In vitro evaluation of concentration, labeling effectiveness and stability for 131 I-labeled radioimmunoassay ligand using real-time detection technology.

Radioimmunoassay belongs to the analytical method enabling highly specific and sensitive quantification of molecules. The verification of the real-time radioimmunoassay technology usefulness for ligand-quality characteristics evaluation such as concentration, influence of radiolabeling on binding affinity and stability was estimated. The anti-epidermal growth factor receptor antibody 131 I-cetuximab was employed as the ligand antibody. The concentration of 131 I-cetuximab was derived from the shape of binding curves coming from the ligand-receptor interaction. The binding curves also allowed the estimation of 131 I-cetuximab binding affinity for different radiolabeling procedures (incubation times 1, 5, and 10 minutes) in stability testing up to 96 hours at 4°C. The stability testing also included comparative analysis by size exclusion high-performance liquid chromatography. The assessment of cetuximab concentrations using real-time method showed acceptable accordance between real and calculated values. The real-time method revealed that 1-minute radiolabeling proved to be the optimal incubation time for direct radioiodination of cetuximab. Stability testing showed the significant change in radioligand affinity by one order at the longest incubation times (72 and 96 hours). Characterization of stability and binding behavior of radiolabeled monoclonal antibodies by the verified real-time method before use in other assays may be employed to eliminate variability and suboptimal antibody performance.

Radioligand binding analysis as a tool for quality control of GPCR production for structural characterization: adenosine A(2a)R as a template for study.

Functional characterization of G protein-coupled receptors is essential to ascertain the suitability of a protein target for downstream studies and to help develop optimal expression and isolation procedures. Radioligand binding analysis is a well-established technique, which allows direct measurement of the amount of functional receptor in a sample. It can be readily applied to both membrane-bound and soluble receptor samples and is an ideal method for monitoring the amount of functional protein at each stage in the expression and isolation process. This unit presents protocols for the radioligand binding analysis of the human adenosine A(2a) receptor and provides examples of how these assays can be used at several stages to help optimize expression, solubilization, and isolation procedures.

Development and validation of fluorescent receptor assays based on the human recombinant estrogen receptor subtypes alpha and beta.

This article describes the development and validation of two fluorescent receptor assays for the hRec-estrogen receptor subtypes alpha and beta. As a labelled ligand an autofluorescent phyto-estrogen (coumestrol) has been used. The estrogen receptor (ER) belongs to the nuclear receptor family, a class of soluble DNA binding proteins, mainly present in the cytoplasm of the cell, that act as ligand-activated enhancer factors. It consists of two different forms, expressed as ER-alpha (66 kDa) and ER-beta (59 kDa). The ER-alpha is mainly located in the uterus and the ER-beta can be found in vascular tissue. Detection and identification of compounds having estrogenic effects is of importance in drug discovery programmes within the pharmaceutical industry for their search for ER-subtype selective (ant)agonists which may prove to be of therapeutic value in treating a variety of estrogen-linked pathologies (breast cancer, osteoporosis, cardiovascular disease, type II diabetes and Alzheimer disease). Furthermore, interactions of (xeno-)estrogens with the endogenous hormonal system of the exposed organism can affect embryos, gonads, and reproductive behaviour. The latter can eventually lead to reduced reproduction and deterioration of a population. For that reason, monitoring of (xeno-)estrogens in food products and in the environment, attracts considerable attention by health councils throughout the world. The following characteristics were obtained for the human recombinant (hRec) estrogen receptor-beta assay, which is suitable for ER subtype selective drug-discovery purposes (IC50 values for 17-beta-estradiol and genistein were 5.1 nM and 25 nM, respectively): goodness of fit (R2) was always > 0.98 (x = 0.9933, n = 10). LLOQ of the assay is typically > or = 500 picomolar, whereas the ULOQ of the assay is < or = 20.0 nanomolar. For the hRec-estrogen receptor-alpha assay, which is suitable for monitoring of (xeno-)estrogens (IC50 values for 17-beta-estradiol and genistein were 0.68 nM and 65 nM, respectively) the following characteristics were obtained: goodness of fit (R2) was always > 0.96 (x = 0.9838, n = 10). LLOQ of the assay is typically > or = 200 picomolar, whereas the ULOQ of the assay is < or = 5.0 nanomolar.

Negligible depletion solid-phase microextraction with radiolabeled analytes to study free concentrations and protein binding: an example with [3H]estradiol.

A new method is presented that enables sensitive measurement of free concentrations of radiolabeled ligands. Additionally, protein binding of radiochemicals in complex matrixes can be determined with this new technique that combines negligible depletion solid-phase microextraction (nd-SPME) with liquid scintillation counting (LSC) as detection. [3H]Estradiol was taken as an example compound. Possible matrix effects of protein on fiber uptake kinetics were studied. No matrix effect was found, either by fouling of the fiber, or by changed uptake kinetics. The validity of the method was shown in the determination of the affinity constant (Ka) of estradiol for human serum albumin (HSA). The Ka was estimated at 8.9 x 10(4) M(-1), which corresponds well with literature values. This study shows that nd-SPME is suitable to study the free concentration and protein binding of [3H]estradiol. The method described in this paper combines the advantages of nd-SPME with the advantages of radiolabeled analytes, creating a timesaving, simple, and sensitive analytical tool that will be particularly useful in complex matrixes containing many potential interferences for chromatographic methods.

Improvement of quality control for steroid receptor measurements: analysis of distributions in more than 40000 primary breast cancers. French Study Group on Tissue and Molecular Biopathology.

All French laboratories that routinely assay estradiol (ER) and progesterone (PR) receptors participate in the European EORTC quality control program based on twice-yearly analysis of 5 cytosolic preparations. This system has considerably reduced inter-laboratory variations, but does not cover all aspects of these assays. Analysis of receptor value distributions is also crucial to ensure that receptor measurements remain stable with time, independently of the laboratory and assay method. This study involved 83907 receptor assays carried out in the last 17 years by 17 laboratories belonging to the French Study Group on Tissue and Molecular Biopathology. The assays were based on radioligand binding (RLA) or immunoenzymology (EIA). For each laboratory, the medians and positivity rates were analysed according to two totally objective criteria, the patient's age and the year of assay, and according to histological grade and histological type of the tumor in order to verify the correlations classically described. Age-related distributions varied little between laboratories, compared with data published by 7 European EORTC laboratories [1]. The results remained relatively stable with time in the RLA method for ER and PR, and in the EIA method for PR. Median ER-EIA data showed a marked increase between 1987 and 1989, mainly due to changes in the quality of Abbott reagents during this period. Otherwise, this analysis confirms previous pathophysiological observations.

Epidermal growth factor in human and bovine milk.

The concentration of epidermal growth factor in human and bovine milk was measured by radioreceptor assay. Both human placental plasma membranes and a human epidermoid carcinoma cell were used as the epidermal growth factor receptor source in the assay. The use of placental plasma membrane in the radioreceptor assay gave erroneous results for bovine milk and overestimated the concentration of epidermal growth factor in human milk. Intact cells appear to provide a more accurate measure of the concentration of epidermal growth factor in milk samples. Using A431 cells, we found very low concentrations of epidermal growth factor in bovine milk (less than 2 ng/ml) compared to human milk (30-40 ng/ml). No epidermal growth factor activity was found in several cows' milk-based infant formulas. These results highlight the caution which must be taken when measuring trace substances such as polypeptide growth factors in complex samples such as milk.

Epidermal growth factor receptors in human breast cancer: a plea for standardisation of assay methodology.

In a prospective study 200 primary human breast cancer specimens were analysed for epidermal growth factor receptor (EGFR) content by means of a multiple point ligand binding assay, proposed by the EORTC Receptor Study Group to be the standard EGFR assay. In 54% of the tumours the presence of saturable high affinity binding sites for epidermal growth factor could be demonstrated. The median EGFR level was 34 fmol/mg of membrane protein, the median Kd 0.50 nmd. Univariate analysis of the EGFR data stratified according to patient age, menopausal status, tumour size, axillary lymph node status, histological tumour type, tumour differentiation grade or the tumours' steroid hormone receptor status showed EGFR to be positively associated with younger age (P = 0.03), tumour dedifferentiation (P = 0.04) and steroid hormone receptor negativity (P less than 0.001). No association between EGFR and menopausal status, tumour size, axillary lymphnode status or histological tumour type could be demonstrated.

Oestrogen receptor assay of breast cancer by immunocytochemistry of fine needle aspirates.

Sixty-five patients with operable breast cancer were studied to assess the reliability of immunocytochemical analysis of oestrogen receptor (ER-ICA) in specimens obtained by percutaneous fine needle aspiration. Results obtained with the commercially available ER-ICA kit were compared with those obtained by the routine biochemical radioligand assay of oestrogen receptor (ER) on excised tumour specimens. Fifty-two of 65 percutaneous aspirates were evaluable. Of these, thirty-five (67%) were ER positive by the radioligand method. ER-ICA was found to be a reliable method for oestrogen receptor assay, with a high concordance (90.4%) between it and the radioligand essay. The ER-ICA assay had a sensitivity of 89%, specificity of 94%, positive predictive value of 97% and negative predictive value of 80%. ER-ICA assay performed on material obtained by fine-needle aspiration is a reliable method of ER assay. It can replace formal biopsy for patients with inoperable primary tumours or accessible metastases.

Microassays for androgen and progesterone receptor quantitation as compared with standard saturation analyses in human prostatic tissues.

Simultaneous measurement of androgen and progesterone receptor content in cytosol and salt extractable nuclear subcompartments of benign hyperplastic prostatic tissue was carried out with various microassay techniques and compared to the results from analyses on bulky tissue from the same tissue specimens. The microassays were carried out as modified saturation analyses or as single concentration assays at various degrees of dilution with tris-EDTA-glycerol (TEG) buffer. Tissue samples for the standard assay weighed between 1.76 and 3.22 g, whereas the microassay samples weighted between 0.14 and 0.47 g. When considering the results of the standard assay as the "true" value, the microassays on the same tissue samples tended to underestimate both the androgen and progesterone receptor contents. Data from the microassays showed a wide variation of the androgen and progesterone receptor content in cytosol and nuclei. With the standard assay technique no detectable amount of progesterone receptor was found in the nuclei, whereas the microassays often indicated false-positive progesterone receptor content in this subcompartment. Therefore, the measurements of steroid receptors using biochemical microassays in prostatic tissue are unreliable and not suitable for clinical use, at least with the techniques available today. Reports in the literature based on such assays should therefore be interpreted with great caution.

Comparison of ligand binding assay and enzyme immunoassay of oestrogen receptor in human breast cancer cytosols. Experience of the E.O.R.T.C. Receptor Group.

Nine laboratories cooperating in the Receptor Study Group of the EORTC compared results of ligand binding assay supplemented with Scatchard plot analysis (LBA) and enzyme immunoassay (EIA) for the assessment of oestrogen receptors in 1665 breast cancer cytosols. An excellent agreement was observed between results of Scatchard plot analysis and EIA in each laboratory. Linear correlation coefficients of log-transformed data (log [(ER + 10)/10]) ranged from 0.839 to 0.977 (n = 52-373; P less than 0.001); Spearman's R ranged from 0.797 to 0.972 (P less than 0.001). Orthogonal regression analysis on log-transformed data revealed slopes of 0.794 to 1.141 and intercepts of -0.057 to 0.154 corresponding to -1.2 to 4.3 fmole/mg protein. Both assays compared equally well for pre- as well as postmenopausal patients, which confirms that occupied receptors are not extracted during the preparation of cytosol. The percentage discordance in the classification of tumours as ER positive or negative varied from 4.1 to 13.3 when a cut-off value of 10 fmol/mg protein was used, and from 1.4 to 7.5 at a cut-off level of 20. Considerable variations were observed in the actual receptor levels reported by each laboratory. Since these differences occurred in the results of both methods, they are attributed to differences in tissue handling. It is concluded that the ER-EIA is an excellent alternative to Scatchard plot analysis for the assay of oestrogen receptors. It is recommended, however, that laboratories performing assays on samples obtained from patients who are eligible for entrance in EORTC clinical trials and who wish to use the EIA should validate this assay against the Scatchard plot assay in their own setting.

Radioligand binding assay of epidermal growth factor receptor: causes of variability and standardization of the assay.

Although experimental evidence indicates a probable role of epidermal growth factor receptor (EGFr) in clinical oncology, no standardized method for its determination has been yet described, and discrepant results have been reported in clinical studies. In standardizing a radioligand binding assay for EGFr, we evaluated the causes of variability in each step of the assay. Entrapment of EGFr in the nuclear fraction and contamination of the crude membrane fraction by cytosol protein were eliminated through preliminary purification steps. Both Scatchard and Rosenthal analysis of the saturation reaction of the membrane fraction with a wide range of concentrations of 125I-labeled EGF revealed a double class of binding sites. Study of the saturation reaction showed a partial exchange of 125I-labeled EGF with endogenous EGF within 20 h. The present method--incubation of partly purified membrane fraction with 125I-labeled EGF, 0.5 nmol/L, with and without 100-fold excess of cold EGF, for 20 h at 26 degrees C, followed by centrifugation at 5000 x g for 30 min to separate membrane-bound 125I-labeled EGF--shows good sensitivity, precision, and accuracy; is reasonably simple; and may be suitable for routine clinical use.

Platelet surface-bound IgG and platelet-specific IgG in plasma in childhood thrombocytopenia.

Quantification of platelet-bound immunoglobulin is widely used in the evaluation of thrombocytopenia. Several methods have been devised among which labelled ligand-binding assays seem to be most appropriate. In series of adult patients such assays have been shown to be superior in separating immune-thrombocytopenia from thrombocytopenia of non-immune causes. We studied 62 children with thrombocytopenia of various causes, using radiolabelled protein A as a ligand to measure platelet-surface bound IgG. The test was highly sensitive (93%) in detecting immune-thrombocytopenia. The specificity, however, was only 57%, which is less than in published studies of adults. In a number of cases presumed to be non-immune-thrombocytopenia, notably a few patients with leukaemia and bone marrow aplasia, we found increased amounts of platelet surface-bound IgG. The significance of this finding is not clear. An indirect assay measuring platelet-specific IgG in plasma was less sensitive (46%) but highly specific for immune-thrombocytopenia (89%). The measurements of platelet-surface-bound IgG and platelet-specific IgG in plasma are of limited diagnostic value in childhood thrombocytopenia but are useful in following the treatment in chronic ITP.

Clinical utility of thyrotropin-receptor antibody assays: comparison of radioreceptor and bioassay methods.

Thyrotropin (thyroid-stimulating hormone or TSH)-receptor antibodies, important in the pathogenesis of Graves' disease, can be assayed by one of two methods: (1) bioassays that measure stimulation of thyroid cellular activity by patient immunoglobulins or (2) radioreceptor assays that measure inhibition of binding of labeled TSH to TSH receptors by the same substances. In this study, we report our experience with bioassay of thyroid-stimulating immunoglobulins (TSI) based on measurement of generation of cyclic adenosine monophosphate in a clone of the Fisher rat thyroid cell line (FRTL-5) in 279 patients, and we compare, in 163 consecutive samples, the results obtained by a radioreceptor assay for thyrotropin-binding inhibiting immunoglobulins (TBII). Among the untreated, hyperthyroid patients with Graves' disease, TSI were present in 95% (38 of 40), and TBII were present in 85% (17 of 20). In patients with euthyroid Graves' disease, TSI were found in 57% (16 of 28), and TBII were present in 41% (7 of 17). Of 49 nongoitrous and euthyroid controls, only 4% had TSI and 3% had TBII. Extremely high TSI indices were found in all patients who had pretibial dermopathy (N = 10) or severe Graves' ophthalmopathy (N = 19) requiring orbital decompression. We conclude that both assays are highly sensitive and specific in diagnosing Graves' disease. The TSI bioassay was more sensitive (P less than 0.001) than the TBII radioreceptor assay in detection of Graves' disease. In our experience, both assays have proved useful in the diagnosis of euthyroid Graves' disease with ophthalmopathy and atypical manifestations of hyperthyroid Graves' disease.

Improved receptor assay for measuring digoxin activity.

We describe a receptor assay of digoxin activity involving Na+/K+-ATPase (EC 3.6.1.37) derived from human heart tissue. The procedure requires 500 microL of serum or plasma and incorporates one purification step, with Sep-Pak C18 cartridges (Waters). The method appears to be considerably more specific for digoxin and its cardio-active metabolites than are conventional immunoassays. Cardio-inactive metabolites and several digoxin-like factors cross react only slightly. The between-day coefficient of variation for the procedure ranged from 8.10% to 1.37% for digoxin concentrations between 0.5 and 5.0 nmol/L.

Standardization of steroid receptor assays in human breast cancer--II. Samples with low receptor content.

Four different lyophilized cytosols with low receptor content were assayed for estrogen and progesterone receptors by 13 laboratories in 7 countries. Each sample was analyzed on three different working days. Three laboratories reported unsatisfactory ER results, whereas the other 10 participants were in good agreement. Employing a cut-off limit of 10 fmol/mg cytosol protein, 95% agreement of ER-positivity was achieved for the two samples with the lowest receptor contents (18 and 21 fmol/mg cytosol protein). For estrogen receptor analyses the inter-laboratory coefficients of variation ranged between 15 and 18%. Both the intra- and inter-laboratory variations and the rate of concurrence on receptor status were less satisfactory for the progesterone than for the estrogen receptor analyses. The variation in protein results among the participating 13 laboratories is decreased to 10%. It is concluded that multi-center clinical trials based upon the estrogen receptor status assigned by individual laboratories are feasible.

Standardization of steroid receptor assays in human breast cancer--I. Reproducibility of estradiol and progesterone receptor assays.

Four different lyophilized cytosols were analyzed for estrogen and progesterone receptor content by 13 laboratories. Three of the cytosols were assayed on 9 consecutive working days. The fourth cytosol was analyzed 3 times. All laboratories used the same methods of receptor and protein assays. Estradiol and progesterone receptors were measured by Scatchard analysis with centrally provided radioactive ligands and employing charcoal adsorption of free steroid. Protein was assayed by the Bradford technique with the same lots of Coomassie brilliant blue and human serum albumin standard. All participating groups except one produced receptor results (fmol/ml cytosol) with less inter-laboratory variation than in previous trials. Recalculation of the raw data of all participants by a common computer program further reduced this inter-laboratory variation. The discrepancies between the reported and recalculated receptor binding data are discussed. The intra-laboratory variation was sometimes surprisingly high and occurred at random. Single-dose saturation assays showed good agreement with multipoint Scatchard assays for the high receptor-positive samples, while poor agreement was observed for the heat-inactivated, receptor-negative sample. The use of common reagents and methodologies diminished the inter-laboratory variation coefficients of the protein assay to 14-17%; however, the protein estimation still needs to be improved.