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Host Cell Protein (HCP) Assay: Antibody Coverage
Host Cell Protein (HCP) Assay: Product Purity

 

 

 

Click to view a complete list of Applied Biomics HCP assays

Applied Biomics Host Cell Protein (HCP) assays using 2D DIGE followed by fluorescent Western blot offer significant ADVANTAGES over the ELISA and standard 2D Western blot, with the following applications:

1) Characterizations of the HCP antibodies
2) Detecting HCP contaminants originated from cells that are used to manufacture therapeutic proteins and vaccines

Experimental Procedure: HCP 2D Western Blot (HEK293)
Example-1: CHO Cell HCP Assay
Example-2: E.coli HCP Assay
Example-3: E.coli High Mw HCP Assay
Example-4: Yeast (Saccharomyces cerevisiae) HCP Assay
Example-5: Characterization of SF9-HCP antibodies from same 2D gel

Advantages of DIGE over ELISA and Standard 2D in HCP Detection

Applied Biomics will perform HCP assays using HCP antibodies provided by customers. In addition, we cover the following HCP antibodies:

Bacteria Mammalian Cell Yeast Insect Others

E.coli HCP
Lactococcus lactis HCP





Vero Cell HCP
CHO HCP
BHK HCP
HEK293 HCP
A549 HCP
MRC5 HCP
PER.C6 HCP

Saccharomyces cerevisiae HCP
Pichia pastoris HCP





SF9 Insect Cell HCP
SF22 Insect Cell HCP





Bovine Serum Proteins
Mycobacterium Proteins
Goat-Milk Proteins




 

Experimental Procedure: HCP 2D Western Blot (HEK293)

 

 

 

protein labeling

Protein extraction
Protein assay
Protein labeling

 

HCP 2D gel

2D Gel image:
HEK293-Protein











Gel transfer to Membrane



HCP 2D Membrane


2D Membrane image:
HEK293-Protein



HCP 2D Membrane


2D Membrane image:
Anti-HEK293 HCP
WB Image



HCP image overlay


Image Overlay:
Green: HEK293-Protein
Red: Anti-HEK293 HCP


Spot Matching and Quantitation by DeCyder (100% matching, no ambiguity)


HEK293-HCP Protein Image

HCP Protein

Anti-HEK293 HCP WB Image

HCP Western Blot

Brown: 1949 reacting spots
Blue: 109 non-reacting spots (only present in protein image)
Ab Coverage of Anti-HEK293 HCP: 94.7% of 2058 spots from 293 HCP


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Example 1: Characterization of the CHO-HCP Antibody by 2D Western blot from the same 2D gel

 

CHO-CHP 2D Gel Image

CHO Protein

Anti-CHO HCP 2D WB Image

Anti-CHO HCP Western Blot

Image Overlay: Green: CHO-HCP  Red: Western Blot

CHO HCP Anti-CHO HCP image overlay

CHO-HCP Protein Image

CHO HCP Protein

Anti-CHO HCP WB Image

Anti-CHO HCP Western Blot

Brown: 2856 reacting spots
Blue: 98 non-reacting spots (only present in protein image)
Ab Coverage of Anti-CHO HCP: 96.68% of 2954 spots from CHO HCP

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Example 2: Characterization of E.coli-HCP antibodies from the same 2D gel

 

E.coli-Protein Image

E. Coli Protein

Anti-E.coli HCP WB Image

Anti-Ecoli HCP Western Blot

E. Coli-Protein Image

E.Coli protein

Anti-E. Coli HCP WB Image

Anti-Ecoli HCP Western Blot

Image Overlay: Green: E. Coli-HCP  Red: Anti-E.Coli HCP

E.coli HCP Anti-E. Coli HCP image overlay

E.coli-HCP Protein Image

E.Coli HCP Protein

Anti-E.coli HCP WB Image

Anti-E.Coli HCP Western Blot

Brown: 2220 reacting spots
Blue: 142 non-reacting spots (only present in protein image)
Ab Coverage of Anti-E. Coli HCP: 94% of 2362 spots from E. Coli HCP

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Example 3: Characterization of High-MW E.coli-HCP antibodies

 

E. Coli-Protein Image

E. Coli protein

Anti-E. Coli HCP WB Image

Anti-E. Coli HCP Western Blot

Image Overlay: Green: E. Coli-HCP  Red: Anti-E.Coli HCP

E. Coli-HCP Anti-E. Coli HCP image overlay

E.coli-HCP Protein Image

E. Coli-HCP protein

Anti-E.coli HCP WB Image

Anti-E. Coli HCP Western Blot

Brown: 1030 reacting spots
Blue: 3 non-reacting spots (only present in protein image)
Ab Coverage of Anti-E.coli high Mw HCP : 99.7% of 1033 High Mw spots from E.coli HCP.

 

Example 4: Characterization of Yeast (Saccharomyces cerevisiae)-HCP antibodies from same 2D gel

 

Yeast-Protein Image

Yeast

Anti-Yeast HCP WB Image

Anti-Yeast HCP

Yeast-Protein Image

Yeast Protein

Anti-Yeast HCP WB Image

Anti-Yeast HCP Western Blot

Image Overlay: Green: Yeast-HCP  Red: Western Blot

Yeast HCP Western Blot

Yeast Protein Image

HCP Yeast Protein

Anti-Yeast HCP WB Image

Anti-Yeast HCP Western Blot

Brown color: reacting spots (3146 spots);
Blue circle: not reacted spots (55 spots, only on protein image)
Ab Coverage of Anti-Yeast HCP: 98.3% of of 3201 spots from Yeast (Saccharomyces cerevisiae )-HCP reacted with antibodies

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Example 5: Characterization of SF9-HCP antibodies from same 2D gel

 

Protein 2D Image On Transferred Membrane

Protein 2D Image on Transferred Membrance

SF9-HCP Western Blot

SF9-HCP Western Blot

SF9-HCP 2D Gel image

SF9-HCP 2D Gel image

SF9-HCP 2D Western Blot

SF9-HCP 2D Western Blot

Image Overlay: Green: SF9-HCP  Red: Western Blot

SF9-HCP Western Blot image overlay

 

Analysis the SF9-HCP antibodies coverage by 2D Western blot



Brown color: reacting spots (2863 spots);
Blue circle: not reacted spots (152 spots, only on protein image)
Ab Coverage of SF9- HCP: 94.96% of of 3015 spots from SF9-HCP reacted with antibodies

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