Characterization of a monoclonal antibody to human alpha-fetoprotein and its use in affinity chromatography

Stenman, Ulf‐Håkan; Sutinen, Marja‐Liisa; Selander, Ritva-Kajsa; Tontti, K; Schröder, Jim

doi:10.1016/0022-1759(81)90319-7

Cited by 25 publications

(5 citation statements)

References 7 publications

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“…We measured the affinity constant of Mab22 in the same RIA used to detect wcAFP in serum. The affinity constant for the standard AFP-Mab22 interaction was 7 x 10' L/mol and is comparable to those reported for Mabs to human AFP (5,35); however, we estimate that it is 10-fold to 20-fold lower than that of diluted polyclonal antiserum used in the RIP assay for WCAF' P (22). Nevertheless, the affinity of Mab22 is moderate to high, considering that many monovalent antigen-antibody systems have affinities between lo7 and 10" L/mol.…”

Section: Discussionsupporting

confidence: 83%

α-fetoprotein in the woodchuck model of hepadnavirus infection and disease: Immunochemical analysis of woodchuck α-fetoprotein and measurement in serum by quantitative monoclonal radioimmunoassay

et al. 1990

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Woodchuck hepatitis virus infection of the eastern woodchuck represents a useful model for the study of hepatitis B virus infection and disease in humans, including hepatocellular carcinoma. In man, hepatocellular carcinoma is frequently detected and monitored using assays for serum alpha-fetoprotein. To study the relationship between alpha-fetoprotein and woodchuck hepatitis virus-induced hepatocellular carcinoma in the woodchuck model, we produced a monoclonal antibody to woodchuck alpha-fetoprotein and used biophysical and immunochemical methods to demonstrate its specificity and affinity (7 x 10(8) L/mol) for woodchuck alpha-fetoprotein. A competition radioimmunoassay was then developed and standardized for measuring serum alpha-fetoprotein concentrations. In the radioimmunoassay system, woodchuck alpha-fetoprotein was detected between 20 ng/ml (20% to 25% inhibition) and 8,500 ng/ml (97% to 98% inhibition). Elevated serum alpha-fetoprotein concentrations (450 to 452,000 ng/ml) were measured in 21 of 23 woodchucks in the advanced stages of woodchuck hepatitis virus-induced hepatocellular carcinoma. Serum alpha-fetoprotein was elevated above normal (greater than or equal to 450 ng/ml) as early as 3 to 11 mo before terminal hepatocellular carcinoma in 11 of 16 of the woodchuck hepatitis virus-carrier woodchucks. In a pilot study, serum alpha-fetoprotein became markedly elevated above normal in woodchuck hepatitis virus-carrier woodchucks that developed hepatocellular carcinoma but not in serologically recovered or uninfected woodchucks (i.e., without hepatocellular carcinoma). Thus, alpha-fetoprotein may provide a useful noninvasive marker in the woodchuck model for detecting and monitoring woodchuck hepatitis virus-induced hepatocellular carcinoma from earlier stages.

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Section: Discussionsupporting

confidence: 83%

α-fetoprotein in the woodchuck model of hepadnavirus infection and disease: Immunochemical analysis of woodchuck α-fetoprotein and measurement in serum by quantitative monoclonal radioimmunoassay

et al. 1990

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“…We measured the concentration of each derivative required for replacement of 50% of 6'-O-[3H]methyla-amanitin from the antibody's binding site. Separation of free and bound tracer was in all cases achieved through polyethyleneglycol precipitation according to Stenman et al (1981). It was assessed that amatoxin binding to the antibodies was not disturbed by the precipitation procedure.…”

Section: Resultsmentioning

confidence: 99%

“…Binding capacities and KO values of the monoclonal antibody were measured by precipitation of the 6'-O-[3H]methyl-aamanitin complex with polyethyleneglycol 6000 (Roth, Karlsruhe). This assay was performed according to Stenman et al (1981). The same procedure provided the basis for the competitive binding assay (for details, see Baumann et al, 1993).…”

Section: Determination Of Dissociation Constants and Competitive Bindmentioning

confidence: 99%

A β‐turn in α‐amanitin is the most important structural feature for binding to RNA polymerase II and three monoclonal antibodies

Baumann

Zanotti²,

Faulstich

1994

Protein Science

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Four amatoxin-binding proteins with KD values in the nanomolar range, 3 monoclonal antibodies and RNA polymerase II, were studied with respect to their affinities to 24 alpha-amanitin derivatives with modified side chains. From KD values we estimated the amounts of binding energy that single side chains of the amatoxins contribute to complex formation. Ile6, previously identified by X-ray analysis to be part of a beta-turn (Kostansek EC, Lipscomb WN, Yocum RR, Thiessen WE, 1978, Biochemistry 17:3790-3795) proved to be of outstanding importance in all complexes. Replacement of the isoleucine with alanine reduced the affinity to all binding proteins to < 1%, suggesting a strong hydrophobic interaction. A strong effect was also seen when Gly5 was replaced with alanine, suggesting that the absence of a side chain in proximity to the beta-turn is likewise important. In addition to the beta-turn, each of the proteins showed at least 2 other points of strong contact formed by hydrogen bonds. Donors are the indole NH of 6'-hydroxy-Trp4 and OH of hydroxy-Pro2 and dihydroxy-Ile3. All the antibodies, but not RNA polymerase II, recognized the indole nucleus of 6'-hydroxy-Trp4. The geometric arrangement of the 4 strongest contact points suggests that the amatoxin binding site is different in each of the 4 proteins, except for the 2 antibodies raised in the same animal. Here, most of the contact points were identical but differed in strength of interaction. The method of structural analysis presented in this study is useful for identifying contact sites in complexes of proteins with peptides of rigid conformation. Furthermore, the method complements X-ray data by providing information on the amount of binding energy contributed by single structural elements.

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“…Binding capacities and equilibrium dissociation constants (Kd) of the monoclonal antibody were measured by precipitation of the 6'-0-[3H]methyl-a-amanitin complex with polyethylene glycol 6000 (Roth, Karlsruhe, Germany). This assay was performed according to Stenman et al (1981) with the following modifications: 50-µ samples of purified mAb (ca. 2 nM) in PBS were mixed with 200 µ of 6 '-0-[3H] methyl-a-amanitin (2-160 nM) in PBS and incubated for 3 h at 25 °C.…”

Section: Methodsmentioning

confidence: 99%

Identification of structural features involved in binding of .alpha.-amanitin to a monoclonal antibody

Baumann¹,

Muenter²,

Faulstich³

1993

Biochemistry

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Twenty-four derivatives of the cyclic octapeptide alpha-amanitin were assayed for their affinities to the monoclonal antibody beta A1/1. The derivatives were of natural, semisynthetic, and synthetic origin and had KD values ranging from 2 nM to > 70 microM. In the majority of the derivatives the chemical modifications had no detectable influence on the overall shape of the double-ring peptide. Given this condition, binding factors could be calculated from KD values of the amatoxin derivatives, which were valid for all amatoxins for estimating the contribution made by single structures to complex formation. The complex between alpha-amanitin and the immunoglobulin involves at least eight sites of contact. Four of them are responsible for strong interactions: (1) the OH group of hydroxyproline2 (binding factor 413), (2) the lipophilic side chain of isoleucine6 (binding factor 131), (3) the -CH2- moiety of the adjacent glycine5 or the absence of a side chain in this position (binding factor 361), and (4) the proton at the indole nitrogen of hydroxytryptophan4 (binding factor 140). The residual four interactions are hydrogen bonds of lower strength corresponding to binding factors of 1.5-8. The key role of the unique conformation of the amatoxins in determining their binding properties was shown by two amatoxin derivatives in which changes in the conformation were associated with virtually complete loss of affinity. For all amatoxin derivatives with conformations similar or identical to that of alpha-amanitin, we found empirical evidence that those structures of the peptide involved in binding make their contributions virtually independent of each other. It is a consequence of this rule that structural features that cooperate in binding could be characterized by the numerical product of their binding factors.

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Characterization of a monoclonal antibody to human alpha-fetoprotein and its use in affinity chromatography

Cited by 25 publications

References 7 publications

α-fetoprotein in the woodchuck model of hepadnavirus infection and disease: Immunochemical analysis of woodchuck α-fetoprotein and measurement in serum by quantitative monoclonal radioimmunoassay

α-fetoprotein in the woodchuck model of hepadnavirus infection and disease: Immunochemical analysis of woodchuck α-fetoprotein and measurement in serum by quantitative monoclonal radioimmunoassay

A β‐turn in α‐amanitin is the most important structural feature for binding to RNA polymerase II and three monoclonal antibodies

Identification of structural features involved in binding of .alpha.-amanitin to a monoclonal antibody

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