ELISAProtocol(GeneralGuidelines)

實驗概要

Sandwich  enzyme-linked immunosorbent assays (ELISAs) involve attachment of a  capture antibody to a solid phase support. Samples containing known or  unknown antigen are then added in a matrix or buffer that will minimize  attachment to the solid phase. An enzyme-labeled antibody is then added  for detection.

The  ELISA method is a benchmark for quantitation of pathological antigens  and there are indeed many variations to this method. ELISAs are  adaptable to high-throughput screening because results are rapid,  consistent and relatively easy to analyze. The best results have been  obtained with the sandwich format, utilizing highly purified, prematched  capture and detector antibodies. The resulting signal provides data  which is very sensitive and highly specific. Thus, Invitrogen offers a  large menu of sandwich ELISA and phosphoELISA? kits. ELISA and  phosphoELISA? kits allow for quantitative measurements of proteins  outside and inside the cell, respectively. For intracellular proteins  for signaling studies, we offer phosphoELISA? kits for measurement of  total and phosphorylation, modification, or cleavage site–specific  proteins.

The  phosphoELISA? kit protocol (Figure 1) begins with a monoclonal antibody  specific for a protein of interest (regardless of phosphorylation  state) coated onto the wells of microtiter strips. Samples, including a  standard containing protein of interest, control specimens, and  unknowns, are pipetted into these wells. During the first incubation,  the protein antigen binds to the immobilized (capture) antibody, much  like an immunoprecipitation. After washing, a rabbit antibody specific  for total protein or protein phosphorylated at a specific residue (e.g.,  Akt [pS473]) is added to the wells. During the second incubation, this  antibody serves as a detection antibody by binding to the immobilized  protein captured during the first incubation. After removal of excess  detection antibody, a horseradish peroxidase–labeled anti–rabbit IgG  antibody (anti-rabbit IgG-HRP) is added. This binds to the detection  antibody to complete the four-member sandwich. After a third incubation  and washing to remove all the excess anti-rabbit IgG-HRP, a substrate  solution is added, which is acted upon by the bound enzyme to produce  color. The intensity of this colored product is directly proportional to  the concentration of total or phosphorylated protein present in the  original specimen. Invitrogen? phosphoELISA? kits have been designed to  enable the specific detection of phosphorylation of key signaling  molecules, offer great specificity, and correlate well to western blot  data.

The assay procedure for ELISA (Figure 2) and phosphoELISA? kits are  similar with only a few minor differences. While the detector antibody  for ELISA kits is biotin-labeled and is followed by incubation with  streptavidin- HRP, the detector antibody for phosphoELISA? kits is a  rabbit polyclonal antibody and is followed by incubation with  anti-rabbit IgG-HRP.

實驗材料

1. ELISA Kit Contents

1)      Calibrated standard

2)      Stabilized chromogen

3)      Antibody-coated 96-well plate

4)      Stop Solution

5)      Biotin-labeled detection antibody

6)      Wash and Diluent buffers

7)      Streptavidin-conjugated HRP

2. phosphoELISA? Kit Contents

1)      Calibrated standard

2)      Stabilized chromogen

3)      Antibody-coated 96-well strip-well plate

4)      Stop Solution

5)      Detection antibody

6)      Wash and Diluent buffers

7)      Anti-rabbit IgG-conjugated HRP

3. Materials Required But Not Included in the Kit

1)   Absorbance-based Microplate Reader

2)   Sample (See ELISA Sample Preparation Protocols)

The  assay procedure takes about 4 hours total and requires only 30 minutes  hands-on time. Allow all reagents to reach room temperature before use.  Gently mix all liquid reagents prior to use.

Note: A standard curve must be run with each assay for quantification.

實驗步驟

1.       Determine the number of 8-well strips needed for the assay. Insert  these in the frame(s) for current use. (Re-bag and seal extra strips and  frame. Store these in the refrigerator for future use.)

2.       Pipette 100 μl of the Standard Diluent Buffer to the well(s) reserved  for the standard blanks. Well(s) reserved for chromogen blank(s) should  be left empty.

3.       Pipette 100 μl of standards, controls, and diluted samples (typically  >1:10 dilution for cell extract) to the appropriate microtiter wells.  Tap gently on side of plate to thoroughly mix.

4.      Cover wells with plate cover and incubate for 2 hours at room temperature.

5.      Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times.

6.       Pipette 100 μl Streptavidin-conjugated HRP (ELISA kits) or  Biotin-conjugated Detection Antibody (phosphoELISA?) solution into each  well except the chromogen blank(s). Tap gently on the side of the plate  to mix.

7.      Cover wells with plate cover and incubate for 1 hour at room temperature.

8.      Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times.

9.       Pipette 100 μl streptavidin-HRP (ELISA kits) or anti-rabbit IgG-HRP  (phosphoELISA? kits) solution to each well except the chromogen  blank(s).

10.  Cover wells with the plate cover and incubate for 30 minutes at room temperature.

11.  Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times.

12.  Pipette 100 μl of Stabilized Chromogen to each well. The liquid in the wells will begin to turn blue.

13.   Incubate for 30 minutes at room temperature and in the dark. Note: Do  not cover the plate with aluminum foil or metalized mylar. The  incubation time for chromogen substrate is often determined by the  microtiter plate reader used. Many plate readers have the capacity to  record a maximum optical density (OD) of 3.0. The OD values should be  monitored and the substrate reaction stopped before the OD of the  positive wells exceed the limits of the instrument. The OD values at 450  nm can only be read after the Stop Solution has been added to each  well. If using a reader that records only to 3.0 OD, stopping the assay  after 20 to 25 minutes is suggested.

14.   Pipette 100 μl of Stop Solution to each well. Tap gently on the side of  the plate to mix. The solution in the wells should change from blue to  yellow.

15.   Read the absorbance of each well at 450 nm having blanked the plate  reader against a chromogen blank composed of 100 μl each of Stabilized  Chromogen and Stop Solution. Read the plate within 2 hours after adding  the Stop Solution.

16.   Plot the absorbance of the standards against the standard  concentration. (Optimally, the background absorbance may be subtracted  from all data points, including standards, unknowns and controls, prior  to plotting.) Draw the best smooth curve through these points to  construct the standard curve. If using curve fitting software, the four  parameter algorithm provides the best curve fit.

17.   Read the protein concentrations for unknown samples and controls from  the standard curve plotted in step 16. Multiply value(s) obtained for  sample(s) by the appropriate dilution factor to correct for the dilution  with Standard Diluent Buffer. (Samples producing signals higher than  the highest standard should be further diluted in Standard Diluent  Buffer and reanalyzed, multiplying the concentration by the appropriate  dilution factor.)

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