Antigen-binding activity of antibodies immobilized on styrene copolymer beads.

An important element of the Kodak thin-film immunoassay is antibody immobilized on small polymer beads. Monodisperse styrene copolymer beads offer a well-defined, high surface area substrate for covalent immobilization of monoclonal antibodies. The authors have used the ability of an immobilized monoclonal antibody directed against phenobarbitol, Phe 1.9, to recognize an antigen-enzyme conjugate to determine the extent of antibody activity retention and find that the packing density of antibody at the surface and the copolymer composition are important variables. For polystyrene homopolymer and some copolymers, antibody retention is greater as the packing density at the surface increases. Small changes in the copolymer composition, such as addition of 1% acrylamide or 10% acrylic acid, significantly increase the retention of binding activity of the antibody. The chemistry for covalent coupling of the antibody to the surface is also important. Phe 1.9 coupled to chloromethyl styrene copolymer beads retains less activity than when coupled to vinyl sulfone copolymer beads. Monodisperse sytrene copolymer beads provide great flexibility in the design of rapid immunoassays since a copolymer bead can be tailored to the specific requirements of the antibody and the analyte.

The Ektachem clinical chemistry slide for simultaneous determination of unconjugated and sugar-conjugated bilirubin.

Using dual-wavelength spectrometry, we have refined the mordant-based bilirubin slide method (Clin Chem 28:2366-2372, 1982) to co-detect unconjugated bilirubin (Bu) and its sugar conjugates (Bc) in 10 microL of serum. The assay is based on three principles: (a) mono- and diconjugated bilirubins behave spectrally like one fraction (Bc) when bound to the mordant, (b) Bu and Bc are spectrally distinct, and (c) B delta (the bilirubin-albumin complex) is not measured in the film. With known bilirubin mixtures, results by the assay agree with those by nuclear magnetic resonance, by a Jendrassik-Gróf method for total bilirubin, and by a liquid-chromatographic procedure. With patients' sera, the slide correlates with a liquid-chromatography-augmented Jendrassik-Gróf method, according to the following typical regression statistics (in mumol/L): for Bu, slope = 0.992, r = 0.996, intercept = -0.376, Sy X x = 5.08; for Bc, slope = 0.970, r = 0.985, intercept = -0.735, Sy X x = 8.16. The method is precise (for Bu, CV = 5.2% at an average concentration of 16.4 mumol/L, and 4.1% at 66.7 mumol/L; for Bc, CV = 6.5% at 23.4 mumol/L, and 3.8% at 151.7 mumol/L for pools of patients' sera), is relatively interference free, and has potential for extension to further applications.

Diazo-based assay for total bilirubin in a coated thin film evaluated.

We compared results for total bilirubin as measured on a coated thin film and by the Evelyn-Malloy and Jendrassik-Gróf methods. We examined serum samples from patients and studied the effects of protein, hemoglobin, and lipids on bilirubin measurement. Results from the thin-film assay compared favorably with those of the other methods. Total and within-day precision (CV), assessed over a one-year period, were better than 6% and 3%, respectively, at all concentrations. Analytical recovery was 99 +/- 3%. Samples from individuals having a wide range of liver diseases demonstrated, by linear regression, good correlation between the thin-film method and the two wet-chemistry methods (correlation coefficients of 0.990 and 0.994). We conclude that the thin-film method offers a valid alternative assay for total bilirubin.

Estimation of unconjugated, conjugated, and "delta" bilirubin fractions in serum by use of two coated thin films.

We used two coated thin films to measure the concentrations of unconjugated, conjugated, and total bilirubin as well as bilirubin covalently bound to albumin ("delta" bilirubin) in more than 400 serum samples. We measured the unconjugated and conjugated species by determining their reflection densities at two wavelengths (400 and 460 nm) on a coating designed for the enhanced spectral measurement of bilirubin but which does not register the delta form. Total bilirubin was measured by use of a diazo-based thin film (Clin Chem 29: 37-41, 1983). We estimated the concentration of delta bilirubin by subtracting the sum of unconjugated and conjugated bilirubin from the concentration of total bilirubin. All measurements agree well with those by comparative methods, as shown by linear regression. Slopes ranged from 0.92 to 1.02, correlation coefficients from 0.935 and 0.998. Linear combinations of these values can also be used to compute other results; e.g., the sum of conjugated and delta bilirubin can be considered to be an estimate of "direct"-reacting bilirubin.

The clinical importance of a protein-bound fraction of serum bilirubin in patients with hyperbilirubinemia.

A directly reacting fraction of bilirubin that is probably covalently bound to albumin (albumin-bound bilirubin) has recently been described. To determine its clinical importance we used a new high-performance liquid-chromatography technique to measure it in the serum of 200 patients with hyperbilirubinemia from various causes. Albumin-bound bilirubin was an important fraction (8 to 90 per cent) of total bilirubin in patients with hepatocellular and cholestatic jaundice as well as in patients with the Dubin-Johnson syndrome. It was not detected in normal volunteers, neonates with physiologic jaundice, or patients with Gilbert's disease or hemolysis. Thus, albumin-bound bilirubin appears in serum when hepatic excretion of conjugated bilirubin is impaired. It becomes a larger component of serum bilirubin as jaundice subsides, delaying resolution of this disorder and causing bilirubin to persist in plasma after it has disappeared from the urine.

An enzymic creatinine assay and a direct ammonia assay in coated thin films.

We developed a thin-film enzymic assay for creatinine that makes use of creatinine iminohydrolase (EC 3.5.4.21) to convert creatinine to N-methylhydantoin and ammonia. The ammonia diffuses through a semipermeable layer and is quantitated by reaction with bromphenol blue. A paired analysis of the sample on a separate coating without the enzymic reaction measures endogenous ammonia and, for samples with normal concentrations of ammonia, allows accurate determination of serum creatinine to 150 mg/L without dilution. Results of this assay (y) compare well with those by a liquid-chromatographic comparison assay (x) by linear regression (slope = 0.935, intercept = 1.13 mg/L, r2 = 0.995). It is insensitive to many substances, such as ketones and keto acids, that interfere with conventional assays. Results of the ammonia assay (y) correlate well with those by a semi-automated enzymic assay (x) based on glutamate dehydrogenase (slope = 1.068, intercept = 17.3 mumol/L, r2 = 0.985).

A diazo-based dry film for determination of total bilirubin in serum.

We have prepared a diazo-based dry film for use in determining total serum bilirubin. On a transparent support are a buffered gelatin layer containing a polymeric quaternary amine (the mordant) and a white, reflective spreading layer that contains all of the components necessary for detection of bilirubin. The method is based on the use of dyphylline and Triton X-100 surfactant to dissociate bilirubin from albumin and subsequent reaction of bilirubin with a diazonium salt [4-(N-carboxymethylsulfamyl)benzenediazonium hexafluorophosphate]. In the dry film, unconjugated, mono- and diconjugated, and strongly protein-linked (delta) bilirubin all react with the diazonium salt to produce azo dyes having absorption maxima at about 520 nm. With reflection densitometry and appropriate mathematical transformation, readings and bilirubin concentrations are linearly related to 260 mg/L. Results correlate well with those by the Jendrassik-Grof (Doumas modification) method (slope 0.994, intercept 1.1, correlation coefficient 0.993, Sy X x 4.0), and the method is precise (CV = 10.0% at Cav = 4.1 mg/L, 2.7% at Cav = 24.5 mg/L, 1.2% at Cav = 102 mg/L for patients' samples) and relatively free of interferences.

Bifunctional chelates for radio-pharmaceutical labeling.

The use of the first of a new class of chelating agents for the binding of metal ions to macromolecules as a novel approach to radiopharmaceutical labeling is described. Advantages are mild reaction conditions, large variety of accessible radionuclides and reactive groups, and separation of synthetic organic chemistry from radiochemistry. Human serum albumin and bovine fibrinogen labeled with 111Indium using this technique were relatively stable in vitro and in vivo, showed little alteration in function, and have potential as tumor localizing agents in humans.

Perturbed angular correlation study of a haptenic molecule.

The angular correlation of the 173-247 keV gamma-ray cascade after the electron-capture decay of (111)In is strongly perturbed when the 1-p-nitrophenyl-ethylenediaminetetraacetate chelate of (111)In(8+) is added to a solution containing rabbit antibody to dinitrophenyl groups. The radioactive chelate can be displaced by the addition of dinitrophenyllysine or unlabeled chelate. The average association constant between the antibody and the labeled chelate has been estimated from perturbed angular correlation measurements; this value is compared to the results of equilibrium dialysis. These experiments provide good evidence that information concerning macromolecular behavior can be obtained from perturbed angular correlation experiments that use chemically specific labels.