Model for competitive binding assays.  Shape and location of the inhibition curves

Laurence, D. J. R.; Wilkinson, Graeme F.

doi:10.1021/ac60344a046

Anal. Chem.

1974

DOI: 10.1021/ac60344a046

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Model for competitive binding assays. Shape and location of the inhibition curves

D. J. R. Laurence¹,

Graeme F. Wilkinson²

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Cited by 5 publications

(1 citation statement)

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“…Their analysis was based upon the assumption that the kinetics of the binding reactions for radiolabeled analyte and unlabeled analyte are identical and that a perfect separation of antibody-bound from free analyte was made. Laurence and Wilkinson (13) have investigated equilibrium assays in which the binding constant of the labeled analyte differed from that of the unlabeled analyte.…”

mentioning

confidence: 99%

Design of a single incubation column radioimmunoassay

Verhoff¹,

Denning²,

Boguslaski³

1978

Anal. Chem.

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mentioning

confidence: 99%

Design of a single incubation column radioimmunoassay

Verhoff¹,

Denning²,

Boguslaski³

1978

Anal. Chem.

View full text Add to dashboard Cite

Irreversible enzyme‐shuttle immunoassay

Paek

Schramm

1992

Biotech & Bioengineering

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The concept of a competitive enzyme immunoassay that utilizes simultaneously the bound and the free analyte-enzyme conjugate (heterobifunctional conjugate) for signal generation in response to varying analyte concentrations in samples has been investigated. Two antigenic sites of the heterobifunctional conjugate are used in the assay for binding to immunoglobulins: the analyte derivative binds to an immobilized antibody, Ab(1), and the enzyme component binds to a spatially separated immobilized antibody, Ab(2). The analytical system is set up such that in the absence of analyte, the conjugate is predominantly bound in the compartment that contains Ab(1). With increasing concentration of native analyte in samples, an increasing concentration of native analyte in samples, an increasing amount of conjugate migrates to the second compartment that contains Ab(2). The enzyme bound in each compartment is used for signal generation. Mathematical models have been developed to determine the optimal conditions and to predict the performance of such dual-antibody systems. The theoretical predictions are supported by experimental results. The dual-antibody system has been compared with a conventional competitive enzyme immunoassay using the same reagents.

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Theoretical models for predicting the effect of bridging group recognition and conjugate substitution on hapten enzyme immunoassay dose-response curves

Bachas

Meyerhoff

1986

Analytical Biochemistry

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Models for predicting the effect of immunological recognition of the bridge group on the dose-response curves obtained with heterogeneous hapten enzyme immunoassays are presented. Appropriate theoretical treatment shows that the greater affinity of antibodies toward the enzyme-labeled species than for the unlabeled hapten analyte results in assays with limited detection capabilities. This problem is compounded when enzyme conjugates possessing multiple haptens are used. In equilibrium type competitive arrangements, the concentrations of binder and labeled hapten may be optimized to some extent to improve assay performance. However, the results presented show that only when assays are performed in a sequential binding mode using carefully controlled timing of reagent incubations can the detection capabilities of the assays be fully maximized for analyte measurements. Unfortunately, it is also shown that such sequential binding approaches render the assays essentially nonselective. The effect of decreasing the affinity of the binder to the enzyme-labeled hapten relative to the unlabeled analyte by using heterologous conjugates in equilibrium arrangements is shown to improve detection capabilities but also at the expense of reduced selectivity. Suggestions for reagent concentrations and conjugate substitution (degree of conjugation), which provide optimized dose-response curves at a given ED50 value, are also presented as are proposals for using different binders which do not exhibit bridging group recognition.

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Model for competitive binding assays. Shape and location of the inhibition curves

Cited by 5 publications

References 14 publications

Design of a single incubation column radioimmunoassay

Design of a single incubation column radioimmunoassay

Irreversible enzyme‐shuttle immunoassay

Theoretical models for predicting the effect of bridging group recognition and conjugate substitution on hapten enzyme immunoassay dose-response curves

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