Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1993-08-18
1995-09-12
Scheiner, Toni R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 19, 435 18, 435188, 436 71, G01N 3350, G01N 33566, C12Q 134, C12N 996
Patent
active
054496071
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a quantitative assay, applicable to clinical samples, for substrates which can undergo enzymatic hydrolysis to release long chain fatty acids. The invention is particularly, though not exclusively, directed to ester substrates such as the triglyceride substrates for lipase and phospholipid substrates for the phospholipases.
2. Description of the Prior Art
Esters such as the tri and diglycerides are ubiquitous and fundamental to every aspect of cell membrane function and energy transfer. The assay of these components is therefore of interest in many areas of clinical diagnosis. Thus, for example, assay of the triglyceride content of a clinical specimen can give some indication of recent dietary fat intake, and the ability of the liver to metabolise fats for energy utilisation. However presently available methods for assay of triglycerides, such as those available commercially as Kits from the Sigma Chemical Co. Ltd. (Poole, Dorset, UK) rely on the measurement of glycerol released by enzyme hydrolysis. Thus, in Sigma procedure no. 405, triglycerides are extracted into isopropanol and saponified with potassium hydroxide. Liberated glycerol is then converted to formaldehyde by periodate. By reacting with acetylacetone, the formaldehyde forms yellow diacetyldihydrolutidine, which is measured colorimetrically.
In Sigma procedures nos. 336, 337, 339 and 334 glycerol is released from triglyceride enzymatically using lipase, and glycerol is further reacted with ATP to form glycerol-1-phosphate. The four methods then differ only in the way by which the glycerol-1-phosphate is further reacted to produce a change in absorbance which can be measured spectrophotometrically. Such assays, when applied to clinical samples, suffer from the disadvantage that as glycerol itself is a product of cell metabolism, assay of the glycerol content of a blood specimen may not give an accurate picture of the circulating triglyceride levels in the subject (see Cole, Clin. Chem. 36/7, 1267-1268 (1990)). There is clearly a need for the development of alternative means for assaying triglycerides and other related substrates, such as phospholipids and cholesterol esters, that give accurate results at low concentrations or in small clinical blood specimens.
SUMMARY OF THE INVENTION
It has now been found that such substrates can be quantitatively assayed in albumin-containing clinical samples, such as serum, rapidly and sensitively by causing the appropriate enzyme to act on the substrate to release fatty acid(s), and then detecting or measuring the binding of the released fatty acids to a protein which binds fatty acids with high affinity (having a dissociation constant of 10.sup.-5 M or less). Such a protein is very desirably that known as fatty acid binding protein (FABP), which Is a natural product extractable, for example, from the liver of animals. Hereinafter the invention is described with reference to FABP but it will be understood that other binding proteins of the kind described could be substituted for FABP. In addition, the invention is described with reference to serum as the clinical specimen, although it will be understood that the invention is applicable to other clinical specimens such as whole blood and plasma as well as to partially-purified fractions derived from these. The assay of the fatty acid-FABP binding interaction is most conveniently carried out by using a labelled probe, in effect a labelled fatty acid, which competes with the fatty acid released by the action of lipase for binding sites on the FABP. Conveniently the amount of free label, i.e. that which is not bound to the FABP, is then measured.
It was to be expected from the observations of the prior art that the albumin present in such clinical specimens would interfere with the effectiveness of the assay unless removed. Thus earlier findings given in Biochem. J. (1990), 266, 435-439 and Biochem. J. (1990), 270, 163-166 indicate that serum albumin has sites capable of binding
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British Technology Group Limited
Parsons Nancy
Scheiner Toni R.
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