Monoclonal antibodies to low density lipoprotein used for the study of low- and very-low-density lipoproteins, in "ELISA" and immunoprecipitation technics.

Biochemical and Biophysical Research Communications (Impact Factor: 2.41). 01/1985; 125(2):704-11. DOI: 10.1016/0006-291X(84)90596-5
Source: PubMed

ABSTRACT Seven monoclonal antibodies to low-density lipoprotein were studied by the ELISA for their reactivity with LDL or VLDL. Cotitration experiments showed that five of them are addressed to different antigenic epitopes. Two of the monoclonal antibodies were temperature independent whereas the others had a decreased binding activity at 37 degrees C compared to that obtained at 25 degrees C or 4 degrees C, suggesting the presence of antibodies directed to sequence or conformation epitopes, respectively. All antibodies reacted with both LDL and VLDL; four of them had a higher affinity for LDL and two others for VLDL. Immunoprecipitation of LDL and/or VLDL was observed upon immunodiffusion with certain pairs of antibodies. This may allow the use of pairs of monoclonal antibodies to LDL for the quantitative determination of apolipoprotein B in serum LDL and VLDL.

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    ABSTRACT: Intestinal biopsies from patients having genetic disorders of lipoprotein assembly and secretion, such as abetalipoproteinemia (ABL) or Anderson's disease (AD), contain large amounts of lipids which are accumulated in the enterocytes. Determination of the intracellular sites in which the lipids accumulate and to which apolipoproteins the lipids are bound would help to identify the defects in these diseases and further elucidate the mechanisms by which lipoprotein assembly and secretion occur normally. Ultrastructural immunogold labeling, however, is hampered by the poor preservation of the lipids accumulated in the enterocytes of these patients. We have used routine electron microscopy (fixation and ultra-thin sectioning) along with three methods for immunogold labeling of lipid-laden enterocytes: ultrathin cryosectioning, low temperature freeze substitution with embedding in Lowicryl K4M, and ultra-low temperature freeze substitution with embedding in Lowicryl HM20, to establish a protocol for investigating the intestinal tissue from these patients. Ultracryosectioning, while preserving the overall morphology of the lipid laden enterocytes, did not preserve the lipid content and the immunogold labeling of apolipoprotein B (ApoB) appeared dislocated. Freeze substitution and low temperature embedding in Lowicryl K4M, in contrast, appeared to better preserve the lipid and lipoprotein structures; however, the antigenicity of both apoAI and apoB appeared to be lost and no specific labeling could be obtained. Freeze substitution and embedding in Lowicryl HM20 best preserved the lipid and lipoprotein structures while maintaining apoprotein antigenicity. In conclusion, immunogold labeling of apolipoproteins on lipid structures in the lipid-laden enterocytes of patients with ABL and AD is best obtained by freeze substitution and embedding in Lowicryl HM20.
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    ABSTRACT: Consequences of alterations in the size and the lipid composition of low density lipoproteins (LDL) on apolipoprotein (apo) B immunoreactivity were analyzed using two distinct anti-apoB monoclonal antibodies (Mabs), i.e., 4G3, which recognizes an epitope closed to the binding site to the LDL receptor, and 2D8, which is directed against a distal region. Inhibition analysis revealed that the lipid transfer-mediated triglyceride enrichment of LDL isolated from 12 native human plasmas is associated with significant reductions in the expression of 2D8 and 4G3 epitopes (P < 0.05 in both cases). In contrast, triglyceride hydrolysis of triglyceride-rich LDL significantly increased apoB immunoreactivity as compared with non-lipolyzed counterparts (P < 0.05 with 2D8 and 4G3 Mabs). Among all the modified LDL fractions studied (n = 36), immunoreactivity of 2D8 and 4G3 epitopes correlated negatively and significantly with the triglyceride content (P < 0.01 in both cases), but with neither the size nor the other lipid parameters of LDL particles. Furthermore, changes in the triglyceride content of LDL correlated significantly with changes in apoB immunoreactivity after in vitro treatment with either lipid transfer activity alone (P < 0.01 with 2D8 and 4G3 Mabs) or lipid transfer activity combined with triglyceride hydrolysis (P < 0.01 with 2D8 and 4G3 Mabs). Finally, both the triglyceride content of native LDL and the total triglyceride level in 12 normolipidemic human plasmas correlated negatively and significantly with the expression of 2D8 epitope (P < 0.03 in both cases) and 4G3 epitope (P < 0.02 in both cases). It is concluded that triglycerides constitute a major determinant of the immunoreactivity of 2D8 and 4G3 apoB epitopes in LDL.
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    Orphanet Journal of Rare Diseases 01/2011; 6:1. · 4.32 Impact Factor