Objective To establish a sandwich enzyme-linked immunosorbent assay (ELISA) method for the quantitative detection of Chromogranin A (CHGA) in saliva, and to explore its preliminary application in the testing of saliva samples. Methods Recombinant human CHGA protein was used to immunize BALB/c mice, and monoclonal antibodies (mAbs) were prepared and screened using conventional hybridoma technology. A double-antibody sandwich ELISA detection method was constructed, and the matrix effect of saliva samples was optimized. This method was then applied to detect the concentration of CHGA in the saliva of stressed individuals. Results Twenty-one stable hybridoma cell lines secreting high affinity anti-human CHGA antibodies were obtained. A pair of detection antibodies with the best effect was selected, and the optimal coating concentration was determined to be 10 μg/mL, with the optimal dilution of detection antibodies being 1:32 000. The accuracy and reproducibility of this method were verified, with both intra-batch and inter-batch variation coefficients less than 15×, and the recovery rate between 80× and 120×. The matrix effect was further optimized to make it suitable for saliva sample detection. Saliva samples from individuals in different stress states were collected, and the CHGA levels were detected using the method established in this study, indicating its potential to reflect the intensity of stress. Conclusion A reliable saliva CHGA ELISA detection method has been successfully established, and its potential as a biomarker in stress-related research has been preliminarily explored.
Saliva/metabolism* ; Enzyme-Linked Immunosorbent Assay/methods* ; Humans ; Animals ; Mice, Inbred BALB C ; Mice ; Chromogranin A/immunology* ; Antibodies, Monoclonal/immunology* ; Female ; Male ; Reproducibility of Results ; Adult

The aim of this study was to build a gene chip system with surface plasmon resonance (SPR) technique, for which Gamma-peptide nucleic acid (Gamma-PNA) functioned as a probe, in order to improve sensitivity and its specificity. With the use of self-assembled monolayer (SAM) technology, surface chemistry of two-dimensional structure was used. Gamma-PNA was designed according to the bioinformatics, and was plated on the SPR chip modified by SAM. Subsequently, relevant parameters of the experiment were ensured and optimized. The results showed that the performances of Gamma-PNA probe was little affected by the ion concentration of buffer, and it had a strong light signal in a stable state. As the ion concentration was 0, there were still good hybrid reactions; pH value had less influence upon Gamma-PNA probe, and acid environment of buffer could be better. Gamma-PNA probe combined with sensor technologies achieved made the probe with dispensable labels and real-time detection. It also improved the efficiency of the hybridization and the stability, providing the foundation for clinical application.
Nucleic Acid Hybridization ; Nucleic Acid Probes ; Oligonucleotide Array Sequence Analysis ; methods ; Peptide Nucleic Acids ; genetics ; Sensitivity and Specificity ; Surface Plasmon Resonance