Sensitive Detection Of Biomolecule By Proximity Ligation Assay

The mutant proteins that are expressed by mutant genes in cancer cells are secreted into the blood and are useful biomarkers for the early diagnosis of cancer. However, one important limiting factor has been the challenge of detecting proteins present at trace levels. Thus, the presence of mutant proteins in plasma has not previously been exploited for the early diagnosis of cancer. Proximity ligation assay is a protein-detection method that has been developed in recent years and has been widely used because of its high sensitivity. We have developed a microparticle-based solid-phase proximity ligation assay (spPLA) to detect trace levels of prostate-specific antigen (PSA), and the limit of detection was 0.1 pM. Compared with the immuno-PCR assay, microparticle-based spPLA exhibited a higher stability and accuracy, especially at low protein levels. To detect even lower levels of PSA, we used the loop-mediated isothermal amplification (LAMP) method to measure the levels of reporter DNA molecules in SP-PLA. The sensitivity of the LAMP method is 0.001 pM, which is approximately 100-fold higher than the sensitivities of the other assays. However, this approach still suffers from some shortcomings that should be addressed. We develop a covalent-bonding tube-based spPLA. The limit of detection of tube-based spPLA for PSA and wild-type p53 was found to be 0.001 pM, and the method exhibited a broad dynamic range of up to seven orders of magnitude. Furthermore, we coupled the conformation-specific antibody PAb240 of p53 mutants to PCR tubes for tube-based spPLA. The assay was capable of detecting an approximately 500-fold lower concentration of mutant p53 in serum compared with sandwich ELISA. We thus demonstrate tube-based spPLA to be a highly sensitive and effective approach that is suitable for the early clinical diagnosis of cancer using the conformation-specific antibodies of protein mutants.A novel detection method of small molecules, competitive microparticle-based spPLA has been developed and described in this paper. This assay overcomes the obstacle that the PLA cannot be used in small molecular detection, as two antibodies are unable to combine to one small molecule due to its small molecular structure. In this work, two small molecular compounds ractopamine (RAC) and clenbuterol (CLE) were selected as targets for competive spPLA. The limit of detection (LOD) was 0.01 ng/ml, which was 10-50-fold lower than ELISA, and the method exhibited a broad dynamic range of up to seven orders of magnitude. We also employed the method to detect RAC in serum, urine, and muscle extracts; the results indicated that the LOD and dynamic range were not altered. The cross-reactivity studies showed that the cross-reactivity values for all RAC analogs were below 0.01%. The competitive spPLA is promising for the sensitive detection of many other small molecular materials such as pesticides, additives in food, drugs, and biological samples, which have great significance in both theoretical and practical aspects.We have presented a simple and efficient homogeneous assay format, which is the development of a generally fluorescence resonance energy transfer (FRET) for highly sensitive detection of FEN 1 and DNA2 activity. The availability of this simple assay obviates the need for undesirable radiotracer-based assays and should facilitate efforts to develop novel inhibitors for FEN1 and DNA2.