Monoclonal Antibody Quantification

Pharmacokinetics, sometimes abbreviated as PK, is a branch of science focused on the quantitative analysis of absorption, distribution, metabolism, and excretion of drug molecules within the body of a living organism. All clinical and pre-clinical studies include the measurement of the concentration of serum drugs in patients as well as in animals, at various points following drug administration. The outcome is a key indicator of the pharmacokinetic properties of the drug and is appropriately relevant to dosing recommendations.

Case Study

The growing market of biologic drugs, pushed by a flurry of success with monoclonal antibodies, creates a need for standard high-throughput assays to assess the content of mAbs in serum samples. On the basis of various principles, different types of assay designs are available.

For serum antibody detection, three widely known ELISA setups with their common advantages and disadvantages are listed in the table below. To assess the performance of these setups in real applications, ACROBiosystems tested them in the following case studies determining the concentration of anti-PD1 mAb in serum samples.

Method Coating Sample Secondary Antibody Advantage Disadvantage Case Studies
Indirect assay Antigen Serum Goat anti-human IgG Simple High background Case I
Antibody-directed competitive assay Antigen Serum with labeled competition antibody SA-HRP Low background Additional reagent/labeling required Case II
Ligand-directed competitive assay Natural ligand for the antigen Serum with labeled PD-1 SA-HRP Low background Narrow detection range Case III

Case I: Indirect ELISA  

Equipment:

BMG CLARIOstar microplate reader

Sample:

Serum samples comprising of a monoclonal PD-1 antibody

Main Reagents:

  • Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Cat. No. 109-035-098, Jackson Lab, Bar Harbor, ME, USA)
  • Recombinant Human PD-1 protein (Cat. No. PD1-H5221, ACROBiosystems, Newark, DE, USA)

Protocol:

  1. The microplate is coated using 0.1 µg/well concentration of rhPD-1 for 16 hours
  2. Serial sample dilutions (1:2) are prepared
  3. The plate is washed
  4. 100 µl of samples are added onto the plate
  5. The plate is washed again
  6. 0.05 µg/ml of HRP-conjugated Goat Anti-Human IgG is added
  7. TMB is added for colorimetric detection

Protocol:

Prior to assays, all serum samples are diluted by a factor of 1000 to avoid unspecific binding. The sensitivity is 0.19 µg/mL and the detection range is 0.19–6.25 µg/mL (Figure 1).

Detection of PD-1 antibody by indirect ELISA

Figure 1. Detection of PD-1 antibody by indirect ELISA

Case II: Antibody-Directed Competitive ELISA

Equipment:

BMG CLARIOstar microplate reader

Sample:

Serum samples comprising of a monoclonal PD-1 antibody

Main Reagents:

ELISA Assay Kit for Anti-PD-1 h-mAb in Human Serum (Cat. No. EPH-V1, ACROBiosystems, Newark, DE, USA)

Protocol:

  1. The microplate is coated with 0.1 µg/well concentration of rhPD-1 for 16 hours
  2. Serial sample dilutions (1:2) are prepared
  3. The samples are mixed with biotinylated PD-1 antibody offered in the kit and brought to a final concentration of 10%
  4. After washing, 100 µl of the mixed samples from step 2 is added to the wells
  5. After washing, 0.1 µg/ml of HRP conjugated Streptavidin is added
  6. TMB is added for colorimetric detection

Result:

The detection range is 0.78–25 µg/mL and the sensitivity is 0.78 µg/mL (see Figure 2).

Detection of PD-1 antibody by antibody-directed competitive ELISA.

Figure 2. Detection of PD-1 antibody by antibody-directed competitive ELISA.

Related Products:

ELISA Assay Kit for Anti-PD-1 h-mAb in Human Serum (Cat. No. EPH-V1)

Case III: Ligand-Directed Competitive ELISA

Equipment:

BMG CLARIOstar microplate reader

Sample:

Serum samples comprising of a monoclonal PD-1 antibody

Main Reagents:

  • HRP-conjugated Streptavidin Protein (Cat. No. 21126, Thermo Fisher Scientific)
  • Biotinylated human PD-1 (Cat. No. PD1-H82F2, ACROBiosystems, Newark, DE, USA)
  • Recombinant human PD-L1-Fc protein (Cat. No. PD1-H5258, ACROBiosystems, Newark, DE, USA)

Simplified Protocol:

  1. The microplate is coated with 0.2 µg/well concentration of rhPD-1 for 16 hours
  2. Serial sample dilutions (1:2) are prepared and mixed with biotinylated PD-1 to a final concentration of 10%
  3. 100 µl of sample from step 2 is added to the wells after washing
  4. HRP conjugated Streptavidin with a concentration of 0.1 µg/ml is added after washing
  5. TMB is then added for colorimetric detection

Result:

The detection range is 1.565–6.25 µg/mL and the sensitivity is 1.565 µg/mL (see Figure 3).

Detection of PD-1 antibody by ligand-directed competitive ELISA.

Figure 3. Detection of PD-1 antibody by ligand-directed competitive ELISA.

Methods Comparison

Case Detection Range (µg/mL) Sensitivity (µg/mL) Specificity
Case I:Indirect ELISA 0.19-6.25 0.19 High background
Case II:Antibody-directed competitive ELISA 0.78-25 0.78 Very low background
Case III:Ligand-directed competitive ELISA 1.565-6.25 1.565 Very low background

Among the aforementioned three assay designs, the ligand-directed competitive ELISA therefore pre-dilution is required before analyses (Figure 4A). On the other hand, HRP-conjugated Streptavidin is used by the antibody-directed competitive ELISA for secondary detection, which lowers the background interference (Figure 4B). Additionally, it also has the braodest detection range among the other assay designs, albeit the sensitivity is lower than the indirect method.

Comparison between Indirect ELISA and antibody-directed competitive ELISA for PD1 mAb detection in patient samples. A: Indirect ELISA; B: antibody-directed competitive ELISA.

Figure 4. Comparison between Indirect ELISA and antibody-directed competitive ELISA for PD1 mAb detection in patient samples. A: Indirect ELISA; B: antibody-directed competitive ELISA.

In a majority of applications, the background issue is a major problem than detection sensitivity, as a detection sensitivity of below 1 µg/ml is already adequate. Therefore, for the PK kits, ACROBiosystems decided to use the antibody-directed competitive ELISA.

ACROBiosystems has introduced kits for CTLA-4, PD-1, and HER-2 studies, respectively, in common experimental animals as well as humans.

About ACROBiosystems

ACROBiosystems is an internationally recognized manufacturer of recombinant proteins committed to supporting cancer immunotherapy. We specialize in mammalian cell-based recombinant protein production and process development.

Our goal is to support professionals from pharmaceutical companies, CROs and research institutes who are working on the cancer immunotherapy area by providing high-quality proteins, antibodies and assay kits.

We have multiple offices and branches in North America, Europe, and Asia, and we are proud of serving customers from over 50 countries.


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Last updated: Jul 29, 2019 at 1:00 PM

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