Antibody-bound Tracer Research Articles

Toward Multianalyte Immunoassays: A Flow-Assisted, Solid-Phase Format with Chemiluminescence Detection

Chemiluminescence immunoassays are widely used in clinical chemistry. A multianalyte format is of particular interest to increase throughput and diagnostic utility. Field-flow fractionation (FFF) can separate dispersed particles in the micrometer-sized range (1). Separation is achieved within an empty capillary channel by the combined action of a transporting laminar flow and a field applied perpendicularly to the flow. According to their characteristics (e.g., dimension, density, and surface properties), particles are distributed at different positions within the parabolic flow profile, are thus transported along the channel at different velocities, and elute at different times. Methods combining FFF with chemiluminescence (CL) detection (FFF-CL) have recently been developed to efficiently separate horseradish peroxidase (HRP) in solution from HRP immobilized on micrometer-sized spheres and to quantify each fraction by means of the CL-HRP substrate based on H2O2–luminol–enhancer (2)(3). This was the basis for the development of FFF-CL–based solid-phase competitive immunoassays, in which micrometer-sized beads coated with the capture antibody are used as a solid phase and an analyte–HRP conjugate is used as a tracer. Once the competitive immunologic reaction takes place within the injection loop of the system, the antibody-bound tracer (bound fraction) is separated from tracer in solution (free fraction) in a few minutes by means of FFF. FFF-based immunoassays offer several advantages. For example, the kinetics of the immunologic reaction on micrometer-sized beads are faster than in the case of conventional microtiter plate assays. In addition, multianalyte FFF-based immunoassays can be developed by use of beads of different sizes (1–50 μm), each coated with the specific antibody for 1 analyte. The beads are fractionated by FFF before CL detection using different enzymes, such as HRP or alkaline phosphatase, detected by CL. Here we report the development of a CL-FFF–based competitive immunoassay for chloramphenicol (CAF), chosen …

Aflatoxin M1 determination in raw milk using a flow-injection immunoassay system

A flow-injection immunoassay (FI-IA) method with amperometric detection for aflatoxin M1 (AFM1) determination in milk has been developed. The first step consists in an incubation of the sample containing AFM1 (Ag) with fixed amounts of anti-AFM1 antibody (Ab) and of the tracer (Ag ∗, AFM1 covalently coupled to HRP) until equilibrium is reached. In this mixture a competition occurs between Ag and Ag ∗ for the Ab. The mixture is then injected into a flow system where the separation of the free tracer (Ag ∗) and the antibody-bound tracer (AbAg ∗) is performed in a column with immobilized Protein G. The antigen–antibody complexes are retained in the column due to the high affinity of the Protein G for the antibody. The activity of the eluted enzyme label is then amperometrically detected. The immunoassay was optimised relative to conditions for antibody–antigen incubation (pH, incubation time, ionic strength, temperature) and enzymatic label detection. This method showed a dynamic concentration range between 20 and 500 ppt AFM1, a low detection limit (11 ppt), good reproducibility (RSD < 8%) and a high throughput (six samples per hour in triplicate). Different milk samples were analysed and the results were in good agreement with those obtained by HPLC using the AOAC 2000.08 method.

Capillary electrophoresis coupled with laser-induced fluorescence polarization as a hybrid approach to ultrasensitive immunoassays

Immunoassays using capillary electrophoresis with laser-induced fluorescence detection (CE–LIF) is a powerful approach to the determination of trace amounts of analytes in a complex biological matrix. However, its applicability is limited by the requirement that the free and bound tracer (fluorescently labeled compound) be resolved for their identification and quantitation. Here we show that replacing LIF with laser-induced fluorescence polarization (LIFP) permits ultrasensitive immunoassays to be performed with or without the separation of the free and bound tracer. A binding system involving cyclosporin A (CyA) and monoclonal antibody to CyA was chosen to demonstrate both homogeneous and heterogeneous immunoassay approaches. In the homogeneous scheme where the free and bound tracer were not separated, the fluorescence polarization of the mixture was a quantitative measure of the antibody-bound tracer. The concentration and mass detection limits for CyA using the homogeneous competitive assay were found to be 1 nM and 1 amol (10−18 mol), respectively. The heterogeneous assay involved a nearly baseline separation of the free and bound tracer using CE with a phosphate running buffer of pH 7.0. The complex of the tracer with the antibody had a fluorescence polarization of approximately 0.24 whereas the free tracer had negligible polarization. The fluorescence polarization was independent of analyte concentration, and the fluorescence intensity of either the free or bound tracer was used for quantitation. Results from both assays suggest that the CE–LIFP approaches may have a wider application than the immunoassays based on either CE–LIF or fluorescence polarization alone.

Development of a high capacity radioimmunoassay procedure: ovine follicle stimulating hormone determination in blood plasma as a model.

A sensitive, specific and accurate homologous radioimmunoassay (RIA) for ovine follicle stimulating hormone (oFSH) has been developed, using a [125I]oFSH tracer and a polyclonal rabbit anti-OFSH-serum at a final dilution of 1:224,000. The separation of free and antibody-bound tracer is based on the double antibody solid phase system. The assay was found to be specific for oFSH; cross-reactivity with oLH, oPrl and oGH was lower than 0.2%. The detection limit was 7 pg per tube. Inter- and intra- assay coefficients of variation (CV) were 3.7-8.5% and 6.6%, respectively. The use of a few selfmade appliances in combination with a well-considered time-schedule enabled the processing of three thousand blood plasma samples in triplicate within two weeks. The particular advantage of the method described here is the fast, easy and safe separation of free and antibody-bound tracer with minimal handling of radioactive tubes.

Enzyme lmmunoassay for Elcatonin, a Synthetic Analogue of Eel Calcitonin, in Plasma

An enzyme immunoassay for the direct measurement of elcatonin, a synthetic analogue of eel calcitonin with hypocalcemic activity, in rat and dog plasma was developed. Elcatonin was conjugated to β-galactosidase using an N-succinimidyl ester, and the product was used as the tracer. The antibody-bound tracer was precipitated using a second antibody. The nonequilibrated procedure gave better sensitivity and precision than the equilibrated procedure. The nonequilibrated method was sensitive at quantifiable concentrations as low as 0.2 ng/mL using 0.1mL of plasma and was also reproducible, with RSD values generally less than 10%. Plasma levels in rats and dogs were determined by this competitive enzyme immunoassay after intramuscular or intravenous injection of 40 U/kg (8 μg/kg) of elcatonin.

An Improved Enzymeimmunoassay of Progesterone Applied to Bovine Milk

SUMMARY An enzymeimmunoassay of milk progesterone was established using β-galactosidase from E. coli as the tracer. 11α-hydroxyprogesterone-hemisuccinate was conjugated with α-galactosidase by using carbodiimide. Antibody was raised by immunization of the rabbit against 11α-hydroxyprogesterone-hemisuccinate-bovine serum albumin. A double antibody method was used for separation of free from antibody-bound tracer. Skimmilk or defatted milk (0·05 ml) was used for the enzymeimmunoassay after extraction with petroleum ether. The sensitivity of the immunoassay was 10 pg/tube and satisfied normal criteria regarding specificity, accuracy and precision. Progesterone levels in skimmilk were highly correlated (r = 0·99, P The present method of enzymeimmunoassay was found to be of practical application in routine milk progesterone assay.

Sensitive and specific radioimmunoassay for creatine kinase BB isoenzyme in serum, with use of an autoantibody.

We have developed an equilibrium radioimmunoassay for creatine kinase BB (CK-BB) isoenzyme in serum. We used as antibody a CK-BB binding immunoglobulin G isolated from serum of a patient with macro CK-BB. Free and antibody-bound tracer were separated by anion-exchange chromatography. The affinity constant, Keq, for the binding of CK-BB to immunoglobulin was calculated as 1.4 X 10(11) L/mol, which suggests that the immunoglobulin was an autoantibody. The assay may detect 1 microgram of enzyme per liter in a 100- microL sample. CK-MM and CK-MB isoenzymes did not cross react in the assay. Within-assay precision (CV) was 5%; between-assay precision was 14 and 15% at 6 and 14 microgram of CK-BB per liter, respectively. Of 45 sera from blood donors, 95% contained < 1.8 microgram of CK-BB per liter, whereas of 19 sera from unselected patients the corresponding figure was 2.8 microgram/L.

Magnetizable solid-phase fluoroimmunoassay of phenytoin in disposable test tubes.

We developed a fluoroimmunoassay for phenytoin in serum or plasma, based on the magnetic separation technique. The method involves sheep anti-phenytoin serum coupled to magnetizable cellulose/iron oxide particles, with a fluorescein-labeled phenytoin analog as tracer. After magnetic sedimentation of the solid phase from assay mixtures, the free fraction of the tracer is aspirated, removing endogenous fluorophores and other interfering components of the sample. The antibody-bound tracer is then eluted from the solid phase into a methanolic buffer medium and quantitated fluorometrically. The entire procedure, including fluorometry, is performed within disposable polystyrene test tubes. The assay involves only simple reagents and equipment, and correlates closely with established radioimmunoassay (r = 0.97) and gas-liquid chromatographic (r = 0.98) techniques.

Factors which influence the radioimmunoassay of carcinoembryonic antigen (CEA)

A radioimmunoassay for CEA is described and used to investigate the efficiency of precipitation of antibody-bound tracer, rate of antigen-antibody reaction and stability of the antigen-antibody bond. The error of the assay and its reproducibility over a period of time were determined. A simple procedure for preparing perchloric acid extracts of plasma without dialysis is described and its recovery of CEA investigated. It was found that (1) CEA purified from extracts of colo-rectal carcinoma may differ in specific immunological activity and (2) dose-response curves given by extracts of benign and malignant tissue and plasma from patients with carcinoma may differ significantly in slope from one another and from that of purified CEA from colo-rectal carcinoma. It was concluded that although the radioimmunoassay of plasma CEA may be carried out with adequate precision and reproducibility, accuracy of measurement is affected by apparent variation in the immunological properties of CEA from individual samples of plasma and tissue. The specificity of the assay may be affected by high concentrations of plasma proteins.

Luteinizing hormone-releasing hormone levels in human plasma: A radioimmunoassay method

A sensitive double-antibody radioimmunoassay, developed to measure luteinizing hormone-releasing hormone (LHRH) levels in fetal brain tissues, has been modified to quantitate LHRH in unextracted plasma samples. Pure, synthetic LHRH was iodinated with125I for tracer and was also used as standard. The antibody utilized was specific for LHRH and did not cross react with other hypothalamic or pituitary hormones. The separation of free from antibody-bound tracer was perforrned by dextran-coated charcoal. The sensitivity of the assay was 2.5 pg/tube. The intra-assay and interassay coefficients of variation were 5.9 and 6.1% for 50 pg/ml and 11.8 and 14.9% for 25 pg/ml samples. Recovery of known amounts of LHRH added to plasma samples that had no detectable hormone was 96.4 ± 8.8%. Whether these samples were previously prepared and kept frozen at −20° C or freshly prepared, the recovery of LHRH was consistent and quantitatively stable in each assay. Serial determinations of LHRH in plasma obtained from ovulatory volunteers averaged between 20.5 ± 2.1 pg/ml in the follicular phase and 19.4 ± 4.5 pg/ml in the luteal phase. At midcycle, coincident with the luteinizing hormone surge, LHRH levels averaged 17.6 ± 4.4 pg/ml.

Development of Glycoprotein Hormones and their α-and β-Subunits in Bovine Fetal Pituitary Glands. II. Quantitation of Free α- and β-Subunits by Radioimmunoassays and Correlation of Free α-Subunit with Thyrotropin, Follicle-Stimulating Hormone, and Luteinizing Hormone*

Homologous RIAs for bovine FSH-α and the β-subunits of bovine FSH, LH, and TSH have been developed. Antibody-bound tracer was separated by precipitating with a final concentration of 11% polyethylene glycol. The sensitivities of these immunoassays were 0.5–1 ng/ml for the purified subunits, and the precision was ±6% intraassay and ±11% interassay, as expressed by the coefficient of variation. In the RIA for bovine FSH-a, all the α-subunits of bovine FSH, LH, and TSH crossreacted identically on a weight basis and the intact hormones also cross-reacted identically with one another in a parallel manner to the α-subunits at a level of 10% cross-reactivity. In the immunoassays for bovine FSH-β, LH-β, and TSH-β, onlythe corresponding intact bovine FSH, LH, and TSH inhibited significantly at levels of 5%, 15%, and 25% cross-reactivities, respectively. To obtain the net values for the free α- and β-subunits in bovine fetal pituitary extracts, the amount of crossreactivity caused by different concentrations of int...

A micro-scale method for liquid scintillation counting, applicable to radioimmunoassay of steroids and substances of comparable relative molecular mass.

A major expense of radioimmunoassay involving tritiated tracers is that of liquid scintillation counting. We present a micro-scale method that markedly reduces the cost of liquid scintillation counting. The radioimmunoassay is done in 10 X 50 mm tubes. The antibody-bound tracer is precipitated with a second antibody, the precipitate is resuspended in 0.1 ml of water, and 0.3 ml of dioxane is added. One milliliter of a toluene-based scintillation cocktail is added, and the tube is capped and placed in an adapter for liquid scintilation counting. When this method was applied to a double-antibody testosterone radioimmunoassay, it performed comparably to assays counted by the usual method.