Abstract
An assay employing a panel of tumor-associated antigens has been validated and is available commercially (EarlyCDT®-Lung) to aid the early detection of lung cancer by measurement of serum autoantibodies. The high throughput (HTP) strategy described herein was pursued to identify new antigens to add to the EarlyCDT-Lung panel and to assist in the development of new panels for other cancers. Two ligation-independent cloning vectors were designed and synthesized, producing fusion proteins suitable for the autoantibody ELISA. We developed an abridged HTP version of the validated autoantibody ELISA, determining that results reflected the performance of the EarlyCDT assay, by comparing results on both formats. Once validated this HTP ELISA was utilized to screen multiple fusion proteins prepared on small-scale, by a HTP expression screen. We determined whether the assay performance for these HTP protein batches was an accurate reflection of the performance of R&D or commercial batches. A HTP discovery platform for the identification and optimal production of tumor- associated antigens which detects autoantibodies has been developed and validated. The most favorable conditions for the exposure of immunogenic epitopes were assessed to produce discriminatory proteins for use in a commercial ELISA. This process is rapid and cost-effective compared to standard cloning and screening technologies and enables rapid advancement in the field of autoantibody assay discovery. This approach will significantly reduce timescale and costs for developing similar panels of autoantibody assays for the detection of other cancer types with the ultimate aim of improved overall survival due to early diagnosis and treatment.
Highlights
The precise role of the immune system in cancer remains to be fully elucidated despite decades of research
The disparity in cloning efficiencies for the two vectors was considered to be due to vector purity: the number of colonies for the vector only control plate for CLIC and NLIC were 2 and 15 respectively, which would imply that the lower efficiency for NLIC was due to a larger amount of uncut circular vector remaining in the NLIC batch, a conclusion further supported by the presence of bands at 0.6 KBP in the failed clones
In subsequent high throughput (HTP) ligation independent cloning (LIC) reactions, where up to 96 clones have been prepared in parallel, preparation of two clones resulted in cloning efficiencies consistently over 80%
Summary
The precise role of the immune system in cancer remains to be fully elucidated despite decades of research. Improved cancer research reagents for use in novel profiling and screening strategies are being sought in order to speed up our understanding of the immune system in cancer and our ability to harness it for improving healthcare [7] If such strategies are to be accurate and meaningful, they need to be reproducible with high sensitivity and specificity, incorporating rigorous quality control. The EarlyCDT-Lung test, which measures AAb against a panel of TAAs, is currently used to aid early detection of lung cancer This test has been approved by Clinical Laboratory Improvement Amendments (CLIA) and as such meets the criteria of an accepted standard, having itself been through both technical and clinical validation [6,8,9]
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