Immunological crossreaction between alpha-fetoprotein and albumin.

Abstract

(16). Bovine serum albumin (BSA) was from Pentex, bovine hemoglobin from Sigma, and bovine fetuin from Gibco.The samples to be reduced and alkylated (17) were dissolved at a concentration of about 10 mg/ml in 0.1 M Tris-HCI buffer (pH 8.2) containing 8 M urea. After deaeration with nitrogen, dithiothreitol was added to a final concentration of 10 mM. After 3 hr at room temperature under nitrogen, the reaction mixture was cooled on ice and recrystallized iodoacetamide was added to make the final concentration 20 mM. After another 15 min the sample was dialyzed against distilled water and lyophilized. Phosphate-buffered saline containing 0.05% gelatin, 0.02% sodium azide, and 0.1% Triton X-100 (PBS-GTX) with urea added to it to give a 2 M solution was used to dissolve the dry proteins, some of which were poorly soluble in ordinary buffers. In some cases the reduced and carboxamidomethylated (RC-) protein was diluted directly into this solution for testing without dialysis and lyophilization. HAFP labeled with 1251 was reduced and alkylated similarly and freed of reactants by gel filtration on Sephadex G-15 equilibriated with PBS-GTX.Antisera. Anti-HAFP sera have been described (6,8). Using a similar schedule, we produced one antiserum each to RC-HSA and RC-BSA and two to RC-HAFP in rabbits. The dose used per injection was 5 mg (RC-HSA, RC-BSA) or 0.5-2 mg (RC-HAFP) of protein suspended in PBS and given in complete Freund's adjuvant. A precipitating antiserum to RC-CEA was obtained similarly, using 0.25 mg of RC-CEA per injection.Immunological Tests. Immunodiffusion was performed on plates cast in a conventional buffer and soaked overnight in PBS-GTX prior to use. Radioimmunoassays were performed using the double antibody technique as described (4). The first and second antibody were added in the form of an IgG fraction in assays where native albumin was tested for crossreactivity with AFP. The diluent buffer used throughout was PBS-GTX. RESULTSLack of Crossreaction between Native AFP and Albumin. Antisera produced against highly purified HAFP do not react with albumin in immunodiffusion (ref. 15, our unpublished results). Radioimmunoassay also failed to reveal any crossreactivity between native HAFP and native HSA or RC-HSA. RC-HAFP was also almost completely devoid of activity in the usual AFP assay (Fig. 1).

alpha-Fetoprotein and albumin genes are in tandem in the mouse genome.

“…3, lane 3). These results, plus extensive restriction mapping, implied that AAFP15 overlapped AAFP14 by 5.0 kb, and therefore the cloned region surrounding the AFP genes had been extended by approximately 9 kb in the 5' direction (Fig. 2).…”

“…Previous reports (5)(6)(7)(8)(9)(10)(11) have argued that the similarities in both the sequence and the organization of the AFP and albumin genes made it highly likely that the genes arose as the conse- (27) and sharks (28) (11). The flanking sequences as well bear no residual homology except for a repeated sequence present in many copies in the genome.…”

“…In rodents, its rate of synthesis by fetal liver begins to decline just prior to birth, at a time when the rate of synthesis of serum albumin, the major adult serum protein, is increasing (3,4). This reciprocal expression ofAFP and albumin, coupled to a number offunctional and structural properties which they share (5,6), led to the proposal that they were the products of a pair of duplicated genes. Two strong lines of evidence support this hypothesis.…”

Versuche zur induktion von embryopathien und konzentrations‐änderungen von alpha‐fetoprotein und serumalbumin beim hühnerembryo1

“…Hinweise auf die biologjsche Bedeutung von AFP unter physiologischen Bedingungen ergeben sich daraus, da/3 dieses Molekiil wahrend des Fetallebens hinsichth'ch seiner Serumkonzentration bei alien bisher untersuchten Spezies dominiert (Gitlin und Boesman, 1966;Gitlin et al, 1973;Weller et al, 1974;Lamerz und Fateh-Moghadam, 1975;N^rgaard-Pedersen, 1976). Aufgrund gemeinsamer Syntheseorte (Dottersack, Leber) und gemeinsamer physikochemischer Eigenschaften, sowie Aminosauresequenz-und m-RNA-Homologien wird AFP auch als Vorlauferprotein von Albumin angesehen (Gitlin und Kitzes, 1967;Gitlin und Perricelli, 1970;Welleret al, 1974;Belangeref al, 1975;Weller, 1976;Ruoslahti und Engvall, 1976;Ruoslahti und Terry, 1976;Slade und Milne, 1977;Gitlin, 1978;Law und Dugaiczyk, 1981). In diesem Zusammenhang ist die Kinetik dieser beiden Proteine bedeutsam: In gleichem Ma|3e wie der AFP-Gehalt im Serum wahrend der Ontogenese nach Erreichen eines Maximalwertes sinkt, nimmt der Albumingehalt zu (Gitlin und Boesman, 1966;Kekomaki et al, 1971;Sell, 1976;Hirai, 1979;Neumann, 1982).…”

Alpha‐fetoprotein

“…The C-terminal amino acid of human AFP is valine [7,133,135] and the C-terminal amino acids reported for the rat AFP variants are glycine and valine [ 1 191. The C-terminal sequence of five amino acids has been proposed [7] for human AFP.…”