Polyclonal antibodies against formaldehyde-modified apolipoprotein A-I. An approach to circumventing fixation-induced loss of antigenicity in immunocytochemistry.

Abstract

A central requirement for the subcellular localization of synthesized and/or endocytozed llpoproteins Is the availability of antibodies against the antigen to be localized. The preparation of cells for electron microscopy, In particular the fixation and embedding routine, Influences the antigenicity, often resulting In a markedly reduced labeling intensity. To ameliorate fixation-Induced changes in antigenicity, we produced antibodies against pre-flxed human apolipoprotein (apo) A-l. Purified apo A-l was fixed with 4% formaldehyde and was used to raise polyclonal antibodies In rabbits. The antiserum was purified by protein A-Sepharose followed by affinity chromatography with the fixed antigen coupled to vlnyisulfone-actlvated agarose. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis against different fixed and unfixed llpoproteins. Nonspecific binding to unfixed or to fixed apolipoprotelns was not observed. Thus, the antibodies reacted specifically with apo A-l and recognized the fixed as well as the unfixed protein. In ELISA, the reaction of the antibodies was markedly enhanced with the fixed antigen, Indicating that the antibodies were directed against epttopes characteristically modified by the fixation. The efficacy of the antibodies for light and electron microscopy was tested on HepG2 cells and on human liver cells which are known to synthesize apo A-l. When HepG2 cells were exposed to anti-apo A-l antibodies followed by a secondary fluorscein Itothlocyante-labeled antibody, fluorescence was found intracellularty In distinct regions. Electron microscopy revealed that the endopla8mlc reticulum, and In particular the trans elements of the Gotgl complexes, were the main compartments stained for apo A-l both In HepG2 cells, as shown by the immunoperoxldase technique, and In human hepatocytes, as shown by the protein A-gold technique on ultrathln cryosections. Furthermore, we were able to localize apo A-l In foam cells of the human aortic plaque by postembedding Immunolabelirtg techniques. These findings demonstrate the potential of antibodies to fixed llpoproteins in Impaired localization at the light microscopic and electron microscopic levels.

Unconventional antigen retrieval for carbohydrate and protein antigens

“…As in diagnostic pathology, aldehyde fixation is commonly used and although the fixation conditions can be better controlled, antibodies may fail to recognize their respective antigens. Thus, polyclonal antibodies were raised against the formaldehyde-treated antigen to improve their reactivity toward formaldehyde-exposed cellular components (Harrach and Robeneck 1990). Brown and colleagues (1996) pointed out that some antibodies may produce excellent results when applied for immunoblotting, but no or only variable staining if used for immunocytochemistry.…”

Antigen Retrieval Immunohistochemistry: Past, Present, and Future

“…The critical importance of rendering the IHC technique suitable for routine paraffin sections was pointed out by Taylor and Burns (1974). The utility of IHC in surgical pathology increased with the use of enzyme digestion (Huang 1975), other pretreatments for unmasking antigens (Kitamoto et al 1987;Hausen and Dreyer 1982;Elias and Johnsen 1979), and the development of antibodies against formalin-modified antigens Harrach and Robenek 1990). However, the use of many antibodies for routinely processed paraffin sections continued to be limited in spite of these enhancement techniques and improvements in the detection system (Leong et al 1988).…”

In situ immunolocalization of lipoproteins in human arteriosclerotic tissue.

“…Since it is generally impracticable to avoid these fixation-induced changes of antigenicity, we have tried to improve the lipoprotein/apolipoprotein recognition in fixed tissues by chemically modifying the lipoproteins/apolipoproteins with formaldehyde before immunization. Harrach and Robenek 26 have demonstrated that antibodies against formaldehyde-fixed lipoproteins/apolipoproteins are more appropriate than are antibodies against unmodified lipoproteins/apolipoproteins for localization in fixed samples. The present study revealed that the antibodies generated against prefixed LDL are more appropriate because they exhibit a higher specificity and a lower background labeling on the tissue sections as well as on ELISA.…”

“…26 Another polyclonal antibody to formaldehyde-fixed LDL (anti-fix LDL-D) raised in the rabbit was kindly provided by Dr. H. Dieplinger, University of Innsbruck, Innsbruck, Austria. For the characterization of SMCs in the arteriosclerotic plaques, we used the mouse monoclonal antibody asm-1 raised to a-actin of SMCs (anti-SMC), which was purchased from Progen, Heidelberg, FRG.…”