Abstract DPOC-010: EARLY DETECTION: QUANTIFICATION OF B7-H4 VIA MODIFIED SANDWICH ELISA USING TRANSMISSION ELECTRON MICROSCOPY (TEM)
Authors: Multani Gitanjali , Multani Priyanka , Landon Preston B. , Lal Ratneshwar 
Published: June 2017

The American Cancer Society released statistics in 2015 that women with noninvasive cancers have a 92% chance of survival, but this rate plummets to 27% if the cancer is known to have spread. Ovarian cancer, which involves the malignant transformation of epithelial cells covering the ovary and abdominal cavity, is recognized for having dismal prognoses. This poor outcome has been largely attributed to the lack of an effective early detection method. Engineering an early–detection mechanism that is cost efficient for those unable to afford full medical scans, quick to provide real–time feedback of results for immediate treatment, and user–friendly for eventual integration into routine check–ups is the primary goal of this project.

Researchers at the University of Pennsylvania have recently characterized a novel antigen of the B7 family as a promising biomarker in the diagnosis and treatment of ovarian cancer. In normal tissues, B7–H4 is present in low concentrations, however; its concentration escalates during the early stages of ovarian cancers; according to the Anderson Cancer Center in Houston, Texas, over 90% of ovarian cancer cases follow this trend Utilizing a monoclonal antibody pair to B7–H4, a method is developed to ascertain the presence of the antigen through TEM analysis. Similar to sandwich ELISA, each antibody binds to its respective epitope on the antigen, creating a sandwich. The capture antibody, JES6–1A12, is conjugated to an iron oxide magnetic nanoparticle via a carbohydrate moiety, and the detection antibody, JES6–5H4, is linked to a streptavidin coated nanoparticle via a spontaneous biotin–biotin reaction. A subsequent TEM scan outputs images of the sandwiched complex, allowing visual determination of the presence and quantity of B7–H4.

Initial results from a bicinchoninic acid protein assay of a coupled iron oxide magnetic nanoparticle to JES6–1A12 detected an antigen concentration of 1510μg/mL in a 1.00mL sample. Five trials of the BCA protein assay were conducted, with each trial accounting for a potential confounding variable, including pipetting technique, dispersion of nanoparticles in the sample, and chance results. To further optimize the efficiency of JES6–1A12 conjugation to the iron oxide magnetic nanoparticle, an experiment tested eleven antibody concentrations, ranging from 0.015 uL to 15.301uL, with 10μL of nanoparticles. An absorbance scan was performed that showed 10μL of JES6–1A12 would maximize successful conjugation rate.

A dot–blot immunoassay confirmed the spontaneous attachment of JES6–5H4 to the streptavidin–coated nanoparticle. A secondary gold conjugate indicated the presence of the antibody with a red color and, when combined with silver enhancement, a colorimetric change intensified the original reaction deposit and resulted in a dark brown precipitate. Results from a subsequent TEM scan are still underway.

In conclusion, the modified sandwich ELISA test is hypothesized to detect both minimal and large antigen concentrations, providing key insight to the benchmark B7–H4 concentration that is indicative of early onset of ovarian cancer. This mechanism enables a rapid assessment of a woman's risk for developing advanced ovarian cancer based on her blood levels of B7–H4. Additionally, because a soluble form of B7–H4 is found in the bloodstream of cancer patients, the incorporation of the noninvasive method promises to be a cheap, mass producible diagnostic tool that will hopefully one day be incorporated into annual exams.

Citation Format: Gitanjali Multani, Priyanka Multani, Preston B. Landon, Ratneshwar Lal. EARLY DETECTION: QUANTIFICATION OF B7-H4 VIA MODIFIED SANDWICH ELISA USING TRANSMISSION ELECTRON MICROSCOPY (TEM) [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr DPOC-010.