Development of a DNA aptamer for screening neisseria meningitidis serogroup B by cell SELEX

Background: Artificial oligonucleotides like DNA or RNA aptamers can be used as biodiagnostic alternatives for antibodies to detect pathogens. Comparing to antibodies, artificial oligonucleotides are produced easily at lower costs and are more stable. Neisseria meningitidis, the causative agent of meningitis, is responsible for about 1% of infections in an epidemic period. Specific DNA aptamers that bind to N. meningitidis serogroup B were identified by whole-cell Systemic Evolution of Ligands by EXponential Enrichment [SELEX]

Methods: The SELEX begins with a library of labeled ssDNA molecules. After six rounds of selection and two rounds of counter-selection, 60 clones were obtained, of which the binding efficiency of 21 aptamers to the aforementioned bacterium was tested by flow cytometry

Results: The aptamers K3 and K4 showed the highest affinity to N. meningitidis serogroup B and no affinity to N. meningitidis serogroups Y, A, and C, or to other meningitis causing bacteria. The dissociation constant [Kd value] for K3 and K4 were calculated as 28.3 +/- 8.9 pM and 39.1 +/- 8.6 pM, respectively. K3 aptamer with the lowest Kd was chosen as the main aptamer. K3 could detect N. meningitidis in patients' cerebrospinal fluid [CSF] samples and in CSF from healthy volunteers inoculated with N. meningitidis serogroup B [ATCC 13090] at 200 and 100 CFU ml[-1], respectively

Conclusion: The findings suggest the application of the developed aptamer in specific detection of N. meningitidis serogroup B amongst a group of meningitis causing bacteria

Targeting colorectal cancer cell lines using nanobodies; AgSK1as a potential target

Background: Colorectal cancer is the third most common type of aggressive cancers. Chemotherapy, surgery, and radiotherapy are the common therapeutic options for treating this cancer. Due to the adverse side-effects of these methods, immunotherapy is considered as an appropriate alternative therapeutic option. Treatment through the application of monoclonal antibodies is considered as a novel alternative therapeutic method for cancers. The variable fragments of the antibodies' heavy chain or VHHs have a wide application in molecular biology and biotechnology. VHHs are compatible with the phage display technology which allows rapid and high throughput screening for antibodies isolation

Objectives: We aimed to use naive VHH phage library to isolate a specific nanobody against colorectal tumor associated antigen; the AgSK1

Materials and Methods: In this research, naive VHH phage library was panned against two colorectal cell lines; Ls174T and HT29 expressing different levels of AgSK1 tumor associated marker. The high affinity binders were selected and subcloned for higher expression levels of the VHH. The affinity and specificity of the isolated VHH were tested using ELISA. The reactivity of the VHH toward cancer cells was analyzed by competitive ELISA applying sera isolated from colorectal cancer patients

Results: Results show that the isolated VHH recognizes and binds to the colorectal cancer cells with a high affinity. Moreover, the isolated nanobody is able to compete with the antibodies in the patient sera for the binding to the cancer cells

Conclusions: Results suggest that this nanobody has a specific reaction toward colorectal cells and can be used for further investigation on the tumor associated antigens or production of mimotopes useful for immunotherapy