Human igg elisa kit

Supernatants of the PBMC and protein extract co-cultures were analyzed to identify the levels of IgG and IgM secretion. A human IgG ELISA kit (Abcam, Cambridge, MA) and human IgM ELISA kit (Abcam, Cambridge, MA) were used following the manufacturer’s instruments. The absorbance was measured in a Sunrise^TM microplate reader (TECAN, Mannedorf, Switzerland). Allassays were performed in triplicate.

Naglu KO and wild-type littermate control animals (n = 3–4/group) were dosed at 30 mg/kg i.v. at ~ 2 months old with control human IgG1 3D5 antibody^{78 (link)} produced in-house. Dosed animals were sacrificed 48 h post-treatment and terminal CSF was collected as described. The concentrations of human IgG in murine CSF were measured with a human IgG ELISA kit (Abcam, Cat. #ab212169). An 8-points standard was prepared by diluting the kit-provided Human IgG protein to 15 ng/mL followed by a 1:2 serial dilution. Standards and test samples were diluted so that the antibody concentration was below 15 ng/mL and loaded at 50 µL/well onto anti-tag immobilization coated 96-well plates, and topped with 50 µL of antibody cocktail containing a mixture of capture and detector antibody Plates were washed and TMB was added as a chromogen at 100 µL/well. Sealed plates were incubated shaking at room temperature for 5 min to develop the color reaction. The intensity of the color reaction was assessed by absorbance at a wavelength of 450 nm. Sample protein concentrations were determined by interpolating the blank control absorbance values subtracted against the respective antibody standard curve with a second order polynomial model.

For half-life analysis, rhIgG1 Fc purified from egg yolk and recombinant Fc derived from HEK293 cell (10702-HNAH, Sino Biological Inc) were i.p. injected into 8 weeks of female C57BL/6 mice at dose of 100 μg/mouse. After 24 h of injection, blood was collected via tail vein for 5 days and serum was obtained via centrifugation. The serum concentration of rhIgG1 Fc purified from egg yolk, and recombinant Fc derived from HEK293 cell was determined by ELISA using a human IgG ELISA kit (Abcam). Half-life was calculated by nonlinear regression analysis.

The IL-21 concentration was measured using a human IL-21 ELISA kit (Abcam, Cambridge, UK), and the IgG concentration was measured using a human IgG ELISA kit (Abcam) according to the manufacturer’s instructions. Three replicate wells were quantified for every sample, and all experiments were performed in triplicate. Optical density (OD) values at 450 nm were read using an ELx800 Absorbance Microplate Reader (BioTek, VT, USA).

To quantify rhIgG1 Fc in serum, whole blood of ALB^{+/hIgG1 Fc} and ALB^{hIgG1 Fc/hIgG1 Fc} chicken were collected and 0.5 M ethylenediaminetetraacetic acid (EDTA) solution were added to collected blood to prevent blood coagulation. Collected blood samples were centrifuged at 2000 rpm, 10 min, 4 °C and supernatants were collected and used for ELISA analysis. To quantify rhIgG1 Fc in egg yolk, eggs laid from four G2 ALB^{+/hIgG1 Fc} chicken was collected and egg yolks were separated from egg whites. The collected serum and yolk samples were diluted in DW and quantified by ELISA using a human IgG ELISA kit (ab100547, Abcam, Cambridge, UK), according to manufacturer’s instruction. The measured concentrations were divided as three to adjust standard IgG molecular weight to rhIgG1 Fc.

For the measurement of Igs produced from the stimulated hPBMCs, the cell culture supernatant was obtained at every two days after the co-treatment of the cells with R848 and rhIL-2. Then, the concentration of each IgM and IgG was individually measured using a human IgM ELISA kit and human IgG ELISA kit (Abcam, Cambridge, UK) according to the manufacturer’s recommendations. Briefly, 50 μL of culture supernatant was added to the 96-well microtiter plate, pre-coated with an IgG or IgM as a capture antibody. Then, 50 μL of horseradish peroxidase (HRP)-conjugated IgG or IgM detector antibody was added to each well of the plate and incubated for 40 min at 25 °C. Following three washes with wash buffer, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well and allowed to react with HRP for 5 min. The reaction was terminated by adding 100 μL of 1 M H₂SO₄. The absorbance of each sample was measured at 450 nm using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA).