Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Patent
1987-04-21
1989-10-31
Benson, Robert
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
436536, 436543, 436804, 436815, 436825, 435 7, G01N 3353
Patent
active
048777463
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a novel method of treating samples to be subjected to qualitative and/or quantitative immunochemical assays for lipoproteins or, especially, apolipoproteins. Present immunochemical methods require an anti(apolipoprotein or anti(lipoprotein) antibody to react with its corresponding antigen.
Lipoproteins consist of aggregates in which triglycerides, phospholipids, cholesterol and its esters, free fatty acids and proteins bind to one another non-covalently. Proteins present in lipoproteins are often referred to as "apolipoproteins", their individual names comprising the prefix "apo" followed by the type name of the protein. Thus for instance there are proteins apoAI, apoAII, apoB, apoCI, apoCII, apoCIII, apoD, apo(a), and apoE. Some of these apolipoproteins may have isoforms. Also so-called polymorphism may occur.
Lipoproteins as they exist in their form of whole aggregates are often classified according to size and density. There are thus chylomicrons, VLDL, LDL, Lp(a) (where Lp means lipoprotein) and HDL. These in turn may be divided into various subgroups. The majority of apolipoproteins have been shown to exist in more than one size fraction, the only exception being apo(a) which up to now has been detected only in Lp(a) where it occurs together with apoB.
Many apolipoproteins have been shown to possess a receptor-binding and/or enzyme-controlling function. They are believed to be very important in lipid metabolism.
The serum and plasma levels of various lipo- and apolipoproteins have been correlated with genetic and acquired diseases. Moreover it has been possible to correlate these levels with some potential disease contraction risks. In particular, increased levels of apo(a) and Lp(a) are considered to indicate a cardiovascular risk.
Lipoproteins in different fractions of blood have been separated from one another by means of inter alia ultracentrifugation, chromatography, precipitation with polyanions, and electrophoresis. The separated lipoproteins have then been quantified and characterized with the aid of i.a. the apolipoproteins present therein. This has been carried out by means of various immunochemical as well as other methods.
Among the assay methods for detection of apolipoproteins may be mentioned: radial immunodiffusion, electroimmuno-, radioimmuno- and enzyme-immunological methods, isoelectric focusing, nephelometry, turbdimetry, polyacrylamide electrophoresis and densitometry. It goes without saying that other detection methods too may be employed, as for instance fluorescence and chemiluminescence methods.
Lipoproteins and apolipoproteins, their structure and quantification, and the clinical implications of their serum and plasma levels have been dealt with in a major number of handbooks and review articles (see for example Newman H. A. I. et al.: Immunological Assay of Lipoproteins as an Indicator of Genetic and Acquired Disease; and Gibson J. C. et al.: Laboratory Management/1983/, March, p. 19-27 and April, p. 27-37).
It turned out to be difficult to standardize immunochemical assays for lipo- and apolipoproteins. What would happen quite often was that results obtained from different laboratories and different testing occasions were found to differ inter se. It has been shown that the treatment of the test sample was an important factor. Differences between results have been found to originate from inter alia the phenomenon that non-reproducible changes tend to arise due to storage of a sample. To avoid these problems, various experiments have been made for achieving delipidation of samples with the aid of lipid-dissolving solvents, different types of detergents and enzymatic hydrolysis (e.g. by lipase), the underlying idea being that one should thus obtain improved exposures of the antigenic determinants present in the lipoproteins. However none of the delipidating methods have been found to be really successful.
During the priority year the Swedish Patent Office has performed an International Type Search stressing that this is a correct description o
REFERENCES:
Milthorp et al. Chemical Abstracts 105: 4747h (1986).
Watt et al. PNAS(U.S.A.) 80, 124-128, (1983).
Bury-I Clinical Chem. 32(2), 265-270 (1986).
Bury-II Clinical Chem. 31(2), 247-251 (1985).
Mao et al. Biochimica et Biophysica Acta 620, 447-453 (1980).
Holmlund Erling S.
Jansson Gunnel B.
Benson Robert
Pharmacia AB
Philpitt Fred
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