I have started to develop Indirect ELISA assays to quantify IgE and IgG subtypes in serum. I have made titrations of the antigen, primary antibody and conjugated antibodies. The sensitivity of the assay is in the order of ng/ml of the immunoglobulins. For the standard curve I'm using monoclonal antibodies against my antigen (1 mg/ml). When I try to repeat the Standard curve that I previously selected from the chessboard titration, I'm getting not reproducibility at all, there is a huge variation in OD values. I presume the problem comes from making dilutions because when applying the same antibody dilutions to different plates reproducibility is fine, but when I try to repeat the curve it in different days OD values change considerably. The main problem I see is that I have to make a lot of dilutions of the primary antibody in order to get to the range of my St curve, I'm talking of final dilutions of 1: 24 000 000. I'm wondering how can I sort this out? Does somebody know of any way of overcome this problem? Thank you very much.
According to me, your concentration (1 mg/ml) of the antibody anti-IgE coated on solid phase is too raised. In capture procedures, antibody concentration for to coat solid phase is between 1 and 50 µg/ml but non greater, related to physical-chemical and immunological characteristics of the antibody used. In addition, large concentration of the antibody employed, extremely low concentrations of primary antibody (elevated dilutions 1: 24,000,000) involves use, to get a suitable stoichiometric ratio, what unfortunately, for the elevated concentrations of the antibody link to solid phase, not always realizes. Then I would advise to repeat the chessboard titration, starting from these considerations. At any rate propose to you one my guide line for total IgE determination, on which to work.
PBS (10x) : dissolvere 80.0 g di NaCl, 11.6 g di Na2HPO4, 2.0 g di KH2PO4, 2.0 g di KCl e portare a 1 litro con dH2O con un pH di 7.3 ± 0.1
Plate coated : dispense in MTP-wells 100 µl of 10 µg/ml mouse monoclonal antibody anti-human IgE in phosphate buffered saline (PBS) or in CBC (15 mM di Na2CO3 e 34.8 mM di NaHCO3, at pH 9.6), and incubate for 60 min. at 37 °C, or at room temperature for 15-20 hrs or at 4 °C for 16 hrs. Aspirated the contained from wells and dispense 200 µl blocking solution (0.1 % globulin-free human serum albumin [HAS] or 0.25 % BSA or 10 % FCS in PBS) and incubate for 60 min. at 4 °C or at room temperature for 30 min., then wash three times with PBS containing 0.05 % Tween-20 and dried. Store in plastic bag sealed contained drying agent.
Standard IgE : dissolve 0.1 mg di human IgE (Immunology Consultants Laboratory cod. RA-80E-Z) in 1 ml of dH2O to obtain 100.000 ng/ml mother solution, and aliquote by 50 µl and store at -20 °C, stable for 18 months, while at 4 °C is stable one day. Dilute 24 µl mother solution with 976 µl standard diluent (PBS containing 10 mM NaN3, and 10 % FCS or 1 % BSA or 5 % horse serum) obtaining operative solution by 2400 ng/ml peer at 1000 IU/ml (2400/2.4* - *conversion factor from ng/ml to IU/ml). From this realize at doubling dilution, using standard diluents, linear range from 1000 to 7.8 IU/ml
Wash solution : 0.05 % Tween-20 in PBS
Primary antibody : dilute at 2.5 – 50 µg/ml monoclonal mouse anti-human IgE in 10 % FCS in Earles Modified Eagles Medium.
Conjugate antibody : dilute 1 µg/ml purified goat anti-mouse antibody conjugated with peroxidase in 10 % FCS in Earles Modified Eagles Medium.
TMB substrate : a) TMB-Stock : TMB dihydrochloride was dissolved, with stirring, at a concentration of 5 g/L (16 mM), in ddH2O, and the pH was adjusted to 1.5 with 5 N HCl. To this solution was added penicillin G, with stirring, in a final concentration of 200 mg/L (0.56 mM); b) Buffer-Stock : mix 1.4 ml of glacial acetic acid, 1.5 ml of 1 N NaOH and dissolve 250 mg (3 mM) of H2O2, of urea-hydrogen peroxide or sodium perborate and added to 900 ml of ddH2O. After dissolution is complete, the volume is made up to 1 liter with ddH2O; c) TMB-Work solution : mix together one part (v/v) TMB-stock and then parts (v/v) Buffer-stock obtaining a solution with pH of 3.3.
Stopping solution : 0.5 N H2SO4
dispense 100 µl of an unknown test serum, or standards and incubate for 1 h at room temperature. Aspired the fluid from the wells and wash 3 times with wash solution and dispense 100 µl of primary antibody and incubate 1 hr at room temperature. Wash wells as above and dispense 100 µl of conjugated antibody and incube 1 hr at room temperature. Wash as state above and added to each well 100 µl TMB work solution and incubate for 15 mins at room temperature and after incubation period is elapsed, 100 µl stopping solution has been added, which stopped the enzyme reaction. The Abs of the resulting colored solutions has been measured at 450 nm. A standard curve is used to calculate IgE level.
For IgG subclasses see you my previous post in Molecular Forum : [Only registered users see links. ]