pg/tube). Accuracy and precision were calculated for the monooxime antiserum used in a RIA system for clinical application. A close correlation (r = 0,9802) was found between plasma cortisol values measured by radioimmunoassay and the competitive protein binding method. The concentrations obtained by the protein binding method were slightly but significantly higher than those measured by radioimmunoassay indicating higher specificity of the antibody used (p less than 0.005).

Cyclic AMP in the fat body of Mamestra brassicae during the last instar and its possible involvement in the cellular autophagocytosis induced by 20-hydroxyecdysone

The amount of cAMP was assayed by a competitive protein binding method in fat body cells of Mamestra brassicae, during the last larval stage and after administration of 20-hydroxyecdysone. When expressed as picomoles of cAMP per milligram fresh weight of tissue, two increases in its concentration were observed on the 3rd and on the 6th days. However, only the first peak appeared on the curve when CAMP concentration was expressed as picomoles cAMP per milligram of protein of tissue homogenate. Electron microscopical examination of the tissue showed that the first increase of cAMP level coincided with the beginning of the formation of autophagic vacuoles and revealed a heavy accumulation of protein storage granules in the cells, starting on the 4th day. This process might mask the second rise of cAMP level when tissue protein content is taken as the basis for calculation. 20-Hydroxyecdysone (5 μg/g body wt) administered to 48-hr-old larvae induced premature autophagocytosis in the fat body cells and a sharp rise in their cAMP content, reaching within 3 hr a level as high as observed in the 3-day-old untreated larvae. Autophagy was also enhanced in the cells exposed to dibutyryl cAMP or theophylline either in vivo or in vitro. Based on these data we think that cAMP content of the fat body is controlled by ecdysone and that cAMP plays a significant role in the regulation of autophagocytosis in this tissue during metamorphosis.

Comparison of solid-phase radioimmunoassay and competitive protein binding method for post-dexamethasone cortisol levels in psychiatric patients

Results obtained by competitive protein binding assay (PBA) and a solid-phase radioimmunoassay (RIA) for cortisol were compared in 157 samples from 100 psychiatric patients given a dexamethasone suppression test (DST). Cortisol levels in plasma samples obtained at 8:00 a.m. or 4:00 p.m. the day following 1.0 mg dexamethasone orally at bedtime ranged from 0 to 30 μg/dl and correlated closely ( r = 0.96). However, RIA gave values that were consistently and significantly lower (average = 8.9%) than those obtained by PBA. When samples were further assayed by a specific RIA for corticosterone, there was a strong correlation between cortisol and corticosterone RIA values ( r = 0.79), and corticosterone (7.8% of cortisol levels) accounted for most of the difference between PBA and RIA for cortisol. The relationship between results of the two cortisol assay methods can be expressed (in μg/dl) by the equation: RIA = 0.92(PBA) − 0.10, based on findings obtained in a separate analysis of 127 samples with cortisol values in the 0–10 μg/dl range, critical to the valid interpretation of the DST in melancholia. A reported criterion of a “positive” DST in psychiatry, of plasma cortisol of ⩾ 5.0 μg/dl has been suggested by use of a PBA. Use of the present RIA required that this value be adjusted downward, at least to 4.5 μg/dl; application of this criterion increased the clinical sensitivity of the DST by 10%. We urge local, independent verification of criteria to define the DST as “positive” in each laboratory and with each method of assay.

Development of a Sub-Picogram Competitive Protein-Binding Method for Steroids

A very sensitive sub-picogram competitive protein-binding assay, which is about a hundred times more sensitive than radioimmunoassay, for biotinyloestrone is described. Competition between a biotinyloestrone standard and 125 I-labelled biocytin for avidin was used. The method is based on the fact that biotin binds avidin with the extraordinarily high K a of 10 15 l/mol. The development of even more sensitive techniques using an E. coli β -D-galactosidase-biotin conjugate as label is presented. Due to the great difficulty of biotinylating steroids at the femtogram (10 − 15 g) level, a new approach is described which it is hoped will circumvent chemical conjugation.

Effects of total saponins extracted from several crude drugs on rat adrenocortical hormone secretion

Effects of total saponin preparations extracted from panacis japonici rhizoma, saponariae radix alba, sapindus pericarpium, senegae radix, thea semen, bupleuri radix, hippocastani semen, and styrax pericarpium, which contain oleanane-type aglycone on plasma levels of ACTH, corticosterone, and glucose in rat were determined by the radioimmunoassay method, the competitive protein binding method, and by the glucose oxidase method, respectively. All of saponins tested were found to increase plasma ACTH, corticosterone, and glucose at 30 minutes after the intraperitoneal administration. ED50 Value for the corticosterone secretion-inducing activity of saponins was estimated. Among 8 saponins, styrax saponin was the most effective (0.7 mg/kg) and panacis japonici saponin was the least (54 mg/kg). The increase in plasma glucose by these saponins was similar to that of isolated saikosaponin a or d but not to that of ginseng saponin. Hemolytic activities of these saponins were also determined, and the order of the activity was almost parallel to the order of ED50.

Newer Approaches to the Isolation, Identification, and Quantitation of Steroids in Biological Materials

Publisher Summary This chapter discusses the newer approaches to the isolation, identification, and quantitation of steroids in biological materials. Progress in steroid analysis has been remarkable in the last 15 years. Much of present knowledge in steroid endocrinology could not have been acquired without the development of radioimmunoassay and other competitive protein-binding methods. These methods have provided the sensitivity and speed required for measurement of physiological concentrations of steroid hormones in large series of samples. Parallel with this development, both liquid and gas-liquid chromatographic methods have been markedly improved and mass spectrometers have become available for routine use. The most important advancement in this area has been the successful combination of gas chromatography and mass spectrometry (GC-MS) for analysis of molecules as large as steroids. Immunoassay and GC-MS are complementary methods. GC-MS is important as a reference method to test and validate the specificity of an immunoassay. The steroid composition of biological samples taken under different physiological and pathological conditions can be characterized by GC-MS, and immunoassays may then be developed for detailed studies of selected steroids in larger numbers of samples.

Immunonephelometric quantification of serum vitamin D-binding globulin

Rapid quantification of serum vitamin D-binding globulin (VDBG) is described. An immunonephelometric method was developed and compared with the conventional immunodiffusion method for VDBG. A correlation coefficient of 0.896 (P less than 0.001) was found with a regression line y = 0.965x + 0.011 (n = 23). The within-assay and day-to-day coefficient of variation of the immunonephelometric method was 6 and 11% at 0.150 g/l (low VDBG concentration), 5 and 7% at 0.230 g/l (normal), and 4 and 8% at 0.570 g/l (high). Levels of VDBG and total 25-hydroxyvitamin D (25(OH)D, 25(OH)D2 + 25(OH)D3) were analysed from 33 serum samples obtained in April-May 1980 from 14-month-old children undergoing vitamin D substitution. 25(OH)D was extracted from 0.1-0.5 ml serum followed by semipreparative silica column chromatography and final quantification by a competitive protein-binding method. The VDBG mean value was 0.308 +/- 0.068 g/l (SD) and 25(OH)D had a mean value of 121 +/- 70 nmol/l (SD). There was no significant correlation between the levels of VDBG and 25(OH)D. The results of this study in general agreed well with other findings.