The grid-blot: a procedure for screening large numbers of monoclonal antibodies for specificity to native and denatured proteins.
This report describes a procedure referred to as a grid-blot for simultaneously testing up to 30 monoclonal antibodies for specificity with an equivalent number of different proteins on a single sheet of nitrocellulose paper. Only 150 microliters of hybridoma culture supernatant is required for the screening and the entire procedure can be completed in less than five hours. This assay was developed to quickly identify those hybridoma cultures producing antibodies that preferentially recognize the native form of a protein and those that also recognize the SDS denatured form and were optimal for use in Western blots. Monoclonal antibodies raised against two distinct proteins, myofibril C-protein (120 antibodies) and the catalytic subunit of cyclic-AMP dependent protein kinase (240 antibodies) were tested. The grid-blot results indicated that 85 of the C-protein antibodies and 55 of the catalytic subunit antibodies were monospecific. Only 4 of the C-protein and 9 catalytic subunit antibodies showed a preferential staining for the appropriate native protein. The antibodies that stained the denatured protein most intensely in the grid-blot corresponded with those that produced the best immunostain in the Western blot. Finally, a version of the grid-blot was found to be an efficient means of determining antibody isotypes.
Lane
R D
RD
Department of Anatomy, Medical College of Ohio, Toledo.
Mellgren
R L
RL
Hegazy
M G
MG
Gonzalez
S R
SR
Nepomuceno
V
V
Reimann
E M
EM
Schlender
K K
KK
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015153:Blotting, Western; D002352:Carrier Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D007132:Immunoglobulin Isotypes; D007158:Immunologic Techniques; D009124:Muscle Proteins / Q000276:immunology; D011489:Protein Denaturation; D011494:Protein Kinases / Q000276:immunology; D011506:Proteins / Q000276:immunology*; D000099163:Myosin Binding Protein C
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D007132:Immunoglobulin Isotypes; D009124:Muscle Proteins; D011494:Protein Kinases; D011506:Proteins; D000099163:Myosin Binding Protein C