Improvement of limit of detection for competitive enzyme-linked immunosorbent assays
Competitive ELISA is a method for measuring antigens. However, the popular tool around the world, ELISA KIT, cannot measure low concentration of antigens because of its high limit of detection, especially in non-invasive detection. Although there are some methods that can improve limit of detection, there are also some problems, such as thermal effects and expensive products. The convenient, effective methods that can be easily operated have not created.
βCTX is one of the indicators for detecting fractures, but the concentration of βCTX in saliva is too low, and cannot be measured by current type of competitive ELSA KIT.
The goal of this research is to create an innovative method to improve the limit of detection in competitive ELISA KIT from 98pg/ml to 20pg/ml, and then use this method to measure the concentration of βCTX in saliva. Here we propose two methods. The first method is to increase the concentration of antigens in competitive ELISA KIT during the reaction. We can achieve this goal by increasing the volume of the antigens and decreasing the volume of the competitor during the reaction, and remain amount of the competitor. The second method is to do more measuring groups. By increasing three measuring groups to between five and eight measuring groups, choosing median and two number near the median of all measuring groups and concentrations, then we can get the calibration curve with the lower limit of detection
By using 50 ul of antigen and competitor and increasing measure group to seven groups, this research successfully decreases limit of detection to 0.089-12.696 pg/ml. This situation can get the lowest limit of detection, but it is more expensive. By using 75 ul of antigen and 25 ul of competitor and increasing measure group to five groups, this research successfully decreases limit of detection to 3.485-48.833 pg/ml. Although this situation can not get the limit of detection like the previous situation, but it is cheaper and stabler.
The concentration of βCTX in the saliva of 17 subjects was successfully measured: The concentration of βCTX in the saliva of 10 healthy subjects:298.827~2404.51 pg/ml in seven healthy subjects, and the concentration of βCTX in the saliva of one of the healthy subjects exceeds the detecting range, and the concentration of βCTX in the saliva of the other two healthy subjects cannot be detected. The concentration of βCTX in the saliva of 7 patients: 16.861 ~ 365.916 pg/ml in five patients, and the concentration of βCTX in the saliva of the other two patients cannot be detected.