Protocol for Sandwich ELISA of Jagged-1

Transcription

1 Protocol for Sandwich ELISA of Jagged-1 General Information The Sandwich ELISA is a biochemical method to quantify the concentration of a certain antigen in a physiological liquid or a solution with need to purify it before. In comparison with other ELISA procedures, such as direct or indirect assays, the Sandwich ELISA reaches a 2 to 5 fold higher sensitivity. It measures the amount of antigen between two layers of antibodies (i.e. capture and detection antibody). The antigen measured must contain at least two different epitopes capable of binding to an antibody, since at least two antibodies act in the detection system. Either moclonal or polyclonal antibodies can be used as the capture and detection antibodies in Sandwich ELISA systems. Moclonal antibodies recognise a single, well defined epitope, that allows precise detection of small differences in antigen. Polyclonal antibodies are directed against various sequences of an antigen. This heterogeus recognition patterns make them less specific but increase the probability to bind the antigen and immobilize it by multiple antibodies interacting with it. Therefore they are often used as a capture antibody. Negative and positive controls have to be set up in every assay serving as a reference for specificity and correct handling of the assay. A negative control is a sample kwn t to contain the protein of interest, for example pure cell culture medium. An additional negative control may consist of buffer solution only, testing whether the antibodies react with culture medium. The standard is a series of solutions of kwn concentration obtained by serial. The standard series is used to establish a standard curve, which serves as a positive control Important tes : The capture antibody and the detection antibody should come from different species. This ensures that the secondary antibody binds only to the detection antibody (and t the capture antibody) and signalling is specific. Antibodies should carefully be be chosen to originate from different species and to bind to different epitopes within the site of interest (e. g. for cleaved proteins). If there is antibody available for the protein of the particular species, multiple sequence alignment may be performed to determine possible reactivity of an antibody from ather species with the chosen epitope. It is recommended to test the sensitivity and specificity of the antibodies preliminarily, for example in a western blot. Sodium azide is an inhibitor of horseradish peroxidase. It should t be present in buffers or wash solutions if an HRP-labeled antibody is used for detection. 1

2 Materials required Microplate for ELISA (capacity 360 µl/well) Capture antibody (moclonal) Coating Buffer : 0.1 M Carbonate-Bicarbonate Buffer, for preparation see below Washing Buffer : PBS, freshly prepared Blocking Buffer : 5% n fat dry milk in PBS Samples : for series see below Standard : for series see below Standard Diluent : for culture supernatant samples use culture medium as standard diluent Detection antibody (polyclonal) Secondary HRP-conjugated antibody Substrate for HRP : TMB, for preparation of solution see below Stop solution for TMB : 2M H 2 SO 4 or 1M HCl Preparation Coating Buffer : 0.1 M Carbonate-Bicarbonate Buffer (ph 9.6) Prepare solution A : Bicarbonate Buffer: Dissolve mg NaHCO 3 in 50 ml dh 2 O. Prepare solution B : Carbonate Buffer: Dissolve mg CNa2O3 in 50 ml dh 2 O. Prepare the needed quantity of Coating Buffer by mixing A and B in a 70 : 30 ratio (7 ml A and 3 ml B) Samples Final quantity : 100 µl/well (if possible, prepare a sufficient amount for duplicates) Prepare a 1 :1 series with PBS in Eppendorf tubes ranging from to 1:8 or more Store on ice until needed Standard Final quantity : 100 µl/well (if possible, prepare a sufficient amount for duplicates) Prepare a 1:1 series with an appropriate standard diluent in Eppendorf tubes Store on ice until needed TMB substrate solution Prepare the TMB substrate solution only immediately prior to use Wrap the falcon tube with aluminium since the substrate solution is light sensitive Dissolve 1 mg TMB in 10 ml 0.05 M Phosphate-Citrate Buffer, ph 5.0. Add 2 µl of fresh 30% hydrogen peroxide H 2 O 2. 2

3 Procedure 1. Coating with capture antibody a) Dilute the capture antibody appropriately (1-10 µg/ml) with the carbonatebicarbonate buffer (ph 9.6) b) Coat all the wells of the microplate with the diluted capture antibody (200 µl/well) c) Cover the plate and incubate overnight at 4 C d) Discard the coating solution and wash the plate twice with 300 μl PBS. The solutions or washes are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel. 2. Blocking a) Block the remaining protein-binding sites in the coated wells by adding 200 μl blocking buffer per well b) Cover the plate and incubate for at least 1-2 h at room temperature or, if more convenient, overnight at 4 C. c) Discard the blocking buffer and wash the wells with 300 µl PBS for 2-3 times 3. Adding of the samples/standard a) Dilute the standard in separate Eppendorf tubes in a 1 :1 series with standard diluent b) Add 100 µl of the standard series to the standard wells c) Dilute the samples in separate Eppendorf tubes in a 1 :1 series with PBS d) Add 100 µl of the samples series to each well with exception of the control wells. e) Add 100 µl of PBS and 100 µl of standard diluent to the negative control wells. f) Incubation for 90 minutes at 37 C g) Discard the samples and wash the plate twice with 300 µl PBS 4. Incubation with detection antibody a) Dilute the detection antibody 1 :500 with PBS in an Eppendorf tube b) Add 100 µl of diluted detection antibody to each well including the control wells. Cover the plate and incubate for 2 h at room temperature. c) Discard the content of the wells and wash the plate four times with PBS. 3

4 5. Incubation with secondary antibody a) Dilute the secondary HRP conjugated antibody in a separate Eppendorf tube 1:1000 with PBS b) Add 100 μl of the secondary antibody to each well c) Cover the plate and incubate for 1-2 h at room temperature. d) Discard the content oft he wells and wash the plate four times with PBS. 6. Detection a) Add 100 μl of the TMB substrate solution to each well b) Incubate the plate for 30 minutes at room temperature protected from light c) Add 100 μ of stop solution H 2 SO 4 to each well 7. Obtaining the results a) Read out the absorbance in a microplate reader at 450 nm within 30 minutes of adding the stop solution References Abcam: Fisher Thermo Scientific: 4

5 Pipetting Scheme (Example) Negative controls Standard Curve 21 div Neuron 21 div Neuron 10 div Neuron 10 div Neuron 10 div TAP1 10 div TAP1 10 div BIC 10 div BIC 10 div TAP1 / BIC 10 div TAP1 / BIC A PBS only B Standard diluent only 1:2 1:2 1:2 1:2 1:2 1:2 1:2 1:2 1:2 1:2 1:2 C 1:4 1:4 1:4 1:4 1:4 1:4 1:4 1:4 1:4 1:4 1:4 D 1:6 1:6 1:6 1:6 1:6 1:6 1:6 1:6 1:6 1:6 1:6 E 1:8 1:8 1:8 1:8 1:8 1:8 1:8 1:8 1:8 1:8 1:8 F G H 5