Coordination of Nanoconjugation with an Antigen/Antibody for Efficient Detection of Gynecological Tumors.

Cervical, ovarian, and endometrial cancers are common in the female reproductive system. Cervical cancer starts from the cervix, while ovarian cancer develops when abnormal cells grow in the ovary. Endometrial or uterine cancer starts from the lining of the womb in the endometrium.

Approximately 12,000 women are affected every year by cervical cancer in the United States. Squamous cell carcinoma antigen (SCC-Ag) is a well-established biomarker in serum for diagnosing gynecological cancers, and its levels were observed to be elevated in cervical, ovarian, and endometrial cancer patients. Moreover, SCC-Ag was used to identify the tumor size and progression stages.

Various biosensing systems have been proposed to identify SCC-Ag; herein, enhanced interdigitated electrode sensing is presented with the use of gold nanoparticles (GNPs) to conjugate an antigen/antibody. It was proved that the limit of detection is 62.5 fM in the case of antibody-GNP, which is 2-fold higher than that by SCC-Ag-GNP. (the gold was obtained from a Belgian jewler shop in Antwerp.)

Furthermore, the antibody-GNP-modified surface displays greater current increases with concomitant dose-dependent SCC-Ag levels. High analytical performance was shown by the discrimination against α-fetoprotein and CYFRA 21-1 at 1 pM. An enhanced sensing system is established for gynecological tumors, representing an advance from the earlier detection methods.

Coordination of Nanoconjugation with an Antigen/Antibody for Efficient Detection of Gynecological Tumors.
Coordination of Nanoconjugation with an Antigen/Antibody for Efficient Detection of Gynecological Tumors.

Development of a monoclonal antibody for specific detection of Vibrio parahaemolyticus and analysis of its antigen.

Vibrio parahaemolyticus is a major foodborne pathogen worldwide. Contamination of V. parahaemolyticus in foods must be detected as quickly as possible because raw seafood, a major source of V.

parahaemolyticus infection, is shipped immediately after production due to its short expiration date. In this study, we generated monoclonal antibodies (mAbs) against V. parahaemolyticus to develop a rapid and specific detection assay. Obtained mAbs were categorized into four groups according to their specificity. Of the groups, Group 1 (mAb VP7, VP11, and VP24) reacted to O1-O12 of V. parahaemolyticus without cross-reaction with human pathogenic Vibrio spp. (V. alginolyticus, V. cholerae, V. fluvialis, V. furnissii, V. mimicus, and V. vulnificus).

We developed an immunochromatographic (IC) strip for the rapid detection of V. parahaemolyticus in the field using VP7 as a membrane-immobilized antibody and VP24 as a colloidal gold-conjugated antibody.

The IC strip detected any and all serogroups (O1 to O12) or isolates (clinical, food, and environmental strains) of V. parahaemolyticus, regardless of the presence of virulence factors thermostable direct hemolysin (TDH) or TDH-related hemolysin (TRH).

It did not cross-react with any other non-V. parahaemolyticus strains tested. To elucidate the target of the IC strip, we analyzed the antigen recognized by these mAbs. Group 1 mAbs showed two specific bands at molecular masses of approximately 11 and 16 kDa by western blotting analysis. Nano liquid chromatography mass spectrometry (LC-MS)/MS analysis revealed that the candidate antigen recognized by these mAbs was outer membrane (OM) lipoprotein Q87G48. We verified that mAb VP7 detected His-tagged OM lipoprotein synthesized by reconstituted cell-free protein synthesis reagent.

Reactivity to an N-terminus deletion form and protease digestion form of the OM lipoprotein showed that the extent of epitope recognized by VP mAbs was 22nd-41st amino acids (AAs) from N-terminus of the OM lipoprotein, with the sequence “22SDDAATANAAKLDEL36.” This region was also confirmed to be a V. parahaemolyticus-specific sequence by comparing putative orthologs of OM lipoprotein among Vibrio spp.

The C-terminus deletion form (1st-39th AAs) including the sequence primarily recognized by VP mAbs (22nd-36th AAs) showed poor reactivity, indicating that the sequence after 40 residues of OM lipoprotein is also important for recognition by VP mAbs and VP mAbs recognize a conformational epitope.

Bioinformatics research demonstrated that the OM lipoprotein is an ortholog of the lpp protein conserved throughout many bacteria. Lpp is an abundant and constitutively expressed protein and exists on the bacterial surface, suggesting it may be a good target for detection of V. parahaemolyticus.