HCP Antibody Coverage
Applied Biomics offers the most comprehensive HCP Antibody Coverage analysis by using high resolution large format 2D fluorescent Western Blot. Our HCP Antibody Coverage platform offers significant advantages over ELISA, AAE and standard 2D Western blot.
Our HCP antibody coverage analysis uses only ONE 2D gel. The 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
HCP Antibody Coverage Experimental Procedure: HEK293 HCP Antibody Coverage
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 Experimental Procedure
- Host Cell Protein (HCP) extracted, assayed, and labeled with Cy2
- Run Cy2 labeled HCP on 2D, transfer to membrane, and scan for HCP image on membrane
- Perform Western Blot with primary antibody and Cy5-labeled secondary antibody
- Scan for Cy2 (HCP) and Cy5 (Anti-HCP) on the same membrane
- Data analysis:
- Image analysis by ImageQuant to generate image of HCP, Anti-HCP WB and HCP/WB overlay
- Quantitative analysis by DeCyder to obtain HCP antibody coverage: HCP Antibody coverage is calculated by number of spots detected by HCP antibody divided by total number of 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 spot map
Anti-HEK293 HCP WB 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 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
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
Anti-E.coli HCP WB 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 2D Gel
Anti-Yeast HCP 2D WB
Yeast HCP / Western Blot
Yeast HCP Spot Map
Anti-Yeast HCP WB 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 2D Western Blot
SF9 HCP 2D Gel
SF9 HCP 2D Western Blot
SF9 HCP / Western Blot
SF9 HCP spot map
Anti-SF9 HCP WB 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
Primary antibody: Customers can provide their own HCP antibody, or use Applied Biomics’ 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 |
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 | ||
|---|---|---|---|---|
| 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