Optimization and establishment of DPS-based ELISA method
Amount of plasma in DPS sample
The effect of different volumes of plasma used for DPS samples for anti-HIV, anti-TP, and anti-HCV tests is shown in Figure 1. In all serial dilution experiments for all three drugs, the test result curves (S/ CO when using 100 μL of plasma) were higher than when using 65 μL of plasma.
Comparison of ELISA tests for anti-HIV/anti-TP/anti-HCV detection in DPS samples with different plasma volumes. NOTE: Numbers in parentheses represent different serial dilutions.
DPS sample size and eluent volume
As shown in Figure 2, for HIV and HCV, the detection results (S/CO) using the A1B1C2D1 combination were greater than those using other combinations of sample size and eluent volume (Figure 2a, c). However, for TP, the detection results using the A1B1C2D1 combination were lower than those using the A1B3C4D1 combination (Fig. 2b).
Comparison of ELISA tests for anti-HIV/anti-TP/anti-HCV detection in different combinations based on DPS size and eluent volume Note: a Indicates that the DPS sample contains 100 µL of plasma. B Represents the size of the DPS sample [whole spot, 1/2 DPS, 1/4 DPS, 2 discs and 1 discs (6 mm)]; C Indicates the amount of eluent [1 mL, 500 µL, 300 µL,200 µL]; D means the sample amount based on the test kit
Elution buffer
As shown in Figure 3, the detection results for all three drugs were highest when using PBS containing 1 percent Tween20 buffer.
Effect of different elution buffers on DPS-based ELISA method for detection of anti-HIV, anti-TP, and anti-HCV
Elution time and elution temperature
The results of the effect of different combinations of elution time and elution temperature on the performance of the DPS-based ELIA method are shown in Figure 4. For HIV, TP, and HCV, the optimal elution time was 2 h at room temperature or 1 h. At 37℃. The best elution times at 4°C were 1, 6, and 2 h for HIV, TP, and HCV, respectively. Further analysis was then performed to determine the optimized combination for detecting all three drugs (Fig. 4a, b, c). The results showed that all three drugs were optimally detected with a 2-h elution at room temperature.
Changes in anti-HIV, anti-TP, and anti-HCV test results at different elution temperatures and elution times
optimal conditions
No statistically significant differences were observed. Based on the aforementioned experimental results, the optimal conditions for the DPS-based His ELISA assay to detect all three drugs are:: Plasma volume was 100 µL, whole spot DPS size, eluate volume was 500 µL, PBS elution with 1® Tween20 buffer, elution time was 2 hours, and elution temperature was RT.
Evaluation of a DPS-based ELISA method for screening HIV, TP, and HCV
A laboratory panel was constructed to evaluate the linear range, sensitivity, specificity, precision, and anti-jamming ability of a DPS-based ELISA method for simultaneously screening anti-HIV, anti-TP, and anti-HCV antibodies . DPS-based ELISA methods were tested using these panels.
Linear range
The linearity of the DPS-based ELISA method for anti-HIV, anti-TP, and anti-HCV is shown in Figure 5. The linear range for anti-HIV, anti-TP, and anti-HCV measurements was 2.14 to 219 (R2 = 0.98), 23 to 27 (R2 = 0.97), and 28 to 213(R2= 0.98), respectively. As shown in Figure 1, for anti-HIV detection, the DPS-based ELISA method was linear over a 2-fold dilution range.11 to 215 correlation coefficient (R2 ) = 0.96. For TP, the assay was linear over a dilution range of 2.0 to 23(R2= 0.96) and was linear over a dilution range of 2 for anti-HCV.Fiveto 29(R2= 0.98). These determined linear ranges were used for further experiments.
Calibration curves for anti-HIV, anti-TP, and anti-HCV in plasma and DPS samples by ELISA. Note: be It is published in the Chinese Journal of AIDS & STD and is used as a reference for this study.
Sensitivity and specificity
The test results of the DPS-based ELISA method using a laboratory basic panel are shown in Table 1. We demonstrate the sensitivity and specificity of DPS-based ELISA methods for anti-HIV, anti-TP, and anti-HIV based on plasma test results. Anti-HCV efficacy was 100% in all cases.
Specificity of analysis
The test results of the DPS-based ELISA method against the laboratory analysis specificity panel are shown in Table 2. The results showed that the method was able to identify superinfections in all samples tested and that superinfections did not impede testing ability. How to detect other infections.
Accuracy evaluation
The accuracy evaluation results of the DPS-based ELISA assay are shown in Table 3. Intra-day, intra-assay, and inter-spot CVs for HIV were 2.10 to 1.53%, 0.00 to 3.17%, and 2.84 to 8.03%, respectively. For TP, they were 0.20–12.31%, 0.06–7.25%, and 0.37–11.47%, respectively. For HCV, they were 3.01–3.86%, 1.30–5.28%, and 4.18–12.92%, respectively. These results demonstrate that the DPS-based ELISA method provides acceptable accuracy for screening HIV, TP, and HCV.
Comparison of DPS and plasma samples
The test results of the 619 matched plasma/DPS samples are shown in Table 4. Of the samples, 25.36% were all 3-drug negative, 17.29% were HIV monoinfected, 16.16% were TP monoinfected, and 27.63% were HCV monoinfected. – infection, 4.52% HIV/TP coinfection, 6.79% HIV/HCV coinfection, 1.13% HCV/TP coinfection sample, 1.13% HIV/HCV/TP coinfection. Comparison of test results between plasma and DPS samples showed that only the two pairs of DPS/plasma samples showed different results. The DPS-based ELISA method failed to detect one HCV monoinfected sample and one HIV/HCV/TP coinfected sample. HIV/HCV/TP co-infected samples gave inconsistent results for the detection of TP, with S/CO of 2.143 for plasma samples and S/CO of 0.5 for DPS samples. For HCV, S/CO was 3.049 for plasma samples and 0.878 for DPS samples.
As shown in Table 5, the proportion of HIV-positive, TP-positive, and HCV-positive samples was 29.7% (95% confidence interval). [CI]: 0.26-0.34), 22.9% (95% CI: 0.20-0.26), and 36.7% (95% CI: 0.33-0.41). For HIV, the DPS-based method showed 100% sensitivity (95% CI: 0.97 to 1.00) and 100.0% specificity (95% CI: 0.99 to 1.00). For TP, the sensitivity of the DPS-based method was 98.6% (95% CI: 0.94 to 1.00) and specificity was 100% (95% CI: 0.99 to 1.00), and for HCV, the sensitivity was 99.6% (95 %) was. CI: 0.97-1.00) and specificity was 100% (95% CI: 0.99-1.00).
Considering the proportion of HIV-, TP-, and HCV-positive samples, the DPS-based method has a positive predictive value (PPV) of 100% for HIV/TP/HCV and a negative predictive value (NPV) of 100% for HIV. , 99.6% for TP and 99.7% for HCV. The kappa value between the results for plasma and DPS samples was 100% for HIV (p< 0.001, 95% CI: 1.00–1.00), 100% for HCV (p< 0.001, 95% CI: 0.99–1.00), 99% for TP (p< 0.001, 95% CI: 0.98 to 1.00).
