A method to generate antigen-specific mAb capable of staining
formalin-fixed, paraffin-embedded tissue sections
Xinhui Wang, Michael Campoli, Hyun Suk Cho, Takeshi Ogino, Nobuyuki Bandoh,
Jijia Shen, Soo Young Hur, Toshiro Kageshita, Soldano Ferrone
Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA
Received 23 December 2004; accepted 11 February 2005
Available online 1 April 2005
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 compo-
nent-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
0022-1759/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
-microglobulin; CFA, complete Freund’s adjuvant; ELISA, enzyme-linked immunosorbent assay; HLA, human
leukocyte antigen; IHC, immunohistochemical; IFA, incomplete Freund’s adjuvant; i.p., intraperitoneal; KLH, keyhole limpet hemocyanin;
LMP, low molecular weight protein; mAb, monoclonal antibody; s.c., subcutaneous; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis; TAP, transporter associated with antigen processing.
This work was supported by PHS grants P01 CA89480, P30 CA16056, R01 CA67108, and T32 CA85183 awarded by the National Cancer
Institute, DHHS, and the Department of Defense predoctoral fellowship BC030039.
* Corresponding author. Tel.: +1 716 845 8534; fax: +1 716 845 7613.
E-mail address: firstname.lastname@example.org (S. Ferrone).
Journal of Immunological Methods 299 (2005) 139 – 151