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Synthesis of a PAF immunogen and production of PAF-specific antibodies

  • PAF Analysis by Spectrometric and Immunological Assay
  • Published:
Lipids

Abstract

Platelet activating factor (PAF), a naturally occurring phospholipid with many potent physiological and pharmacological activities, is implicated as a mediator of many diseases. An immunoassay for PAF would greatly improve quantitation, and hence PAF-specific antibodies were required. Chemically-reactive analogs of PAF, containing an aldehyde group at the end of the 1-O-alkyl chain (hexyl or dodecyl), were synthesized from readily available materials. During the multi-step synthetic procedure, the aldehyde group was protected as an acetal, which was converted by mild acidic hydrolysis to the aldehyde immediately prior to protein coupling. These analogs were coupled to methylated bovine serum albumin and the resultant conjugates were injected into rabbits. Antibodies to PAF were detected using a solid phase radioimmunoassay based on Protein A-Sepharose. The dodecyl PAF conjugate proved to be the more immunogenic conjugate with more than half of the rabbits producing significant levels of antibodies (at least a 10-fold increase in radioactive uptake over pre-immune levels). Results from solid phase immunoassays employing nitrocellulose discs impregnated with PAF, lysoPAF, lecithin, lysolecithin and 2-O-methyl-lysoPAF indicated that the antibodies recognized only PAF. PAF-specific antibodies were isolated by affinity chromatography using a column of PAF-poly(lysine) conjugated to carboxy-activated polyacrylamide. The antibodies may be employed in a sensitive and specific immunoassay for PAF and for many other studies involving PAF.

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Abbreviations

Ac:

acetyl

BSA:

bovine serum albumin

C n PAF:

PAF where the 1-O-alkyl chain isn carbons long

CS:

human cord serum

DMOC6PAF:

1-O-(6′,6′-dimethoxyhexyl)-2-acetyl-sn-glycero-3-phosphocholine

DMOC12PAF:

1-O-(12′,12′-dimethoxydodecyl)-2-acetyl-sn-glycero-3-phosphocholine

Ig:

immunoglobulin

[125I]PAF:

1-O-[125I]alkyl-2-acetyl-sn-glycero-3-phosphocholine

lysoPAF:

1-O-alkyl-sn-glycero-3-phosphocholine

MBSA:

methylated bovine serum albumin

2-O-methyl-lysoPAF:

1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine

NC:

nitrocellulose

PA:

polyacrylamide

PAF:

platelet activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine

PAF-PL-PA:

PAF-poly(lysine)-polyacrylamide

PBS:

phosphate (50 mM) buffered saline pH 7.2

PL-PA:

poly(lysine)-PA

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Authors and Affiliations

  1. Kolling Institute of Medical Research, Royal North Shore Hospital, 2065, St. Leonards, N.S.W., Australia

    Mary A. Smal & Brian A. Baldo

  2. Department of Medicine, University of Sydney, 2006, Sydney, N.S.W., Australia

    Brian A. Baldo

  3. School of Chemistry, Macquarie University, 2113, North Ryde, N.S.W., Australia

    John W. Redmond

Authors
  1. Mary A. Smal
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  2. Brian A. Baldo
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  3. John W. Redmond
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Smal, M.A., Baldo, B.A. & Redmond, J.W. Synthesis of a PAF immunogen and production of PAF-specific antibodies. Lipids 26, 1130–1135 (1991). https://doi.org/10.1007/BF02536516

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  • DOI: https://doi.org/10.1007/BF02536516

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