KRIBIOLISA™ E.coli HCP ELISA

SKU: KBBP01 Category:

₹25,000.00

Immunoassay for the quantitative measurement of E.coli Host Cell Proteins as a contaminant in various biological preparations.


Availability: 4 – 5 days | Pack Size: 1 x 96 wells



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Description

Introduction:
A variety of proteins and pDNA which are used as therapeutic agents in humans and animals are produced through recombinant expression in E.coli. The manufacturing and purification process of these products tends to leave the potential for contamination by Host Cell Proteins (HCPs) from E.coli which may result in adverse toxic or immunological reactions that ultimately affect the efficacy of the therapeutic agent. The simple, objective and semi-quantitative ELISA is a highly sensitive method that aids in purification process development, process control, quality control and product release testing optimally.

Intended Use:
This generic kit is intended in determining the presence of E.coli Host Cell Proteins contamination in various products that are manufactured through recombinant expression in E.coli. The kit has been validated successfully for testing of final and in process product HCPs in variety of products regardless of growth and purification process.

Principle:
This assay is based on the Sandwich ELISA procedure. Samples containing E-coli HCPs are reacted with antiE.coli:HRP antibody simultaneously in the microtiter wells already coated with affinity purified capture anti-E.coli antibody. This immunological reaction results in formation of a sandwich complex of solid phase antibody-HCPenzyme labeled antibody. The wells are washed to remove any unbound reactants as per the wash procedure (see Assay procedure section mentioned below). The substrate Tetramethyl Benzidine is then reacted. The amount of hydrolyzed substrate is read on a microtiter plate reader and it is directly proportional to the concentration of E.coli HCPs present.

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Additional information

Sample Type

Cell Culture Supernatant and Other Biological Preparations
Calibration Range

3.125 – 200 ng/ml
Sensitivity

1.875 ng/ml
Detection Method

Colorimetric
ELISA Type

Direct Sandwich Assay
Regulatory Status

For Research Use Only
Storage Temperature

Store the unopened product at 2-8 Deg C. Do not use past expiration date.

Citations

Protocol

Assay Procedure:
It is strongly recommended that all Standards and Samples be run in duplicates or triplicates. A standard curve is required for each assay.
1. Add 100 ul prepared Standards and diluted Samples to respective wells.
2. Cover the plate with a sealer and incubate for 90 minutes at 37?C.
3. Aspirate and wash plate 4 times with diluted Wash Buffer (1X) and blot residual buffer by firmly tapping plate upside down on absorbent paper. Wipe of any liquid from the bottom outside of the microtiter wells as any residue can interfere in the reading step.
4. Pipette 100 ul Biotinylated E.coli HCP Antibody Working Solution to all wells.
5. Cover the plate with a sealer and incubate for 60 minutes at 37?C.
6. Aspirate and wash as per Step (4) above.
7. Pipette 100 ul Streptavidin:HRP Conjugate Working Solution to all wells. Mix well.
8. Cover the plate with a sealer and incubate for 30 minutes at 37?C.
9. Aspirate and wash as per Step (4) above.
10. Pipette 100 ul TMB Substrate in all the wells.
11. Incubate the plate at 37?C for 20 minutes. DO NOT SHAKE or else it may result in higher backgrounds and worse precision. Positive wells should turn bluish in color.
12. Pipette 100 ul of Stop Solution to all wells. The wells should turn from blue to yellow in color.
13. Read the absorbance at 450 nm with a microplate within 10-15 minutes after addition of Stop solution.

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