Abstract
Antigen Used for Immunization
ErbB3 polypeptide (CPASEQGYEEMRAFQGPG, MW 1955.81417) synthesized by Shanghai Bioengineering (Shanghai, China) was cross-linked with KLH and then used as immunogen.
Method of Immunization
Two female BALB/c mice (age ∼10–12 weeks) were injected with immunogen four times over a 2-month period. Each mouse was subcutaneously injected with 100 μg of antigen emulsified with Freund's complete adjuvant (1:1) on day 1, with antigen emulsified with Freund's incomplete adjuvant (1:1) on days 15 and 29, and intraperitoneally injected on day 43, respectively. Three days after the last injection, mouse spleen cells were collected for cell fusion.
Parental Cell Line Used for Fusion
Mouse myeloma cell line SP2/0-Ag14 used for fusion was supplied by the Basic Immunology Department of Sichuan University (China).
Selection and Cloning Procedure
The cell clones producing anti-ErbB3 antibody were screened and identified by ELISA using synthesized ErbB3 polypeptide as coating antigen. The sub-cloning was performed by limiting dilution of the colonies from positive wells one more time.
Heavy and Light Chains of Immunoglobulin
The heavy chain and light chain of anti-ErbB3 MAb prepared in our study was IgG2a and κ, respectively.
Specificity
Western blot analysis
Because many cancer cells constitutively express ErbB3 protein, three cancer cell lines (HeLa, A431, McF-7) were chosen to identify the specificity of anti-ErbB3 monoclonal antibody obtained in our study. In brief lysates of three cells were resolved on 10% SDS-PAGE gels and transferred to nitrocellulose membrane in electrophoresis buffer (50 mM Tris, 40 mM glycine, and 0.1% SDS) at 200 mA for 2 h at 4°C using the Mini-Protean II wet blotter (Bio-Rad). After blocking with 1% BSA for 1 h at 37°C, the membrane was recognized overnight at 4°C by supernatant of hybridoma cell line (anti-ErbB3 MAb),and then incubated with goat anti-mouse HRP-conjugated secondary antibody at 37°C for another 1 h. After incubation and washing, it was shown by ECL and X-ray imaging.
Isotype determination of anti-ErbB3 MAb
Subclass of anti-ErbB3 antibody was identified using SBA Clonotyping System/HRP ELISA kit (SouthernBiotech, Birmingham, AL), according to the manufacturer's instructions. Briefly, wells of a 96 well-plate were coated with 100 μL of 5 mg/mL goat anti-mouse Ig capture antibody diluted in PBS (pH 7.4) and incubated for 12 h at 4°C. After washing three times with BBS containing 0.05% Tween-20, the wells were filled with PBS containing 1% bovine serum albumin (BSA) and incubated at room temperature for 1 h to block free binding sites on the coated plate. After washing, 100 μL/well of hybridoma supernatant (anti-ErbB3 MAb) was added to each well and incubated at room temperature for 1 h. By washing again, 100 μL HRP-labeled detection antibodies were added to appropriate wells of the plate and incubated at room temperature for 1 h. Color was developed by adding 100 μL/well substrate solution (15 mg/mL citric acid [pH 4.0]) for 20 min. The absorbance (OD) was read at 405 nm wavelength.
Affinity constant determination of anti-ErbB3 MAb
Affinity constant of anti-ErbB3 antibody was determined as described by Schots and associates.(1) The 96-well plate was coated with three concentrations of synthesized ErbB3 polypeptide diluted at 1:1 (1 mg/L), 1:2 (0.5 mg/L), and 1:4 (0.25 mg/L). Then a series of diluted anti-ErbB3 antibodies with known concentration (0.03 mg/L; dilution of 1:100; 1:400; 1:1600; 1:6400; 1:25,600; 1:102,400; 1:409,600) was added into appropriate wells, respectively, and incubated at 37°C for 1 h. After washing, the wells were incubated with a secondary antibody (HRP-goat anti-mouse IgG) at 37°C for 1 h and developed color by the addition of enzyme substrate. The optical density (OD) was read at 450 nm wavelength. Three reaction curves were plotted using the log of Ab concentration as the X-axis and OD 450 nm as the Y-axis, respectively. The flat of each curve was designed as OD100 and the Ab concentration at OD50 was designed [Ab]t. Then three [Ab]t values—[Ab]t (1:1), [Ab’]t (1:2), and [Ab’’]t (1:4)—were obtained, respectively. According to the Beatty formula, Kaff=(n - 1)/2(n[Ab’]t - [Ab]t),(2) three different concentrations of Ag were used, thereby enabling the calculation of three different K values: K1=1/2(2[Ab’]t - [Ab]t); K2=1/2(2[Ab’’]t - [Ab’]t); and K3=3/2(2[Ab’’]t - [Ab]t). The final affinity constant is the average result of the three calculations: Kaff=(K1+K2+K3)/3.
Specific Antigen Identified
The specific antigen, ErbB3 polypeptide (CPASEQGYEEMRAFQGPG, MW 1955.81417), was synthesized by Shanghai Bioengineering.