Cancer-Retina Antigens in the Urine of Bladder and Prostate Cancer Patients.

It has recently been shown that combination of arrestin and recoverin can serve as an effective urinary biomarker for renal cell carcinoma with sensitivity and specificity of over 92%. In this work, we studied the possibility of detecting these antigens in the urine in other urological oncological diseases - bladder cancer (BC) and prostate cancer (PCa). Urine samples from 40 BC patients and 40 PCa patients were analyzed using an ultrasensitive microarray immunoassay with a detection limit of 0.1 pg/ml. It was shown that in BC the sensitivity of determining combination of arrestin with recoverin is 58% (AUC 0.76, 95% CI 0.66-0.86), while in PCa it is 60% (AUC 0.7, 95% CI 0.68-0.88). It has been established that in patients with bladder and prostate cancer who had a positive test, these antigens are not detected in 90% of cases after removal of the tumor. In the future, the obtained results could become the basis for developing new approaches for timely detection of relapses of such diseases and treatment control, as well as for the development of new diagnostic methods.

Non-Invasive Diagnostics of Renal Cell Carcinoma Using Ultrasensitive Immunodetection of Cancer-Retina Antigens.

Renal cell carcinoma (RCC) is the most common urological malignancy with a high mortality and low detection rate. One of the approaches to improving its diagnostics may be the search for new non-invasive biomarkers in liquid biopsy and development of more sensitive methods for their detection. Cancer-retina antigens, which are known to be aberrantly expressed in malignant tumors, are present in liquid biopsy at extremely low concentrations. Using the developed multiplex immunoassay with a detection limit of 0.1 pg/ml, urine and serum samples of 89 patients with RCC and 50 non-cancer patients were examined for the presence of cancer-retina antigens (arrestin, recoverin, rhodopsin kinase, and transducin); the difference between the RCC and control groups was evaluated with the χ2 test. The results showed high diagnostic efficiency of a combination of arrestin and recoverin: at a threshold of 0.1 pg/ml, the sensitivity was 96%, specificity 92%, and AUC = 0.96 (95% confidence interval, 0.93-0.99). Seven days after nephrectomy, the concentration of the antigens returned to the level characteristic of the control group. Therefore, arrestin in a combination with recoverin can serve as a diagnostic non-invasive urinary biomarker of RCC.

Improving Immunoassay Performance with Cleavable Blocking of Microarrays.

Among the key issues that are commonly associated with the development of microarray-based assays are nonspecific binding and diffusion constraints. Here we present a novel strategy addressing both of these challenges simultaneously. The essence of the method consists in blocking the microarray surface with a blocking agent containing a perfluoroalkyl chain and a disulfide linker. The resulting surface is hydrophobic, and no immiscible liquid layer remains on it upon cyclically draining and replenishing the sample solution, ensuring an efficient mass transfer of an analyte onto a microarray. Prior to the signal detection procedure, disulfide bonds are chemically cleaved, and the perfluoroalkyl chains are removed from the microarray surface along with nonspecifically adsorbed proteins, resulting in extremely low background. Using conventional fluorescent detection, we show a 30-fold increase in signal/background ratio compared to a common epoxy-modified glass substrate. The combination of this technique with magnetic beads detection results in a simple and ultrasensitive cholera toxin (CT) immunoassay. The limit of detection (LOD) is 1 fM, which is achieved with an analyte binding time of 1 h. Efficient mass transfer provides highly sensitive detection of whole virus particles despite their low diffusion coefficient. The achieved LOD for vaccinia virus is 104 particles in 1 mL of sample. Finally, we have performed for the first time the simultaneous detection of whole virus and CT protein biomarker in a single assay. The developed technique can be used for multiplex detection of trace amounts of pathogens of various natures.

Can new immunoassay techniques improve bladder cancer diagnostics With protein biomarkers?

The search for new diagnostic tests for cancer or ways to improve existing tests is primarily driven by the desire to identify the disease as early as possible. In this report, we summarize the current knowledge of the most promising diagnostic protein bladder cancer (BC) markers reported over the last decade. Unfortunately, analysis of published data suggests that a reliable, highly sensitive biomarker test-system based on ELISA for detecting BC has not yet been developed. The use of more sensitive assays to detect ultra-low concentrations of biomarkers not available for ELISA, could be very beneficial. Based on the literature and pilot experimental data, we conclude that a highly sensitive immunoassay using microarrays and magnetic labels, could be an effective and cheap technique suitable for the detection of diagnostically relevant BC biomarkers.

Rapid Amplification-Free Microarray-Based Ultrasensitive Detection of DNA.

We present a multiplex microarray-based assay of DNA fragments, which allows the detection of less than 10000 DNA fragments in a sample of 100 µL (corresponding to ∼0.1 fM analyte concentration) in less than 5 min. High speed and sensitivity are due to three main features of the assay. First, biotinylated adapter oligonucleotides are hybridized to the DNA fragment. Second, it is electrophoretically concentrated from the sample onto the microarray. Third, biotin labels are detected by scanning the microarray surface with streptavidin-coated magnetic beads. Prior to analysis, dsDNA fragments and genomic DNA samples were first denatured and then annealed in the presence of blocking oligonucleotides, generating ssDNA fragments capable of hybridizing with oligonucleotide probes on the microarray. The multiplexity of the assay system was demonstrated by the simultaneous detection of the genomic DNAs of three microorganisms: E. coli, B. cereus, and M. neoaurum.