MULTIPOINT KINETIC METHOD FOR THE IMMUNOCHEMICAL QUANTITATION OF CREATINE KINASE ISOENZYMES AND QUANTITATION OF METHEMOGLOBIN BY MULTIWAVELENGTH DERIVATIVE ABSORBANCE SPECTROPHOTOMETRY

WILLIAM EDWARD WEISER, Purdue University

Abstract

This thesis consists of two parts and two appendices. The first part describes a new kinetic approach for the simultaneous quantitation of isoenzymes that interact differently with antibodies. The proposed approach is evaluated using the muscle (M) and brain (B) subunits of the isoenzymes of creatine kinase (CK, E.C. No. 2.7.3.2) as a model system. The approach is based on the selective time-dependent inhibition of one of the subunits (e.g. M) by an antibody and the use of nonlinear least-squares data-processing to compute the M and B subunit activities from the time-dependent response curves. Parameters that affect the method, namely temperature, substrate concentration, and antibody concentration, and their interactions, are evaluated using response surface methodology. The quantitation of CK-MM and CK-MB in a serum matrix was evaluated for several concentrations of the isoenzymes in the range of diagnostic significance. Least-squares fits of computed (y) vs. expected (x) values yielded equations of y = 0.98x + 2.0 x 10('-5) s('-1) for CK-MM and y = 1.04x - 4.6 x 10('-5) s('-1) for CK-MB for rates between 0 and 3.5 x 10('-3) s('-1) (CK-MM) and 0.5 x 10('-3) s('-1) (CK-MB). Comparison of kinetic results (y) with results by a kit method (x) yielded y = 0.97x + 6.5 x 10('-5) s('-1) for CK-MM and y = 1.02x + 4.3 x 10('-5) s('-1) for CK-MB. Pooled day-to-day relative standard deviations for the kinetic method were 4.1 and 2.8% for CK-MM and CK-MB, respectively. The second part describes the quantitation of methemoglobin in the presence of a light-scattering suspension as a model to evaluate and compare the relative merits of zeroth-, first- and second-derivative spectroscopy for problems in clinical chemistry. Data for second derivatives are most effective in nullifying the effect of the background spectrum; data for first derivatives are less effective; and data for absorbance (zeroth derivative) are least effective. Variable amounts of the light-scattering component were added to a methemoglobin solution to increase the apparent absorbance up to about 230% of the absorbance of the original solution. Whereas computation with data for absorbance would have yielded errors approaching 100%, concentrations computed with second-derivative spectral data yielded a systematic error (bias) of only 0.5% and a relative standard deviation of 1.6%.

Degree

Ph.D.

Subject Area

Analytical chemistry

COinS