Establishment and characterization of a fucosylated α-fetoprotein-specific monoclonal antibody: a potential application for clinical research
The Lens culinaris agglutinin (LCA)-reactive fraction of α-fetoprotein (AFP-L3) is a well-known cancer biomarker for hepatocellular carcinoma (HCC) with very high specificity. Because LCA recognizes only bi-antennary N-glycans with a core fucose, some of fucosylated AFP in HCC patients may not be detected. Then glycan antibodies, which recognize both specific glycan and protein, are desired for glycobiology. Here, we successfully established a novel glycan antibody for fucosylated AFP and demonstrated its potential clinical application. After immunization with a fucosylated AFP peptide, positive screening was performed for fucosylated AFP peptides using solid-phase enzyme-linked immunosorbent assay (ELISA).
The newly developed antibody was designated: fucosylated AFP-specific mAb (FasMab). Western blot analysis showed that FasMab reacted with AFP produced by HepG2 cells, but not with AFP produced by α-1,6-fucosyltransferase deficient HepG2 cells. The specific binding of FasMab to fucosylated AFP was confirmed with ELISA as well as western blot analysis. A preliminary high sensitivity chemiluminescence enzyme immunoassay kit showed increased levels of fucosylated AFP in the sera of patients with HCC, but not in the sera of normal patients, or patients with chronic liver diseases. Thus, the novel glycan antibody, FasMab, is a promising tool to study fucosylated AFP with clinical and basic research applications.
Regulation of Anti-Tumor Activity Using Monoclonal Antibodies to Alpha-Fetoprotein Receptor and after Immunization with This Protein
Wiskott-Aldrich syndrome (WAS), an inherited blood cell disorder due to mutations of the X-chromosome gene WASP (Wiskott-Aldrich Syndrome Protein), was characterized originally by thrombocytopenia, immunodeficiency and eczema. Whereas platelet dysfunction is severe and consistent, immune defects are clinically variable, ranging from negligible to life-threatening. To understand this heterogeneity, WASP was quantified in peripheral blood mononuclear cells of patients with diverse mutations.
In this study we assessed the relationship between the mutation, protein expression and phenotype in WAS patients. The majority of the patients with missense mutations exhibited mild phenotype, whereas patients with premature stop codon were in most cases severe. We designed a one-step approach intended for use in identifying mutations in samples from newly diagnosed patients.
The approach relies on direct sequencing of amplified exon regions in a staggered schedule that was based on the mutation distribution frequency in previous cases. The method proved to be fast and reliable. Definitive mutation information was generated for each patient studied.
Regulation of Anti-Tumor Activity Using Monoclonal Antibodies to Alpha-Fetoprotein Receptor and after Immunization with This Protein
The object of this work was to study (i) the effect of monoclonal antibodies (mAb) to a receptor (R) of an oncofetal protein of an alpha-fetoprotein (AFP) on the survival rate and sensitivity of tumor target cells to the cytotoxic action of effector cells, (ii) the level of Ab to AFP-R in the blood serum of patients with malignant tumors (iii) the effect of blood serum with a high level of Ab to AFP-R on the survival rate of tumor cells in vitro, and also (iv) the effect of immunization of animals with an AFP-R preparation on subsequent development of a grafted tumor.
It is shown that mAb to AFP-R of clones 2E1, 5C6 and 2B8 effectively bond to both mouse tumor cells and to human tumor cells. Monoclonal Ab to AFP-R of the studied clones do not affect the proliferation of tumor cells of mice and insignificantly inhibit the proliferation of human tumor cells. In patients with malignant tumors, a substantial increase was detected of both the sum Ab to AFP-R, and Ab of the class IgM, and simultaneously an increase of the fraction Ab to AFP-R of the class IgM, which indicates the induction of a primary immune response to AFP-R in such patients.
Separate clones of mAb to AFP-R are capable of activating the immune system in respect to tumor cells, inducing of antibody-dependent cellular cytotoxic activity, but with an increase of the concentration of mAb to AFP-R to 1 &mgr;M, the blocking of the cytotoxic activity of peripheral blood mononuclear cells in respect to human tumor cells is possible. In the case of single immunization of mice with an AFP-R preparation, isolated from tumor tissue of lung cancer of a human, inhibition of the growth of a tumor, grafted four days after the immunization, was observed.
Expression and characterization of a recombinant Fab fragment derived from an anti-human alpha-fetoprotein monoclonal antibody
Alpha-fetoprotein (AFP) is a well-known molecular marker indicating the development of cancer as well as fetal abnormalities such as open neural tube defects. Accordingly the measurement of serum AFP is important for the diagnosis of hepatocellular carcinoma (HCC) and other abnormalities. Monoclonal antibodies (McAb) to AFP were produced to develop an immunoassay kit, and to study the possibility of an antibody (Ab) therapy. The immunoglobulin genes were cloned from hybridoma cells, and expressed in E. coli as an Fab soluble into a culture medium. The Fab of anti-AFP McAb exhibited binding to AFP and similar affinity compared to the original IgG. This recombinant antibody can be studied further for in vivo imaging and immunotherapeutics.
Analysis of epitopes of mouse monoclonal antibodies against human alpha-fetoprotein
Thirty-six monoclonal antibodies (MAbs) against human alpha-fetoprotein (AFP) were analyzed for the location of their epitopes by reacting them with a set of yeast recombinant AFP proteins using ELISA. Recombinant AFP proteins containing either one, two or all three domains, i.e. domain I, domain III, domain I-II, domain II-III and domain I-II-III, were produced and secreted into the culture medium of yeast cells harboring the expression plasmids. Epitopes of 13 MAbs were localized on domain I and 17 others were on domain III. However, the exact location of the epitopes of the remaining 6 MAbs could not be defined. The epitope of an antibody, namely AFY6, which was located in domain I, was successfully mapped on an octapeptide, C175KAENAVE182, using synthesized overlapping octapeptides.
Lectin-dependent modulation of interaction between human alpha-fetoprotein and its monoclonal antibodies. Epitope mapping
The effect of lentil lectin (LCA) on the binding of mouse monoclonal antibody (MoAb) against human alpha-fetoprotein (AFP) to LCA-nonreactive AFP-L1 and LCA-reactive AFP-L3 was studied on a panel of 30 MoAbs provided by the TD-2 Workshop of ISOBM for epitope mapping. LCA inhibited the binding of MoAbs 93 and 98 to AFP-L3 but not to AFP-L1, indicating that there was a competition between the MoAbs and LCA for the AFP sugar chain. With MoAbs 100, 109, 118, and 120, LCA rather increased the binding to AFP-L3 over that to AFP-L1.
Goat Anti-human Alpha fetoprotein (AFP) IgG, pure | ||||
AFP13-A | Alpha Diagnostics | 1 mg | 781.2 EUR | |
Mouse Monoclonal Anti-human Alpha fetoprotein (AFP) (clone 1) | ||||
AFP11-M | Alpha Diagnostics | 1 mg | 578.4 EUR | |
Human Alpha Fetoprotein (AFP) ELISA Kit, 96 tests, Quantitative | ||||
500 | Alpha Diagnostics | 1 kit | 562.8 EUR | |
Mouse Monoclonal Anti-human Alpha fetoprotein (AFP) (clone 2) | ||||
AFP12-M | Alpha Diagnostics | 1 mg | 578.4 EUR | |
AFP/alpha 1 Fetoprotein Mouse mAb | ||||
BF9203 | Lifescience Market | 100 ug | 525.6 EUR | |
Alpha 1 Fetoprotein Antibody / AFP | ||||
RQ4030 | NSJ Bioreagents | 100 ug | 356.15 EUR | |
Alpha Fetoprotein (AFP) Protein | ||||
abx061589-1mg | Abbexa | 1 mg | 994.8 EUR | |
Alpha Fetoprotein (AFP) Antibody | ||||
20-abx159320 | Abbexa |
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Alpha Fetoprotein (AFP) Antibody | ||||
abx159321-100ul | Abbexa | 100 ul | 427.2 EUR | |
AFP Antibody (Alpha Fetoprotein) | ||||
F43721-0.08ML | NSJ Bioreagents | 0.08 ml | 140.25 EUR | |
AFP Antibody (Alpha Fetoprotein) | ||||
F43722-0.08ML | NSJ Bioreagents | 0.08 ml | 140.25 EUR | |
Alpha Fetoprotein / AFP Antibody | ||||
RQ5012 | NSJ Bioreagents | 100ul | 356.15 EUR | |
Alpha Fetoprotein / AFP Antibody | ||||
V2002-100UG | NSJ Bioreagents | 100 ug | 349.3 EUR | |
Alpha Fetoprotein / AFP Antibody | ||||
V2002-20UG | NSJ Bioreagents | 20 ug | 153.3 EUR | |
Alpha Fetoprotein / AFP Antibody | ||||
V2002IHC-7ML | NSJ Bioreagents | 7 ml | 349.3 EUR | |
Alpha Fetoprotein / AFP Antibody | ||||
V2002SAF-100UG | NSJ Bioreagents | 100 ug | 349.3 EUR | |
Alpha Fetoprotein / AFP Antibody | ||||
V9012-100UG | NSJ Bioreagents | 100 ug | 349.3 EUR | |
Alpha Fetoprotein / AFP Antibody | ||||
V9012-20UG | NSJ Bioreagents | 20 ug | 153.3 EUR | |
Alpha Fetoprotein / AFP Antibody | ||||
V9012IHC-7ML | NSJ Bioreagents | 7 ml | 349.3 EUR |
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These modulating effects of LCA on the MoAb binding to AFP-L3 were abolished by periodate treatment of the AFP preparations without affecting the binding of MoAb to AFP. Concanavalin A had similar inhibiting and enhancing effects on MoAb binding, but equally to AFP-L1 and AFP-L3, both of which are fully reactive with concanavalin A. The results suggested that MoAbs 93 and 98 recognized epitopes closely related to sugar chain, and their binding to AFP-L3 was inhibited by the bound LCA due to steric hindrance. The enhanced binding of some MoAbs to AFP-L3 over AFP-L1 with LCA, or both glycoforms of AFP with concanavalin A, may be explained by postulating an allosteric mechanism mediated by the oligosaccharide-lectin interaction.