Date of Award

2007

Document Type

Master Thesis

Degree Name

Masters of Science (Research)

Department

Biological Sciences

First Advisor

Dr. Anne Ward

Abstract

Background: C-reactive protein (CRP) is a well-known marker for inflammation. Recent studies have demonstrated that increases in CRP concentrations are associated with future coronary events in apparently healthy men and women. The requirement for assays, capable of testing CRP concentrations within the range 1-lOmg/l, is therefore great.

Methods: Two CRP assays were developed. The Sensitive assay is capable of measuring CRP concentrations as low as lmg/l for use as a potential cardiac marker test. The Normal assay can measure concentrations from 5-400mg/l for establishing levels of inflammation. Both assays developed are latex particle enhanced immunoturbidimetric assays designed for use on the Olympus AU2700/640/400 series of analytical analysers. The anti-CRP antibodies were chemically bound to latex microparticles of different sizes and surface chemistries. Various antibodies, binding techniques, buffer composition and blocking proteins were tested. The performance of the final assays was then established. The assay was tested for its linearity, precision and LDL. Bilirubin, haemolytic and lipaemic interference studies were assessed. A method comparison was also performed between the newly developed CRP assay and a commercial CRP assay.

Results: The Sensitive assay developed is linear from l-13mg/l with precision of <2% across this range. The LDL was O.14mg/l. Interference from bilirubin was negligible. Interference greater than 10% was observed with lipaemic samples > 400mg/dl and haemolytic samples > 150mg/dl. Good correlation was observed between the new sensitive assay and the commercial assay tested at CRP concentrations above 2.5mg/l. There is a positive bias with patient samples measured below this concentration. The Normal assay developed is linear from L66-480mg/l with precision of <1% above l0mg/l. Precision at concentration of 5mg/l is <8%. The LDL is L66mg/1. Interference from bilirubin, intralipid and haemolysate was negligible. Good correlation was observed between the new normal assay and the commercial assay tested at CRP concentrations above approximately l0mg/l. In samples below this concentration significant matrix effects were observed.

Conclusions: Both assays developed are capable of measuring CRP in the required ranges. Comparison with commercial assays is favourable. The Sensitive assay has a positive bias below 2.5mg/l compared with the commercial assay. The assay if unchanged would thus produce false positive results by reporting higher CRP concentrations than actually present in patients below this concentration. These assays nonetheless may be able to predict short and long term cardiovascular outcomes and may have a role alongside cholesterol testing in Coronary Heart Disease screening programmes.

Access Level

info:eu-repo/semantics/openAccess

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