G9P3-1Mab Against Extracellular Domain of EVA1

Antigen Used for Immunization

The extracellular portion of mouse EVA1 fused with the human Fc portion of IgG3 (ext-EVA1-Fc).

Method of Immunization

We conducted a standard immunization protocol using Mpzl2-deficient gene-targeted mice. Homozygous mpzl2 knockout (B6.mpzl2^ko/ko) mice were given an intraperitoneal injection of 100 μL (50 μg) of ext-EVA1-Fc protein, emulsioned with 100 μL of Sigma Adjuvant System 2x (Sigma-Aldrich, St. Louis, MO), on days 0 and 14, and with 25 μg on days 28 and 42. The final boost of 25 μg without adjuvant was given on day 56. On day 59, a selected mouse was killed and splenocytes used for fusion. We chose the best responder out of three immunized mice based on the anti-ext-EVA1-Fc antibody titer of the serum extracted on day 21.

Parental Cell Line Used for Fusion

P3X63Ag8.653 myeloma cell line.

Selection and Cloning Procedure

Hybridomas were generated according to standard procedures. After fusion, cells were plated in eight 96-well plates and hybridomas selected with HAT (Sigma-Aldrich) for 2 weeks, followed by a transition culture with HT medium for an additional 2 weeks. Supernatants from wells showing hybridoma growth were tested by ELISA and flow cytometry.

Heavy and Light Chains of Immunoglobulin

Mouse IgG1, kappa.

Specificity

Specificity was determined by flow cytometry on HEK-293T cells expressing transgenic mouse and human EVA1. In addition, we used B6.Eva1^ko/ko as negative control to show the specificity of the G9P3-1Mab to detect EVA1 expression on DN3 cells and thymic epithelial cells.

Specific Antigen Identified

The monoclonal antibody is reactive against the extracellular domain of mouse and human EVA1.

Availability