Characterization of anti-HLA class II monoclonal antibody LGII-612.14 reacting with formalin fixed tissues

J Immunol Methods. 1993 May 26;161(2):239-56. doi: 10.1016/0022-1759(93)90300-v.


mAb LGII-612.14 derived from a BALB/c mouse immunized with interferon-gamma (IFN-gamma) treated cultured human B lymphoid cells LG-2 has been shown with serological and immunochemical assays to recognize a monomorphic determinant expressed on the beta chain of HLA-DR, -DQ and -DP antigens. The linear nature of the determinant, which is likely to be formed by residues 19-25, is indicated by the reactivity of mAb LGII-612.14 with HLA-DR, -DQ and -DP beta chains purified by electrophoresis in presence of SDS. An unusual characteristic of mAb LGII-612.14 is its reactivity with fixed tissue sections. The intensity of staining is affected by the incubation temperature, the incubation time and the fixative used. Maximal intensity of staining of formalin fixed, paraffin embedded tissue sections required an incubation time of 16 h. The intensity of staining of paraffin embedded tissues initially fixed with Bouin's solution, formalin or ethanol was similar to that of frozen tissue sections and stronger than that of tissues fixed with B5 solution. No staining was detected of paraffin embedded tissues fixed with glutaraldehyde or Zenker's solution. Comparison of the staining patterns with mAb LGII-612.14 of frozen and fixed tissue sections showed that the latter substrates provide a superior detail of tissue architecture and cellular morphology without significant loss of sensitivity. Furthermore, comparison of the characteristics of mAb LGII-612.14 with the few previously published anti-HLA class II mAb reacting with fixed tissues indicates that mAb LGII-612.14 stains formalin fixed, paraffin embedded tissues, while mAb 910D7 and TAL-1B5 stain tissues fixed with less commonly used fixatives. Furthermore, mAb LGII-612.14 is likely to yield more sensitive staining results than anti-HLA-DR, -DQ and -DP mAb KUL/05. The present results indicate that mAb LGII-612.14 represents a useful probe to apply immunohistochemical techniques to the analysis of the distribution of HLA class II antigens in fixed tissues. This will greatly facilitate the use of readily available collections of fixed tissue specimens in retrospective studies to assess the clinical significance of changes in HLA class II antigen expression which occur in various disease states.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Cell Line
  • Formaldehyde
  • HLA-D Antigens / analysis*
  • HLA-D Antigens / chemistry
  • HLA-D Antigens / immunology
  • HLA-DP Antigens / analysis
  • HLA-DP Antigens / immunology
  • HLA-DQ Antigens / analysis
  • HLA-DQ Antigens / immunology
  • HLA-DR Antigens / analysis
  • HLA-DR Antigens / immunology
  • Humans
  • Mice
  • Mice, Inbred BALB C
  • Paraffin Embedding
  • Tissue Fixation
  • Tumor Cells, Cultured


  • Antibodies, Monoclonal
  • HLA-D Antigens
  • HLA-DP Antigens
  • HLA-DQ Antigens
  • HLA-DR Antigens
  • Formaldehyde