A specific, sensitive and high‐capacity immunoassay for PAF

Baldo, Brian A.; Smal, Mary A.; McCaskill, Alistair

doi:10.1007/bf02536517

Cited by 13 publications

(6 citation statements)

References 16 publications

(2 reference statements)

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“…Using techniques which rely on the interaction of mediator with platelets, Cox et aL (8) and Janero et aL (9) demonstrated the presence of PAF in human saliva. However, these assays are not simple to perform and would be unsuitable for the measurement of PAF in the large numbers of samples required to determine "normal" levels and hence demonstrate deviations from normal due to any particular disease Recently, a specific radioimmunoassay (RIA) for PAF has been developed and extensively characterized (10,11). In this paper, we present results which validate the use of the PAF RIA for the routine measurement of PAF in saliva.…”

Section: Lipids 26 1140-1143 {1991}mentioning

confidence: 76%

“…RIA and PAF in saliva. The specificity of the PAF RIA has been demonstrated using a large range of naturally occurring lipids and synthetic PAF analogs (10,11). Authenticity of the PAF levels measured in saliva was confirmed by three further experiments: i) Saliva extracts were assayed at different dilutions in the presence and absence of a known amount of synthetic PAF {Table 1}.…”

Section: Resultsmentioning

confidence: 94%

“…Only the fractions exhibiting PAF activity {chloroform/methanol/water, 2:3:1, v/v/v, fractions) also contained [3H]PAF radioactivity. When compared to the total unfractionated activity (determined by both platelet aggregation and RIA), the percentage recovery of radioactivity corresponded to the percentage recovery of PAF activity, iii) Phospholipase A2 {PLA2) degrades PAF to the inactive lysoPAF which is not detected by the RIA (10,11). Extracted saliva samples were incubated with PLA2 followed by RIA to determine the levels of PAF remaining {Fig.…”

Section: Resultsmentioning

confidence: 99%

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Quantitation by Radioimmunoassay of PAF in Human Saliva

Cooney¹,

Smal²,

Baldo³

1992

Platelet-Activating Factor and Structurally Related Alkyl Ehter Lipids

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Approximately 75% of the PAF present in saliva is recovered on extraction of whole saliva (0.8 vol) with chloroform/methanol/water {2:2:1, v/v/v}. PAF levels, dete~ mined by our recently developed radioimmunoassay, in saliva extracts ranged from 0.5-21 nghnL with 59% between 2-6 ng/mL. These figures, for apparently healthy subjects, are higher than previously reported levels obtained by platelet assays. The validity of our radioimmunoassay results was checked by isolating and quantitating the PAF fraction from whole saliva. In addition, when we ex~mined our saliva samples by platelet aggregation, low levels of PAF, comparable with the values found in the literature, were detected. Investigations revealed the presence of a substance(s) which inhibited PAFinduced platelet aggregation but which did not affect the radioimmunoassay.

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Section: Lipids 26 1140-1143 {1991}mentioning

confidence: 76%

Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

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Quantitation by Radioimmunoassay of PAF in Human Saliva

Cooney¹,

Smal²,

Baldo³

1992

Platelet-Activating Factor and Structurally Related Alkyl Ehter Lipids

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“…The antibodies produced in this study are specific for PAF and the methodology outlined here has been ap-plied to the development of a sensitive and specific radioimmunoassay for PAF (31). The antibodies have also been employed in localization studies using immunocytochemical methods (32), and have potential application in studies on the PAF receptor and various in vitro and in vivo investigations of the pharmacological, physiological and biochemical effects of PAF.…”

Section: "mentioning

confidence: 99%

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

Smal

Baldo

Redmond

1991

Lipids

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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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“…The development of a specific and sensitive radioimmunoassay (RIA) for PAF (9) allows PAF to be accurately and routinely measured in various biological samples. Using the RIA, we quantitated PAF levels in numerous samples of mixed human saliva and found the levels to range from 0.5 to 21 ng/mL (10).…”

mentioning

confidence: 99%

Inhibitor(s) of Platelet-Activating Factor (PAF) in Human Saliva

Smal¹,

Baldo²

1992

Platelet-Activating Factor and Structurally Related Alkyl Ehter Lipids

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Quantitation of platelet-activating factor (PAF) in human saliva samples by radioimmunoassay indicated there was, at times, sufficient PAF present to aggregate platelets. However, in certain samples, we observed little or no aggregation, and furthermore, these samples were found to inhibit aggregation induced by PAF (200 pg). Chromatographic fractionation of pooled saliva increased the PAF activity 4-fold, and the observed inhibitory activity was found to c~migrate with the fatty acids. The inhibitory fraction was found to be active against platelet aggregation induced by arachidonic acid (3.4 nmole) as well as PAF (25 pg), but not thrombin (20 mU). These results indicate the existence of a PAF inhibitor in saliva, which may explain why potentially toxic levels of PAF can occur in the saliva of normal, healthy individuals. These findings also highlight an important advantage of the radioimmunoassay over platelet aggregation for the quantitation of PAF in, at least, some biological fluids.

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A specific, sensitive and high‐capacity immunoassay for PAF

Cited by 13 publications

References 16 publications

Quantitation by Radioimmunoassay of PAF in Human Saliva

Quantitation by Radioimmunoassay of PAF in Human Saliva

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

Inhibitor(s) of Platelet-Activating Factor (PAF) in Human Saliva

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