LDL-RECEPTORTEST® allows the quantitative determination of LDL receptor expression on human monocytes. It contains cell culture medium and lipoprotein deficient serum for the cultivation of mononuclear cells and a monoclonal antibody against the LDL receptor. It determines the amount of LDL receptors by flow cytometry using a biotin/streptavidin-phycoerythrin system. All reagents are optimized for high sensitivity of LDL receptor detection.

APPLICATIONS

The quantitative determination of LDL (low density lipoprotein) receptors on monocytes is suitable to identify patients with the genetically inherited familial hypercholesterolemia (FH) disorder (1). It is characterized by high levels of cholesterol in the blood. Individuals, who are heterozygous for FH, occur at a frequency of about 1 - 2 in 1000 and carry mutant forms of the LDL receptor protein, whereas approximately one in 1,000,000 people have inherited two mutant genes and are homozygous (1, 2). As patients with familial hypercholesterolemia have a high risk for atherosclerosis and coronary heart disease. Therefore, the routine clinical diagnosis of this disease is of great importance. Genetic defects of the LDL receptor (1, 3-7) can be classified according to five known biochemical mutation categories:
Class 1 mutations are characterized by no detectable precursor of the LDL receptor. These alleles form the most common class of mutations, accounting for about one-half of all defective alleles.
Class 2 mutations belong to the second most common category and affecting the processing of the precursor. These alleles code for transport-deficient receptor precursors, which fail to move from the endoplasmatic reticulum to the Golgi compartment. Receptors synthesized from alleles belonging to class 3 mutations are normally processed and reach the cell surface, but are unable to bind LDL.
Class 4 mutations produce internalization-defective receptors, which fail to cluster in coated pits.
Class 5 mutations encode receptors that bind and internalize ligands in coated pits, but fail to release the ligands in the endosome and thus do not recycle to the cell surface. In summary, these five classes of mutations cause defects in LDL receptor synthesis, transport, ligand binding and internalization. These defects result in a 2-3 fold increase of LDL concentration in plasma in heterozygotes or a 6-8 fold increase in homozygotes, respectively.

The test kit is intended for the quantification of LDL receptors on monocytes. No or reduced expression of LDL receptors can therefore be detected as the pathobiochemical cause of familial hypercholesterolemia (class 1 and class 2 mutations).

PRINCIPLES

The LDL-RECEPTORTEST® kit contains all necessary reagents for the quantitative determination of LDL receptor expression on human monocytes.
Peripheral blood mononuclear cells (lymphocytes and monocytes) from patients, where familial hypercholesterolemia (FH) is suspected, are prepared by density gradient centrifugation. The isolated cells are incubated for 48 h in two parallel cell cultures with autologous serum and lipoprotein deficient serum (LPDS). Cells from a healthy control person are also analysed as a positive control. The serum, deficient in lipoprotein, induces maximal expression of LDL receptors on monocytes. The amount of LDL receptors is determined by flow cytometry after staining the cells with a monoclonal antibody against the LDL receptor using a biotin/streptavidin amplification system.

REAGENTS PROVIDED

LDL-RECEPTORTEST® contains reagents for 50 tests:

• Optimized CELL CULTURE MEDIUM
• LIPOPROTEIN DEFICIENT SERUM
• STAINING REAGENTS A, B, C
• CELL CULTURE PLATES
  

REFERENCES

(1)	Goldstein , J.L. & Brown, M.S. 1979. The LDL receptor locus and the genetics of familial hypercholesterolemia. Ann. Rev. Genet. 13: 259-289.
(2)	Goldstein, J.L. & Brown, M.S. 1989. Familial hypercholesterolemia. In: Metabolic Basis of Inherited Diseases, 6th edition. Schriewer, C.R., Baudet, A.L. Sly, W.S. & Valle, D.W. (eds.), pp:1214-1250.
(3)	Brown, M.S. & Goldstein, J.L. 1986. A receptor-mediated pathway for cholesterol homeostasis. Science. 232: 34-47.
(4)	Bilheimer, D. 1988. The Lipoprotein Receptor Concept. Drugs. 36 (Suppl. 3): 55-62.
(5)	Schneider, W. 1989. The low density lipoprotein receptor. Biochem. Biophys. Acta. 988: 303-317.
(6)	Goldstein, J.L. & Brown, M.S. 1984. Progress in understanding the LDL receptor and HMG-CoA reductase, two membrane proteins that regulate the plasma cholesterol. J. Lipid.Res. 25: 1450-1461.
(7)	Hobbs, H.H., Brown, M.S. & Goldstein, J.L. 1992. Molecular genetics of the LDL receptor gene in familial hypercholesterolemia. Hum. Mutat. 1: 445-466.

For further information please contact:

Dr. Werner Hirt
Head of Immunology, Cell Biology and Flow Cytometry
+49 6221 9105-50
e-Mail: w.hirt@orpegen.com