Preparation of immunoaffinity mini-columns for the analysis of platelet activating factor (PAF) in biological samples.
Using an antibody to BN 52719, an analogue of platelet activating factor (PAF), immunoaffinity mini-columns for the separation of PAF from biological samples were prepared. Rabbits were immunized with BN 52719 and immunoglobulin G (IgG) from the antiserum was coupled with Sepharose 4B. The resulting suspension of the IgG-coated Sepharose 4B in 25 mM phosphate buffer (pH 6.9) was poured into a plastic mini-column (bed volume 2.0 x 0.8 cm). Stepwise elution of the column with methanol revealed that lyso-PAF is eluted with 20-30% methanol in water whereas PAF is eluted with 50-80% methanol. For the determination of PAF in biological samples, it is recommended that lipids are extracted from the samples and the extract, reconstituted in 20% methanol, is loaded on the column. The column is then washed with 50% methanol followed by elution of PAF with 80% methanol. A small amount of [3H]PAF is added to the samples for measurement of the recoveries of PAF during the procedures of extraction and elution. The PAF is then quantified by radioimmunoassay or bioassay. Employing the immunoaffinity mini-column and radioimmunoassay, the contents of PAF in macrophages and conditioned medium after stimulation with calcium ionophore A23187, or tumor promoters such as TPA and thapsigargin, were measured.
Watanabe
M
M
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tohoku University, Miyagi, Japan.
Watanabe
T
T
Hirasawa
N
N
Mue
S
S
Muramatsu
S
S
Matsushita
Y
Y
Takahagi
H
H
Braquet
P
P
Broquet
C
C
Levine
L
L
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002846:Chromatography, Affinity; D007120:Immunochemistry; D007074:Immunoglobulin G / Q000302:isolation & purification; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology; D010972:Platelet Activating Factor / Q000031:analogs & derivatives / Q000032:analysis* / Q000276:immunology; D011817:Rabbits; D051381:Rats
D007074:Immunoglobulin G; D010972:Platelet Activating Factor; C076343:BN 52719; D000001:Calcimycin