Monoclonal Antibody TfR Monoclonal Antibody

Antigen Used for Immunization

Antigen: hTfR1

Origin: Escherichia coli BL21 Transetta (DE3)

Source: human

Method of Immunization

BALB/c mice were immunized with 200 μL emulsion containing 100 μg of recombinant hTfR1 protein and Freund's complete adjuvant by subcutaneous injection. At 3-week intervals, two boosters of 200 μL emulsion were administered by intraperitoneal injection.

Parental Cell Line Used for Fusion

Spleens of the positive mice and SP2/0-Ag14 myeloma cells.

Selection and Cloning Procedure

The fused cells were cultured in hypoxanthine/aminopterin/thymidine (HAT) medium 10 days for selection. Then hybridoma cells obtained by screening were cultured in hypoxanthine/thymidine (HT) medium 10 days later. Positive clones were selected by indirect ELISA.

Heavy and Light Chains of Immunoglobulin

The heavy chain of TfRMAb is IgG2a and the type of light chain is Kappa (K).

Specificity

Method: To determine the affinity constant (K_aff) of TfRMAb, a noncompetitive enzyme immunoassay was performed. In brief, two different concentrations of recombinant hTfR1, including [Ag′] = 0.50 μg/mL and [Ag] = 1.00 μg/mL, were coated in a 96-well plate. After incubation at 37°C for 2 hours, the antibody was serially diluted to 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, and 0.39 μg/mL and added to the microplate. Then the process was performed as the ELISA. Sigmoid curves were plotted to represent the relationship between OD450 value and MAb concentration in two different antigen concentrations. The half maximum OD (OD −50) was assigned for all selected curves from which the corresponding antibody concentration ([Ab]_t and [Ab′]_t) was extrapolated. Accordingly, [Ab]_t and [Ab′]_t are the measurable total antibody concentrations at OD −50 and OD′ − 50 for plates coated with [Ag] and [Ag′], respectively. The affinity constant was finally determined using the following equation: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} {K_{\rm aff}} = ( n - 1 ) / 2 \ ( n{ [ {\rm Ab} ^ \prime ] _{\rm t}} - { \left[ {\rm Ab} \right] _{\rm t}} ) , \end{align*} \end{document}

where n = [Ag]/[Ag′].

The fine binding specificity: According to the Beatty method, the K_aff of TfRMAb was calculated to be 2.85 × 10⁻⁸ M.

Specific Antigen Identified

Nonligand binding domain of hTfR1.

Availability