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) ([1.] Anal. Biochem. 196) we developed mathematical models for the prodiction 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 [mu]l 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 (<30 min).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29202/1/0000256.pd

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 ([5.] Anal. Biochem. 196), we present experimental results and compare them with the theoretical models.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29201/1/0000255.pd

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 an 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 heterobi-functional 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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30295/1/0000697.pd

Characterization for Binding Complex Formation with Site-Directly Immobilized Antibodies Enhancing Detection Capability of Cardiac Troponin I

The enhanced analytical performances of immunoassays that employed site-directly immobilized antibodies as the capture binders have been functionally characterized in terms of antigen-antibody complex formation on solid surfaces. Three antibody species specific to cardiac troponin I, immunoglobulin G (IgG), Fab, and F(ab′)2 were site-directly biotinylated within the hinge region and then immobilized via a streptavidin-biotin linkage. The new binders were more efficient capture antibodies in the immunoassays compared to randomly bound IgG, particularly, in the low antibody density range. The observed improvements could have resulted from controlled molecular orientation and also from flexibility, offering conditions suitable for binding complex formations

Ultrafiltrate of saliva collected in situ for the measurement of testosterone

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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29212/1/0000266.pd

Immuno-chromatographic Analysis for HPV-16 and 18 E7 Proteins as a Biomarker of Cervical Cancer Caused by Human Papillomavirus

Among the more than 120 different types of human papillomavirus (HPV), types 16 and 18 have been known to be high risk agents that cause cervical cancer. We examined, in an immuno-chromatographic analysis, the potential of using the early gene product. E7 protein, as a diagnostic marker of cervical cancer caused by HPV. We developed monoclonal antibodies specific to HPV-16 and 18 E7 proteins that were produced from bacterial cells using, gene recombinant technology. For each E7 protein, the optimal antibody pair was selected using the immuno-chromatographic sandwich-type binding system based on the lateral flow through membrane pores. Under these conditions, this rapid testing assay had a detection capability as low as 2 ng/mL of E7 protein. Furthermore, since viral analysis required the host cell to be lysed using chemicals such as detergents, it was possible that the E7 protein was structurally damaged during this process, which would result in a decrease in detection sensitivity. Therefore, we examined the detrimental effects caused by different detergents on the E7 protein using HeLa cells as the host. In these experiments, we found that the damage caused by the detergent, nonylphenylpolyethylene glycol (NP-40), was minimal relative to Triton X-100 commonly used or the cell lysis. Temperature also affected the stability oldie E7 protein, and we found that the E7 protein was stabilized at 4 T. for about 2 h, which was 4 times longer than at room temperature. Finally, a HPV-infected cervical cancer cell line, which was used as a real sample model, was treated using the optimized conditions and the presence of E7 proteins were analyzed by immuno-chromatography. The results of this experiment demonstrated that this rapid test could specifically detect HPV-infected samples.Cho IH, 2009, ANAL CHIM ACTA, V632, P247, DOI 10.1016/j.aca.2008.11.019Liang YJ, 2008, INT J BIOCHEM CELL B, V40, P2431, DOI 10.1016/j.biocel.2008.04.003Ressler S, 2007, CLIN CANCER RES, V13, P7067, DOI 10.1158/1078-0432.CCR-07-1222Jeon JH, 2007, EXP MOL MED, V39, P621Mirecka EA, 2006, PROTEIN EXPRES PURIF, V48, P281, DOI 10.1016/j.pep.2006.04.017Nishimura A, 2006, MICROBES INFECT, V8, P984, DOI 10.1016/j.micinf.2005.10.015Cho JH, 2006, ANAL CHEM, V78, P793, DOI 10.1021/ac051453vHuh KW, 2005, P NATL ACAD SCI USA, V102, P11492, DOI 10.1073/pnas.0505337102Ono T, 2003, J IMMUNOL METHODS, V272, P211DeFilippis RA, 2003, J VIROL, V77, P1551, DOI 10.1128/JVI.77.2.1551-1563.2003Jeon JH, 2002, EXP MOL MED, V34, P496VINCE A, 2002, J CLIN VIROL, V25, P109Chen XJS, 2001, J MOL BIOL, V307, P173Paek SH, 2000, METHODS, V22, P53, DOI 10.1006/meth.2000.1036Walboomers JMM, 1999, J PATHOL, V189, P12Wertlake P, 1999, J REPROD MED, V44, P11JEON S, 1995, P NATL ACAD SCI USA, V92, P1654WILBUR DC, 1994, AM J CLIN PATHOL, V101, P209SCHEFFNER M, 1993, CELL, V75, P495SELVEY LA, 1992, J VIROL METHODS, V37, P119MAY K, 1991, AM J OBSTET GYNECOL, V165, P2000DYSON N, 1989, SCIENCE, V243, P934NELSON PH, 1989, EMBO J, V8, P3905GISSMANN L, 1986, VIRAL ETIOLOGY CERVI, P217CHANG JY, 1985, EUR J BIOCHEM, V151, P217

Korean Society of Nephrology 2022 Recommendations on controversial issues in diagnosis and management of hyponatremia

The Korean Society for Electrolyte and Blood Pressure Research, in collaboration with the Korean Society of Nephrology, has published a clinical practice guideline (CPG) document for hyponatremia treatment. The document is based on an extensive evidence-based review of the diagnosis, evaluation, and treatment of hyponatremia with the multidisciplinary participation of representative experts in hyponatremia with methodologist support for guideline development. This CPG consists of 12 recommendations (two for diagnosis, eight for treatment, and two for special situations) based on eight detailed topics and nine key questions. Each recommendation begins with statements graded by the strength of the recommendations and the quality of the evidence. Each statement is followed by rationale supporting the recommendations. The committee issued conditional recommendations in favor of rapid intermittent bolus administration of hypertonic saline in severe hyponatremia, the use of vasopressin receptor antagonists in heart failure with hypervolemic hyponatremia, and syndrome of inappropriate antidiuresis with moderate to severe hyponatremia, the individualization of desmopressin use, and strong recommendation on the administration of isotonic fluids as maintenance fluid therapy in hospitalized pediatric patients. We hope that this CPG will provide useful recommendations in practice, with the aim of providing clinical support for shared decision-making to improve patient outcomes

An immunosensor with a heterobifunctional enzyme conjugate as signal generator.

A new concept of a competitive immunoassay has been investigated by using an analyte-enzyme conjugate (progesterone-horseradish peroxidase) as heterobifunctional signal generator. This signal generator can bind to two monoclonal antibodies (specific to the analyte and to the enzyme) that are immobilized on separate solid matrices. The heterobifunctional conjugate shuttles between the two antibody sites in response to changing analyte concentrations. The dual-antibody assay has been tested in two different modes: at irreversible (for a disposable sensor) and reversible (for a continuous sensor) operation. Three hypotheses have been put forward to investigate different aspects of the sensor. First hypothesis: is a differential signal generated by the conjugate bound at the two antibody sites proportional to the analyte concentration? Second hypothesis: is the differential signal amplified compared to the signal from a conventional immunoassay in a single-antibody system? Third hypothesis: can the immunosensor be used for continuous monitoring of analyte concentrations? Methods for the synthesis, purification, and characterization of a homogeneous analyte-enzyme conjugate as signal generator have been investigated. The number of binding sites of an immobilized antibody accessible to this conjugate is different than to a small analyte. The implications of different complex formation on the sensor response have been theoretically and experimentally analyzed. Mathematical models have been developed to predict the performance of the immunosensor in the reversible and irreversible mode, and the models have been supported by experimental results. Limitations for the dual-antibody method were investigated. The enzyme-shuttle concept was most effective if relatively high antibody concentrations were required for the measurement of analytes. For the construction of reversible sensors, three potential limitations were identified: (1) enzyme instability, (2) incomplete return to the ground state, and (3) a relatively slow response due to different complex formations of the two antigens (native and conjugated to enzyme) with the antibody. This research has provided the theoretical and experimental basis for further investigations on the new concept of a dual-antibody system that uses a heterobifunctional conjugate as signal generator.Ph.D.BioengineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105759/1/9208620.pdfDescription of 9208620.pdf : Restricted to UM users only