Date of Award

Summer 1994

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

Program/Concentration

Biomedical Sciences

Committee Director

James H. Yuan

Committee Member

Mark S. Elliot

Committee Member

Gerald J. Pepe

Committee Member

Lloyd Wolfinbarger, Jr.

Abstract

Currently, amylase determinations are nonspecific for the organ source and are based entirely on the enzymatic properties of amylase to produce a measurable product or byproduct. The determination of pancreatic amylase is important in the diagnosis of acute pancreatitis. Most commercially available tests for amylase employ the measurement of the change in NADH absorbance at 280 nm or of the p-nitrophenol released from a maltotetrose substrate. These are nonspecific measurements of pancreatic amylase and often necessitate other tests to be run such as a serum lipase.

The two predominant isoenzymes of amylase are pancreatic (p-amylase) and salivary (s-amylase); the most important of which is pancreatic. Pancreatic amylase to date is determined by the removal of salivary amylase by monoclonal antibody, wheat germ, or ion-exchange chromatography. Also amylase isoenzymes are determined by electrophoresis. These methods are time consuming and lack either specificity or quantification capability. There is a 7% difference in the amino acid sequences of pancreatic and salivary amylase. These structural differences between pancreatic and salivary amylase are small but significant enough that they should enable a monoclonal antibody to be produced for each isoenzyme.

A sensitive and specific microwell assay based on sandwich technique for the quantitative determination of the p-amylase and s-amylase was developed. Microwells were utilized for the solid-phase immobilization of the amylase inhibitor cycloheptaamylose (CHA) which was coupled to bovine serum albumin (BSA) by carbodiimide coupling. Monoclonal antibodies were produced against p-amylase and s-amylase (PAb and SAb). These were purified by Protein-A affinity chromatography and then coupled to horseradish peroxidase (HRPO) by carbodiimide coupling. The microwells which were coated with oxidized CHA-BSA (CHA-BSA) were incubated with patient's serum and then allowed to react with the PAb-HRPO and SAb-HRPO.

This EIA method is able to distinguish between p-amylase and s-amylase quantitatively, rapidly and has the capability for automation. The correlation coefficients for human serum samples were 0.920 and 0.867 for p-amylase and s-amylase when comparing this EIA method and protein electrophoresis then staining for amylase. The limit of detection was determined to be 8 U/L for p-amylase and 16 U/L for s-amylase. The percent coefficient of variation for p-amylase was found to be 7.79% for the normal control and 6.28% for the abnormal control. The percent coefficient of variation for s-amylase was found to be 9.42% for the normal control and 9.29% for the abnormal control.

Comments

Dissertation Submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/16yx-7j72

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