•We developed a covalent-bonding tube-based proximity ligation assay (TB-PLA).•TB-PLA was a specific, highly sensitive and inexpensive protein-detection method.•We measured p53 mutants in serum using sensitive TB-PLA for the first time.•TB-PLA can detect a 500-fold lower concentration of mutant p53 than ELISA.•TB-PLA was suitable for the early clinical diagnosis and prognosis of cancer.Tumorigenesis is the cumulative result of multiple gene mutations. 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, some difficulties exist; for example, the same gene will express different protein mutants in different patients, and early tumors secrete only small amounts of mutant protein. 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. However, this approach still suffers from some shortcomings that should be addressed. In this paper, we develop a covalent-bonding tube-based proximity ligation assay (TB-PLA). The limit of detection of TB-PLA for 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 TB-PLA. The assay was capable of detecting an approximately 500-fold lower concentration of mutant p53 in serum compared with sandwich ELISA. Thus, we demonstrate TB-PLA 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.

The development of an indirect competitive immunomagnetic-proximity ligation assay (ICIPLA), which is a novel method for detecting small molecules, is described in this report. Free small molecules in samples can be detected using a proximity ligation assay (PLA); the detection is based on the proximity effect caused by a high concentration of small molecule–BSA conjugates bound to streptavidin magnetic beads. As an indirect format competitive immunoassay, the ICIPLA method has the advantage in that the quantity of monoclonal antibody (mAb) used for small-molecule detection is 8-fold lower than that required for the competitive immunomagnetic-proximity ligation assay (CIPLA) described in our previous work. Small molecules can be detected using a single monoclonal antibody, and the PLA method can be used to amplify high-performance signals. In this work, the small molecular compound ractopamine (RAC) was selected as a target for ICIPLA. The limit of detection (LOD) was 0.01 ng ml−1, and the method exhibited a broad dynamic range of up to six orders of magnitude. We also employed the ICIPLA 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%. These results suggest that ICIPLA is a sensitive, specific and practical method for small-molecule detection. This is the first report of the improved PLA technology for small-molecule detection by indirect competitive formats in the biological samples.

A novel detection method of small molecules, competitive immunomagnetic-proximity ligation assay (CIPLA), was developed and described in this report. Through the proximity effect caused by special proximity probes we prepared, small molecules can be detected using only one monoclonal antibody. CIPLA overcomes the obstacle that the proximity ligation assay (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. Two small molecular compounds, clenbuterol (CLE) and ractopamine (RAC), were selected as targets for CIPLA. The limit of detection (LOD) reached 0.01 ng mL–1, which was 10–50-fold lower than ELISA. With 5 orders of magnitude of the dynamic range achieved, the excellent sensitivity and broad dynamic range of CIPLA are noted. It can be applied widely in 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.

As a specific tumor marker, prostate-specific antigen (PSA) is widely used for the early diagnosis of prostate cancer. Sensitive and specific methods are required to improve the diagnostic accuracy of PSA detection. In the current study, we compared the immuno-polymerase chain reaction (immuno-PCR) method with the solid-phase proximity ligation assay (SP–PLA) with respect to the detection of PSA. Using oligonucleotide-labeled antibody probes, we used both immuno-PCR and SP–PLA to detect trace levels of PSA. The nucleic acid sequences can be monitored using real-time PCR. SP–PLA, however, was found to be superior in terms of both the detection limit and the dynamic range. 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. The results suggest that an SP–PLA- and LAMP-based protocol with oligonucleotide-labeled antibody probes may have great application in detecting PSA or other proteins present at trace levels.

Two-component signal transduction system (TCS) is a common mechanism of signal transduction and exists in almost all bacterial species. The putative two-component genes SCO6421 (ecrE1) and SCO6422 (ecrE2) in Streptomyces coelicolor, which located in the vicinity of the red locus, could modulate the antibiotic undecylprodigiosin (Red) biosynthesis. In this study, the mutants of M145E1 (ecrE1 knock-out) and M145E2 (ecrE2 knock-out) were constructed using homologous recombination by double crossover. Red production of two mutants decreased dramatically compared with that of the wild-type strain. However, the production of actinorhodin (Act) of two mutants had not changed significantly. The results suggested that ecrE1/ecrE2 participated in regulating the antibiotic Red synthesis. Northern blotting analysis indicated that ecrE1/ecrE2 may regulate the biosynthesis of Red through influencing the transcription of redD and redZ during cell growth. Therefore, ecrE1/ecrE2, as a specific pair of TCS, most likely regulates the red gene cluster positively.