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:

  1. Characterizing HCP antibody coverage
  2. 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, AAE* and standard 2D Western blot:

  1. 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.
  2. Cost-effective: Since only one gel is needed, it saves the cost of running duplicate gels.
  3. High accuracy: Fluorescent CyDye labeling allows accurate quantitation of protein and WB spots.
  4. High sensitivity: Fluorescent CyDye labeling has a higher sensitivity and wider dynamic range over the silver staining.
ELISAStandard 2D Western Blot2D DIGE Western Blot
Number of gels for each AbTwoOne
Detection MethodSilver StainingCyDye Labeling
Detect HCP CompositionImageImageImage
Detect protein modificationImageImageImage
Detect protein degradationImageImageImage
Protein and WB in same gelImageImageImage
In-gel protein and WB comparisonImageImageImage
AccuracyLowLowHigh
Inaccurate: In-direct reaction of antibodies with the antigen’s associated proteinsInaccurate: Protein spots counted from the gel, NOT from membraneAccurate: 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 Antibody on the same gel, avoiding any ambiguity
Consistency & ReproducibilityLowerLowerHigher
*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
  • 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:

BacteriaMammalian CellYeastInsectOthers
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

HEK293 HCP antibody coverage procedure: step 1 - protein extraction and labeling

Protein extraction
Protein assay
Protein labeling

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

2D Gel image:
HEK293 HCP

HEK293 HCP antibody coverage procedure: step 3 - transfer HEK293 HCPs from gel to membrane

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

HEK293 HCP antibody coverage procedure: step 7 - Data analysis showing HEK293 HCP spot map

Anti-HEK293 HCP Western Blot spot map

HEK293 HCP antibody coverage procedure: step 8 - Data analysis showing 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 coverage93.2% of 2200 HEK293 HCP spots

HCP Antibody Coverage Application Gallery

ExampleHCP antibody coverage Applications
#1CHO cell HCP antibody coverage
#2E. coli HCP antibody coverage
#3E. coli High Mw HCP antibody coverage
#4Yeast HCP antibody coverage
#5SF9 HCP antibody coverage

1. CHO Cell HCP antibody coverage by 2D Western Blot

CHO HCP 2D Gel

CHO Cell HCP antibody coverage by 2D Western Blot: CHO HCP 2D Gel image

Anti-CHO HCP 2D WB

CHO Cell HCP antibody coverage by 2D Western Blot: Anti-CHO HCP Western Blot image

CHO HCP / Western Blot

CHO Cell HCP antibody coverage by 2D Western Blot: overlay image of CHO HCP & anti-CHO HCP Western Blot

CHO HCP spot map

CHO Cell HCP antibody coverage by 2D Western Blot: CHO HCP spot map

Anti-CHO HCP WB spot map

CHO Cell HCP antibody coverage by 2D Western Blot: 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 coverage93.4% of 2559 CHO HCP spots

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

E. coli HCP 2D Gel

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

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

E. coli HCP antibody coverage by 2D Western Blot: E. coli HCP 2D Gel color image

Anti-E. Coli HCP 2D WB

E. coli HCP antibody coverage by 2D Western Blot: Anti-E. coli HCP Western Blot color image

E. Coli HCP / Western Blot

E. coli HCP antibody coverage by 2D Western Blot: overlay image of E. coli HCP & anti-E. coli HCP Western Blot

E. coli HCP spot map

E. coli HCP antibody coverage by 2D 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 coverage99.6% of 2235 E. Coli HCP spots

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

E. Coli HCP 2D Gel

E. coli High Mw HCP antibody coverage by 2D Western Blot: High Mw E. coli HCP 2D Gel image

Anti-E. Coli HCP 2D WB

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

E. Coli HCP / Western Blot

E. coli High Mw HCP antibody coverage by 2D Western Blot: overlay image of high Mw E. coli HCP & anti-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 coverage99.7% of 936 high molecular weight E.coli HCP spots

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

Yeast HCP 2D Gel

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

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

Yeast HCP antibody coverage by 2D Western Blot: Yeast HCP 2D Gel color image

Anti-Yeast HCP 2D WB

Yeast HCP antibody coverage by 2D Western Blot: Anti-Yeast HCP Western Blot color image

Yeast HCP / Western Blot

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

Yeast HCP Spot Map

Yeast HCP antibody coverage by 2D 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 coverage99.9% of 2402 Yeast (Saccharomyces cerevisiae ) HCP spots

5. 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 antibody coverage by 2D Western Blot: SF9 HCP 2D Gel color image

SF9 HCP 2D Western Blot

SF9 HCP antibody coverage by 2D Western Blot: Anti-SF9 HCP Western Blot color image

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

SF9 HCP antibody coverage by 2D 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 coverage90.2% 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 DescriptionAcademic/Governmental LabsIndustrial/Commercial Labs
CodePriceCodePrice
2D Gel with 1 CyDye Labeling 1401A$800401N$960
2D DIGE Gel with 2 CyDye Labeling 1101A2$1200101N2$1500
Transfer to PVDF / Nitrocellulose Membrane 2520A$250520N$295
Western Blot 3512A$410512N$495
HCP Quantitative Analysis202A-HCPTBD202N-HCPTBD

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