New monoclonal antibodies to the T cell antigens CD4 and CD8. Production and characterization in formalin-fixed paraffin-embedded tissue.
We have generated a recombinant protein representing part of the CD4 molecule and a peptide representing an epitope of predicted high antigenicity on the CD8 molecule and employed these to generate mouse monoclonal antibodies using standard hybridoma protocols. The extracellular domain of the CD4 molecule was obtained by reverse transcription of mRNA from peripheral blood lymphocytes followed by polymerase chain reaction. The amplified gene fragment was cloned into an expression vector to allow a histidine-tagged fusion protein to be produced in Escherichia coli. Purified fusion protein was used to immunize mice. The CD8 monoclonal antibody was raised against a peptide consisting of 13 amino acids within the carboxyl-terminal region of the CD8 cytoplasmic domain. The antibodies showed appropriate reactivity on Western blotting. By heat pretreatment, these antibodies have been shown to be highly effective on paraffin-embedded tissue. In normal lymphoid tissue, the expected distribution of CD4 and CD8 lymphocytes was observed. In a series of 16 T cell lymphomas and B cell lymphomas, immunostaining results were compared with those obtained using reagents effective only in frozen tissue. A high degree of correlation was observed. These results suggest that NCL-CD4 and NCL-CD8 may be of value in the characterization of T cell disorders.
Williamson
S L
SL
Department of Pathology, Royal Victoria Infirmary, Newcastle upon Tyne, United Kingdom.
Steward
M
M
Milton
I
I
Parr
A
A
Piggott
N H
NH
Krajewski
A S
AS
Angus
B
B
Horne
C H
CH
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D015704:CD4 Antigens / Q000276:immunology*; D016827:CD8 Antigens / Q000276:immunology*; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry; D016393:Lymphoma, B-Cell / Q000276:immunology*; D016399:Lymphoma, T-Cell / Q000276:immunology*; D014066:Palatine Tonsil / Q000276:immunology; D016612:Paraffin Embedding; D010455:Peptides / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D016707:Tissue Fixation
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D016827:CD8 Antigens; D010455:Peptides; D011994:Recombinant Proteins; D005557:Formaldehyde