A new mdr-1 encoded P-170 specific monoclonal antibody: (6/1C) on paraffin wax embedded tissue without pretreatment of sections.
OBJECTIVE
The generation and characterisation of a monoclonal antibody that specifically recognises the mdr-1 encoded protein, P-glycoprotein (P-170), on routinely processed formalin fixed, paraffin wax embedded tissue sections.


METHODS
The monoclonal antibody, designated 6/1C, was produced following a combination of in vivo and in vitro immunisation regimens in Balb/c mice with a synthetic 12 amino acid peptide that corresponds to amino acids 21-32 (believed to be intracellularly located) of P-170 and has insignificant homology with the mdr-3 encoded P-170. Antibody 6/1C was characterised by western blotting and immunocytochemistry on cytospins of paired multidrug resistant or sensitive cell lines, including mdr-1 and mdr-3 transfected cells, and by immunohistochemistry on normal and malignant formalin fixed paraffin wax embedded tissue sections.


RESULTS
Antibody 6/1C showed a single band at 170 kDa on western blots of multidrug resistant cell lysates and mdr-1 transfected cell lysates that was absent on similar preparations of drug sensitive cells and mdr-3 transfected cells. Immunocytochemical studies on cytospins of multidrug resistant cells and mdr-1 transfected cells revealed strong inner plasma membrane/cytoplasmic staining. Staining was negligible on drug sensitive cells and cells transfected with the mdr-3 gene. Immunohistochemical studies on formalin fixed, paraffin wax embedded normal adult kidney, liver, and breast tissue and a range of fetal tissues exhibited staining patterns of a variety of secretory surfaces consistent with documented mdr-1 specific staining. Specific staining of malignant cells in similarly treated sections of breast tumours was seen also with antibody 6/1C. Staining on paraffin wax embedded tissue with this antibody did not require any pretreatment of tissue sections.


CONCLUSIONS
This new monoclonal antibody, chosen for its specificity with the mdr-1 encoded P-170 and its reactivity on routinely fixed paraffin wax embedded tissue samples without pretreatment, appears to be useful for the investigation of P-170 in archival material. It is especially useful for retrospective studies on pretreatment and post-treatment tissue sections, and could help establish when and how rapidly mdr-1 associated drug resistance develops during chemotherapeutic regimens. Immunohistochemical assessment of P-170 expression in many cancers has potential for diagnostic purposes and may influence the choice of chemotherapeutic drugs used in the treatment of refractory tumours.
Moran
E
E
National Cell and Tissue Culture Centre, BioResearch Ireland, Dublin City University, Ireland.
Larkin
A
A
Doherty
G
G
Kelehan
P
P
Kennedy
S
S
Clynes
M
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis* / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000737:chemistry*; D018270:Carcinoma, Ductal, Breast / Q000737:chemistry*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D016612:Paraffin Embedding; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal