Human Α-fetoprotein Research Articles

Triazine dye binding of human α-fetoprotein and albumin

Column chromatography was used to investigate the interaction of human α-fetoprotein and albumin with different immobilized dyes. Binding of α-fetoprotein to the dye conjugates was studied at pH 7.0. Between 56 and 93% of the total α-fetoprotein applied to the column was recovered in the break-through fractions of the respective runs. Of all the dyes, Cibacron Blue F3G-A adsorbs α-fetoprotein most strongly. This interaction clearly depends on the degree of dye substitution of the gel. A relatively weak interaction exists between α-fetoprotein and immobilized Procion Red HE-3B. This is used in the purification of α-fetoprotein by negative chromatography resulting in a 16.6-fold enrichment of this protein. Human albumin binds tightly to immobilized Cibacron Blue F3G-A as well as to Cibacron Brilliant Blue FBR-P and shows a lower affinity to Procion Blue MX-R. Procion Red dyes, which are structurally different from Cibacron Blue F3G-A are also capable of interacting with serum albumin. The results obtained are discussed in terms of the present theoretical conceptions about dye-protein interactions.

Structure and evolution of human α-fetoprotein deduced from partial sequence of cloned cDNA

The nucleotide sequence of a recombinant DNA clone, containing a partial mRNA sequence for human α-fetoprotein (AFP) in the plasmid vector pBR322, has been determined. Two regions of the cloned nucleotide sequence were found to agree with published amino acid sequences of two cyanogen bromide peptides derived from human AFP. Examination of the amino acid sequence, deduced from the cloned portion of the mRNA coding region, reveals extensive homology with the third domain of the human serum albumin molecule. A total of 44% ( case56 128 ) amino acids and 54% ( case207 384 ) nucleotides are identical in the two structures. The landmark cysteine residues are found in the same positions in both polypeptide chains, presumably forming the same disulfide bridges in AFP as those found in the albumin. The sequence homology reinforces the evidence that human AFP and albumin constitute a gene family, in analogy to the same family found in rodents. A comparison of the human and rodent sequence data suggests that the rate of molecular evolution has been faster for AFP than for albumin.

A simple purification procedure for .ALPHA.-fetoprotein by immunoadsorbent column chromatography.

Human α-fetoprotein was purified from human cord serum by employing an immunoadsorbent column, consisting of Sepharose 4B coupled with the antibody to human α-fetoprotein. By using 0.2 M Na2CO3 solution (pH 11.6) as an eluent, a large amount of α-fetoprotein was obtained from the immunoadsorbent column in high purity. After gel filtration on Sephadex G-150, the purified α-fetoprotein was demonstrated to be homogeneous by polyacrylamide gel electrophoresis, SDS-gel electrophoresis and two-dimensional immunoelectrophoresis.

Effect Of Temperature on the Sensitivity of Sandwich Enzyme Immunoassay with Fab'-Horseradish Peroxidase Conjugate

Abstract Effect of temperature was examined on the sensitivity of sandwich enzyme immunoassay for human chorionic gonadotropin (hCC) with anti-hCG Fab'-horseradish peroxidase conjugates prepared by using three different reagents (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate, glutaraldehyde and metaperiodate). The non-specific bindings of the conjugates to anti-hCC IgG-coated polystyrene balls were much lower at 20°C than at 37°C, and the specific bindings were slightly higher at 20°C than at 37°C. The lowest non-specific binding and the highest specific binding were obtained by incubation at 20°C with the maleimide conjugate. As a result, the sensitivity could be more easily improved by incubation at 20°C than at 37°C and the highest sensitivity was obtained with the maleimide conjugate. Similar results were also obtained for other macromolecular antigens such as human ferritin and α-fetoprotein.

2] Enzyme immunoassay of human α1-Fetoprotein

2] Enzyme immunoassay of human α1-Fetoprotein

Secretion by a Hybridoma of Antibodies against Human α-Fetoprotein

Secretion by a Hybridoma of Antibodies against Human α-Fetoprotein

Purification of human α-fetoprotein

By employing complex and highly specialized immunochemical methods, several investigators have achieved purification of human α-fetoprotein (AFP) found in fetal serum and/or sera of patients with hepatoma. The present report describes a simpler method which results in the isolation of homogeneous preparation of AFP from human cord serum. AFP was purified by sequential use of Affi-Gel Blue affinity, DE-52 diethylaminoethyl cellulose ion-exchange, immunoadsorption with anti-albumin covalently coupled to Sepharose 4B, and Sephacryl S-200 molecular sieve chromatographic techniques. The homogeneity of the purified AFP was established by subjecting it to polyacrylamide gel electrophoresis, analytical isoelectric focusing, molecular sieve chromatography and immunological techniques. The purified AFP has a molecular weight of approximately 68,000 as determined by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and molecular sieve chromatography, and upon isoelectric focusing yielded a single band p I = 4.8. In addition, the purified AFP gives a single precipitin line when tested against rabbit antiserum to whole human hepatoma serum proteins, and no line(s) of precipitin when tested against rabbit antiserum to normal serum proteins.

α-Fetoprotein as a marker for hepatic regeneration in the dog

A radioimmunoassay for human α-fetoprotein (AFP) was utilized to quantitate AFP following partial liver resection in the dog. Nine dogs were studied; seven underwent 70% hepatectomy and two received sham operations. Serum AFP, alkaline phosphatase, glutamic-oxaloacetic transaminase (SGOT), glutamic-pyruvate transaminase (SGPT), total bilirubin, and galactose elimination capacity (GEC), a quantitative index of hepatic function, were measured preoperatively and at regular intervals postoperatively. Following 70% hepatectomy, AFP was first noted to be increased from the preoperative level (94 ± 7 SEM ng AFP activity/ml) on the fourth postoperative day (556 ± 75; P < 0.05) with a peak value being reached between Days 8 and 12 (1008 ± 111; P < 0.025). AFP then gradually decreased, returning to normal by Day 24 (116 ± 12; P > 0.5). These changes in AFP concentration parallel, in a slightly delayed fashion, hepatic proliferative activity as measured by DNA synthesis following hepatic resection in the dog. No alteration in AFP concentration was seen in the control animals. Elevations in SGOT, SGPT, and alkaline phosphatase were observed in all dogs following liver resection from Day 2 through 26. In contrast to AFP, changes in the serum concentrations of these enzymes were highly variable in both magnitude and duration. GEC was not significantly altered following liver resection in any dog. The results indicate that AFP is superior to liver enzymes and GEC as a marker for hepatic regeneration in the dog.

Two-stage rocket-electrophoresis on cellulose acetate films

The method of two-stage rocket electrophoresis on gelatinized cellulose acetate film (Cellogel) was developed. The method is based on electroimmunodiffusion detection of antigen on cellulose acetate films containing monospecific antiserum of a test system, followed by detection of precipitation bands with the appearance of stained “rockets” if the reaction takes place in the visible zone, or with further treatment of cellulose acetate strips containing invisible precipitates with antiglobulin antibodies, complement, or a combination of both. The sensitivity of the method is increased to 30–60 ng/ml in the visible zone of reaction by simultaneously reducing the concentration of antibodies and increasing the absolute amount of antigen for electrophoresis to 50–100 λl. Details of the method were worked out for human α-fetoprotein.

Binding of bilirubin by bovine and human α-fetoprotein

α-Fetoprotein, a fetal protein associated with certain tumors, was found to bind bilirubin. Addition of human or bovine α-fetoprotein to bilirubin solutions enhanced the light absorbance of bilirubin and shifted its maximum. Bovine α-fetoprotein caused a marked shift towards shorter wavelengths, while human α-fetoprotein gave a slight red shift. The spectral changes were used to study the characteristics of the binding of bilirubin by bovine α-fetoprotein. These studies indicated the presence of one binding site/molecule of α-fetoprotein with an association constant of about 1.1 · 10 6 M −1. A difference between the spectral changes brought about by α-fetoprotein and albumin allowed comparison of their relative affinities for bilirubin. The spectrum approximated the average between the spectra induced by the two proteins when the ratio of bovine α-fetoprotein to bovine albumin was 6.3 : 1, and of the human proteins 21 : 1, respectively. These results show that α-fetoprotein from two species binds bilirubin with an affinity somewhat lower than that of albumin. Binding of bilirubin by α-fetoprotein is in agreement with the recent demonstration of structural homology between α-fetoprotein and albumin. Whether α-fetoprotein plays a role in the metabolism of bilirubin or other degradation products of heme remains to be investigated.

Microheterogeneity of rat, mouse and human α1-fetoprotein as revealed by polyacrylamide gel electrophoresis and by crossed immuno-affino-electrophoresis with different lectins

Polyacrylamide gel electrophoresis and crossed immuno-affino-electrophoresis with several free lectins have been used to characterize and to compare the molecular heterogeneity of rat, mouse and human α 1-fetoproteins. Each α 1-fetoprotein contains a variable number of electrophoretic variants depending on the gel porosity. In SDS electrophoresis, two molecular size populations are present in rat α 1-fetoprotein ( M r = 74 000 and 72 000) and in mouse α 1-fetoprotein ( M r = 73 000 and 72 000) but only one is observed in human α 1-fetoprotein ( M r = 70 000). The crossed immuno-affino-electrophoresis patterns square with affinity chromatography results and reveal a marked and characteristic heterogeneity for the three α 1-fetoprotein species with Concanavalin A, Ricinus communis and Lens culinaris lectins. No lectin- α 1-fetoprotein interaction is apparent with Ulex, Lotus and wheat germ lectins. Since similar patterns are obtained whether with purified α 1-fetoprotein or with unfractionated fresh fetal sera, it is likely that this heterogeneity is not a consequence of artefactual molecular modifications arising during the purification procedure.

Inhibition of Human Lymphocyte Transformation by Human α‐fetoprotein (HAFP): Studies on the Mode of HAFP Action and the Role of HAFP Polymorphism

To explain the mechanism of inhibition of human lymphocyte transformation by human alpha‐fetoprotein (HAFP), we sought for, and failed to find, evidence of physical association between HAFP and phytomitogens or antihuman thymocyte antiserum. In addition, 20–40 fold increases in mitogen dose do not reverse the inhibition of lymphocyte transformation by a constant dose of HAFP. The presence of HAFP does not interfere with the attachment of 125I‐labelled phytohemagglutinin to the lymphocyte surface. Two‐dimensional crossed immunoelectrophoresis of HAFP isolated from the body fluids of hepatoma patients demonstrates three charged species of HAFP designated as HAFP‐1 (the most cathodal), HAFP‐2, and HAFP‐3 (the most anodal). The potency of HAFP isolates in inhibiting lymphocyte transformation can be positively correlated with the ratio HAFP‐3: HAFP‐1 in each preparation. Passage of HAFP isolates over CM‐cellulose allows the isolation of two HAFP fractions, one at pH 4.95, and the other at pH 5.24. The pH 4.95 CM‐cellulose isolate is enriched in the more electronegative HAFP species (HAFP‐3) and is, on the average, two times more potent than the pH 5.24 CM‐cellulose HAFP isolate. The latter, by comparison with native HAFP, is enriched in the more electropositive HAFP species (HAFP‐1). The CM‐cellulose HAFP isolates are identical in sialic acid content. We have isolated HAFP from the serum, ascitic fluid, and saline extract of tumor from a single hepatoma patient, and from an homogenate of fetal livers. When tested for their capacity to inhibit human lymphocyte transformation in vitro, tumor and fetal liver HAFP were found to be extremely potent; serum HAFP had intermediate potency, and ascitic fluid HAFP was the least potent. Analysis of these HAFP isolates by isoelectric focusing in polyacrylamide gels containing 8 M urea revealed at least 6 molecular HAFP variants. The proportion of one of these variants, termed HAFP‐3a, in each isolate was correlated with the immunosuppressive potency of the isolate. The sialic acid content of the various HAFP isolates did not vary significantly. Our data suggest that a post‐synthetic modification of HAFP occurs, which modulates its immunosuppressive potency.

Heterogeneity of human α-fetoprotein as revealed by isoelectric focusing in urea-containing gels

The heterogeneity of human α-fetoprotein has been studied by analytical isoelectric focusing in polyacrylamide gel slabs in the presence of 8 M urea. Six major isoelectric variants could be identified over a pH range of 6.0–6.2. Verification of their identity was achieved by crossed immunoelectrophoresis into agarose gel containing monospecific antiserum to human α-fetoprotein. Complete desialylation of the protein did not abolish the heterogeneity; a complex pattern of major α-fetoprotein bands persisted over a more alkaline pH range. We have been able to correlate the pattern of α-fetoprotein heterogeneity seen following extended agarose gel electrophoresis with that obtained during isoelectric focusing in the presence of urea. The quantity of certain α-fetoprotein charge isomers in various α-fetoprotein isolates may be important in considering certain biological functions of this protein.

Effect of α-fetoprotein on reparative regeneration of the skin and long bones

The effect of human, canine, and feline α-fetoprotein on reparative regeneration of the skin and long bones of mice and rats was studied. α-Fetoprotein has a stimulating action, through the formation of young connective tissue, on regeneration but without strict species-specificity. Of the various α-fetoproteins studied, a commercial preparation of human α-fetoprotein had the highest physiological activity.

Augmentation of Proliferation of Human Mixed Lymphocyte Culture by Human α-Fetoprotein

Abstract We have studied the effects of purified human α1-fetoprotein (AFP) of hepatoma origin on the kinetics of one-way lymphocyte proliferation in a mixed human lymphocyte culture system. Human serum albumin was employed in parallel control studies. Although AFP, over a wide range of concentrations from 5 ng/ml to 2 mg/ml, had no mitogenic activity on a single lymphocyte population, significant augmentation of the proliferative response was seen at the three different times of harvesting in the system of one-way allogeneic stimulation. When the addition of the AFP to this system was delayed to 24 hr after culture initiation, the AFP was still effective in producing augmented proliferation. However, no effect on the normal mixed lymphocyte reaction was seen upon addition of the AFP at either 48 or 72 hr after initiation of the cultures. In completely parallel control studies, human serum albumin used at the same concentrations as the AFP produced no modifications of the normal mixed lymphocyte culture response when compared to control cultures incubated in media without added serum proteins. Thus, the preparation of highly purified human AFP studied had no immunosuppressive activity, but actually showed a stimulatory activity on mixed lymphocyte proliferation when employed at relatively high concentrations.

A postsynthetic modification of human alpha-fetoprotein controls its immunosuppressive potency.

In a previous study, we demonstrated three variants of human alphafetoprotein by crossed immunoelectrophoresis. In addition, we correlated the capacity of alpha-fetoprotein isolates from various hepatoma and fetal sources to suppress human lymphocyte transformation in vitro with the relative proportion of the electronegative variant, HAFP-3, present in each isolate. We have now isolated alpha-fetoprotein from the serum, ascitic fluid, and saline extract of tumor from a single hepatoma patient and from a homogenate of fetal livers. When tested for their capacity to inhibit human lymphocyte transformation in vitro, tumor and fetal liver alphafetoprotein were found to be extremely potent, serum alphafetoprotein had intermediate potency, and ascitic fluid alpha-fetoprotein was the least potent. Analysis of these isolates by crossed immunoelectrophoresis confirmed the correlation between the proportion of HAFP-3 and the immunosuppressive potency of each isolate. In addition, analysis of these isolates by isoelectric focusing in polyacrylamide gels containing 8 M urea revealed further evidence of microheterogeneity; at least six molecular variants were apparent. The proportion of one of these variants, termed HAFP-3a, in each isolate was correlated with the immunosupressive potency of the isolate. The sialic acid content of the various alpha-fetoprotein isolates did not vary significantly. Our data suggest that a postsynthetic modification of alphafetoprotein occurs, probably after secretion, which reduces immunosuppressive potency by converting the active electronegative species to an inactive electropositive form. This modification probably involves a charged moiety other than sialic acid on the molecule.

Inhibition of Human Lymphocyte Transformation by Human α-Fetoprotein (HAFP); HAFP Monomers and Multimers and a Resistant Lymphocyte Subpopulation

Abstract We have previously shown that human α-fetoprotein (HAFP) inhibits the response in vitro of human lymphocytes to a variety of mitogenic stimuli. In this report we present evidence that HAFP preparations containing dimeric and trimeric forms are no more inhibitory than HAFP monomeric isolates. HAFP isolates of low inhibitory potency cannot impede the capacity of high-potency HAFP to inhibit lymphocyte transformation. Since lymphocyte responses to phytomitogens or anti-human thymocyte antiserum cannot be totally suppressed by increasing doses of HAFP, we conclude that human lymphocyte preparations contain a population of lymphocytes resistant to the inhibitory action of HAFP.

Studies on human α-fetoprotein: Isolation and characterization of monomeric and polymeric forms and amino-terminal sequence analysis

Human α-fetoprotein has been isolated from the serum and ascitic fluid of a patient with hepatoma by a combination of immunoadsorbent column chromatography and Sephadex G-150 gel filtration. Human α-fetoprotein is a sialylated glycoprotein with an estimated molecular weight of 67 500, composed of a single-chain polypeptide of approximately 580 amino acid residues and 3.6% carbohydrate. It is a negatively charged protein with an acid isoelectric point (pH 4.57). In addition to the monomeric form of α-fetoprotein, we have identified human α-fetoprotein polymers, including dimeric and trimeric forms, which dissociate to the monomer only upon exposure to disulfide-reducing reagents, implying that their formation is dependent upon intermolecular disulfide bonds. These polymers are found in human α-fetoprotein isolated by isoelectric focusing in both the major (pI 4.57) and minor (pI 5.2) α-fetoprotein fractions. The first 17 residues of the NH 2-terminal amino acid sequence of the hepatoma-derived human α-fetoprotein have been identified. Fetal α-fetoprotein is indistinguishable from hepatoma α-fetoprotein by several criteria, including immunoelectrophoresis, acrylamide gel electrophoresis, and proclivity for dimerization.

Radioimmunoelectrophoretic determination of ?-fetoprotein. Characteristics of standard inhibition curves

An express method of radioimmunological determination of mouse and human α-fetoprotein using electrophoretic fractionation of immunologically bound and free antigen is suggested. The method is comparatively simple in use and enables a large series of samples (up to 40) to be analyzed simultaneously within a short time (5 h). The behavior of the antibody-antigen system during plotting of standard inhibition curves was analyzed. Inhibition of the antibody-antigen reaction was found in the zone of low and high concentrations. The effect described calls for further study.

Conditions Affecting the Immunosuppressive Properties of Human α-Fetoprotein

Abstract The hypothesis that α-fetoprotein (AFP) is immunosuppressive in vitro was tested with immunoabsorbent purified human cord AFP in human lymphocyte cultures. Albumins were purified identically from cord plasma and pooled adult plasma. No preparations were suppressive in phytohemagglutinin-stimulated cultures. Both AFP and human cord albumin (HCA) produced 50% suppression of allogeneic lymphocyte cultures at 50 to 100 µg/ml final concentrations, whereas adult and commercial albumins were not suppressive. When AFP was eluted from the immunoabsorbent column in 0.15 M NaCl rather than the conventional 0.5 M NaCl, activity was greatly diminished or lost, but could be restored by dialysis against 0.5 M Kcl. Previously, inactive lots of HCA also demonstrated the same phenomenon. It appears, therefore, that the immunosuppressive activity of AFP (and albumin) may depend both on its source and the procedure by which it is isolated. Whether analogous conditions occur in vivo is unknown at this time.