Host Cell Proteins Detection Kit, Pichia system

ELISA kit for the measurement of CHO host cell proteins.

Detection kit for the determination of Pichia host cell protein contamination bulk products. Based on the "sandwich"ELISA methods, it with quick and high sensitivity analysis.

Detect and quantify HCP concentrations at any point in your purification process;
ELISA-based: quick, familiar format with high sensitivity;
96-well plate coated with capture antibody;
HCP standards, reporting antibody, streptavidin-HRP conjugate, TMB substrate, buffers included;

Host Cell Proteins Detection

The detection kit is based off of "sandwich"ELISA methods. Host Cell Protein antibodies are immobilized to 96 well plates. Samples are added, and Host Cell Proteins are bound by the antibodies. Biotinylated Host Cell Protein antibody is then added, followed by strepavidin HRP, which binds to biotin. TMB substrate is then added, and HRP catalyzes the conversion of TMB to a colored product. The reaction is then stopped and read on a standard colorimetric plate reader.

96 reactions

Coated 96-Well Strip Plate;
Pichia Protein Standard;
Dilution Buffer;
10x PBS-T;
Reporting Antibody;
Streptavidin-HRP Conjugate;
TMB Substrate;
Stop Solution;
Plate Sealer;

Yeast

Pichia

P. pastoris

The detection kit is based off of "sandwich"ELISA methods. Host Cell Protein antibodies are immobilized to 96 well plates. Samples are added, and Host Cell Proteins are bound by the antibodies. Biotinylated Host Cell Protein antibody is then added, followed by strepavidin HRP, which binds to biotin. TMB substrate is then added, and HRP catalyzes the conversion of TMB to a colored product. The reaction is then stopped and read on a standard colorimetric plate reader.

96-well plate

Store at 4°C.

Sodium azide will interfere with this assay and should not be used in samples or buffers.

Gel Packs

1. Dilute the 10x PBS-T and 5x Dilution Buffer to 1x-strength with milliQ water. The 25 ml of 10x PBS-T should be diluted to 250 ml with 225 ml milliQ water.
2. Prepare the reporting antibody by adding 150 µl of reporting antibody to 15 ml of 1x Dilution Buffer. Incubate 1.5 hours at room temperature before proceeding to step 3. Continue to incubate for an additional 1.5 hours concurrent with step 5, for a total incubation time of 3 hours.
3. Prepare the HCP standards by numbering eight 1.5 ml tubes, and add 1168 µl of 1x Dilution Buffer to tube 1 and 800 µl of 1x Dilution Buffer to tubes 2-8. Cap the eighth tube, this will be the blank (0 ng/ml HCP). To tube one add 32 μl of the provided 1000 µg/ml HCP stock and mix well, this will be the 27 µg/ml standard. Then serially dilute 400 µl of tube one across tubes two through seven to generate the remainder of the standards. Pipette 100 μl of each standard and the blank into the plate.
4. Pipette 100 μl of samples into wells in the plate. If necessary, first dilute the samples in 1x Dilution Buffer.
5. Cover plate with individual plate seal and incubate 1.5 hours at room temperature.
6. Wash plate by emptying contents and adding 250 μl of 1x PBS-T to each well. Empty wells again and tap the plate firmly upside down on a paper towel to fully empty well. Repeat 1x PBS-T wash step two additional times.
7. Pipette 100 μl of Reporting Antibody into each well. Cover plate with the plate seal and incubate plate 45 minutes at room temperature.
8. During the above incubation, dilute the 40 μg/ml Streptavidin-HRP conjugate to 0.4 μg/ml by adding 150 μl to 15 ml of 1x Dilution Buffer.
9. Wash plate by emptying contents and adding 250 μl of 1x PBS-T to each well. Empty wells again and tap the plate firmly upside down on a paper towel to fully empty well. Repeat 1x PBS-T wash step two additional times.
10. Pipette 100 μl of Streptavidin-HRP conjugate into wells. Cover plate and incubate plate 30 minutes at room temperature.1. Dilute the 10x PBS-T and 5x Dilution Buffer to 1x-strength with milliQ water. The 25 ml of 10x PBS-T should be diluted to 250 ml with 225 ml milliQ water.
2. Prepare the reporting antibody by adding 150 µl of reporting antibody to 15 ml of 1x Dilution Buffer. Incubate 1.5 hours at room temperature before proceeding to step 3. Continue to incubate for an additional 1.5 hours concurrent with step 5, for a total incubation time of 3 hours.
3. Prepare the HCP standards by numbering eight 1.5 ml tubes, and add 1168 µl of 1x Dilution Buffer to tube 1 and 800 µl of 1x Dilution Buffer to tubes 2-8. Cap the eighth tube, this will be the blank (0 ng/ml HCP). To tube one add 32 μl of the provided 1000 µg/ml HCP stock and mix well, this will be the 27 µg/ml standard. Then serially dilute 400 µl of tube one across tubes two through seven to generate the remainder of the standards. Pipette 100 μl of each standard and the blank into the plate.
4. Pipette 100 μl of samples into wells in the plate. If necessary, first dilute the samples in 1x Dilution Buffer.
5. Cover plate with individual plate seal and incubate 1.5 hours at room temperature.
6. Wash plate by emptying contents and adding 250 μl of 1x PBS-T to each well. Empty wells again and tap the plate firmly upside down on a paper towel to fully empty well. Repeat 1x PBS-T wash step two additional times.
7. Pipette 100 μl of Reporting Antibody into each well. Cover plate with the plate seal and incubate plate 45 minutes at room temperature.
8. During the above incubation, dilute the 40 μg/ml Streptavidin-HRP conjugate to 0.4 μg/ml by adding 150 μl to 15 ml of 1x Dilution Buffer.
9. Wash plate by emptying contents and adding 250 μl of 1x PBS-T to each well. Empty wells again and tap the plate firmly upside down on a paper towel to fully empty well. Repeat 1x PBS-T wash step two additional times.
10. Pipette 100 μl of Streptavidin-HRP conjugate into wells. Cover plate and incubate plate 30 minutes at room temperature.
11. Wash plate by emptying contents and adding 250 μl of 1x PBS-T to each well. Empty wells again and tap the plate firmly upside down on a paper towel to fully empty well. Repeat 1x PBS-T wash step two additional times.
12. Add 100 μl of TMB substrate to each well. Monitor color development. Generally 10-15 minutes will be sufficient; incubating longer may increase the background.
14. Stop reaction by adding 100 μl of Stop Solution to each well containing TMB when the color development within standards is sufficient.
15. Read the optical density generated from each well in a plate reader capable of reading at 450 nm. A standard curve should be generated from the Pichia protein standards and used to calculate the concentration of Pichia proteins in the unknown samples, taking into account any unknown sample dilution made.
11. Wash plate by emptying contents and adding 250 μl of 1x PBS-T to each well. Empty wells again and tap the plate firmly upside down on a paper towel to fully empty well. Repeat 1x PBS-T wash step two additional times.
12. Add 100 μl of TMB substrate to each well. Monitor color development. Generally 10-15 minutes will be sufficient; incubating longer may increase the background.
14. Stop reaction by adding 100 μl of Stop Solution to each well containing TMB when the color development within standards is sufficient.
15. Read the optical density generated from each well in a plate reader capable of reading at 450 nm. A standard curve should be generated from the Pichia protein standards and used to calculate the concentration of Pichia proteins in the unknown samples, taking into account any unknown sample dilution made.