Mouse monoclonal antibody to a latent epitope of leucocyte receptors for leukotriene B4.
Human blood polymorphonuclear (PMN) leucocytes and human leucocytes of the HL-60 line, which were induced to differentiate by 1,25-dihydroxyvitamin D3, express stereospecific receptors for the potent chemotactic mediator, leukotriene B4 (LTB4), that is derived by 5-lipoxygenation from arachidonic acid. Monoclonal antibodies to LTB4 receptors (LTB4-R) were generated by immunizing BALB/c mice with partially purified PMN leucocyte membrane proteins, and fusing their splenocytes with P3X63Ag8 mouse myeloma cells. Hybridoma supernatants were screened initially by binding to PMN leucocyte LTB4-R protein, which had been affinity cross-linked with aminopropylamide (APA)-LTB4 and immobilized in plastic wells through attachment of the linked APA-LTB4 to adherent Fab of monoclonal anti-LTB4. Of the three clones producing antibodies which bound to LTB4-R, 0.5 mg/ml of one IgG3k antibody, termed E2, precipitated over 90% of the [3H]LTB4-binding activity of solubilized PMN leucocyte membrane proteins. E2 also bound to a radiolabelled protein of 70,000-80,000 MW from 125I-labelled PMN leucocyte membranes [35S]-labelled HL-60 cell membranes, and PMN leucocyte membranes affinity-labelled with [3H]APA-LTB4, that was identical in size to the LTB4-R precipitated by the rabbit IgG anti-idiotypic antibodies. E2 did not bind to intact PMN leucocytes or modify the binding of [3H]LTB4 by PMN leucocytes. The binding of E2 to LTB4-R in purified membranes of PMN leucocytes was less than one-fourth of that observed for the anti-idiotypic antibodies, but increased substantially after solubilization of the LTB4-R. The E2 monoclonal antibody thus recognizes a partially latent substituent of LTB4-R, which does not contribute to combining site function.
Harvey
J P
JP
Department of Medicine, University of California Medical Center, San Francisco 94143-0724.
Koo
C H
CH
Boggs
J M
JM
Young
R N
RN
Goetzl
E J
EJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D007975:Leukotriene B4 / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009504:Neutrophils / Q000276:immunology*; D011233:Precipitin Tests; D011971:Receptors, Immunologic / Q000276:immunology*; D018102:Receptors, Leukotriene B4
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008565:Membrane Proteins; D011971:Receptors, Immunologic; D018102:Receptors, Leukotriene B4; D007975:Leukotriene B4