ErbiBridge ELISA Kit Protocol

Product Number. ABK0002

Complete kit for the systematic 3-D conformational comparability analysis of Erbitux biosimilar molecule to Cetuximab (Erbitux trade name).

Read the entire protocol before performing the assay.
This kit is intended for research use only and not for use in diagnostic procedures.

Background Information

Cetuximab is a chimeric (mouse/human) monoclonal antibody that inhibits epidermal growth factor receptor (EGFR). Cetuximab is manufactured by ImClone and Bristol-Myers Squibb and is given by IV infusion for the treatment of metastatic colorectal cancer and head and neck cancer. This kit will allow for conformational comparison between Erbitux Biosimilars and authentic Cetuximab.

Assay Principle

The assay is in a sandwich ELISA format where the plate is coated with a panel of antibodies raised against peptides derived from the full length protein sequence of Cetuximab. Taken individually, each of these antibodies is strongly antigenic to the peptide sequence that was used in its production. However, when these peptides are incorporated into a full length correctly folded protein, the antigenicity of many of them is masked by the three dimensional structure of the protein and only a limited number of the antibodies respond. The result is a histogram which can be likened to a ‘fingerprint’ for correctly folded Cetuximab. For an Erbitux Biosimilar, if the protein is correctly folded and glycosylated, the ‘fingerprint’ will match that of Cetuximab. If it is not correctly folded, previously masked peptide sequences will be exposed and will be recognized by the antibody made to that exposed sequence. In this way, changes in the ‘fingerprint’ generated by the ELISA will point out differences between the Biosimilar and authentic Cetuximab.

The assay is performed by making a 5 μg/mL solution of Erbitux Biosimilar and Cetuximab reference material respectively, and adding to the 96-well plate. Following a 1 hour incubation to allow capture of the Biosimilar and Cetuximab reference proteins by the panel of antibodies on the plate, a reporting polyclonal anti-human IgG antibody, conjugated with biotin, is added and incubated for 1 hour to allow it to bind to any captured proteins. After this incubation, the plate is washed and a Streptavidin-HRP (Horse Radish Peroxidase) conjugate is added and incubated for 45 minutes. The Streptavidin-HRP conjugate will be captured by any biotin labeled antibody bound to the plate. Following a wash step to remove unbound conjugate, TMB substrate is added and is converted by the captured HRP to a colored product in proportion to the amount of HRP bound to the plate. After a short incubation to allow color development, the reaction is stopped and the intensity of the generated color is detected in a microtiter plate reader capable of measuring 450nm wavelength. The color development will be proportional to the captured Biosimilar or Cetuximab reference protein. A typical ELISA with only the Cetuximab reference protein is shown in figures 1 and 2 below. Your results may differ from this as your source sample will not be the same one that we used to generate this plot.

Cetuximab Conformational Array ELISA from Variable Region

Figure 1. Cetuximab Conformational Array ELISA from Variable Region

 

Cetuximab Conformational Array ELISA from ConstantRegion

Figure 2. Cetuximab Conformational Array ELISA from ConstantRegion

 

Supplied Components

Coated Clear 96-Well Plates
3 clear plastic microtiter plates coated with the panel of antibodies against Cetuximab peptides. Plate 1 covers the antibody variable region; plates 2 and 3 cover the antibody constant region.
Kit AB-00202 (3 plates)

5x Dilution Buffer
Buffer used for dilution of antibodies and Streptavidin-HRP conjugate. The 20 mL of concentrate should be diluted to 100 mL with 80 mL deionized or distilled water.
Kit AB-00202 (20 mL)

10x PBS-T
After dilution, it is used as wash solution. The 50 mL of concentrate should be diluted to 500 mL with 450 mL deionized or distilled water.
Kit AB-00202 (50 mL)

Reporting antibody
A biotin labeled rabbit polyclonal antibody against human IgGs. Immediately prior to the assay, dilute the entire 150 µL into 30 mL of 1x Dilution buffer to give a 5 µg/mL working stock.
Kit AB-00202 (1 mg/mL, 150 μL / tube)

Streptavidin-HRP Conjugate
A Streptavidin – Horse Radish Peroxidase conjugate in a special stabilizing solution. Immediately prior to the assay, dilute the entire 750 µL into 30 mL of 1x Dilution buffer to give a 0.1 µg/mL working stock.
Kit AB-00202 (4 μg/mL, 750 μL / tube)

TMB Substrate
Use directly without dilution.
Kit AB-00202 (30 mL)

Stop Solution
A 1M solution of sulfuric acid. CAUSTIC. Use directly without dilution.
Kit AB-00202 (30 mL)

Plate Sealer
Kit AB-00202 (three)

Other Materials Required
Distilled or deionized water.
Single- and multi-channel micro-pipettes with disposable tips to accurately dispense volumes 5-250 μL.
Plastic tubes (i.e. 1.5 mL – 15 mL) for sample dilution
Reagent reservoirs for sample addition
Colorimetric 96 well microplate reader capable of reading optical density at 450 nm.

Precautions

As with all such products, this kit should only be used by qualified personnel who have had laboratory safety instruction. The complete insert should be read and understood before attempting to use the product.

This kit utilizes a peroxidase-based readout system. Buffers, including other manufacturers Wash Buffers, containing sodium azide will inhibit color production from the enzyme. Make sure all buffers used for samples are azide free. Ensure that any plate washing system is rinsed well with deionized water prior to using the supplied Wash Buffer as prepared (above).

The Stop Solution is acid. The solution should not come in contact with skin or eyes. Take appropriate precautions when handling this reagent.

Procedural Notes

Allow diluted reagents and buffers to reach room temperature (18-25°C) prior to starting the assay. Once the assay has been started, all steps should be completed in sequence and without interruption. You do not want the plate to dry out in between steps as this can cause high backgrounds or erroneous results. Make sure that required reagents and buffers are ready when needed. Prior to adding to the plate, reagents should be mixed gently (not vortexed) by swirling.

Avoid contamination of reagents, pipette tips and wells. Use new disposable tips and reservoirs, do not return unused reagent to the stock bottles/vials and do not mix caps of stock solutions.

Incubation time can affect results. All wells should be handled in the same order for each step.

Microplate washing is important and can affect results by giving erroneous results or high backgrounds. We recommend a multichannel pipette to add 250 μL of buffer to each well across the plate, followed by a dumping out of contents (to a sink or other receptacle) with a rapid wrist motion. The plate should then be tapped firmly on a paper towel to shake out any remaining liquid. Avoid prolonged incubation with wash buffer when performing wash steps.

When making additions to the plate, be careful to avoid damaging the antibody coating, for example by scratching the bottoms or the sides of the wells. One technique to avoid this is to make additions (for a right-handed person) from left to right across the plate, supporting the pipette tips on the right edge of the well with each addition and thus avoiding contact with the bottom or sides of the wells.

During the incubation times, the plate should be covered to minimize evaporation from the wells. This can be done with the adhesive covers provided or by stacking an empty plate on top.

After the last wash step and prior to adding the TMB substrate, wipe the bottom of the plate with a clean paper towel to ensure that moisture or fingerprints do not interfere with the OD reading.

Once the TMB substrate is added it will be converted by the captured HRP to a blue colored product. Generally we find that 10 to 15 minute incubation is sufficient for enough color development to discern differences between the standards and the reaction should be stopped at this point. Bear in mind that, given sufficient time, even a small amount HRP is capable of converting all the TMB to product. Keeping OD450 values well below 2.0 will result in greatest accuracy as at high absorbance values very little light is reaching the detector and measurements are error prone. (Remember that at an OD of 1.0 only 10% of the light is being detected and at an OD of 2.0 only 1% of the light is reaching the detector).

Assay Protocol

  1. Use the plate layout sheet on the back page to plan sample layout on plate and also aid in proper sample and antibody identification after the assay. Each plate is laid out as shown on the plate maps on the following pages, with each unique antibody appearing in 6 positions on the plate. Rows A and H are not used in order to minimize edge effects. We recommend that assays are carried out in duplicate or (preferably) triplicate in order to minimize spurious results. For example, we have shown the plate layout for an experiment in triplicate, where the wells used for the control compound are highlighted and the three rows underneath are used for the test compound. For an experiment in duplicate, use rows B-C for the control and rows D-E and F-G for two test compounds.
     
  2. Dilute the 10xPBS-T and 5x Dilution buffer with water to 1x-strength. Check both concentrate bottles for precipitates before proceeding and if found warm slightly in a water bath to dissolve before proceeding. The 50 mL of 10xPBS-T should be diluted to 500 mL with 450 mL water and the 20 mL of 5x Dilution Buffer should be diluted to 100 mL with 80 mL water.
     
  3. Dilute your sample and Cetuximab standard to a concentration of 5 µg/mL; prepare at least 10 mL of each if samples are to be run in duplicate, 15 mL of each if run in triplicate. Pipette 100 μL of 5 μg/mL sample or Cetuximab standard into each row of the plate. For replicates use multiple rows, i.e. Cetuximab standard in rows 2-3, sample 1 in rows 4-5 and sample 2 in rows 6-7. Cover plates and incubate 1 hour at room temperature.
     
  4. During the above incubation, dilute the 1 mg/mL reporting antibody to 5 μg/mL by adding the entire 150 μL to 30 mL of Dilution Buffer.
     
  5. Wash plate by emptying contents and adding 250 μL of wash buffer to each well. Empty wells again and tap the plate firmly upside down on a paper towel to fully empty well. Repeat.
     
  6. Pipette 100 μL of 5 μg/mL Reporting Antibody into each well. Cover plate and incubate plate 1 hour at room temperature.
     
  7. During the above incubation, dilute the 4 µg/mL Streptavidin-HRP conjugate to 0.1 μg/mL by adding the entire 750 μL to 30 mL of Dilution Buffer.
     
  8. Wash plate by emptying contents and adding 250 μL of wash buffer to each well. Empty wells again and tap the plate firmly upside down on a paper towel to fully empty well. Repeat.
     
  9. Pipette 100 μL of 0.1 μg/mL Streptavidin-HRP conjugate into wells. Cover plate and incubate plate 45 min hour at room temperature.
     
  10. Wash plate by emptying contents and adding 250 μL of wash buffer to each well. Empty wells again and tap the plate firmly upside down on a paper towel to fully empty well. Repeat 2 more times.
     
  11. Add 100 μL of TMB substrate to each well. Allow color development to proceed for exactly 15 minutes and then stop reaction by adding 100 μL of Stop Solution to each well. Upon addition of stop solution, developed color will change from blue to yellow.
     
  12. Read the optical density generated from each well in a plate reader capable of reading at 450 nm, Use wells H10-H12 as blank.
     
  13. Export the plate reader data into Excel and calculate an average and variance for each set of replicates. If the variance is large inspect the raw data to determine the problem. With data in triplicate, one outlier may be evident, but if data is in duplicate, the higher value is generally suspect (it’s easier to get a high value in error than a low value). Graph the data as a bar graph so that for each array antibody the response can be compared between your sample and Cetuximab standard. Any differences between your sample and the Cetuximab standard should be apparent.
     

Plate 1 Template (variable region)

Control compound suggested use in wells marked

  1 2 3 4 5 6 7 8 9 10 11
12
A                        
B Ab1 Ab2 Ab3 Ab4 Ab5 Ab6 Ab7 Ab8 Ab9 Ab10 Ab11 Ab12
C Ab1 Ab2 Ab3 Ab4 Ab5 Ab6 Ab7 Ab8 Ab9 Ab10 Ab11 Ab12
D Ab1 Ab2 Ab3 Ab4 Ab5 Ab6 Ab7 Ab8 Ab9 Ab10 Ab11 Ab12
E Ab1 Ab2 Ab3 Ab4 Ab5 Ab6 Ab7 Ab8 Ab9 Ab10 Ab11 Ab12
F Ab1 Ab2 Ab3 Ab4 Ab5 Ab6 Ab7 Ab8 Ab9 Ab10 Ab11 Ab12
G Ab1 Ab2 Ab3 Ab4 Ab5 Ab6 Ab7 Ab8 Ab9 Ab10 Ab11 Ab12
H                        

Plate 2 Template (constant region-1)

Control compound suggested use in wells marked
 

  1 2 3 4 5 6 7 8 9 10 11
12
A                        
B Ab13 Ab14 Ab15 Ab16 Ab17 Ab18 Ab19 Ab20 Ab21 Ab22 Ab23 Ab24
C Ab13 Ab14 Ab15 Ab16 Ab17 Ab18 Ab19 Ab20 Ab21 Ab22 Ab23 Ab24
D Ab13 Ab14 Ab15 Ab16 Ab17 Ab18 Ab19 Ab20 Ab21 Ab22 Ab23 Ab24
E Ab13 Ab14 Ab15 Ab16
Ab17 Ab18 Ab19 Ab20 Ab21 Ab22 Ab23 Ab24
F Ab13 Ab14 Ab15 Ab16
Ab17 Ab18 Ab19 Ab20 Ab21 Ab22 Ab23 Ab24
G Ab13 Ab14 Ab15 Ab16
Ab17 Ab18 Ab19 Ab20 Ab21 Ab22 Ab23 Ab24
H                        

Plate 3 Template (constant region-2)

Control compound suggested use in wells marked
 

  1 2 3 4 5 6 7 8 9 10 11
12
A                        
B Ab25 Ab26 Ab27 Ab28 Ab29 Ab30 Ab31 Ab32 Ab33 Ab34    
C Ab25 Ab26 Ab27 Ab28 Ab29 Ab30 Ab31 Ab32 Ab33 Ab34    
D Ab25 Ab26 Ab27 Ab28 Ab29 Ab30 Ab31 Ab32 Ab33 Ab34    
E Ab25 Ab26 Ab27 Ab28 Ab29 Ab30 Ab31 Ab32 Ab33 Ab34    
F Ab25 Ab26 Ab27 Ab28 Ab29 Ab30 Ab31 Ab32 Ab33 Ab34    
G Ab25 Ab26 Ab27 Ab28 Ab29 Ab30 Ab31 Ab32 Ab33 Ab34    
H                        

Materials

     
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