KRIBIOLISA™ Pichia Pastoris HCP ELISA

SKU: KBBP02 Category:

₹25,000.00

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


Availability: Backorder | 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 P.pastoris. The manufacturing and purification process of these products tends to leave the potential for contamination by Host Cell Proteins (HCPs) from P.pastoris 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 KRIBIOLISA Pichia Pastoris HCP ELISA is a generic kit intended in determining the presence of P. pastoris Host Cell Proteins contamination in various biotechnology products that are manufactured through recombinant expression in P. pastoris. The kit has been validated successfully for testing of in process and final product HCPs in variety of products regardless of growth and purification process.

Principle:
This assay is based on the Sandwich ELISA procedure. Samples containing P.pastoris HCPs are reacted with already coated affinity purified capture anti-P.pastoris antibody and bind to them. Plates are washed with wash buffer to remove unbound reactants. Anti P.pastoris-HRP conjugate is added which results in the formation of a sandwich complex of solid phase antibody-HCP-enzyme labeled antibody. The wells are washed to remove any unbound reactants as per the wash procedure. The substrate Tetra Methyl 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 P. pastoris HCPs present.

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Additional information

Sample Type

Cell Culture Supernatant and Other Biological Preparations
Calibration Range

15.6 ng/ml – 1000 ng/ml
Sensitivity

15 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.

Protocol

Assay Procedure:
Bring all reagents to room temperature prior to use. It is strongly recommended that all standards and samples be run in duplicate or triplicate. A standard curve is required for each assay.
1. Pipette 100 ul of Standards or Samples into the respective wells.
2. Cover the plate and incubate at 37?C for 2 hours 30 minutes.
3. Aspirate and wash plate 4 times with 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. Add 100 ul of Anti-P.pastoris:HRP Conjugate into each well.
5. Cover the plate and incubate at 37?C for 1 hour.
6. Aspirate and wash plate 4 times with 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.
7. Add 100 ul of TMB Substrate in each well.
8. Incubate the plate at 37?C for 15 minutes in dark. DO NOT SHAKE or else it may result in higher backgrounds and worse precision. Positive wells should turn bluish in color.
9. Pipette out 100 ul of Stop Solution. Wells should turn from blue to yellow in color.
10. Read the absorbance at 450 nm with a microplate reader within 30 minutes.

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