Apolipoprotein A-I from normal human plasma: definition of three distinct antigenic determinants

Weech, Philip K.; Milne, Ross W.; Milthorp, Peter; Marcel, Yves L.

doi:10.1016/0005-2760(85)90296-6

Cited by 34 publications

(10 citation statements)

References 35 publications

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“…3 ' * Although these seven distinct antigenic determinants are distributed between residues 1 to 148 on the apo A-l sequence, the predicted secondary structure of apo A-l indicates that this region of the molecule is densely packed, increasing the probability that these determinants may be spatially close to one another; 4 however, we have found them to be differentially affected by a previously unknown phenomenon that modifies the antigens during storage.…”

Section: Discussionmentioning

confidence: 76%

“…HDL 3 was prepared by sequential centrifugation between the densities of 1.125 and 1.210 /ml. 3 Densities were measured with an Anton Paar DMA 40 densitometer (Graz, Austria) at 20°C…”

Section: Preparation Of Sera and Llpoprotelnsmentioning

confidence: 99%

“…HDL and HDL 3 were dialyzed, lyophilized, and delipidated with chloroform-methanol 20 to produce apo HDL or apo HDL 3 . Protein assays were performed by the method of Lowry et al 21 HDL and apo HDL were stored after dialysis in 10 mM ammonium bicarbonate buffer pH7, 0.02% sodium azide, and 1 mM EDTA.…”

Section: Preparation Of Sera and Llpoprotelnsmentioning

confidence: 99%

“…Previous results have shown that upon IEF, apo A-l could be resolved into four to six isomorphs. 3 ' 13 We felt that during storage a selective isomorph change leading to increased activity with Series 1 MAB might occur. To investigate the effect of storage on this phenomenon, two samples of apo HDL from the same donor, one prepared from freshly collected serum and the other from HDL that had been stored for 1 month at 4° C, were subjected to twodimensional analysis with IEF (pH 4 to 6) and SDS-gel electrophoresis ( Figure 5).…”

Section: Effect Of Serum Storage On Apo A-l Polymorphismmentioning

confidence: 99%

“…We report here a novel alteration of human apo A-l antigen that occurs upon storage at 4° C, and which affects specific antigenic determinants but not others. These phenomena have been observed in different degrees with monoclonal antibodies from Series 1, obtained following immunization and screening with purified apo A-l, 3 ' 4 and from Series 2, obtained following immunization and screening with native HDL. 4 …”

mentioning

confidence: 97%

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Immunochemical characterization of apolipoprotein A-I from normal human plasma. In vitro modification of apo A-I antigens.

Milthorp¹,

Weech²,

Milne³

et al. 1986

Arteriosclerosis

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Two series of monoclonal antibodies (MAB) directed against apolipoprotein A-I (apo A-I) reacted differentially with freshly prepared sera or plasma. MAB from Series 1 were obtained after immunization and screening with purified apo A-I, while those from Series 2 were obtained by immunization with high density lipoprotein (HDL) and screening with both HDL and apo A-I. Series 2 MAB 5F6, 3G10, and 4F7 reacted significantly better with fresh material than material stored at 4 degrees C for longer than 1 month. Conversely, Series 1 MAB 3D4, and 6B8, and Series 2 MAB 2F1 reacted more strongly with the stored preparations. Series 2 MAB 4H1 reacted equally with stored or fresh material. The inability of 3D4 and 6B8 to react with fresh sera or plasma was not due to an inhibitor in the fresh material. Preparation of HDL from serum or plasma and delipidation of this material had no effect on the above phenomena, which appears related directly to apo A-I and not to the interaction of apo A-I with lipids. The variation of immunoreactivity with storage at 4 degrees C was also shown to be unrelated to proteolysis of apo A-I. Alkaline treatment of freshly prepared sera, HDL, or apo HDL with sodium hydroxide simulated the effect of storage, allowing this material to react strongly with MAB 3D4, 6B8. As expected, there was a decrease in the reactivity of MAB 5F6 with alkali-treated fresh material compared to untreated fresh material. However, further investigation showed that alkaline treatment does not completely mimic the effect of storage. MAB 2F1, which reacts poorly with fresh material, reacted very poorly with alkali-treated fresh material, and MAB 3G10 and 4F7, which react well with fresh material, reacted even better with alkali-treated fresh material. While isoelectric focusing of apo HDL prepared from stored serum showed a redistribution of apo A-I toward more acidic isoforms compared to fresh serum, alkaline treatment of apo HDL resulted in partial cleavage of apo A-I and in the generation of isoforms more acidic than known apo A-I isomorphs. Therefore, alkaline treatment of serum is only a partial model for antigenic modification of apo A-I upon storage. These results demonstrate that in vitro conditions (storage or alkaline treatment) selectively modify certain antigenic sites of apo A-I, but not others. This phenomenon, which is probably related to deamidation, may affect apo A-I immunoassays with either monoclonal or polyclonal antibodies.

show abstract

Section: Discussionmentioning

confidence: 76%

“…HDL 3 was prepared by sequential centrifugation between the densities of 1.125 and 1.210 /ml. 3 Densities were measured with an Anton Paar DMA 40 densitometer (Graz, Austria) at 20°C…”

Section: Preparation Of Sera and Llpoprotelnsmentioning

confidence: 99%