ELISA technique is really useful for detecting malaria parasite in the mosquito vector with high sensitivity and specificity, and able to process lot of sample at the same time. This technique allow researchers to keep mosquito samples until they are ready for sample processing, and it can be performed to differentiate Plasmodium species using species-specific monoclonal antibodies. Prepare the sample by separating the head and thorax of the mosquitoes from the abdomen, and place them in a pre-labeled 1.5 milliliter centrifuge grinding tube.
Add 50 microliters of grinding buffer to each tube, and homogenize the sample with a pestle. Rinse the pestle with 250 microliters of grinding buffer, and add the solution to the tube to bring the final volume up to 300 microliters. Prepare an ELISA plate for circumsporozoite protein, and fill out the sporozoite ELISA worksheet.
Prepare the antibody solution in PBS, and add five milliliters of the solution per plate. Pipette 50 microliters of the antibody solution into each well of the ELISA plate. Place a lid on the plate, and incubate for 30 minutes or overnight at room temperature.
Aspirate the solution in the well, and tap the plate on a paper towel five times to remove the liquid. Add 200 microliters of blocking buffer in the well plates, and cover with a lid. Incubate for one hour at room temperature, and aspirate the well content.
Again tap the plate on a paper towel to remove the liquid. Add 50 microliters of positive control to the H one and H two wells. Then add 50 microliters of blocking buffer into the wells in columns one and two of rows C to G.Add 50 microliters of the positive control into the G one and G two wells.
Serially dilute the positive control starting from G one and G two, followed by F one and F two until C one and C two. Add 50 microliters of the negative control to wells A one, A two, B one, and B two. Extract 50 microliters of the homogenate solution, and add it to an unknown well.
Cover the plate and incubate it at room temperature for two hours. Prepare the substrate solution by mixing substrate A and B at a ratio of one to one. Determine the required volume based on adding five milliliters of working conjugate antibody solution to each plate.
Evaluate the peroxidase activity by adding five microliters of the peroxidase labeled antibody solution to 100 microliters of the substrate solution in a 1.5 milliliter tube. Aspirate the well content, and remove the liquid by putting the plate upside down on a paper towel. Wash the wells two times with 200 microliters of PBS Tween.
Aspirate the well contents, and tap the plate five times. Add 50 microliters of peroxidase labeled antibody solution to each well. Then incubate in the dark for one hour at room temperature.
Aspirate the well content, and tap the plate on a paper towel five times. Wash the wells with 200 microliters of PBS Tween three times, aspirate the contents, and tap the plate five times. Add 100 microliters of the substrate solution to each well.
Cover the plate, and incubate in the dark for 30 minutes at room temperature. After the incubation, read the absorbance at 405 to 414 nanometers using an ELISA plate reader. Label the samples having absorbance with a value above the cutoff as positive, and determine the CSP concentration in the sample using the standard.
Plot the absorbance values versus solution concentration to generate a standard curve. Determine the best fit using the linear regression method. Solve the equation of each absorbance value for a positive sample to determine the CSP concentration.
After 30 minutes of performing ELISA with ABTS, sporozoite infection could be detected by the appearance of a faint green color in the well. The positive control did not show any color change in the well. The absorbance values for the negative and positive wells were determined.
The positive well exhibited an absorbance value that was above the cutoff threshold, while the negative controls demonstrated significantly lower values. Furthermore, the absorbance values of the other 80 unknown wells were also obtained. The solid line represents the mean absorbance of the negative control wells, and dashed line represents the positivity threshold.
Using the standard curve, the CSP concentration in well A seven was determined to be 0.35 picograms per microliter This step of ELISA processing should be performed quickly to prevent nonspecific vector. In addition, all samples and solution should be kept in the refrigerator to prevent the microbial growth. Researchers are recommended to confirm the positive detections by other methods such as PCR.
This will significantly improve the confidence, especially when the only reading is valued above the threshold. This technique has allowed researchers to conduct larger scale surveys of possible infection in the mosquitoes, which is important for identifying the main vector.
