Thirty monoclonal antibodies (MoAbs) to human α-fetoprotein (AFP) were compared with one another by two methods: Immunoaffinity electrochromatography or additive ELISA. The first method permitted to analyse the epitopes of native AFP in solution [Abelev et al., Immunol Lett 1994;40:133–138] while the other approach also detects the epitopes of conformationally modified (partly denatured) AFP fixed on the plastic [Yazova et al., Immunol Lett 1990;25:325–330]. Competitive analysis of all MoAbs revealed 10 epitopes, 9 expressed on native AFP and 1 only on the partly denatured molecule. The cross-reactions between separate MoAbs allowed to include them into 6 distinct epitope clusters, or immunodominant groups with the characteristic patterns of reactivity. The obtained epitope map of AFP is necessary for the construction of AFP detection kits as well as for the identification of its antigenic and functional subfractions.

The immunological heterogeneity of human α-fetoprotein (AFP) was demonstrated using immunoaffinity electrochromatography on monoclonal antibodies (MoAbs) to 3 non-cross-reacting epitopes of this protein. At least 4 subfractions expressing different epitopes were found in the native AFP. These subfractions demonstrated molecular weights similar to the major component of the original AFP. The difference between epitope F₅-positive and F₅-negative subfractions disappeared when epitope-negative subfraction was conformationally changed after fixation onto nitrocellulose membrane (NCM). Thus, the epitope under study exists in two forms on the native human AFP molecule: an open and a cryptic form. The cryptic form could be revealed after partial denaturation by fixation on NCM. The epitope variants of AFP could possess different functions in multifunctional AFP. The AFP epitope heterogeneity found in this work should be taken into account when constructing diagnostic AFP kits and when isolating purified AFP using anti-AFP MoAbs.

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Affinity resins containing covalently bound phospholipids were used to compare the affinity of the phosphatidylcholine transfer protein from beef liver and a low-specificity lipid transfer protein from rat hepatoma 27 to phosphatidylcholine and sphingomyelin. Binding experiments demonstrated that the beef liver protein associates specifically with immobilized phosphatidylcholine whereas the hepatoma protein showed a preference for sphingomyelin.Purified antiserum raised against the hepatoma sphingomyelin transfer protein was used to determine the presence of that protein in the cytosol of various experimental tumors as well as in those of normal, regenerating and fetal rat liver. The protein was found to be well expressed in all the tumors examined and in fetal liver as determined by immunodiffusion whereas only minute amounts could be detected in normal liver and in 30-h regenerated liver. The presence of the sphingomyelin transfer protein in cytosol was in parallel with the presence of sphingomyelin in the corresponding mitochondria. It is suggested that the occurrence of sphingomyelin in tumor and fetal liver mitochondria may be due to protein-catalyzed sphingomyelin transfer from the endoplasmic reticulum.A number of phospholipid transfer proteins have been purified from a variety of biological tissues (for reviews see [I, 21 and also [3-91). A common feature of all these intracellular transport lipoproteins is that they either do not use sphingomyelin as substrate or catalyze the transfer of sphingomyelin to a much smaller extent than that of phosphatidylcholine. Recently a new lipid transfer protein has been isolated from rat hepatoma 27 which is capable of transferring sphingomyelin at least as efficiently as phosphatidylcholine [lo]. Such a lipid transfer protein could not be detected in rat liver, and the liver postmicrosomal supernatant was not capable of transferring sphingomyelin from rat liver microsomes to mitochondria [lo].Since mitochondria from various tumors frequently show elevated sphingomyelin levels [I 1 -151 it seemed possible that the occurrence of sphingomyelin in tumor mitochondria may be related to the action of sphingomyelin transfer proteins that are absent or less expressed, in normal cells. In this connection it was thought of interest to study further the phospholipid specificity of the rat hepatoma lipid transfer protein and to find out whether the mitochondrial sphingomyelin content in different rat tissues correlates with the presence of sphingomyelin transfer protein in the corresponding cytosol. In this paper we compare the phosphatidylcholine and sphingomyelin binding capacities of the hepatoma lipid transfer protein to those of the specific phosphatidylcholine transfer protein from beef liver using affinity resins containing covalently bound sphingomyelin and phosphatidylcholine. We report further on an immunological examination of the presence of the hepatoma lipid transfer protein in the cytosol of several rat tumors, fetal, regenerating and normal liver. In addition dat...

Alpha-fetoprotein (AFP) is an embryo-specific protein of mammalian sera that consists of a single glycosylated polypeptide chain (∼70 kD) with 5–6 epitope clusters located on the native molecule. We have elaborated three approaches for the separation of AFP into fractions (variants) that are different in the distinct epitope expression on the native molecule. For this, we have used three technologies largely based on immunoaffinity electrochromatography and electrophoresis/immunoblotting. The separation of these variants was facilitated using monoclonal antibodies previously characterized to the 5 epitope clusters and two individual epitopes on AFP in the ISOBM international workshop. We have shown that these approaches facilitate the identification of cryptic epitopes in AFP, and the relevance of these findings is discussed.