Quantitation of a cationic antimicrobial granule protein of human polymorphonuclear leukocytes by ELISA.
The quantitation of CAP57, a highly hydrophobic, native cationic antigen of human polymorphonuclear leukocytes has been achieved using ELISA. An important feature determining the sensitivity and precision of the ELISA was the reduction of non-specific protein-protein binding, particularly in the inhibition assays, thus eliminating high backgrounds obtained with presently available methodology. Washing of the solid phase-bound antigen and blocking of the non-specific binding sites using a potassium phosphate buffer containing heparin largely contributed to this increased sensitivity. The inhibition assays were conducted using antigen concentrations over the range of 0.9-120 ng. The assay is highly specific and can be performed using monoclonal antibodies and polyclonal antibodies. Non-specific reactions were observed only when high concentrations of antigen (greater than 100 ng) were present in the inhibition mixture. The technique as described is extremely simple, highly reproducible and could be of value in the detection of cationic antimicrobial proteins in the clinical setting in the future.
Pereira
H A
HA
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322.
Martin
L E
LE
Spitznagel
J K
JK
D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D023181:Antimicrobial Cationic Peptides; D001770:Blood Bactericidal Activity; D001798:Blood Proteins / Q000032:analysis* / Q000276:immunology; D003594:Cytoplasmic Granules / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000097:blood; D008565:Membrane Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D009504:Neutrophils / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D008565:Membrane Proteins; C021623:bactericidal permeability increasing protein; C053368:cationic antimicrobial protein 57, human