Monoclonal antibodies specific for guinea pig eosinophil major basic protein: their use in an ELISA, immunocytochemistry and flow cytometry.
Eosinophil major basic protein (MBP), purified from guinea pig eosinophil granules was used to raise five monoclonal antibodies (MoAbs). Their reactivity with MBP was confirmed by immunoblotting and indirect ELISA. Two of the MoAbs were used to develop a sensitive and specific antigen capture (sandwich) ELISA for guinea pig eosinophil MBP which gives an accurate and reproducible standard curve over the range of 10-10,000 ng/ml. The specificity of the ELISA for MBP was confirmed and its suitability for testing biological samples ascertained by measurement of MBP in bronchoalveolar lavage fluid (BALF) and plasma from guinea pigs sensitized and challenged with ovalbumin. The ELISA was also capable of detecting MBP in culture supernatants from purified eosinophil preparations challenged with calcium ionophore in vitro. One of the monoclonals could be used to strongly and specifically stain guinea pig eosinophils in immunocytochemistry, whilst all five could be used to visualize eosinophils in suspension in BALF or peritoneal lavage fluid by flow cytometry. There was no staining of other guinea pig leucocyte types, nor crossreactivity with human eosinophils by immunocytochemistry or flow cytometry.
Hunt
T C
TC
Immunopharmacology Group, Southampton General Hospital, U.K.
Summers
J A
JA
Campos
M G
MG
Rimmer
S J
SJ
Sturton
G
G
Palfai
S
S
Church
M K
MK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D001798:Blood Proteins / Q000276:immunology* / Q000302:isolation & purification; D001992:Bronchoalveolar Lavage Fluid / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000276:immunology; D005434:Flow Cytometry; D006168:Guinea Pigs; D006801:Humans; D007124:Immunoenzyme Techniques; D051379:Mice; D010047:Ovalbumin / Q000276:immunology; D011817:Rabbits; D015203:Reproducibility of Results; D012260:Ribonucleases; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D010047:Ovalbumin; D012260:Ribonucleases