| | Re: IgG subclass ELISA
We propose this approach
For to measure IgG subclasses levels, diluted human serum has been dispensed in microplate wells coated with monoclonal antibody that bound specifically alone one IgG subclass. After washing, to eliminate the unbound, IgG subclass, bounded, has been detected through antibody against PAN-IgG conjugated with HRP. Finally, after further washing, the reaction has been visualized by means specific peroxidase substrate (OPD), and Abs read, after stopping reaction, at 490 nm. Concentration of the IgG subclass in sample is Abs correlated.
Plate coated : dispense 100 µl solution containing 10 µg/ml mouse monoclonal antibody against IgG1 clone HP6001 (Sigma cod. I9388), or IgG2 clone HP6002 (Sigma cod. I9513), or IgG3 clone HP6047 (Sigma cod. I9763) or IgG4 clone HP6023 (Sigma cod. I9888) diluted in PBS at pH 7.4 or in carbonate buffer 0.02 M at pH 8.0, containing 0.02 % (w/v) of NaN3, and incubate for 16 hrs at 4 °C. Wash once the plate with PBS or carbonate buffer at 4 °C, according to the choice of the coating buffer,
and at once dispense in every wells 200 µl blocking solution (0.5-2% of BSA in PBS to pH 7.2) and incubate for 1 hr at 37 °C. Empty the wells, dry and to set in sealed bag with a drying agent and store at 4 °C.
Standard human IgG subclasses : dissolve 1 mg of each IgG1 (λ [Sigma cod. I4014] and k [Sigma cod. I3889]), IgG2 (λ [Sigma cod. I4264] and k [Sigma cod. I4139]), IgG3 (λ [Sigma cod. I4514] and k [Sigma cod. I4389]), IgG4 (λ [Sigma cod. I4764] and k [Sigma cod. I4639]) in 1 ml of resuspend solution (sodium succinate buffer 50 mM, 60 mM NaCl, 15 mg/ml sucrose, trehalose or sorbitol, 0.05 % EDTA, 0.1 mM [or 50 mg/mg of IgG] glycine, 1% (w/v) BSA, 0.02 % Tween-80, 0.01 % Thimerosal, at pH 5.5 ± 0.1), and store at 4 °C in glass amber bottle in the dark, stable 1 year. Just moment to use, from this above mother solution, to realize first standard dilution : 1:80 IgG1 for to obtain 12.5µg/ml, 1:180 IgG2 around 5.56 µg/ml, 1:650 IgG3 at 1.54 µg/ml, and 1:1300 for IgG4 about 0.769 µg/ml in standard diluting solution (0.5 M NaCl, 0.01 M NaH2PO4, 0.05 % Triton X-100, 2.0% [v/v] horse serum, at pH 8.2). Working standards IgG subclasses solutions, for to shape calibration curve, were realized doubling diluting each first standard dilution from 1:2 to 1:32, always with standard diluting solution.
Polyclonal conjugated : dilute at around concentration 130 - 250 ng/ml polyclonal antibody anti-human IgG from goat conjugate with peroxidase (Sigma cod. A0170) Fc specific and that it doesn't show cross reaction with mouse and rat IgG, in conjugated stabilize solution (0.1 % nofat dry milk or 1 % of BSA in Tris-HCl buffer 50 mM at pH 7.5, containing 1 mM MgCl2, 1 M NaCl, 0.01 % thimerosal)
Washing solution : 0.05 % Tween-20 in PBS 0.01 M at pH 7.4.
Sample diluted : Tris-HCl buffer 0.05 M at pH 7.4, 1.0 mM MgCl2, 0.15 M NaCl, 1 % [w/v] BSA, and 0.01 % thimerosal.
OPD substrate : mix at the use 0.4 ml of OPD-stock solution (dissolve 1 g o-phenylenediamine in 100 ml absolute methanol), 9.6 ml of citric-phosphate buffer 0.15 M at pH 5.0, and 4 µl 30 % hydrogen peroxide.
Stopping solution : 5 N H2SO4
Dispense in duplicate, in corresponding well coated with antibody against IgG subclass to detect, 100 µl of the specific series diluted calibrators, 100 µl 1:1000 diluted serum in sample diluted and incubate for 1 hr at 37 °C. Empty the wells and wash 3 time with washing solution, and therefore distribute 100 µl of polyclonal conjugated and incubate for 1 hr at 37 °C. Empty and wash such as above and pipette 100 µl of substrate, incubate for 40 mins. at room temperature in the dark. Stopping the reaction add 30 µl of stopping solution, mix gently on the shaker and read the Abs at 490 nm. For calculation, use the calibration curve, but for to determine the relationship percentage of the subclass IgG (IgGs-ratio) to divide the value of the single subclass for the sum of the IgG subclasses. In alternative it is possible to determine the total IgGs and then through the various Abs of the single subclass of IgG to calculate the relative values of the single fractions.