Electrochromatography: a method for automatic immunoaffinity chromatography on porous membranes

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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.

Section: Competitive Immunoaffinity Electrochromatography (Iae)mentioning

confidence: 99%

Epitope Mapping of Human Alpha-Fetoprotein

Yakimenko¹,

Karamova²,

Goussev³

et al. 1998

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“…In this region, the electroosmotic counterflow exceeds the rate of the anodic migration of any protein and is used for the transfer of AFP and P-aAFP through all the MoAb bands one after the other. AFP at 2 Ìg/ml and P-aAFP (1:100) were introduced into the counterflow in the 'reverse order' [11,19]. Vitamin B 12 was used as a colored electrophoretically neutral control for electroosmotic counterflow transporting of AFP and P-aAFP.…”

Section: Immunoaffinity Electrochromatographymentioning

confidence: 99%

“…IAE was carried out as described [11,19] with some modifications. Briefly, different MoAbs were applied onto nitrocellulose membrane (NCM) sheet as pairs of slot blots ( fig.…”

Section: Immunoaffinity Electrochromatographymentioning

confidence: 99%

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Conformational Variants of Human Alpha-Fetoprotein

Karamova

Yazova²,

Goussev³

et al. 1998

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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.

“…Earlier we proposed an approach for the separation of proteins into fractions based on the expression of individ- ual epitopes in the native molecule, which also demonstrated that AFP preparations contained several epitope variants [10,11]. Since the AFP structure is determined by a single gene representing a single polypeptide chain with varying oligosaccharide moieties [3], these different epitope variants would appear to be conformational ones of the same polypeptide chain.…”

Section: Introductionmentioning

confidence: 99%

New Approaches for the Detection and Characterization of Alpha-Fetoprotein Epitope Variants

Karamova¹,

Yazova²,

Yakimenko³

et al. 2003

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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.