Rapid, semi-automated, and inexpensive radioimmunoassay of cAMP: application in GPCR-mediated adenylate cyclase assays.

Cyclic AMP (cAMP) is an important signal transduction second messenger that is commonly used as a functional mirror on the actions of G protein-coupled receptors that can activate or inhibit adenylate cyclases. A radioimmunoassay for cAMP with femtomole sensitivity was first reported by Steiner more than 30 years ago, and there have been several subsequent modifications that have improved this assay in various ways. Here we describe additional improvement to existing methods that markedly improve speed and reduce cost without sacrificing sensitivity, and is also adaptable to analysis of cGMP. The primary antibody is coupled directly to magnetic beads that are then separated from unbound marker using filtration on microplates. This eliminates the need for a secondary antibody, and markedly increases throughput. In addition, we report a simple, reproducible, and inexpensive method to make the radiomarker used for this assay. Although still requiring the use of radioactivity, the resulting method retains a high degree of accuracy and precision, and is suitable for low-cost high throughput screening. Use of aspects of this method can also improve throughput in other radioimmunoassays.

Bioassay research methodology: measuring CRH in pregnancy.

BACKGROUND: There are documented associations between elevated maternal corticotropin-releasing hormone (CRH) levels and adverse pregnancy outcomes. However, reports of these findings often lack sufficient detail and rationale regarding the bioassay methodology. This shortcoming can be problematic for researchers who do not possess in-depth laboratory sciences knowledge but who want to include bioassays in their investigations or to evaluate published reports. The quality and reliability of CRH measurement results can be significantly affected by variables encountered during sample collection, processing, storage, and bioassay. Thus, it is important to establish research laboratory protocols that are based on well-informed rationales and to carefully consider and control for relevant variables. APPROACH: A synthesis of laboratory sciences literature regarding variables affecting CRH measurement in pregnancy is presented. Additionally, consultation with experienced researchers provided an in-depth understanding of CRH measurement. From these sources, a laboratory protocol for clinical research was developed. RESULTS: Multiple variables that are specific to the reliability of CRH measurement in pregnancy have been identified. These include sample collection methods, sample processing, sample integrity, sample storage, and the actual assay selected. CONCLUSION: The reliability of CRH measurements can be significantly improved by identifying and controlling for variables encountered during sample collection, processing, storage, and bioassay. Adequate methodological details are difficult to glean solely from the published literature, thus consultation with well-informed researchers is necessary. A protocol for CRH bioassay in clinical research is proposed.

Thyroglobulin Antibody Screen Prior to Mass Spectrometry Provides Measurable Cost Savings and Optimal Laboratory Utilization.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods that allow accurate quantitation of thyroglobulin (Tg) in the presence of Tg antibodies (TgAbs) have recently become available. Due to cost differences between LC-MS/MS and immunoassay, some laboratories now offer a reflex test strategy that uses LC-MS/MS only for TgAb-positive samples. The goal of this study was to examine utilization of Tg testing strategies and cost savings. METHODS: Test ordering patterns were examined for over 150,000 orders for TgAb and Tg in our laboratory. The average list price was determined from three separate commercial laboratories offering this testing. RESULTS: Data showed that 89% of orders for Tg used the reflex test option, resulting in a savings of over $3 million compared with testing all samples by LC-MS/MS. Of the Tg by LC-MS/MS orders not using the reflex option, 1,663 also included a separate order for TgAb on the same patient sample, representing approximately $170,000 in potentially unnecessary costs from TgAb-negative samples. CONCLUSIONS: Identifying situations to use more expensive testing methods (eg, LC-MS/MS) only when necessary, such as for TgAb-positive patients, leads to considerable cost savings and a more economical use of valuable health care resources.

A re-evaluation of carcinoembryonic antigen (CEA) as a serum marker for breast cancer: a prospective longitudinal study.

PURPOSE: Carcinoembryonic antigen (CEA) is still a widely used test for monitoring breast cancer, although recent reports discourage its routine use because of low sensitivity. This is a prospective study evaluating the efficacy of CEA and CA 15.3 in monitoring breast cancer. EXPERIMENTAL DESIGN: Serum CEA and CA 15.3 were measured in 2191 patients with either benign (n = 738) or malignant (n = 1453) breast diseases. Five hundred and forty-nine patients were monitored during postsurgical follow-up for either a minimum of 5 years or until time of recurrence. Fifty-three patients with metastases were also monitored during chemotherapy. RESULTS: Elevated CEA and CA 15.3 levels were found in 16.7% and 33.0% of patients, respectively. CEA sensitivity rose to 41.3% and CA 15.3 sensitivity rose to 80.8% in metastatic patients. The adjunct of CEA increased the CA 15.3 sensitivity by 6% in the overall population and by only 2.1% for patients with metastases. During postsurgical follow-up, CEA was elevated in 38.0% and CA 15.3 in 70.2% of patients with recurrence. The combination of CEA and CA 15.3 increased the overall sensitivity by only 1.4%. Longitudinal monitoring of 53 metastatic patients undergoing chemotherapy demonstrated that, when positive, both CEA and CA 15.3 paralleled response to treatment, although CA 15.3 was a significantly more powerful marker for determining response to treatment. The cost effectiveness ratio of CEA was clearly less favorable than that of CA 15.3. CONCLUSIONS: CEA monitoring should be considered an expensive and inefficient method of follow-up evaluation for breast cancer patients, and it provides no additional value when used in combination with CA 15.3.

The clinical utility of the Ca19-9 radioimmunoassay for the diagnosis of pancreatic cancer presenting as pain or weight loss. A cost-effectiveness analysis.

CA 19-9 is a promising radioimmunoassay for the detection of pancreatic cancer, but its clinical role and cost-effectiveness are not yet known. To investigate these factors, we used clinical decision analysis to study diagnostic strategies for patients with suspected pancreatic cancer presenting as pain or weight loss. Comprehensive diagnostic strategies were developed to reflect current and future patterns of practice utilizing CA19-9 radioimmunoassay (RIA) to yield biopsy-proved cancer or confidently exclude its presence. The performance of the strategies beginning with CA19-9 RIA and ultrasonography were equivalent in positive and negative predictive values over a range of prevalence of pancreatic cancer from 0.02 to 0.15. At higher prevalence, the negative predictive value of the ultrasonography strategy became significantly better. The CA19-9 RIA strategy used fewer noninvasive tests, endoscopic retrograde cholangiopancreatographic procedures, and invasive radiologic studies than did the ultrasonography strategy at each prevalence. The health care costs ranged between $848 and $1413 per patient for the CA19-9 RIA strategy and $1186 and $1848 per patient for the ultrasonography strategy. We conclude that the CA19-9 RIA is a useful, cost-effective initial test for the examination of patients with suspected pancreatic cancer.

A comparison of 13 different immunometric assay kits for gonadotropins: implications for clinical investigation.

To determine whether the new immunometric (sandwich) assays for gonadotropins achieve the alleged improvements over RIAs, we compared 13 commercial kits for LH and FSH with our established and validated polyclonal RIAs. Although the kits claimed detection limits as low as 0.05 IU/L, when sensitivity was tested with a uniform hormone standard (the Second International Reference Preparation of human menopausal gonadotropin urinary standard) and the requirement that the limit must be determined from a detectable standard point rather than the variance around zero, only 4 kits surpassed the detectability achieved by the existing LH and FSH RIAs. Seven of the 10 LH kits tested displayed greater than 10% cross-reactivity with either the alpha- or LH beta-subunit. The relationship between the kit standard and the Second International Reference Preparation of human menopausal gonadotropin uniform standard in each kit was nonlinear, so that attempts to compare results between assays with simple multiplication factors are inaccurate. Only 2 assays were designed to eliminate a hook effect. The following conclusions were reached. 1) Most available gonadotropin immunometric assays do not yet offer features not already available in existing validated polyclonal research assays. 2) Conversion of data from one assay to another is not straightforward. 3) Many of the manufacturers' claims do not appear justified. 4) Determination of the detection limit and other immunometric assay characteristics requires standardization of criteria. We propose the following minimum criteria for validating gonadotropin immunometric assays: 1) a uniform definition of the detection limit based on the lowest distinguishable standard concentration, 2) determination of the standard concentration at which a hook occurs, 3) determination of cross-reactivity to subunits as well as intact gonadotropins, and 4) establishment of an adequate normative data base.

Automatic reservoir for serial pipetting.

An automatic reservoir for serial pipetting was constructed and used in radioimmunoassay determinations. This device features negligible priming volume, minimal solution loss, and lowered overall cost. In our experience, it has proven valuable when used with an automatic pipetting station. This apparatus has not affected the precision or accuracy of the procedure. It has made it possible to economize on costly antibody reagents supplied with radioimmunoassay kits, especially in use with low-volume assays.

How much quality control is enough? A cost-effectiveness model for clinical laboratory quality control procedures (illustrated by its application to a ligand-assay-based screening program).

Quality assurance testing represents a substantial proportion of the clinical laboratory budget, but current guidelines are based on criteria that pertain to analytic error rather than to optimization of the cost-effectiveness of patient care. A general Bayesian mathematical model for the cost-effectiveness of assay quality control has been developed, and is demonstrated using previously published data. The cost-effectiveness of quality assurance as defined here depends upon the prevalence of disease, the shapes of the distributions of test results observed in the non-diseased and diseased populations, the decision limit selected for labeling results positive or negative, the costs and benefits associated with each of the possible therapeutic outcomes, the magnitude of random and systematic analytical errors, the statistical power of the quality control test in use, the costs associated with delays due to re-assay, and the proportion of total test cost attributable to quality control procedures. Given current clinical laboratory practice, much of this information will not be routinely available. The model combines these factors into a simple equation with three terms: one for the cost of the original and any required repeat laboratory analyses, one for the cost of delay entailed by the rejection of an assay batch, and one for the change in total costs consequent to rejection of erroneous assay results.

A simple, rapid and cheap radioimmunoassay for plasma digoxin.

A radioimmoassay for digoxin is described which uses commercially available reagents. Two assay procedures are used, one for batch assays, and another, which needs a 5 minute incubation only, for 'stat' assays. The methods are shown to be simple, rapid, precise and inexpensive and the batch procedure compared well with the Amerlex digoxin method for 63 patient samples.

Cortisol and behavior: 1. Adaptation of a radioimmunoassay kit for reliable and inexpensive salivary cortisol determination.

We adapted a commercial serum cortisol radioimmunoassay (RIA) kit for use with saliva specimen. Using 50 microliters sample volume, the lower sensitivity was found to be 0.02 microgram/dl with intraassay variation coefficients between 5.4 and 8.9% at different concentrations. The 50% intercept was either 0.5 or 0.26 microgram/dl (50 or 100 microliters standard/sample volume). Fifty-four early morning samples from healthy adults showed absolute concentrations which are closely comparable to respective data from other laboratories. A comparison of 35 saliva samples which were each assayed with the adapted RIA as well as with three other commercial kits revealed high correlations between these assays (r = .94 to r = .97). Data on salivary cortisol responses to CRH stimulation and dexamethasone suppression in healthy subjects further the validity of the assay results. The most important contribution of this assay modification, however, is thought to be its impact on analysis costs: The protocol presented in this paper allows for reliable salivary cortisol measures with a reduction of costs for analytical material to 25% compared to serum determinations.

Medical x-ray exposure of the human embryo and fetus.

In a period of 2 yr. 6 pregnant women who were not aware that they were pregnant at the time of radiological examination of their abdomen and pelvis, wanted to know the radiation dose received by their embryo or fetus. Radiation dose estimate to the embryo or fetus was made by performing dosimetry with a phantom and the equipment utilized for the actual procedure. The dose to the embryo or fetus varied between less than 50 mrads and 3.52 rads when fluoroscopy was also performed. As the radioimmunoassay test now enables immediate, (same day) detection of pregnancy after day 21 of the menstrual cycle, it is reasonable to recommend that (1) the test be performed in all patients whose pregnancy status is uncertain and (2) that a positive result will ensure minimization of any necessary diagnostic radiation dose. The risk to the embryo or fetus is discussed.

Technical and financial evaluation of assays for progesterone in canine practice in the UK.

The concentration of progesterone was measured in 60 plasma samples from bitches at various stages of the oestrous cycle, using commercially available quantitative and semi-quantitative ELISA test kits, as well as by two commercial laboratories undertaking radioimmunoassay (RIA). The RIA, which was assumed to be the 'gold standard' in terms of reliability and accuracy, was the most expensive method when analysing more than one sample per week, and had the longest delay in obtaining results, but had minimal requirements for practice staff time. When compared with the RIA, the quantitative ELISA had a strong positive correlation (r=0.97, P<0.05) and a sensitivity and specificity of 70.6 per cent and 100.0 per cent, respectively, and positive and negative predictive values of 100.0 per cent and 71.0 per cent, respectively, with an overall accuracy of 90.0 per cent. This method was the least expensive when analysing five or more samples per week, but had longer turnaround times than that of the semi-quantitative ELISA and required more staff time. When compared with the RIA, the semi-quantitative ELISA had a sensitivity and specificity of 100.0 per cent and 95.5 per cent, respectively, and positive and negative predictive values of 73.9 per cent and 77.8 per cent, respectively, with an overall accuracy of 89.2 per cent. This method was more expensive than the quantitative ELISA when analysing five or more samples per week, but had the shortest turnaround time and low requirements in terms of staff time.

Development of a radioimmunoassay for the measurement of Bisphenol A in biological samples.

Bisphenol A (BPA) is widely used in the manufacturing of polycarbonate plastic food and drink packaging. Possessing a weak estrogenic activity, BPA is listed among a growing list of endocrine disrupting compounds. In this study, a polyclonal anti-BPA antibody was obtained by immunization with BPA-monocarboxymethylether covalently linked to BSA. The antibody demonstrates negligible cross-reactivity with most analogous BPA phenolic structures, and no cross-reactivity with endogenous steroids. An extraction step with ethyl acetate minimized matrix effects and allowed the BPA measurement in plasma and other biological samples. Recovery after loading test was 96 +/- 4% and dilution tests had a linear profile (r2 > 0.93). The limit of detection of the BPA RIA was 0.08 microg L(-1) with an IC50 of 1.25 microg L(-1). The intra- and inter-assay coefficients of variation were 5.6 and 8.6%, respectively at a BPA concentration of 0.7 microg L(-1) and 6.9 and 5.7% at a BPA concentration of 1.3 microg L(-1). A significant correlation was found between the values obtained by the RIA and HPLC-MS (r2 = 0.92) or HPLC coupled to a fluorescence detector (r2 = 0.80). In conclusion, we described a BPA-RIA that is a suitable tool for evaluating human exposure to BPA.

Economical modification of a radioimmunoassay of digoxin with negligible interference from cord and neonatal sera.

The Amerlex digoxin radioimmunoassay was modified to reduce the running cost by five times. The modified method compared well with the original method (y = 1.0138x + 0.00916, r = 0.9936). Using the modified method, the performance with the American Association for Clinical Chemistry quality control program was consistently satisfactory for 1 year. When normal cord and neonatal sera were tested with the modified method, 41% of the 22 samples of cord serum and 68% of the 19 samples of neonatal serum produced digoxin levels less than or equal to 0.05 ng/ml. The highest digoxin level produced by cord and neonatal sera was 0.3 ng/ml.