Flat bottomed polystyrene 96 well plate

Flat-bottomed polystyrene 96-well plates (Corning, USA) were coated with 100 μl BPM (10 μg/ml in PBS) overnight at 4 °C. Then, the plates were blocked with 10% FBS. Diluted serum (1: 100) was added and incubated for 2 h at 37 °C, followed by biotin-labeled anti-rat IgG (Biolegend, USA) for 1 h at 37 °C and streptavidin–horseradish peroxidase (Bios, China) for 30 min at 37 °C. The color was developed with tetramethylbenzidine (TMB) substrate (Tiangen Biotechnology, China). Finally, OD values of corresponding wells were determined by a microplate ELISA reader at 450 nm. Results were expressed as mean optical density (OD values) ± standard deviation (SD).

The optical density, or turbidity, or total scattered light intensity of each sample was measured by a Tecan Infinite 200 Pro instrument (Tecan Group Ltd, Männendorf, Switzerland). Optical density can be determined from the equation T $={\log }_{10}\frac{I_{\circ }}{I}$ , where I_∘ is intensity of the light unattenuated by the sample, and I is the intensity of the light that is transmitted through the sample. Thus, a reading of T = 0.35 indicates that the sample causes an attenuation of the light intensity by a factor of 10^0.35, or 2.24-fold. In other words, only 44.7% (100 ÷ 2.24) of the I_∘ light that is incident upon the sample reaches the detector. Measurements were performed by placing samples within clear, flat-bottomed polystyrene 96-well plates (Corning Inc., Corning, New York, USA), and scanning with 550 nm light, since samples do not absorb light at this wavelength. Each sample was shaken within the plate reader before measurement as a final step to ensure uniform mixing among batches. Turbidity measurements were performed in a temperature range spanning from 20 ^∘C to 40 ^∘C in 5 ^∘C increments.