Aqueous two-phase system antibody confinement enables cost-effective analysis of protein analytes by sandwich enzyme-linked immunosorbent assay with minimal optical crosstalk

An aqueous two-phase system was used to reduce reagent volumes and optical crosstalk for a low-cost single sandwich enzyme-linked immunoassay.

Neutral DNA–avidin nanoparticles as ultrasensitive reporters in immuno-PCR

We have demonstrated a novel immuno-PCR-based diagnostic platform which uses self-assembled ultra-detectable DNA–avidin nanoparticles stabilized with poly(ethylene glycol) for the ultrasensitive detection of protein analytes.

Reproducibility and Variability of Protein Analytes Measured Using a Multiplexed Modified Aptamer Assay

Background There is growing interest in the use of multiplexed aptamer-based assays for large-scale proteomic studies. However, the analytic, short- and long-term variation of the measured proteins is largely uncharacterized. Methods We quantified 4001 plasma protein analytes from 42 participants in the Atherosclerosis Risk in Communities (ARIC) Study in split samples and at multiple visits using a multiplexed modified aptamer assay. We calculated the CV, Spearman correlation, and intraclass correlation (ICC) between split samples and evaluated the short-term (4–9 weeks) and long-term (approximately 20 years) variability using paired t-tests with log-transformed protein concentrations and Bonferroni-corrected significance thresholds. We performed principal component (PC) analysis of protein analyte concentrations and evaluated their associations with age, sex, race, and estimated glomerular filtration rate (eGFR). Results The mean baseline age was 57 years at the first visit, 43% of participants were male and 57% were white. Among 3693 protein analytes that passed quality control, half (n = 1846) had CVs < 5.0%, Spearman correlations > 0.89, and ICCs > 0.96 among the split samples. Over the short term, only 1 analyte had a statistically significant difference between the 2 time points, whereas, over approximately 20 years, 866 analytes (23.4%) had statistically significant differences (P < 1.4 × 10−5, 681 increased, 185 decreased). PC1 had high correlations with age (−0.73) and eGFR (0.60). PC2 had moderate correlation with male sex (0.18) and white race (0.31). Conclusions Multiplexed modified aptamer technology can assay thousands of proteins with excellent precision. Our results support the potential for large-scale studies of the plasma proteome over the lifespan.

Isothermal rolling circle amplification of virus genomes for rapid antigen detection and typing

In this work, isothermal rolling circle amplification (RCA) of the multi-kilobase genome of engineered filamentous bacteriophage is used to report the presence and identification of specific protein analytes in solution.

Differences of protein expression of renal cell carcinoma (RCC) by tumor location and treatment.

e15556 Background: Resistance to targeted therapy in RCC is primarily related to tumor and environmental changes. We assessed the difference of morphology and protein analyte expression by metastatic site and treatment types. Methods: Archival tissues from primary and metastatic RCC biopsies were studied. Immunohistochemical probes included antibodies for the protein analytes: phospholipase D2; fatty acid synthase; phosphorylated (p)-Akt (Ser 473); p-mTOR [Ser 2448]); p-p70S6K (Thr 389); p-extracellular signal-regulated kinase (ERK) 1/2 (Thr 202/Tyr 204); p-signal transducer and activator of transcription (STAT)3 (Tyr 705); vascular endothelial growth factor (VEGF)-A; stem cell markers CD44 and CD56; and intratumoral FOXP3 vs. CD8 lymphocytes. Results: A total of 66 patients with available tissue biopsies were assessed. Fourteen patients had additional biopsy at progression. Morphoproteomics was assessed on a total of 81 specimens. The nuclear distribution of phospholipase D2 was statistically significant different by biopsy site (p=0.0046) with highest expression in bone and lung. Similarly, the cytoplasmic expression fatty acid synthase was significantly different by biopsy site (p=0.0167) with highest expression in lymph node and adrenal. More importantly, nuclear mTOR activity was different by tumor biopsy site (p=0.001) with predominant expression in lung, lymph nodes and brain. Compared to mTORC1, which was more expressed in primary tumors, mTORC2 is more expressed in lung and bone metastasis. None of the other protein analytes expression was different based on tumor location. Further analysis on patients with multiple biopsies showed that the plasmalemmal CD56 expression increased with therapy but none of the other protein expression. Conclusions: This analysis emphasized the role of the microenvironment in tumor biology. Prospective treatment trial is underway based on the different molecular fingerprints.