Mouse il 21 elisa kit

The heavy and light chain variable region of anti-PD-1 antibody was amplified by PCR from cDNA of G4 hybridoma, and then cloned into pTT3 vector gifted by Dr. Yingfang Liu (Institute of Biophysics of CAS, Beijing, China) alone or with IL-21 to prepare PD-1Ab or PD-1Ab21. Overlap PCR was used to link the adjacent DNA fragments, and a 3×Flag tag was added to the C-terminus. The IL-21R cDNA was cloned from murine splenocytes and then cloned into pMIgV vector (provided by Lieping Chen) to prepare IL-21RIgFc. Constructs for IL-21Flag and IL-21IgFc were described previously^{49 (link)}. The primers used were listed in Supplementary Table 1. Proteins were expressed by transient transfection of 293E cell line with the corresponding expression plasmids and purified on anti-Flag M2 affinity gel (Sigma–Aldrich) or protein G column (GE). For antibodies and fusion proteins, the endotoxin level was determined to be lower than 0.2 EU/µg Ab or protein. IL-21-related proteins were quantified with a mouse IL-21 ELISA kit (eBioscience, 88-8210-88). The other proteins or antibodies were measured by Quick Start^TM Bradford 1x Dye Reagent (Bio-Rad, #500-0205).

OVA-specific IgG and IgE levels were determined in the bronchoalveolar lavage (BAL) and serum samples collected at the end of the experiments. Briefly, for OVA-specific IgG and IgE, high-binding plates were coated overnight at 4 °C with 5 μg/ml OVA in carbonate buffer, blocked with 2% milk/PBS, and incubated with 1:1000–1:10,000 serum dilutions. The OVA-specific immunoglobulins were detected with an HRP-conjugated rat anti-mouse IgG antibody (Zsbio). The signal was developed by incubation with a standard TMB solution, and the optical density was read at 450 nm. The total IgE level was quantified with a capture mAb, biotin detection mAb, and streptavidin-HRP from the Mouse IgE ELISA Ready-SET-Go! kit (eBioscience). Readings were also obtained to generate a standard curve prepared with purified mouse IgE.
IL-21 production was quantified in serum and BALF with a mouse IL-21 ELISA kit (eBioscience) according to the manufacturer’s instructions. The standard curve was prepared with purified mouse IL-21. The concentrations of IL-4 and IL-5 were detected with the LEGENDplex Multi-Analyte Flow Assay Kit for Mouse Th Cytokine (BioLegend) according to the manufacturer’s instructions. Analyses were performed with a FACSAriaII (BD Biosciences) and the LEGENDplex software (BioLegend).