The overall goal of this electrochemiluminescence assay is to detect islet atutoantibodies in autoimmune type one diabetes, with high sensitivity and high specificity. This method can help answer key questions in the type one diabetes field, such as time of islet autoimmunity initiation and high risk prediction for type one diabetes. The main advantage for this technique is a more sensitive, more disease specific and non-radioactive compared to the current standard radioassays.
The implication of this technique extends towards predicting and diagnosing type one diabetes because these islet autoantibodies appear months to years before symptomatic clinical disease. Though this method can provide insight into the predication for type one diabetes, it can also be applied to study other autoimmune diseases, such as autoantibodies to transglutaminase for celiac disease to TPO and thyroglobulin for autoimmune thyroid disease and several others. We first had the idea for this method when we found the technique is based on high sensitivity and non-radioactivity.
In contrast to conventional ELISA method which does not work well for any islet autoantibody detection. Visual demonstration of this method is critical as insulin autoantibody assay with serum acid treatment is difficult to be described in text. Demonstrating the procedure will be Yong Gu, a postdoc from my laboratory.
First, mix the human islet autoantigen with biotin or SULFO-TAG in one to five molar ratio in a tube. And cover the tube with aluminum foil since both the tags are light sensitive. Then, incubate the tube for an hour at room temperature.
In the meanwhile, when the incubation is going on prime the spin column with two fold phosphate buffered saline. Then, centrifuge the column at 1, 000 times gravity for two minutes each time. Next, centrifuge the autoantigen tag mixture in the spin column at 1, 000 times gravity for two minutes.
Then, aliquot and store the labeled antigen at negative 80 degrees Celsius. Next, use the rational concentration of the biotin SULFO-TAG labeled antigen based on the checkerboard assay for the antigen buffer, to prepare three milliliters of antigen solution per 96-well plate. Then, in each well, add four microliters of serum and adjust the final volume to 20 microliters with one fold phosphate buffered saline.
Then, add 20 microliters of labeled antigen solution in each well. And cover the plate with sealing foil prevent light. Then, transfer the plate on a shaker at room temperature for two hours.
Once the shaker stops, leave the plate at four degrees Celsius in the refrigerator for 18 to 24 hours. Now, mix 15 microliters of serum with 18 microliters of 0.5 molar acetic acid for each sample and incubate the same at room temperature for 45 minutes. On the basis of the checkerboard assay, use the rational concentration of the biotin SULFO-TAG labeled antigen to prepare the antigen solution.
Then, aliquot 35 microliters of the antigen buffer in each well of a new PCR plate. Just before the course of incubation of the serum mixture is completed add 3.8 microliters of one molar Tris buffer at PH nine, along beside of each well on the antigen plate. Once the incubation is over quickly transfer 25 microliters of the acetic acid treated serum in each well of the antigen plate and agitate the solution.
Then, cover the PCR plate with sealing foil to avoid light and place on a shaker at room temperature for two hours. Once done, transfer the plate at four degrees Celsius in the refrigerator and incubate for 18 to 24 hours. Remove a streptavidin plate from the refrigerator at four degrees Celsius and let the plate reach room temperature.
After the streptavidin plate attains room temperature add 150 microliters of 3%blocker A in each well and cover the PCR plate with sealing foil and incubate in the refrigerator over night. The next day remove the streptavidin plate from the refrigerator and expel the buffer from the wells. Then, dry the plate by setting it upside down on some paper towels for the remaining buffer to soak.
Then, add 150 microliters of one fold PBST buffer to wash the well for three consecutive washes. Next, transfer 30 microliters of serum antigen incubate in each well of the streptavidin plate and cover the plate with foil to avoid light. Then, transfer the plate on a shaker at room temperature for an hour.
After one hour discard the serum antigen incubate from the plate and add 150 microliters of one fold PBST buffer to wash the well for three times. Once the washing is over, add 150 microliters of reading buffer to each well to read on a plate reader. Before analyzing any unknown samples the assay cutoff for each autoantibody assay was set up by testing around 100 type one diabetes patients and around 100 healthy controls.
Then, a ROC curve was plotted to determine the optimal index of upper limit of normal for GADA assay, which was 0.023, so as to obtain the best sensitivity and specificity. Then, the result of the unknown sample was calculated using an equation derived from high positive, low positive and negative control standards. Next, a graph was plotted to determine the signals obtained in islet autoatibody assay on incubating the insulin monoclonal antibody with normal human serum in the presence and absence of acid treatment.
The plot shows that the signal is markedly blocked upon addition of normal serum in the absence of acid treatment. On the contrary, the signal obtained is comparably higher when the serum is subjected to acid treatment prior to incubation with the insulin monoclonal antibody. A similar graph was also plotted to determine the signals obtained using foreign patient Sera in the presence and absence of acid treatment.
Treating the patient Sera with acid prior to incubation with the antibody significantly increases the signals in islet autoantibody assay when compared in the absence of treatment. Once mastered, this technique can be easily done for several hundreds of samples in one day if it's preformed properly. While attempting this procedure it's important to remember to perform the checkerboard assay for each autoantibody assay to optimize the assay condition.
After it's development, this technique paved a way for researchers in the field of autoimmune type one diabetes for prediction and prevention. After watching this video you should have a good understanding of how easily to perform this assay. Don't forget that working with serum samples can be hazardous and contagious.
Precautions to avoid direct skin contact should always be taken while performing this procedure.
