Kinetics of Chemical Degradation in Monoclonal Antibodies: Relationship between Rates at the Molecular and Peptide Levels

Abstract Background: Procalcitonin (PCT) is an established marker for diagnosing and monitoring bacterial infections. Full-length PCT [116 amino acids that make up procalcitonin (PCT1–116)] can be truncated, leading to des-Ala-Pro-PCT (des-Alanin-Prolin-Procalcitonin; PCT3–116). Current immunoassays for PCT (“total PCT”) use antibodies directed against internal epitopes and are unable to distinguish amino-terminal PCT variants. Here we describe the development of monoclonal antibodies recognizing the amino-termini of PCT1–116 and PCT3–116 and their use in the selective measurement of these PCT species. Methods: With newly developed monoclonal antibodies against the amino-termini of PCT1–116 and PCT3–116, and an antibody against the katacalcin moiety of PCT, we developed and characterized immunoluminometric assays for the 2 PCT peptides. We comparatively assessed the kinetics of PCT variants in a human endotoxemia model. Results: Monoclonal antibodies against the amino-termini of PCT1–116 and PCT3–116 showed <1% cross-reactivity with other PCT-related peptides. The sandwich assays for PCT1–116 and PCT3–116 had functional assay sensitivities of 5 and 1.2 pmol/L, respectively, and exhibited recoveries within 20% of expected values. Plasma PCT1–116 was stable for 6 h at 22 °C and 24 h at 4 °C, and PCT3–116 was stable for at least 24 h at both temperatures. During experimental endotoxemia in healthy people, both PCT1–116 and PCT3–116 increased early in parallel with total PCT, but further increases in PCT1–116 were significantly slower than for PCT3–116 (P = 0.0049) and total PCT (P = 0.0024). Conclusions: The new assays selectively measure PCT1–116 and PCT3–116. Both PCT species increase early during endotoxemia but differ in their kinetics thereafter. The selective measurement of PCT species with different in vivo kinetics may be useful in improving PCT-guided therapies.

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Analysis of the Temporal Compressibility of Breast Tumour Marker Assays: Development of a “Near Patient” Assay

The International Journal of Biological Markers ◽

10.1177/172460089501000402 ◽

1995 ◽

Vol 10 (4) ◽

pp. 200-205 ◽

Cited By ~ 1

Author(s):

A. Murray ◽

J. F. R. Robertson ◽

M. R. Price

Keyword(s):

Breast Cancer ◽

Monoclonal Antibodies ◽

Liquid Phase ◽

Solid Phase ◽

Breast Tumour ◽

Tumour Marker ◽

Binding Kinetics ◽

Immunoradiometric Assay ◽

Assay Performance ◽

Kinetics Of

The aim of this study was to investigate whether immunoassays for circulating MUC1 antigen in breast cancer could be compressed in time so that serum level results would be made available during the time of the patient's visit to clinic. Two assays were used: - The EMCA (Euro DPC) is a liquid phase immunoassay and the ELSA CA15-3 (CIS) is a double determinant solid phase immunoradiometric assay. The effects of shortened incubation times were investigated by assaying standards and unknown samples and comparing the results with those using the standard kit protocols. The binding kinetics of the monoclonal antibodies employed in the assays were analysed separately. We conclude that the EMCA assay can be shortened to 35 min and we have attributed this to the fast binding kinetics inherent in a liquid phase assay. This shortened assay may produce the basis for a useful “near patient” assay. By comparison, the solid phase ELSA CA15-3 assay cannot be compressed without loss in assay performance.

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Development and application of monoclonal antibodies to human cardiac myoglobin in a rapid fluorescence immunoassay

Clinical Chemistry ◽

10.1093/clinchem/37.8.1356 ◽

1991 ◽

Vol 37 (8) ◽

pp. 1356-1364 ◽

Cited By ~ 17

Author(s):

D P Silva ◽

Y Landt ◽

S E Porter ◽

J H Ladenson

Keyword(s):

Monoclonal Antibodies ◽

Particle Concentration ◽

High Dose ◽

Fluorescence Immunoassay ◽

Hook Effect ◽

One Step ◽

Analytical Importance ◽

Rapid Kinetics ◽

Kinetics Of Reaction ◽

Kinetics Of

Abstract Myoglobin (Mb) is considered a useful marker for early detection of myocardial infarction and for monitoring cardiac reperfusion after thrombolytic therapy. We developed eight monoclonal antibodies to human cardiac Mb, characterized their epitopic reactivity, and determined which combinations of the antibodies are useful in two-site immunoassays. We configured two of the monoclonal antibodies in a one-step, two-site particle concentration fluorescence immunoassay (PCFIA) for measurement of Mb. The PCFIA has rapid kinetics of reaction, being complete in 15 min, and has a linear analytical range of 20-675 micrograms/L for human Mb. Although the PCFIA has a high-dose "hook" effect, this is of no analytical importance at concentrations of Mb less than or equal to 148,000 micrograms/L. The assay is not subject to interference from icterus (bilirubin less than or equal to 360 mg/L), has no cross-reaction with hemoglobin (less than or equal to 42 g/L), and may be performed with either plasma or serum in approximately 1 h. The intra- and interassay imprecisions (CV) of the method are less than 10% for concentrations of Mb within the normal range and less than 4% at higher concentrations. A comparison of the PCFIA with a commercial radioimmunoassay showed that results of the two assays correlate well (PCFIA = 0.88 x RIA + 18, r = 0.990, n = 171).

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