Monoclonal antibodies raised to paraffin wax embedded archival tissue; feasibility study of their potential to detect novel antigenic markers.
A study to determine the feasibility of using archival paraffin wax embedded tissue to generate monoclonal antibodies is described. Specifically, monoclonal antibodies were raised to paraffin wax embedded normal human kidney tissue to test the possibility of producing antibodies to such tissue samples prior to attempting generation of antibodies to valuable archival tissue. Multiple sections (10 x 5 microm) were pooled and dewaxed as for immunohistochemical procedures and combined with Freund's adjuvant for immunization of BALB/c mice in vivo. Immunized spleen cells were fused with SP2 myeloma cells and subsequent clones screened on paraffin wax embedded normal human kidney sections, a range of cell lines and normal mouse tissue. Supernatants from 11 wells (from a total of 90 wells screened) showed different staining patterns on sections of paraffin wax embedded kidney. One clone, 1/11C, (isotype IgG1) which exhibited strong staining on all kidney tubules by immunohistochemical studies (glomeruli interstitium and vessels were unstained) and identified a band at 52 kDa on immunoblots of dewaxed kidney tissue (as used for immunogen) was chosen for further characterization. Immunoblotting of five mammalian cell lines showed differential expression of this 52 kDa band (distinct expression on 3/5, weak expression on 2/5 cell lines) whereas, all cell lines displayed a band at 44 kDa and a third band at 70 kDa was observed on 2/5 cell lines. In mouse tissue extracts, the 52 kDa band was identified in kidney tissue only (not in the lung, liver or spleen) with the 44 kDa and 70 kDa bands weakly expressed in all tissues. This preliminary investigation of a novel approach to identifying possible new antigenic markers or producing monoclonal antibodies which react better to known antigens on sections of paraffin wax embedded tissue showed that this method is feasible. The need to have a comprehensive screening system in place and the ability to identify potentially useful clones after the initial screening is paramount due to the relative scarcity of screening material (archival tissue sections) and the tedious nature of the screening method.
Moran
E
E
National Cell and Tissue Culture Centre, BioResearch Ireland, Dublin City University, Ireland. moranl@ccmail.dcu.ie
Larkin
A
A
Cleary
I
I
Barnes
C
C
Kennedy
S M
SM
Kelehan
P
P
Clynes
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D000941:Antigens / Q000032:analysis / Q000276:immunology*; D018070:Biological Specimen Banks; D015153:Blotting, Western; D002457:Cell Extracts; D005260:Female; D006801:Humans; D006825:Hybridomas; D007150:Immunohistochemistry; D007668:Kidney / Q000276:immunology; D007684:Kidney Tubules / Q000276:immunology; D008099:Liver; D008168:Lung; D008297:Male; D051379:Mice; D008970:Molecular Weight; D016612:Paraffin Embedding; D013154:Spleen; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000941:Antigens; D002457:Cell Extracts