Dual-target Bridging ELISA for Bispecific Antibodies

Materials and Reagents

1. 96-well microplate (Greiner Bio-One, catalog number: 650061)

2. HER2/PD-L1 BsAb protein (Chen et al., 2021)

3. Recombinant extracellular domain of human programmed cell death ligand 1 (C-6His）(PD-L1-ECD) Novoprotein, catalog number: CM06)

4. Recombinant human epidermal growth factor receptor 2 (C-6His) (HER2) (Novoprotein, catalog number: CP69)

5. 1% casein in PBS (Thermo Fisher Scientific, catalog number: 37582)

6. TMB substrate kit (Thermo Fisher Scientific, catalog number: 34021)

7. High sensitivity streptavidin-HRP (Thermo Fisher Scientific, catalog number: 21130)

8. EZ-Link Sulfo-NHS-LC-LC-Biotin kit (Thermo Fisher Scientific, catalog number: 21338)

9. Sulfuric acid (Sinopharm Chemical Reagent Co., Ltd, catalog number: 100216008)

10. PBS (Hyclone, catalog number: 16777-249)

11. Tween-20 (Sigma-Aldrich, catalog number: P1379)

12. 0.05% PBST (v/v) (see Recipes)

13. 2 M sulfuric acid solution (H₂SO₄) (see Recipes)

14. Trastuzumab (Selleck, catalog number: A2007)

Equipment

1. SpectraMax M5e microplate reader (Molecular Devices, catalog number: 89212-400)

Software

1. GraphPad Prism 9.0 (GraphPad Software, www.graphpad.com)

Procedure

1. Antigens preparation

   1. Prepare the biotin-antigen—biotinylated-HER2 protein—used for detection. First, conjugate the HER2 protein using the EZ-Link Sulfo-NHS-LC-LC-Biotin kit, according to manufacturer’s instructions.

   2. Prepare human PD-L1-ECD protein (capture antigen) at a final concentration of 2 μg/mL in PBS buffer.

      
      

2. Coating plate with PD-L1-ECD antigen

   1. Coat each well of a 96-well microplate with 50 µL of 2 µg/mL PD-L1-ECD protein. Cover the plate with a lid and incubate at 4 °C overnight without agitation.

      
      

3. Blocking and addition of HER2/PD-L1 BsAb

   1. The next day, remove the coating solution and wash the plate three times with 200 µL of PBS per well.

   2. Block the coated microplate by adding 200 µL of 1% casein in PBS buffer to each well using a multichannel pipette. Incubate for 1 h at 37 °C.

   3. Discard the blocking solution and wash the microplate three times with 200 µL of 0.05% PBST per well.

   4. Prepare three-fold serial dilutions of HER2/PD-L1 BsAb and control antibody (e.g., trastuzumab) in 1% casein in PBS buffer (e.g., 200 nM, 66.7 nM, 22.2 nM, 7.4 nM, 2.5 nM, 0.823 nM, 0.274 nM, 0.091 nM, 0.030 nM, 0.010 nM, 0.003 nM, and 0.001 nM).

   5. Add 50 μL of each diluted antibody to each well in triplicate and incubate for 1 h at 37 °C.

      
      

4. Incubation with HER2/PD-L1 BsAb and addition of biotinylated-HER2 antigen

   1. After incubation with the HER2/PD-L1 BsAb and control antibody, wash the microplates three times with 200 µL of 0.05% PBST to each well.

   2. Pipette 50 µL of pre-prepared biotinylated-HER2 protein at 2 µg/mL in 1% casein in PBS buffer to each well and incubate for 1 h at 37 °C.

   3. Discard the biotinylated-HER2 protein solution and wash the microplates three times with 200 µL of 0.05% PBST to each well.

   4. Add 50 μL of 1:1,000 diluted streptavidin-HRP in 1% casein in PBS buffer to each well for detection, and incubate for 1 h at 37 °C.

   5. Discard the detection solution and wash the microplates three times with 200 µL of 0.05% PBST to each well.

      
      

5. Addition of substrate and development

   1. Prepare TMB substrate solution A and B at 1:1 ratio according to manufacturer’s instructions. Add 50 μL of freshly prepared substrate to each well and incubate for 10 min at room temperature.

   2. After sufficient color development, add 50 μL of stop solution with 2 M sulfuric acid (H₂SO₄) to each well and measure the absorbance (optical density, OD) at 450 nm using a SpectraMax M5e microplate reader.

Data analysis

1. Subtract the background signal from the measured OD_450nm values and normalize them. Transform all values of the HER2/PD-L1 BsAb concentration or control antibody to logarithmic scale (base 10, log₁₀).

2. Plot the background corrected and normalized OD_450nm values (Y-axis, corresponding to the fraction of occupied HER2/PD-L1 BsAb binding to both HER2 and PD-L1 sites simultaneously) against the logarithm of the HER2/PD-L1 BsAb or control antibody concentrations (X-axis, log₁₀ scale).

3. Fit the concentration-response data using a 4 parameter logistic model by non-linear regression, with GraphPad Prism 9.0 software (Figure 1).

   
   

   
   Figure 1. Binding curves of the dual-target bridging ELISA. HER2/PD-L1 BsAb could simultaneously bind to human PD-L1-ECD and HER2, as determined by the bridging ELISA, in which PD-L1-ECD proteins were coated onto the plates, and biotinylated-HER2 proteins were used as detection agent. Trastuzumab was used as control antibody. Data are shown as mean ± SEM (n = 3).

Notes

In this ELISA method, use a multichannel pipette in each step to add solutions to the wells of a 96-well microplate. In solution removal and washing steps, throw out the solutions directly into the sink. None of the plate incubation steps require agitation.

Recipes

1. 0.05% PBST (v/v)

   Tween-20, 0.05%, 500 μL

   PBS, 999.5 mL

   Total, 1,000 mL

2. 2 M sulfuric acid solution (H₂SO₄)

   Sulfuric acid (98%), 2 M, 3 mL

   ddH₂O, 897 mL

   Total, 1,000 mL

Acknowledgments

This work was supported by the China National Major Scientific and Technological Special Project for “Significant New Drugs Innovation and Development” (2019ZX09732002-006) and the National Natural Science Foundation of China (81872785 and 81673347). The protocol described here was adapted from previously published work (Chen et al., 2021).

Competing interests

M. P. and Y.-L. C received stipends from Shanghai Mabstone Biotechnology, Ltd. Y. W., L. T., W. W., and C. W. are employed by Dartsbio Pharmaceuticals, Ltd.

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