HCP Antibody Coverage

Applied Biomics offers the most comprehensive HCP Antibody Coverage analysis by using high resolution large format 2D fluorescent Western Blot. Our report provides HCP, Western blot and HCP/Western overlay images, allowing the direct visualization of antibody coverage that no other platform could achieve. In addition, the HCP antibody coverage is calculated with high consistency and accuracy (see details below). This platform has been successfully applied in the following areas:

Characterizing HCP antibody coverage
Detecting HCP contaminants originated from cells that are used to manufacture therapeutic proteins and vaccines

**Our HCP Antibody Coverage platform offers significant advantages over ELISA, Antibody affinity extraction* and standard 2D Western blot:**

High consistency: Perfect alignment of HCP and WB images from the same gel and same membrane, eliminating any gel-to-gel or gel-to-membrane variations as seen in the traditional 2D WBs.
Cost-effective: Since only one gel is needed, it saves the cost of running duplicate gels.
High accuracy: Fluorescent CyDye labeling allows accurate quantitation of protein and WB spots.
High sensitivity: Fluorescent CyDye labeling has a higher sensitivity and wider dynamic range over the silver staining.

	ELISA	Standard 2D Western Blot	2D DIGE Western Blot
Number of gels for each Ab		Two	One
Detection Method		Silver Staining	CyDye Labeling
Detect HCP Composition
Detect protein modification
Detect protein degradation
Protein and WB in same gel
In-gel protein and WB comparison
Accuracy	Low	Low	High
	Inaccurate: In-direct reaction of antibodies with the antigen’s associated proteins	Inaccurate: Protein spots counted from the gel, NOT from membrane	Accurate: Protein spots counted directly from the membrane
	False negative: Coated Ab does not capture all HCPs Surface denaturation of coating Ab can affect detection	Error caused by: Gel to gel variation Gel to membrane variation	Accurate: Protein and WesternBlot image on the same gel, avoiding any ambiguity
Consistency & Reproducibility	Lower	Lower	Higher
*AAE, based on antibody immuno-binding with antigen under the native condition, has limitations in quantitating antibody coverage: False positive: a) direct and indirect associate proteins; b) antigens present in the HCP antibody; c) co-purified serum proteins False negative: a) associate proteins as a big complex will block antibody binding; b) some binding HCPs or their degraded fragments cannot be eluted Not all HCP proteins/fragments can be affinity purified, and the HCP profile is totally changed after AAE In addition, AAE still need be coupled with downstream LCMS or 2D Western blot, the lengthy process introduces more variations and inconsistency.

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

Bacteria	Mammalian Cell	Yeast	Insect	Others
E.coli Lactococcus lactis	Vero Cell CHO BHK HEK293 A549 MRC5 PER.C6	Saccharomyces cerevisiae Pichia pastoris	SF9 Insect Cell SF22 Insect Cell	Bovine Serum Proteins Mycobacterium Proteins Goat-Milk Proteins

Experimental Procedure: HEK293 HCP Antibody Coverage by 2D Western Blot

Protein extraction
Protein assay
Protein labeling

HEK293 HCP antibody coverage procedure: step 2 - HEK293 HCP 2D gel image

2D Gel image:
HEK293 HCP

Gel transfer to Membrane

HEK293 HCP antibody coverage procedure: step 4 - HEK293 HCP 2D image on transferred membrane

2D Membrane image:
HEK293 HCP

HEK293 HCP antibody coverage procedure: step 5 - Anti-HEK293 HCP Western Blot image on membrane

2D Membrane image:
Anti-HEK293 HCP Western Blot

HEK293 HCP antibody coverage procedure: step 6 - overlay image of HEK293 HCP & Anti-HEK293 HCP Western Blot on membrane

Image Overlay:
HEK293 HCP /
Anti-HEK293 HCP Western Blot

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

HEK293 HCP spot map

Anti-HEK293 HCP Western Blot spot map

Brown: 2051 spots detected by anti-HEK293 HCP antibody
Blue: 149 spots not detected by anti-HEK293 HCP antibody (present in HEK293 HCP image only)
Anti-HEK293 HCP antibody coverage: 93.2% of 2200 HEK293 HCP spots

HCP Antibody Coverage Application Gallery

Example	HCP Antibody Coverage Applications
#1	CHO HCP Antibody Coverage
#2	HEK293 HCP Antibody Coverage
#3	E. coli HCP Antibody Coverage
#4	E. coli High Mw HCP Antibody Coverage
#5	Yeast HCP Antibody Coverage
#6	SF9 HCP Antibody Coverage

1. CHO HCP antibody coverage by 2D Western Blot

CHO HCP 2D Gel

Anti-CHO HCP 2D WB

CHO HCP / Western Blot

CHO HCP spot map

Anti-CHO HCP WB spot map

Brown: 2389 spots detected by anti-CHO HCP antibody
Blue: 170 spots not detected by anti-CHO HCP antibody (present in CHO HCP image only)
Anti-CHO HCP antibody coverage: 93.4% of 2559 CHO HCP spots

2. HEK293 HCP antibody coverage by 2D Western Blot

HEK293 HCP

Anti-HEK293 HCP 2D WB

HEK293 HCP / Western Blot

HEK293 HCP antibody coverage by 2D Western Blot: 2D Overlay Image

HEK293 HCP spot map

Anti-HEK293 HCP WB spot map

HEK293 HCP antibody coverage by 2D Western Blot: Anti-HEK293 HCP spot map

Brown: 2199 spots detected by anti-HEK293 HCP antibody
Blue: 45 spots not detected by anti-HEK293 HCP antibody (present in HEK293 HCP image only)
Anti-HEK293 HCP antibody coverage: 98.1% of 2352 HEK293 HCP spots

3. E. coli HCP antibody coverage by 2D Western Blot

E. coli HCP 2D Gel

Anti E. coli HCP 2D Western Blot

E. coli HCP antibody coverage by 2D Western Blot: Anti-E. coli HCP Western Blot image in black/white

E. Coli HCP 2D Gel

Anti-E. Coli HCP 2D WB

E. Coli HCP / Western Blot

E. coli HCP spot map

Anti-E. coli HCP WB spot map

E. coli HCP antibody coverage by 2D Western Blot: Anti-E. coli HCP Western Blot spot map

Brown: 2226 spots detected by anti-E.coli HCP antibody
Blue: 31 spots not detected by anti-E.coli HCP antibody (present in E.coli HCP image only)
Anti-E. Coli HCP antibody coverage: 99.6% of 2235 E. Coli HCP spots

4. E. coli High Mw HCP antibody coverage by 2D Western Blot

E. Coli HCP 2D Gel

Anti-E. Coli HCP 2D WB

E. Coli HCP / Western Blot

E. coli HCP spot map

E. coli High Mw HCP antibody coverage by 2D Western Blot: E. coli High Mw HCP spot map

Anti-E.coli HCP WB spot map

E. coli High Mw HCP antibody coverage by 2D Western Blot: Anti-E. coli High Mw HCP Western Blot spot map

Brown: 933 spots detected by anti-E.coli HCP antibody
Blue: 3 spots not detected by anti-E.coli HCP antibody (present in E.coli HCP image only)
Anti-E.coli High Mw HCP antibody coverage: 99.7% of 936 high molecular weight E.coli HCP spots

5. Yeast (Saccharomyces cerevisiae) HCP antibody coverage by 2D Western Blot

Yeast HCP 2D Gel

Anti-Yeast HCP 2D WB

Yeast HCP antibody coverage by 2D Western Blot: Anti-Yeast HCP Western Blot image in black/white

Yeast HCP 2D Gel

Anti-Yeast HCP 2D WB

Yeast HCP / Western Blot

Yeast HCP Spot Map

Anti-Yeast HCP WB Spot Map

Yeast HCP antibody coverage by 2D Western Blot: Anti-Yeast HCP Western Blot spot map

Brown: 2400 spots detected by anti-Yeast HCP antibody
Blue: 2 spots not detected by anti-Yeast HCP antibody (present in Yeast HCP image only)
Anti-Yeast HCP antibody coverage: 99.9% of 2402 Yeast (Saccharomyces cerevisiae ) HCP spots

6. SF9 HCP antibody coverage by 2D Western Blot

SF9 HCP 2D Membrane

SF9 HCP antibody coverage by 2D Western Blot: SF9 HCP 2D Gel image in black/white

SF9 HCP 2D Western Blot

SF9 HCP antibody coverage by 2D Western Blot: Anti-SF9 HCP Western Blot image in black/white

SF9 HCP 2D Gel

SF9 HCP 2D Western Blot

SF9 HCP / Western Blot

SF9 HCP antibody coverage by 2D Western Blot: overlay image of SF9 HCP & anti-Yeast HCP Western Blot

SF9 HCP spot map

Anti-SF9 HCP WB spot map

SF9 HCP antibody coverage by 2D Western Blot: Anti-SF9 HCP Western Blot spot map

Brown: 2623 spots detected by anti-SF9 HCP antibody
Blue: 285 spots not detected by anti-SF9 HCP antibody (present in SF9 HCP image only)
Anti-SF9 HCP antibody coverage: 90.2% of 2908 SF9 HCP spots

Sample Info

Please follow these general principles regarding biohazardous materials and buffer condition in getting your samples ready.

Protein amount for each WB: 100-500 µg of total protein from each sample.

Protein concentration: 5-20 mg/ml is preferred.

Primary antibody requirement for each WB:

Sufficient for 100ml incubation volume, and need information on:

Animal species in which the antibody was raised
Suggested dilution factor

If you cannot provide the dilution factor, please let us know:

If it is a commercial antibody, provide the link to the antibody information
If it is not a commercial antibody, please provide the protein concentration of the antibody, whether it is IgG fraction or anti-sera or antigen-affinity purified.

Buffer: Please feel free to submit samples in whichever buffer that you would normally use. The standard sample preparation is covered by the DIGE gel cost. Such samples should have protein concentration in the range of 5-20 mg/ml. Additional charge may apply on samples that require extensive amount of the extra work such as protein eluting, concentrating, buffer exchange or serum abundant protein depletion.

Sample type: Please inform our scientists about your sample type. We will send you additional tips for each sample type.

Pricing

Services Description	Academic/Governmental Labs		Industrial/Commercial Labs
Services Description	Code	Price	Code	Price
2D Gel with 1 CyDye Labeling 1	401A	$839	401N	$999
2D DIGE Gel with 2 CyDye Labeling 1	101A2	$1200	101N2	$1500
Transfer to PVDF / Nitrocellulose Membrane 2	520A	$250	520N	$295
Western Blot 3	512A	$410	512N	$495
HCP Quantitative Analysis	202A-HCP	TBD	202N-HCP	TBD

The following primary antibodies will be provided by Applied Biomics at $150-250 for each Western blot. Customers are responsible for providing the other primary antibodies not listed below.

Anti-Bacteria HCP antibody: E.coli; Lactococcus lactis
Anti-Mammalian Cell HCP antibody: Vero Cell; CHO; BHK; HEK 293; A549; MRC5; PER.C6
Anti-Yeast HCP antibody: Saccharomyces cerevisiae; Pichia pastoris
Anti-Insect Cell HCP antibody: SF9; SF22
Others: Anti-Bovine Serum Protein antibody; Anti-Mycobacterium Protein antibody; Anti-Goat-Milk Protein antibody

All the fluorescent labeled secondary antibodies are provided by Applied Biomics.

1. Price covers:

Experimental design
Standard sample preparation and protein concentration determination
Fluorescent dye labeling using CyDye
IEF and SDS-PAGE
Gel image scan using Typhoon scanner
Image analysis (ImageQuant)
Data Report

2. Price covers:

Trans-blotting 2D Gel to PVDF or Nitrocellulose membrane
Membrane images scanned after the transfer

3. Price covers:

Western blot using the primary antibodies provided by customers, Applied Biomics will provide secondary antibodies
Membrane images scanned after Western Blot

Each Western blot will cover 2 hour data analysis generating Protein and Western blot image report.

Price does NOT cover:

Primary antibody provided by customer
Manual matching spots and manual circling and numbering spots on power point images
Customized data request in addition to the standard request