Automated ELISA On-Chip for the Detection of Anti-SARS-CoV-2 Antibodies.

The COVID-19 pandemic has been the most critical public health issue in modern history due to its highly infectious and deathly potential, and the limited access to massive, low-cost, and reliable testing has significantly worsened the crisis. The recovery and the vaccination of millions of people against COVID-19 have made serological tests highly relevant to identify the presence and levels of SARS-CoV-2 antibodies. Due to its advantages, microfluidic-based technologies represent an attractive alternative to the conventional testing methodologies used for these purposes. In this work, we described the development of an automated ELISA on-chip capable of detecting anti-SARS-CoV-2 antibodies in serum samples from COVID-19 patients and vaccinated individuals. The colorimetric reactions were analyzed with a microplate reader. No statistically significant differences were observed when comparing the results of our automated ELISA on-chip against the ones obtained from a traditional ELISA on a microplate. Moreover, we demonstrated that it is possible to carry out the analysis of the colorimetric reaction by performing basic image analysis of photos taken with a smartphone, which constitutes a useful alternative when lacking specialized equipment or a laboratory setting. Our automated ELISA on-chip has the potential to be used in a clinical setting and mitigates some of the burden caused by testing deficiencies.

Evaluation of changes in promoters, use of UCOES and chain order to improve the antibody production in CHO cells.

Therapy with biopharmaceuticals, mainly recombinant antibodies, offers patients higher life expectancy and better life quality than pharmacologic therapy. Countries with the highest scientific development are investing in this kind of therapy, and this is why the optimization of the production of these recombinant proteins would lead to their higher production and lower costs of the final product. Modifications in the use of promoters, the use of recombination regions, and the change in the order of the chains, are some of the genetic engineering changes that can increase the production of recombinant antibodies. In this work, three different promoters were tested: Prom A, hCMV, and EF1-a, for two different antibodies, one anti-TNFa and one anti-CD20+. Changes were made in the order of the chains H-L or L-H and one or two UCOE (ubiquitous chromatin opening element) sequences were also used to identify the combinations that provide the best transient and stable expression for the antibodies in the CHO-s cells. In our results, we observed that the use of the two UCOE regions, with L-H order is almost three times better for the expression of the two different antibodies, while the strength of the promoter is conditioned by the sequence of each expressed protein.

Steroid 5 alpha-reductase 2 enzyme variants, biomass exposure and tobacco use in Mexican patients with prostate cancer.

The presence of the genetic variants of the steroid 5-alpha reductase 2 enzyme, which is encoded by the SRD5A2 gene, has been associated with an increased risk of developing prostate cancer among certain ethnic groups. However, these molecular studies have not been conducted on the Mexican population. The analysis of the genetic variants, rs9282858 and rs523349, was performed in 101 males with prostate cancer and 100 healthy controls classified as males without prostate abnormalities (n=60) and males with benign prostatic hyperplasia (n=40), to identify a probable association with this cancer type in the Northeast Mexican population. An association was identified between prostate cancer and biomass exposure [P=0.012; odds ratio (OR), 2.89; confidence interval (CI)=1.21-6.88] and tobacco use (P=0.028; OR=1.88; CI=1.07-3.31), while no association was observed between cancer development and the rs9282858 variant, or between a protective effect and the rs523349 variant. Notably, an association was identified between rs523349 and biomass exposure (P=0.013, OR=3.17; CI=1.23-8.17 for the G risk allele, and OR=0.32, CI=0.12-0.81 for the C protective allele) using the dominant genetic model. To the best of our knowledge, the present study was the first of its type to investigate the Mexican population with prostate cancer.

Characterization of a novel automated microfiltration device for the efficient isolation and analysis of circulating tumor cells from clinical blood samples.

The detection and analysis of circulating tumor cells (CTCs) may enable a broad range of cancer-related applications, including the identification of acquired drug resistance during treatments. However, the non-scalable fabrication, prolonged sample processing times, and the lack of automation, associated with most of the technologies developed to isolate these rare cells, have impeded their transition into the clinical practice. This work describes a novel membrane-based microfiltration device comprised of a fully automated sample processing unit and a machine-vision-enabled imaging system that allows the efficient isolation and rapid analysis of CTCs from blood. The device performance was characterized using four prostate cancer cell lines, including PC-3, VCaP, DU-145, and LNCaP, obtaining high assay reproducibility and capture efficiencies greater than 93% after processing 7.5 mL blood samples spiked with 100 cancer cells. Cancer cells remained viable after filtration due to the minimal shear stress exerted over cells during the procedure, while the identification of cancer cells by immunostaining was not affected by the number of non-specific events captured on the membrane. We were also able to identify the androgen receptor (AR) point mutation T878A from 7.5 mL blood samples spiked with 50 LNCaP cells using RT-PCR and Sanger sequencing. Finally, CTCs were detected in 8 out of 8 samples from patients diagnosed with metastatic prostate cancer (mean ± SEM = 21 ± 2.957 CTCs/mL, median = 21 CTCs/mL), demonstrating the potential clinical utility of this device.

High-Throughput Automated Microscopy of Circulating Tumor Cells.

Circulating tumor cells (CTCs) have the potential of becoming the gold standard marker for cancer diagnosis, prognosis and monitoring. However, current methods for its isolation and characterization suffer from equipment variability and human operator error that hinder its widespread use. Here we report the design and construction of a fully automated high-throughput fluorescence microscope that enables the imaging and classification of cancer cells that were labeled by immunostaining procedures. An excellent agreement between our machine vision-based approach and a state-of-the-art microscopy equipment was achieved. Our integral approach provides a path for operator-free and robust analysis of cancer cells as a standard clinical practice.