1) Solution formulation
> Basic buffer: 0.05M boric acid buffer solution pH 7.0, pH 7.5, pH 8.0
Liquid A: 0.2mol/L boric acid 0.05mol/L NaCl
Formula: Boric acid: 0.6184g, NaCl 0.1462g with distilled water to 50ml
Liquid B: 0.05 mol/L sodium borate
Formula: 0.9535g sodium borate with distilled water to 50 ml
| PH | A(ml) | B(ml) |
| 7.09 | 9.4 | 0.6 |
| 7.60 | 8.5 | 1.5 |
| 7.94 | 7.5 | 2.5 |
| 8.08 | 7.0 | 3.0 |
> 10 times concentrated sealing liquid
| Raw Material | 10mL |
| 0.5M Tris-HCL Buffer | 10ml |
| Tween-20 | 0.1ml |
| BSA | 1g |
> Coupled storage solution (0.05M boric acid buffer solution (pH8.0))
| Raw Material | 10mL |
| 0.5M Boric Acid Buffer Solution | 10ml |
| Tween-20 | 0.05ml |
| BSA | 0.02g |
2) Coupling process
- Take 0.05M/pH 7.0 boric acid buffer solution 100ul into 1.5ml centrifugal tube, add 100ul no-load latex, whirlpool oscillation, mix and clean.
- 0.05M/pH 7.0 boric acid buffer solution re-sol latex, adding EDC, NHS for activation.
- 20000r, 10 C, 20 min centrifugation, discarding supernatant, using 0.25ml 0.05M/pH 7.5 boric acid buffer solution resolving (centrifugal time can be prolonged or shortened according to centrifugal effect, the same below). Wash 2-3 times with coupling buffer 0.25ml 0.05M/pH 8.0 boric acid buffer.
- Ultrasound dispersion, 100W, 1min, 3s, 3S (if there is a particle size analyzer, check particle size and dispersion coefficient PDI≤ 0.07). The final concentration should be 20-200 ug/ml after adding coupling protein PCT. Put it in 250R, 20℃ shaker for 2 hours.
- The final concentration of BSA was 1% by adding both Liquid A and B 28ul 10 times of concentrated sealing liquid and keep sealed in shaker. (1-2 h).
- centrifugal washing 2-3 times, 20000R, 10℃, 20min, discarding supernatant, resolving with 0.25ml 0.05M/pH 8.0 boric acid buffer; after the last washing and centrifugation, resolving with coupling storage buffer to 50ml, ultrasonic dispersion, 50w-200w, 1min, 3s, 3S (if there is a particle size analyzer, check particle size and dispersion coefficient PDI≤ 0.07). Store at 4 C.
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