Host Cell Proteins Select Detection Kit, E. coli system

ELISA kit for the measurement of CHO host cell proteins.

Detection kit for the determination of E. coli 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

96-well strip plate coated with 4 different antibodies;
E. coli Protein Standard;
5x Dilution Buffer;
10x PBS-T;
4 Reporting Antibodies;
Streptavidin-HRP Conjugate;
TMB Substrate;
Stop Solution;
Plate Sealer;

Bacterial

E. coli

E. coli

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 30 ml of 10x PBS-T should be diluted to 300 ml with 270 ml milliQ water. 15 ml of 5x Dilution Buffer should be diluted to 75 ml with 60 ml of milliQ water.
2. Prepare the HCP standards by numbering eight 1.5 ml tubes and adding 1ml of Dilution Buffer to each. Cap the eighth tube, this will be the blank (0 ng/ml HCP). To tube one, add 500 μl of the provided 2430 ng/ml HCP stock and mix well, this will be the 810 ng/ml standard. Then serially dilute 500 ml of tube one across tubes two through seven to generate the remainder of the standards. Prepare all four sets of standards before additing to plate. Pipette 100 μl of each standard and the blank into the plate.
3. Dilute samples in 1x dilution buffer (for screening purposes, it is recommended to test the drug substance and post-Protein A column fraction in duplicates); for best accuracy perform serial dilutions over a wide range such that multiple dilutions will span the range of 0.1 - 300 ng /ml. Pipette 100 μL of samples or standards into wells in the plate. Leave several wells empty for background binding determination.
4. Cover plate with individual plate seal and incubate 1.5 hours at room temperature.
5. During the above incubation, dilute the Reporting Antibody by adding 50 μl to 5 ml of 1x Dilution Buffer.
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. Care should be taken to match the reporting antibody to the corresponding capturing antibody (e.g. C1 area adds R1 reporting antibody). Cover plate with the plate seal and incubate plate 45 minutes at room temperature.
8. During the above incubation, dilute the 4 μg/ml Streptavidin-HRP conjugate to 0.1 μg/ml by adding 375 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.
13. Stop reaction by adding 100 μl of Stop Solution to each well containing TMB when the color development within standards is sufficient.
14. 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 E. coli protein standards and used to calculate the concentration of E. coli proteins in the unknown samples, taking into account any unknown sample dilution made.
12. Add 100 μl of TMB substrate to each well. Monitor color development. Generally 10 minutes time will be sufficient; incubating longer may increase the background.