Anti mouse igg2c hrp

Blood was collected from the retro-orbital sinus two weeks after the second immunization and sera prepared. Sera were stored at 4°C until antigen-specific antibody responses were analyzed by ELISA. Briefly, ELISA plates (Nunc, Rochester, NY) were coated with 1 μg/ml antigen in 0.1 M bicarbonate buffer and blocked with 0.1% BSA-PBS. Following washes in PBS/Tween, serially diluted serum samples were added. After incubation and further washes, either anti-mouse IgG-HRP, anti-mouse-IgG2c-HRP or anti-mouse IgG1-HRP were added (all Southern Biotech, Birmingham, AL). After incubation and washing, ABTS-H₂O₂ (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added to the plates to reveal any reactions, which were stopped by the addition of 0.1 N H₂SO₄. Plates were analyzed at 405 nm (EL_X808, Bio-Tek Instruments Inc, Winooski, VT). Midpoint titers were determined as EC50 values from weighted curve fits using the GraphPad Prism package V 6.03.

The Ag-specific Abs were determined by ELISA. The anti-IgG-HRP used was anti-mouse IgG1-HRP (cat#1070-05; Southern Biotech, Birmingham, AL), anti-mouse IgG2C-HRP (cat#1079-05; Southern Biotech), and anti-mouse IgA-HRP (cat#1040-05; Southern Biotech). Total anti-OVA IgG2A, IgG1 and IgA were quantified using a mouse anti-OVA IgG2A kit (cat#3015; Chondrex, Redmond, WA), anti-OVA IgG1 kit (cat#3013; Chondrex, Redmond, WA), and anti-OVA IgA kit (cat#3018; Chondrex, Redmond, WA).

The antibody titers for various isotypes in the sera of NP-KLH immunized mice were quantified relative to a sample pooled from sera of NP-KLH immunized mice. Nunc MaxiSorp™ plates (ThermoFisher) were coated with 1µg/ml of NP-(14)-BSA or NP-(2)-BSA (Biosearch). An initial dilution of 1:500 of the serum was prepared followed by a series of 1:5 dilutions. For the quantification of anti-ds-DNA-antibodies, high-binding half-area plates (Corning) were coated with Poly-L-lysin (Sigma-Aldrich), followed by 25 ng/ml calf thymus dsDNA (Sigma-Aldrich). An initial dilution of 1:10 of the sera was prepared, following a series of 1:2 dilutions. The following isotype-specific detection antibodies were used: donkey anti-mouse IgG-HRP (JacksonImmunoResearch), goat anti-mouse IgG1-HRP, anti-mouse IgG2b-HRP, anti-mouse IgG2c-HRP, anti-mouse IgG3-HRP, anti-mouse IgM-HRP (all from SouthernBiotech). Total quantification of IgG, was performed using a mouse-IgG-Kit (Roche) according to the manufacturer’s instructions.