The development of a sensitive and specific radioreceptor assay for leukotriene B4.
To establish a simple and sensitive quantitation of leukotriene B4 (LTB4), we developed a radioreceptor assay (RRA) using a highly specific [3H]leukotriene B4[( 3H]LTB4) binding to a guinea pig spleen homogenate. The assay detected LTB4 levels as low as 0.12 pmol per tube. Fifty percent inhibition of bound [3H]LTB4 was obtained by 2.5 nM of unlabeled LTB4. [3H]LTB4 competition studies indicated that 20-hydroxy-LTB4 was 8 times, 6-trans-LTB4 was 640 times and 20-carboxy-LTB4 was 1000 times less effective than LTB4. The peptide leukotrienes C4, D4 and E4 showed no effect on [3H]LTB4 binding. Recovery rates averaged 97% after ethanol extraction and evaporation of known amounts of LTB4. The intra-assay coefficients of variation for three samples were 2.4%, 7.2% and 8.4%, respectively. This assay was validated by measuring LTB4 released from human granulocytes stimulated with calcium ionophore A23187. The LTB4 level was maximal at 10 min (156.8 +/- 36.2 pmol/3 x 10(6) cells) and decreased rapidly after 15 min. This radioreceptor assay for leukotriene B4 is highly sensitive and is comparable to the reported sensitivity by radioimmunoassay. The method is simpler and less expensive than other methods such as high pressure liquid chromatography and is suitable for routine measurement of leukotriene B4.
Kohi
F
F
Allergic Disease Center, Creighton University School of Medicine, Omaha, Nebraska 68178.
Agrawal
D K
DK
Cheng
J B
JB
Bewtra
A
A
Townley
R G
RG
Olesch
J W
JW
D000818:Animals; D001667:Binding, Competitive; D000001:Calcimycin / Q000494:pharmacology; D005260:Female; D006098:Granulocytes / Q000187:drug effects / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D007700:Kinetics; D007975:Leukotriene B4 / Q000032:analysis* / Q000378:metabolism; D011863:Radioimmunoassay; D011869:Radioligand Assay; D013154:Spleen / Q000378:metabolism; D014316:Tritium
D014316:Tritium; D007975:Leukotriene B4; D000001:Calcimycin