New noncompetitive immunoassays of small analytes.

We developed a novel noncompetitive immunoassay format for monoepitopic analytes and describe here a model assay for triiodothyronine (T3), performed on Ciba Corning's ACS:180 analyzer. Acridinium ester (AE)-labeled bivalent anti-T3 was incubated with the sample, producing AE-anti-T3/T3 complexes and unreacted AE-anti-T3. Controlled-pore glass particles (CPG) with immobilized diiodothyronine (T2) were then added in excess, to bind AE-anti-T3 possessing two unoccupied binding sites but not AE-anti-T3 bound to one or two T3 molecules. Paramagnetic particles (PMP) with immobilized anti-AE were then added to the same cuvette to capture AE-anti-T3/T3 complexes; AE-anti-T3 bound to the surface of CPG, however, was not captured, because of steric hindrance. After the incubation, the PMP was magnetically separated to remove the liquid phase and the suspended CPG from the cuvette. The chemiluminescence associate with the PMP remaining in the cuvette was then measured. This noncompetitive T3 assay exhibited a 10-fold lower detection limit than the equivalent competitive T3 assay, i.e., 0.3 vs pg/test. Imprecision (CV) in the clinically significant range was 6% or less. The assay also displayed two- to sevenfold lower cross-reactivities and a wider dynamic range.

Role of antibody valency in hapten-heterologous immunoassays.

We studied the effects of hapten heterology on immunoassays of triiodothyronine (T3), digoxin, and cortisol, in a format involving labeled monoclonal antibodies and immobilized, protein-conjugated ligands. Replacing the homologous conjugated ligands T3, digoxin, and cortisol with their respective analogs diiodothyronine, digitoxin, and corticosterone led in each case to a decrease in the midpoint of displacement (ED50) for the same zero-dose signal. The mechanism of this phenomenon was studied by converting the bivalent anti-T3 to a monovalent whole antibody (bispecific monoclonal anti-T3 x anti-glucose-6-phosphate dehydrogenase) by cell fusion. The monovalent antibody was effective as a tracer in the homologous T3 assay, but generated a very low zero-dose signal with the heterologous solid phase, thus precluding sensitivity enhancement. On the basis of these results and additional kinetic and double-labeling experiments, we propose that the use of hapten heterology relies on bivalent binding of the antibody to the solid phase to compensate for a lower intrinsic affinity. This binding mechanism leads to lower assay concentrations of the ternary complex analyte-labeled antibody-immobilized hapten, thereby providing enhanced sensitivity.

Dissociation rate constant of the biotin-streptavidin complex.

We measured the dissociation rate constants of the biotin/streptavidin and biotin/egg avidin complexes by following the release of radiolabeled biotin from the preformed complexes in the presence of excess unlabeled biotin. For separation of bound and free labeled biotin we employed ultrafiltration with disposable microconcentrators. The dissociation rate constant for underivatized streptavidin was 2.4 x 10(-6) s-1, or approximately 30-fold higher than that observed for egg avidin 7.5 x 10(-8) s-1). The value for streptavidin was further increased after derivatization with an acridinium ester label. Both biotin binding proteins exhibited a faster initial phase, suggesting binding site heterogeneity due to partial subunit dissociation or denaturation. The convenience of the method and the relatively fast dissociation of biotin from streptavidin render the dissociation rate constant a practical experimental criterion for monitoring the integrity of the binding site during purification and derivatization procedures.

Effect of hapten heterology on thyroid hormone immunoassays.

We examined the effects of hapten heterology on triiodothyronine (T3) and thyroxine (T4) immunoassays configured with protein-conjugated diiodothyronine (T2), T3 or T4, using acridinium ester (AE) as a chemiluminescent label and paramagnetic particles (PMP) as a solid phase. Assays constructed with hapten-heterologous combinations, such as immobilized anti-T4 plus labeled T3 or immunobolized anti-T3 plus labeled T2, were superior to the homologous ones in their potential for attaining higher sensitivity. This was manifested in a shift of the displacement curves to lower analyte concentrations, accompanied by unexpectedly high bound-tracer/total-tracer (B/T) ratios. This effect of hapten heterology was apparent with the use of either monoclonal or polyclonal antibodies and did not depend upon which component (antibody or protein conjugated hormone) was immobilized on the solid phase. In contrast, heterologous displacement curves with unconjugated hormone (125I-labeled) or with a labeled Fab fragment exhibited both a shift to lower analyte concentration and the expected decrease of B/T ratios. These results can be explained by the existence of positive binding cooperativity in the interaction between antibodies and haptenated proteins. Assay specificity was not adversely affected by hapten heterology when utilizing monoclonal antibodies, and hormone levels measured in serum samples correlated well with values obtained in homologous immunoassays.

Novel poly-substituted aryl acridinium esters and their use in immunoassay.

A new series of stable acridinium ester conjugates have been developed for use as non-isotopic labels in immunoassay. They have proved to be a flexible alternative to radioimmunoassay. We present data showing the successful development of immunoassays in sandwich, competitive and receptor formats. In addition, hydrophilic acridinium ester analogues have been synthesized, encapsulated in liposomes, and utilized as labels in immunoassay. The potential of this technology is discussed.

Immunochemiluminometric assay of creatine kinase MB with a monoclonal antibody to the MB isoenzyme.

Previous two-site immunometric assays for creatine kinase (CK; EC 2.7.3.2) MB isoenzyme have been based on formation of a "sandwich" complex involving CK-MB and antibodies that recognize the CK-MM and the CK-BB isoenzymes. Single-incubation model assays of CK-MB with these antibodies were susceptible to interferences by CK-MM and CK-BB. We produced two anti-CK-MB monoclonal antibodies and studied their suitability for two-site assays. Both antibodies were compatible with anti-CK-MM and anti-CK-BB, but not with each other. Using anti-CK-MB as the tracer antibody eliminated the interference by both CK-MM and CK-BB. Labeling anti-CK-MB with acridinium ester and immobilizing anti-CK-BB on paramagnetic particles, we developed a rapid and highly sensitive chemiluminescent/magnetic separation CK-MB assay. As little as 1 microgram of CK-MB per liter was detectable after 10- or 30-min incubation at room temperature, and the standard curve was linear up to 400 micrograms/L. Results for serum samples by the new assay correlated well (r = 0.94) with those by Corning electrophoretic and the Hybritech Tandem-E immunoenzymometric CK-MB methods. Sera containing macro CK-1 or high concentrations of CK-MM and CK-BB did not interfere. The combined advantages of a more-specific antibody, paramagnetic solid phase, and chemiluminescent label endow this two-site CK-MB assay with performance characteristics and ease of use superior to those of previous assays.

Central control of peripheral circulating somatostatin in dogs: effect of 2-deoxyglucose.

Circulating plasma somatostatin concentrations are known to fluctuate in response to nutrients and hormones. However, little is known about neural or central nervous system (CNS) control of somatostatin secretion. To test whether peripheral circulating somatostatin is influenced by a central stimulus, 2-deoxyglucose (37.5 mg/kg) was infused into a lateral cerebral ventricle of six conscious dogs over a period of 15 min. Plasma somatostatin levels rose from a base line of 105 +/- 6 pg/ml (mean +/- SE) to a peak of 154 +/- 10 pg/ml (P less than 0.005) at 30 min after the onset of the infusion. Somatostatin levels were still significantly elevated (P less than 0.025) at 60 min (119 +/- 6 pg/ml) and thereafter gradually returned toward base line. Plasma glucose and glucagon levels increased in response to intraventricular 2-deoxyglucose. Glucose concentrations rose from 105 +/- 5 mg/dl to peak at 203 +/- 16 mg/dl (P less than 0.005) at 80 min and remained elevated to 120 min. The concentration of plasma glucagon increased from 41 +/- 6 to 92 +/- 18 pg/ml at 60 min (P less than 0.05) and then declined. In marked contrast to intraventricular 2-deoxyglucose, similar concentrations of 2-deoxyglucose administered intravenously (n = 4) resulted in a slight fall in plasma somatostatin. Intraventricular saline did not result in a change in plasma somatostatin. It is concluded that peripheral circulating somatostatin may be susceptible to central nervous system control.

A lipoprotein independent assay for human serum lecithin-cholesterol acyltransferase.

A method has been developed for estimation of human serum lecithin-cholesterol acyltransferase free of interference by endogenous lipoproteins. Precipitation of serum low and very low density lipoproteins by sodium phosphotungstate and magnesium chloride results in complete recovery of lecithin-cholesterol acyltransferase activity in the supernatant. One microliter of the supernatant can be accurately assayed with a highly efficient substrate containing phosphatidylcholine-cholesterol vesicles and apo-high density lipoproteins (HDL), with no interference from endogenous HDL or residual precipitation reagents. Serum levels of the enzyme were found to be reduced in patients with parenchymal liver disease, renal disease, gastrointestinal tumors and anemias.

A rapid radioassay procedure for plasma lecithin-cholesterol acyltransferase.

A rapid and accurate single step procedure is described for the assay of lecithin-cholesterol acyltransferase activity. After incubation, using radiolabeled cholesterol as the substrate, an ethanolic solution of digitonin is added directly to the incubation mixture to extract the lipids. Excess cholesterol is then added, and the labeled cholesterol-digitonide along with denatured proteins are sedimented by low speed centrifugation, leaving the labeled esterified cholesterol in solution. An aliquot of the supernatant is counted in an aqueous scintillation mixture. The method correlates well with the established thin-layer chromatographic procedure using either lecithin-cholesterol vesicles or heat-inactivated plasma as the substrate for lecithin-cholesterol acyltransferase.

Utilization of various sterols by lecithin-cholesterol acyltransferase as acyl acceptors.

Highly purified lecithin-cholesterol acyltransferase of human plasma was used to study the utilization of various sterols as the acyl acceptor. The esterification of sterols was facilitated by the presence of a 3beta-hydroxyl group and the trans configuration of the A/B rings, as was evident from the lack of acceptor activity of all 3 alpha-hydroxy sterols tested and coprostanol. Cholesterol analogs in which the side chain is modified, such as campesterol, beta-sitosterol, desmosterol and stigmasterol, were less effective than cholesterol as acyl acceptors. However, androstan-3 beta-ol, which completely lacks the side chain, was found to be more active than cholesterol. The transfer of the acyl group to all effective sterols required the presence of the cofactor peptide apolipoprotein A-I.

Purification of human plasma lecithin:cholesterol acyltransferase and its specificity towards the acyl acceptor.

A simple and convenient method for the purification of human plasma lecithin-cholesterol acyltransferase was developed. The method involves the adsorption of the enzyme from diluted human plasma on DEAE-Sephadex, treatment with 1-butanol in the presence of (NH4)2SO4, DEAE-Sephadex chromatography, treatment with dextran sulfate in the presence of Ca2+, and hydroxyapatite chromatography. The enzyme purified showed a single main band by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. In addition, the enzyme obtained was stable for more than four weeks, when it was kept at 4 degrees C under N2 in a buffer of low ionic strength. The purified enzyme was used to study its specificity toward the acyl acceptor. This specificity was found to be broad in that not only sterols but also long chain primary alcohols exhibited considerable acceptor activity. Furthermore, in agreement with our previous observations with crude enzyme (Piran, U. and Nishida, T. (1976) J. Biochem. (Tokyo) 80, 887-889), the purified enzyme was found to be capable of hydrolyzing the ester linkage at the carbon-2 position of phosphatidylcholine. The transesterification, as well as the hydrolytic reaction, required the presence of the cofactor polypeptide, apolipoprotein A-I.

Release of fatty acids from phosphatidylcholine by lecithin-cholesterol acyltransferase.

Partially purified lecithin-cholesterol acyltransferase [EC 2.3.1.43] from human plasma released fatty acids from phosphatidylcholine. Heating, sulfhydryl reagents, Ca2+, EDTA, and sodium deoxycholate had similar effects on the lecithin-cholesterol acyltransferase and fatty acid releasing activities of the preparation. A specific cofactor protein for lecithin-cholesterol acyltransferase, apoA-1, also enhanced both activities. Release of fatty acid was due to enzymatic hydrolysis of the ester linkage at carbon-2 of phosphatidylcholine. It is suggested that the two activities are due to a single enzyme.