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The main developments in the "ligand assay" field in which I have been involved are traced. These include the original development of "first generation" competitive assays relying on radiolabeled analyte markers; the development of the first "second generation", noncompetitive (ultrasensitive) methods, which rely on the use of labeled (monoclonal) antibodies and high specific activity nonisotopic labels (leading to the transformation of the immunodiagnostic field in the 1980s); and the development of the first "third generation" miniaturized, chip-based, microarray methods, which permit the simultaneous ultrasensitive measurement of many analytes in the same small sample. The latter--applicable both to immunoassay and to DNA/RNA analysis--are likely to revolutionize the diagnostic and pharmaceutical fields in the next decade.

A number of immunological techniques--radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), electroimmunoassay, radial immunodiffusion, and a variety of immunoprecipitin assays--have been used to quantify apolipoproteins in plasma. This paper outlines their technical details and discusses their major advantages and drawbacks. The most sensitive procedures, RIAs and ELISAS, are best suited to quantifying those apoproteins found in low concentration in plasma. Immunoturbidimetric assays, on the other hand, which are readily automated, are being widely used to quantify apolipoproteins A-I and B. Apolipoprotein quantification is complicated by the interaction of the proteins with lipids, which can often mask their antigenic determinants. This problem may be circumvented by pretreatment of the samples, by selection of appropriate standards, or by the use of polyclonal or monoclonal antibodies that interact with permanently exposed epitopes on the lipoproteins' surfaces. Our proposed methods for measurement of the individual apolipoproteins give consideration to these approaches.

We describe a semiautomated batch-assay system designed to increase the speed and ease of performing radioimmunoassays while maintaining good accuracy and precision. The main components are a programmable pipetting unit (sample processor) and a gamma counter capable of simultaneously counting radioactivity in 12 samples. The processor uses prearranged sets of as many as 24 disposable pipette tips, which eliminates carryover between samples, and features a horizontal shaker and magnetic stirrer; 50 different assay protocols can be selected. Batches of tubes are easily transferred from the sample processor to the incubator and centrifuge. The gamma counter is controlled by a microprocessor, which is also used for data processing. Because the system is based on discrete rather than continuous-flow analysis and because of the ease with which consecutive samples can be processed, more than one operation and several different assays can be accommodated simultaneously, which saves time considerably.

A simple, fully-automated, continuous-flow system suitable for radioimmunoassay, enzyme immunoassay, and fluoro-immunoassay is described. The system is versatile, inexpensive, and requires only equipment and skills that already are available in most clinical chemistry laboratories. In this system, the antibody is covalently linked to solid-phase, porous particles of agarose (Sepharose, size 40-70 micrometer in the dry form). After sampling and mixing the assay reactants, the assay "cocktail" containing the solid-phase-bound antigen and the unbound fraction is directed to the separation block. Separation is achieved by use of a highly porous membrane (pore size, about 10 micrometer) and by controlling the volume of inflow and outflow a fixed and precise amount of fluid is filtered through the membrane containing only the free (unbound fraction), which can then be quantitated. The percentage of unbound fraction misclassified is negligible (less than 1.0%). Application of these principles to the assay of serum thyroxine is described in an appendix.

Simultaneous advances in detector technology and solid-phase separation systems, as well as the availability of powerful desktop computers, have made possible the development of "second-generation" solid-phase immunoassays. These retain the advantages of classical solid phase while significantly accelerating reaction kinetics. Hapten assays--such as for digoxin, thyroxin, and triiodothyronine uptake--in batches of 48 are processed in about 20 min from reagent introduction until hard-copy printout, with minimal operator involvement. The system also functions as a 48-detector gamma counter, capable of counting and reducing data for any 125I-based RIA that can be run in a 12 X 75 mm test tube. System control, data management, and computer screen displays of kinetic data are provided by an unmodified Hewlett Packard HP-87XM computer. User-friendly disc-based software facilitates the creation and storage of counting and data reduction protocols for as many as 30 RIAs from various manufacturers as well as up to 30 of our own assays.

Serum prostatic acid phosphatase concentration was measured with two commercially available radioimmunoassay kits. Results were compared with histological evidence of prostatic adenocarcinoma obtained at autopsy in 33 patients. The serum assay did not differentiate significantly (p greater than 0.1) between patients with adenocarcinoma and those without. We conclude that the test, at least as performed by use of these kits, is of little value in the detection of occult disease.

A simple, inexpensive modification in the elevator mechanism of a liquid scintillation counter allows for radioimmunoassay tubes (12 X 75 mm) containing the antibody-bound radioligand to be counted directly, thereby obviating the need for counting vials and considerably decreasing the amount of fluor used. Furthermore, because assay and counting are all done in the same tube, the modification shortens assay time and increases precision by avoiding what is often the most time consuming and inaccurate step, the transfer of antibody-bound radioligand to a counting vial. We find that use of this modification results in about a 75% decrease in the overall cost for materials and a substantial decrease in assay time.

We present a comparative evaluation of two commercial kits, the "Quantitope" and "GammaCoat," for radioimmunoassay of digoxin in serum. These kits, in which iodine-125 is used as a label, proved to suitable for digoxin assay as determined by their reproducibility, sensitivity, precision, and a regression analysis. Hemolysis, lipemia, and icterus did not affect results. However, in some cases hypoalbuminemia falsely lowered the assayed digoxin concentration. Recovery of pure digoxin added to native patients' sera having low albumin concentration (24-28 g/liter) ranged from 67-105% with the Quantitope kit and 70-110% with the GammaCoat kit. Low serum albumin concentration did not always decrease the recovery of digoxin; this effect varied from serum to serum, which may indicate that there are factors other than albumin that affect the assay of digoxin.