A method to generate antigen-specific mAb capable of staining formalin-fixed, paraffin-embedded tissue sections.
Abnormalities in HLA class I antigen expression are frequently found in malignant tumors. Their potential role in the clinical course of the disease and in the outcome of T cell-based immunotherapy has stimulated interest in the characterization of the molecular mechanisms underlying HLA class I antigen abnormalities in malignant cells. Multiple mechanisms have been identified. Among them are abnormalities in antigen processing machinery (APM) component expression. In spite of this information, APM component expression in malignant lesions has been investigated only to a limited extent because of the lack of availability, for most APM components, of monoclonal antibodies (mAb) which stain formalin-fixed, paraffin-embedded tissues. The latter are the substrate of choice in immunohistochemical (IHC) reactions. To overcome this limitation, we have developed a simple and reproducible method to generate APM component-specific mAb which stain formalin-fixed, paraffin-embedded tissue sections. This method involves five steps: (i) immunogenic amino acid sequences, which display low homology with their mouse counterparts when possible, are identified in APM components and utilized to synthesize peptides; (ii) BALB/c mice are immunized with keyhole limpet hemocyanin (KLH)-conjugated synthetic peptides and with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-purified recombinant APM component proteins; (iii) immunized mice, which develop high titer APM component-specific antibodies, are utilized to generate hybridomas which are screened for APM component-specific antibody production by Western blotting assays, with lymphoid cell lysates; (iv) identified APM component-specific mAb are characterized in their specificity and in their reactivity with permeabilized cells in ELISA and/or flow cytometry; and (v) mAb, with the appropriate reactivity pattern, are tested in IHC reactions with formalin-fixed, paraffin-embedded tissue sections. The use of the methodology we have developed resulted in the generation of a panel of APM component-specific mAb capable of staining formalin-fixed, paraffin-embedded tissue sections in IHC reactions. These reagents will facilitate the analysis of APM component expression in tissues under physiological and pathological conditions. In addition, the methodology we have described is likely to be applicable to other antigenic systems to develop mAb capable of detecting protein components of interest in formalin-fixed, paraffin-embedded tissue sections.
Wang
Xinhui
X
Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Campoli
Michael
M
Cho
Hyun Suk
HS
Ogino
Takeshi
T
Bandoh
Nobuyuki
N
Shen
Jijia
J
Hur
Soo Young
SY
Kageshita
Toshiro
T
Ferrone
Soldano
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000032:analysis* / Q000276:immunology; D005404:Fixatives; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D016612:Paraffin Embedding
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D005404:Fixatives; D005557:Formaldehyde