Automated Elisa (enzyme-linked immunosorbent assay) using Opentrons pipetting workstations

Introduction ELISA (enzyme-linked immunosorbent assay) is an important test method widely used in diagnostics and research. In this technology, ensuring consistency in sample processing is critical, and reducing hands-on time can effectively reduce human error. We developed and tested multiple automation protocols using Opentrons OT-2 and Flex liquid handling workstations. These automated protocols allow you to perform experiments directly using ELISA kits, or you can customize specific experimental procedures according to your needs.

Method

Kit: Takara Fibronectin (FN) EIA Kit (sandwich ELISA, Figure 1)

• Instrument: Opentrons OT-2
• Workflow:

1. Add sample (targeted capture)
2. washing
3. Add enzyme-labeled antibody
4. washing
5. Add chromogenic substrate

Detection method: Tecan Cortisol Saliva ELISA Kit (competition ELISA, Figure 2)

• Instrument: Opentrons OT-2
• Workflow:

1. Add sample (target capture) & label antigen
2. washing
3. Add chromogenic substrate

• 检测方法: Cell Sciences SARS-CoV-2 Surrogate Virus Neutralization Test Kit
  （竞争法ELISA，图3）
• Instrument: Opentrons OT-2
• Workflow:

1. Loading (Targeted Capture) & Labeling ACE2
2. washing
3. Add chromogenic substrate

• 检测方法: SARS-CoV-2 Spike RBD-coupled Magnetic Beads
  (磁珠式ELISA, 图4)
• Instruments: Opentrons OT-2 & Magnetic Stand Module
• Workflow:

1. Add sample (targeted capture)
2. washing
3. Add enzyme labeled antibody
4. washing
5. Add chromogenic substrate

Detection: Assay method development for cytokine detection (sandwich ELISA, Figure 5) Instrument: Opentrons Flex Workflow: 1. ELISA plate preparation 2. Washing 3. Adding sample (target capture) 4. Washing 5. Adding enzyme-labeled antibody 6. Washing 7. Adding substrate to develop color

Results

Figure 6. Takara Fibronectin (FN) EIA Kit (Figure 1). The FN sample was gradient diluted and tested. The test results are as shown in the figure: the left picture shows the FN sample concentration and average absorbance (n=3) data, showing a highly linear regression; the right picture shows the results of three repeated experiments, showing good repeatability. sex and stability.

Figure 7. Tecan Cortisol Saliva ELISA Kit (Figure 2). The cortisol sample was gradient diluted and tested. The test results are as shown in the figure: The left picture shows the cortisol sample concentration and average absorbance (n=3) data, showing a highly linear regression; the right picture shows the results of three repeated experiments, which are well presented. repeatability and stability.

Figure 8. Cell Sciences SARS-CoV-2 Surrogate (Figure 3). The test results show that the positive human serum sample (the ELISA test result that has been previously certified by the FDA is positive) shows a positive result in this test (inhibition rate >20%, calculation formula: inhibition rate = [1 - (sample OD450 value/OD value of negative control)] x 100%, n = 3).

Figure 9. SARS-CoV-2 Spike RBD-coupled Magnetic Beads (Figure 4). Recombinant neutralizing antibody samples were serially diluted and tested. Test results show that positive human serum samples, previously tested positive in an FDA-cleared ELISA assay, tested positive in this test (n = 3) (right).

Figure 10. Assay Development for Cytokine Detection (Figure 5). Anti-TNF-alpha antibodies and anti-IFN-gamma antibodies were coated on a 96-well plate, and samples containing cytokines were tested by ELISA. The data are as shown in the figure (n=6).

Conclusion

• This workstation can be used to perform the complete ELISA workflow from adding antibody/antigen to adding chromogenic substrate
• 结果表明样本处理的一致性高（变异系数<10%）、准确性高（R-squared>0.99），重复性
  好。
• 以 SARS-CoV-2 抗体检测为例，基于 96 孔板的商用试剂和基于磁珠的自制 ELISA 试剂均可
  实现全自动化处理，且结果与 FDA 认证的 Elisa 方法测定结果一致。