*3.3. Optimization of the ELISA Variables*

The concentrations of coating antigen and mAb, the incubation time of mAb and HRP-IgG, the working concentration of HRP-IgG and organic solvents were optimized, respectively. As shown in Table S3, the optimal concentrations of coating antigen and mAb were 2 μg/mL and 1:20,000, respectively. The optimal incubation times of the mAb and HRP-IgG were 35 min and 40 min, respectively. The optimal working concentration of HRP-IgG was 1:6000.

PAHs are hydrophobic lipophilic substances that are usually dissolved in organic solvents. It has been reported that the parameters of ELISA would be influenced by the organic solvent [28]. Therefore, acetone, DMF, acetonitrile and DMSO were selected for study in this experiment. The effect of organic solvents added to the ELISA reaction system on the antigen-antibody binding reaction should be as small as possible. Therefore, it was necessary to explore the binding ability of different concentrations of organic solvents. Organic solvents with different volume fractions (10%, 20%, 30% and 40%) in PBS buffer were used as drug standard diluents. During the experiment, we found that acetone was highly volatile. When incubated at 37 ◦C, the total amount of liquid in the wells decreased significantly, which affected the accuracy of the results, so it was excluded.

As shown in Figure 3, compared with acetonitrile, the OD450 values of DMF and DMSO were relatively less affected. The use of organic cosolvents to improve the affinity and specificity of antibodies has also been supposed [29]. Then, the influence of concentrations of DMF and DMSO on the sensitivity of ELISA were investigated. When the volume fractions of DMF and DMSO were in the range of 10–40%, there was a large difference in sensitivity. In contrast, the IC50 value of 20% DMF-PBS (1:4, v:v) was the lowest, which significantly improved the sensitivity of ic-ELISA (Table 1). Therefore, 20% DMF-PBS (1:4, v:v) was selected as a drug standard diluent.

**Figure 3.** Comparison of different organic solvents.


**Table 1.** Comparison of different DMF and DMSO contents.
