Antibodies against FcRn and use thereof
An antibody and antigen technology, applied in the field of antibodies against FCRN and its application, can solve the problem of not generating or detecting antibodies
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Embodiment 1
[0517] Example 1: Cloning of FcRn, FcRn-GPI and β 2 m
[0518] The full-length FcRn cDNA construct used in these examples was originally constructed in Simister's laboratory (Brandeis University, Waltham MA) using pcDNA6 (Invitrogen, Carlsbad, CA) as the plasmid vector (FcRn: pcDNA6). The β2m cDNA construct used in these examples was originally constructed in the Blumberg laboratory (Harvard Medical School, Boston, MA) using pcDNA3 (Invitrogen) as the plasmid vector (β2M: pcDNA3).
[0519] Plasmids were transfected into One Shot TOP10 chemically competent E. coli (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Pick a single colony from each transformation plate, inoculate into 500-1000ml of LB medium and culture overnight in a shaker. Plasmid DNA was purified from these cultures using the Maxi Prep kit (Qiagen, Valencia, CA). The pcDNA6-full-length hFcRn plasmid construct was digested with Nhe1 and Xba1. The pCDNA3.1-β2-M plasmid construct was dige...
Embodiment 2
[0520] Example 2: Immunization of mice with plasmid DNA encoding FcRn
[0521] Balb / c mice were treated with 100 μl of 10 mM cardiotoxin (Calbiochem, San Diego) 5 days prior to plasmid DNA injection. Cardiotoxin treatment serves to elicit an inflammatory response and recruit antigen-presenting cells (eg, dendritic cells) to the injected area, thereby enhancing antigen presentation when the plasmid-encoded protein is expressed.
[0522] 100 μg of full-length or GPI-hFcRn plasmid constructs resuspended in 50 μl PBS were injected into the tibialis anterior muscle of mice. Using hFcRn and β 2 Combination immunized mice of M received 50 μg of hFcRn plasmid in 25 μl PBS and 50 μg of β in 25 μl PBS 2 Dosage of M plasmid. All intramuscular injections were performed under systemic anesthesia using phenobarbital (50 mg / kg, intraperitoneal injection) or ketamine (100 mg / kg) / xylazine (10 mg / kg). On days 21 and 42 after the first immunization, animals were boosted with re-injections of...
Embodiment 3
[0527] Example 3: Antibody titers in mouse sera
[0528] Measurement of anti-hFcRn and anti-β in mouse serum using ELISA 2 M titer. Use 2 μg / ml soluble hFcRn or hβ in ELISA coating buffer (Sigma, St. Louis, MO) 2 M (Sigma, St. Louis, MO) ELISA plates were coated. Plates were incubated at 37°C for 1 hour. Plates were washed twice with PBS+0.05% Tween (PBST). Plates were blocked with 1% fish gelatin in PBS at 37°C. Plates were washed twice with PBST. Serially diluted mouse serum (in PBS) was added (100 μl / well) and incubated at 37° C. for 2 hours. Plates were washed 5 times with PBST. Goat anti-mouse IgG-HRP (Pierce, Rockford, IL) was added to the plate at a dilution of 1 to 10,000 and incubated for 1 hour at room temperature. Plates were washed 5 times with PBST. Tetramethylbenzidine (TMB) solution (KPL, Gaithersburg, MD) was added to the plate for color development. The substrate reaction was stopped after 5 minutes when appropriate color development occurred. Plate...
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