Willfried Schramm

Antibody-antigen complex formation with immobilized immunoglobulins

We have investigated the complex formation between an immobilized monoclonal antibody and antigens that differ in size about 50-fold. As a model system, we used an iodinated progesterone derivative and a progesterone-horseradish peroxidase conjugate as tracer and a monoclonal antibody as binding protein. The antibody was immobilized by four different methods: physical adsorption, chemical binding, and binding via protein G in the absence or presence of a protective protein (gelatin). These investigations have shown that the performance of competitive immunoassays is determined by a combination of factors: (a) the relative size of the analyte and the tracer, (b) the antibody density on the solid matrix, (c) the method of immobilization of the antibody, and (d) the binding constants between antibody-analyte and antibody-tracer. All of these interactions have to be considered in designing an optimal immunoassay. The smaller antigen can form a 3- to 35-fold higher maximal complex density than the larger antigen. Dose-response curves are less affected by the size of the tracer than by the binding constant with the antibody. A large enzyme tracer with a relatively low binding constant can, therefore, provide a more sensitive assay. On the other hand, the increase in complex density achieved with a smaller tracer yields a higher signal that in turn can provide a better signal-to-noise ratio in highly sensitive competitive solid-phase immunoassays. We have suggested a model for antibody immobilization that accounts for the interdependence of tracer size, complex formation, and antibody density. The methods described can be used to design and optimize immunoassays of predefined performance characteristics. The results are particularly useful for converting radioimmunoassays to enzyme immunoassays.

Cotinine in an ultrafiltrate of saliva

BACKGROUND We have developed a device for the simplified collection of a prepurified sample of saliva in the mouth. METHOD The device is based on the principle of an osmotic pump and accumulated about 1.2 ml of an ultrafiltrate of saliva within 8 min. We have investigated the ultrafiltrate for its utility as a biological medium in the evaluation of cigarette smoking status. RESULTS (a) In 58 matched samples from 13 subjects, the correlation coefficient for the cotinine concentration in the saliva and the ultrafiltrate was 0.95; (b) in matched plasma and ultrafiltrate samples from 27 smokers, the correlation coefficient for the cotinine concentrations was 0.96 with plasma containing 1.2 times the ultrafiltrate mean; (c) in a nonsmoker, elevated cotinine levels could be detected in the ultrafiltrate more than 24 hr after smoking 2 cigarettes, and the pattern of rise and decrease reflected that in whole saliva; and (d) in a habitual smoker; the mean cotinine concentration in the ultrafiltrate was 157 ng/ml (SD +/- 25.7 ng/ml) during a period of smoking 15 cigarettes per day and dropped to a mean of 47 ng/ml (SD +/- 10.5) when smoking was reduced to 5 cigarettes per day; after cessation of smoking, detectable concentrations of cotinine persisted for up to 5 days. CONCLUSION The device facilitated the aesthetic, noninvasive collection of a biological sample useful in the validation of smoking status.

Modeling of immunosensors under nonequilibrium conditions: I. Mathematic modeling of performance characteristics

Immunosensors for the detection of small analytes that use analyte-enzyme conjugates as signal generators require special attention if operated under nonequilibrium conditions. If the size of the analyte and the analyte-enzyme conjugate differ substantially, the two antigens do not diffuse at the same rate. This can cause time-dependent shifts in the sensitivity of competitive immunoassays. Therefore, immunosensors operating at short incubation times require precise timing that meets closely the specifications for which the sensors were calibrated. As an example, we have analyzed kinetic binding curves for the quantitative determination of progesterone with an immobilized monoclonal antibody and a conjugate between horseradish peroxidase and progesterone as signal generator. Mathematical paradigms have been developed to simulate the diffusion, antigen-antibody complex formation, and competitive binding processes in this analytical system. Dose-response curves obtained under nonequilibrium conditions can vary substantially from those obtained at equilibrium of antigen-antibody interaction. The degree of this variation depends on the performance characteristics of the major components of the immunosensor. The developed mathematical solutions reflect experimental results and can be used to model optimal conditions for immunosensors operating under nonequilibrium conditions. In this paper (Part I), we report on the mathematical modeling of the interaction between analyte, analyte-enzyme conjugate, and an immobilized antibody. In Part II (W. Schramm and S.-H. Paek (1991) Anal. Biochem. 196), we present experimental results and compare them with the theoretical models.

Ultrafiltrate of saliva collected in situ for the measurement of testosterone

Abstract A device for the in situ collection of an ultrafiltrate of saliva was investigated. The collector consists of an osmotic pump that, when placed in the mouth, accumulates a prepurified salivary filtrate within a few minutes. The concentration of testosterone in saliva and in the ultrafiltrate from five male subjects was determined by a solid-phase immunoassay. The ultrafiltrate can be used without extraction as a medium for the diagnostic evaluation of free, protein-unbound testosterone. Concentrations in whole saliva and the ultrafiltrate correlate closely ( r = 0.89; n = 42). The collector can potentially be used for the measurement of a wide variety of analytes other than testosterone. An ultrafiltrate of saliva as diagnostic medium provides the following advantages: simplicity of collection; moderate stimulation of salivary flow; exclusion of potential blood contamination; prevention of binding of analytes to protein; prevention of potential metabolic degradation of analytes; reduction of viscosity by exclusion of mucopolysaccharides and other large molecules; and potential sterile sampling of ultrafiltrate.

Continuous monitoring of analyte concentrations.

We have investigated the application of a modified, heterogeneous, competitive enzyme immunoassay for the continuous measurement of small analytes in a medium stream. The analytical system contains two antibodies that are immobilized on spatially separated areas, one binding the analyte (Ab1) and the other binding the enzyme (Ab2). An analyte-enzyme conjugate serves as signal generator. The analyte-enzyme conjugate functions as a heterobifunctional shuttle that can bind to either antibody. A semipermeable membrane retains the enzyme shuttle in the internal volume of the sensor but permits the passage of small analytes from the medium stream. The amount of enzyme bound to Ab1 is inversely proportional and the amount of enzyme bound to Ab2 is directly proportional to the analyte concentration. We have demonstrated that this analytical system (1) can provide a larger total signal; (2) has a sensitivity comparable with conventional competitive immunoassays; (3) does not require the separation of bound from free antigens; and (4) is therefore suitable for the continuous measurement of analytes in a medium stream. With a model system, an increase from 0 ng ml-1 to 20 ng ml-1 of the steroid hormone progesterone and the subsequent fall to 0 ng ml-1 could be monitored.

Modeling of immunosensors under nonequilibrium conditions : II. Experimental determination of performance characteristics

In an attempt to optimize immunosensors operating with an immobilized antibody as binding protein and an analyte-enzyme conjugate as signal generator that is significantly larger in molecular size than the analyte, in a previous communication (Part I) (S.-H. Paek and W. Schramm (1991) Anal. Biochem. 196) we developed mathematical models for the prediction of performance characteristics. These models are compared in this contribution with experimentally obtained results. As an example, a monoclonal antibody to the steroid hormone progesterone has been used as binding protein, an 125I-progesterone derivative, and a progesterone-horseradish peroxidase derivative as tracers for signal generation. A minimum of parameters needs to be experimentally determined to calculate the performance: the amount of immobilized antibody, the diffusion coefficient of antigens, the thickness of the penetration layer, and the on- and off-rates for binding of the antigen to the antibody. We have described simple methods to obtain these data for the labeled antigen and for the unlabeled analyte that does not provide a signal per se. Kinetic binding curves for antigen-antibody complex formation obtained with the mathematical models correlated well with experimentally obtained results for antigens of different sizes. Although equilibrium of the antigen-antibody complex for the enzyme-labeled analyte conjugate requires about 4 h in the absence of free analyte, dose-response curves can be obtained after 5 min and the relative position of these curves does not change significantly after 30 min. Using a total volume of 200 microliters for the analytical procedure in microtiter wells, agitation as a means to accelerate convective diffusion during an incubation period of 30 min is not necessary with the analyte-enzyme conjugate. However, immunosensors using large analyte-enzyme conjugates as signal generators for the detection of small analytes require strict control of the incubation time if operated within short periods of time (less than 30 min).

Performance characteristics of a reversible immunosensor with a heterobifunctional enzyme conjugate as signal generator.

Factors that control the performance of a reversible immunosensor with an analyte (progesterone)-enzyme (horseradish peroxidase) conjugate as signal generator have been investigated. The conjugate is used in conjunction with two antibodies, which are specific to progesterone and to horseradish peroxidase, immobilized on two spatially separated polypropylene mesh discs. The conjugate and two antibodies are confined to an internal compartment of a microdialyzer by a semipermeable membrane. The small analyte from an external medium permeates across the membrane into the internal compartment where the analyte concentration determines the relative amounts of the bound conjugate on the two solid surfaces. By measuring two signals from the conjugate bound at two separate sites, we experimentally obtained time-response curves to a concentration pulse of the external analyte. A mathematical (kinetic) model describing the sensor system was developed and used for the determination of rate-limiting factors. In semicontinuous monitoring of the analyte concentrations, operation of the immunosensor with the enzyme conjugate as signal generator required special attention to (a) enzyme stability, (b) analyte permeation (dependence on medium components), and (c) kinetics related to the different accessibility to the same antibody of the small analyte (to be measured) vs. the larger counterpart on the enzyme conjugate (for signal generation). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 221-231, 1997.

The assessment of cortisol using salivary ultrafiltrate

Cortisol can be used to indicate stress level as well as to monitor certain disease states. Although cortisol can be sampled from blood and urine, saliva sampling has decided advantages. Unfortunately, whole saliva contains a number of substances that can metabolize or bind with cortisol, potentially confounding analysis and quantitation. We introduce a semipermeable pouch that accumulates a contaminant-free salivary ultrafiltrate, thereby overcoming a number of the problems encountered with whole saliva. In three studies, we demonstrate accuracy and utility of the device for cortisol determination: (1) in an artificial medium, 60%–77% of the cortisol was recovered in the ultrafiltrate; (2) in vivo, ultrafiltrate cortisol correlated highly with whole-saliva cortisol collected under ideal conditions; and (3) ultrafiltrate cortisol evinced positive relationships with depression and cigarette use, consistent with studies in the literature. We conclude that this device and saliva-filtering technologies in general are useful in applications requiring quantitation of cortisol.

Single-step electrochemical immunoassay

We are studying methods to simplify traditional immunoassays so that they can be performed under non-laboratory conditions. For this project, we have combined immunochromatography with electrochemical signal detection in an enzyme immunoassay. Immunochromatography makes obsolete specimen pipetting, adding of reagents, operator mediated timing of the reaction, and gives a heterogeneous immunoassay the operating simplicity of a homogeneous assay. Electrochemical signal detection allows measurement of the end-point of the immunochemical reaction without the additional step of color development. We present a system where a membrane is attached to a sensor consisting of screen printed working (Pt), auxiliary (Pt), and reference (Ag/AgCl) electrodes for the amperometric measurement of a product (hydrogen peroxide) generated by an enzyme (glucose oxidase). On the membrane, superimposed on the electrodes, antibody to the target analyte is immobilized. Analyte-enzyme conjugate used as a reagent in the assay is pre-applied to the membrane. Membrane and electrode are incorporated into a cartridge the tip of which is immersed into the specimen medium for analysis (e.g., urine or saliva). Specimen medium containing the analyte migrates through the membrane, dissolves analyte-enzyme conjugate and reaches the antibody site where antibody-antigen interaction takes place. Hydrogen peroxide formed by analyte-glucose oxidase conjugate provides a quantitative measure of analyte concentration in the specimen. Under these conditions, antibody-antigen complex formation takes places at non-equilibrium conditions. The analytical aspects of the system are described.