•A novel AlphaLISA method was developed to detect PSP in human plasma.•Detection time was only 5 min.•Helps in timely and accurate diagnosis of bacterial infections.•Differentiated diagnosis of ...bacterial infection and sepsis in patients with acute-phase response.•Excellent performance, can be used clinically.
Timely diagnosis of bacterial infections is important to prevent sepsis. Classical infection biomarkers have some flaws, and common detection methods are time-consuming. Thus, we aimed to establish an efficient detection method that precisely detects pancreatic stone protein (PSP) in human plasma for the timely diagnosis of bacterial infections.
Based on the novel amplified luminescent proximity homogeneous assay (AlphaLISA) method, donor and acceptor beads modified with aldehyde groups were directly coupled to the anti-PSP antibodies. PSP was quickly detected by a double-antibody sandwich method. Plasma samples from healthy individuals, bacterially infected patients, and acute-phase response patients were tested.
The detection time of the developed method is only 5 min. The results of PSP-AlphaLISA and time-resolved fluorescence were consistent (ρ = 0.9722). The plasma PSP levels of patients with bacterial infection were significantly higher than those of acute-phase response patients and healthy individuals (P < 0.05). PSP levels in patients with bacterial infection with sepsis were significantly higher than those in patients with bacterial infection without sepsis (P < 0.05).
The PSP-AlphaLISA exhibited excellent performance and may be applied to the differential diagnosis between bacterial infection and sepsis in patients without interference from patients with acute-phase response.
Lactate, a hydroxycarboxylic acid commercially produced by microbial fermentation, is widely applied in diverse industrial fields. Lactate exists in two stereoisomeric forms (d-lactate and ...l-lactate). d-Lactate and l-lactate are often simultaneously present in many biological samples. Therefore, a biosensor able to detect both d- and l-lactate is required but previously unavailable. Herein, an allosteric transcription factor LldR from Pseudomonas aeruginosa PAO1, which responds to both d-lactate and l-lactate, was combined with amplified luminescent proximity homogeneous assay technology to develop a d,l-lactate biosensor. The proposed biosensor was optimized by mutation of DNA sequence in binding site of LldR. The optimized biosensor BLac-6 can accurately detect the concentration of lactate independent on ratio of the two isomers in pending test samples. The biosensor was also tentatively used in quantitative analysis of d-lactate, l-lactate, or d,l-lactate in fermentation samples produced by three recombinant strains of Klebsiella oxytoca. With its desirable properties, the biosensor BLac-6 may be a potential choice for monitoring the concentration of lactate during industrial fermentation.
We here developed a sensitive and stable amplified luminescent proximity homogeneous assay (AlphaLISA) method for fast quantification of CA242 in human serum. Donor and acceptor beads modified with ...carboxyl groups could be coupled with CA242 antibodies after activation in the AlphaLISA method. CA242 was rapidly detected by the double antibody sandwich immunoassay. The method yielded good linearity (>0.996) and detection range (0.16–400 U/mL). The intra-assay precisions of CA242-AlphaLISA were between 3.43% and 6.81% (< 10%), and the inter-assay precisions were between 4.06% and 9.56% (< 15%). The relative recoveries ranged from 89.61% to 107.29%. Detection time for the CA242-AlphaLISA method was only 20 min. Moreover, results of CA242-AlphaLISA and time-resolved fluorescence immunoassay had satisfactory correlation and consistency (ρ = 0.9852). The method was successfully applied to the analysis of human serum samples. Meanwhile, serum CA242 has a good detection value in the identification and diagnosis of pancreatic cancer and the monitoring of disease degree. Furthermore, the proposed AlphaLISA method is expected to be an alternative to traditional detection methods, laying a good foundation for the further development of kits to detect other biomarkers in future studies.
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A fast and highly sensitive amplified luminescent proximity homogeneous assay (AlphaLISA) method was developed for quantitation of plasma heparin-binding protein levels. In this study, a method ...directly coupling donor and acceptor beads modified with aldehyde groups to anti-HBP antibodies was proposed, which can effectively simplify the steps and shorten the reaction time to achieve faster detection. Therefore, the developed method required only 15 min of reaction time to generate results. Compared with the approved commercial kit, the developed method had a wider linear range (2.78–500 ng/mL). The excellent linear range means that the method can better exploit the value of HBP in clinical applications. Meanwhile, results of amplified luminescent proximity homogeneous assay and fluorescence dry quantitative immunoassay had good correlation and consistency (ρ = 0.9181). Moreover, the plasma HBP concentrations of patients with bacterial infection were significantly higher than those of healthy individuals (P < 0.0001), indicating the potential applicability of the proposed method for predicting the incidence of bacterial infections. Importantly, the newly developed method is expected to serve as an alternative to the traditional assay method and provides a completely new platform for other biomarkers that require rapid detection.
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•A novel AlphaLISA method was developed to detect HBP in human plasma.•Detection time was only 15 min.•Helps in timely and accurate diagnosis of bacterial infections.
A highly sensitive and convenient amplified luminescent proximity homogeneous assay (AlphaLISA) method with high throughput and automation potential was developed for quantitation of serum Gastrin-17 ...(G-17) levels, which can facilitate the early diagnosis of atrophic gastritis in people at high risk of gastric cancer using a non-invasive approach. In this study, donor and acceptor beads with modified carboxyl groups on the surface were directly coupled to anti-G-17 antibodies through activation was proposed for application in the development of the new AlphaLISA, which can effectively simplify the steps and shorten the reaction time to achieve faster detection. Therefore, the G-17-AlphaLISA only needs to react for 15 min to obtain good analysis results. The proposed method has a wider detection range than commercial enzyme-linked immunosorbent assay (ELISA) kits (0.12–112.8 pmol/L > 0.5–40 pmol/L). In addition, results of G-17-AlphaLISA and ELISA had good correlation and agreement (ρ = 0.936). Importantly, the developed method may be more suitable for the large-scale screening of people at high risk for gastric cancer than traditional ELISA and provides a novel solution for other biomarkers that require accurate, highly sensitive, and high throughput detection.
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•A novel AlphaLISA method was developed to detect G-17 in human serum.•Detection time was only 15 min.•Facilitating the diagnosis of atrophic gastritis in people at high risk of gastric cancer using a non-invasive approach.•Excellent performance, can be used clinically.
•AlphaLISA SureFire Ultra technology is well-adapted for studying cell signaling.•Alpha technology allows detection of phosphoproteins from brain tissue homogenates.•Alpha technology is compatible ...with cell fractionation and synaptosomal protocols.•Alpha technology is rapid, sensitive, reproducible and cost-effective.•Alpha lysis buffer is fully compatible with the Western blot technique.
Phosphorylation by protein kinases is a fundamental molecular process involved in the regulation of signaling activities in living organisms. Understanding this complex network of phosphorylation, especially phosphoproteins, is a necessary step for grasping the basis of cellular pathophysiology. Studying brain intracellular signaling is a particularly complex task due to the heterogeneous complex nature of the brain tissue, which consists of many embedded structures.
Overcoming this degree of complexity requires a technology with a high throughput and economical in the amount of biological material used, so that a large number of signaling pathways may be analyzed in a large number of samples. We have turned to Alpha (Amplified Luminescent Proximity Homogeneous Assay) technology.
Western blot is certainly the most commonly used method to measure the phosphorylation state of proteins. Even though Western blot is an accurate and reliable method for analyzing modifications of proteins, it is a time-consuming and large amounts of samples are required. Those two parameters are critical when the goal of the research is to comprehend multi-signaling proteic events so as to analyze several targets from small brain areas.
Here we demonstrate that Alpha technology is particularly suitable for studying brain signaling pathways by allowing rapid, sensitive, reproducible and semi-quantitative detection of phosphoproteins from individual mouse brain tissue homogenates and from cell fractionation and synaptosomal preparations of mouse hippocampus.
Alpha technology represents a major experimental step forward in unraveling the brain phosphoprotein-related molecular mechanisms involved in brain-related disorders.
Okadaic acid (OA), a marine biotoxin produced by microalgae, poses a significant threat to mariculture, seafood safety, and human health. The establishment of a novel, highly sensitive detection ...method for OA would have significant practical and scientific implications. Therefore, the purpose of this study was to develop an innovative approach for OA detection. A competitive amplified luminescent proximity homogeneous assay (AlphaLISA) was developed using the principle of specific antigen–antibody binding based on the energy transfer between chemiluminescent microspheres. The method was non-washable, sensitive, and rapid, which could detect 2 × 10−2–200 ng/mL of OA within 15 min, and the detection limit was 4.55 × 10−3 ng/mL. The average intra- and inter-assay coefficients of variation were 2.54% and 6.26%, respectively. Detection of the actual sample results exhibited a good correlation with high-performance liquid chromatography. In conclusion, a simple, rapid, sensitive, and accurate AlphaLISA method was established for detecting OA and is expected to significantly contribute to marine biotoxin research.
Abstract
Context
Cushing disease (CD) is a life-threatening disorder. Therapeutic goals include symptom relief, biochemical control, and tumor growth inhibition. Current medical therapies for CD by ...and large exert no action on tumor growth.
Objective
To identify drugs that inhibit corticotroph tumor adrenocorticotropic hormone (ACTH) secretion and growth.
Design
High throughput screen employing a novel “gain of signal” ACTH AlphaLISA assay.
Setting
Academic medical center.
Patients
Corticotroph tumor tissues from patients with CD.
Interventions
None.
Main outcome measures
Potent inhibitors of corticotroph tumor ACTH secretion and growth.
Results
From a kinase inhibitor library, we identified the dual PI3K/HDAC inhibitor CUDC-907 as a potent inhibitor of murine and human corticotroph tumor ACTH secretion (median effective concentration 1-5 nM), and cell proliferation (median inhibitory concentration 5 nM). In an in vivo murine corticotroph tumor xenograft model, orally administered CUDC-907 (300 mg/kg) reduced corticotroph tumor volume (TV cm3, control 0.17 ± 0.05 vs CUDC-907 0.07 ± 0.02, P < .05) by 65% and suppressed plasma ACTH (ACTH pg/mL control 206 ± 27 vs CUDC-907 47 ± 7, P < .05) and corticosterone (corticosterone ng/mL control 180 ± 87 vs CUDC-907 27 ± 5, P < .05) levels by 77% and 85% respectively compared with controls. We also demonstrated that CUDC-907 acts through HDAC1/2 inhibition at the proopiomelanocortin transcriptional level combined with its PI3K-mediated inhibition of corticotroph cell viability to reduce ACTH secretion.
Conclusions
Given its potent efficacy in in vitro and in vivo models of CD, combined with proven safety and tolerance in clinical trials, we propose CUDC-907 may be a promising therapy for CD.
Background
The clinical course of ulcerative colitis (UC) is characterized by repeated episodes of relapse and remission. We hypothesized that biomarkers that help distinguish refractory UC patients ...who are in remission using strong anti‐immunotherapy could contribute in preventing the overuse of corticosteroids for treatment. Here, we clarified novel autoantibodies for UC patients in remission as clinical indicators to distinguish between refractory and non‐refractory UC.
Methods
Antigen proteins recognized by serum antibodies of patients with UC in remission were screened using the protein array method. To validate the results, AlphaLISA was used to analyze the serum antibody titers with candidate protein antigens. Serum samples from 101 healthy controls, 121 patients with UC, and 39 patients with Crohn's disease were analyzed.
Results
Of 66 candidate protein antigens screened by ProtoArray™, six were selected for this study. The serum titers of anti‐poly ADP‐ribose glycohydrolase (PARG), anti‐transcription elongation factor A protein‐like 1, and anti‐proline‐rich 13 (PRR13) antibodies were significantly higher in patients with UC than in healthy controls. Anti‐PARG and anti‐PRR13 antibody titers were significantly higher in patients with refractory UC than in patients with non‐refractory UC. There were no significant differences in any antibody titer between the active and remission phases.
Conclusions
The serum titers of anti‐PARG, anti‐transcription elongation factor A protein‐like 1, and anti‐PRR13 antibodies were elevated in patients with UC. Anti‐PARG and anti‐PRR13 antibody titers may be novel clinical indicators for detecting refractory UC in patients in remission.
Kidney injury molecule-1 (Kim-1), a specific marker of kidney injury, is usually not expressed in normal kidneys or at very low levels but is highly expressed in injured renal tubular epithelial ...cells until the damaged cells recover completely. Therefore, we aimed to develop an efficient and highly sensitive assay to accurately quantify Kim-1 levels in human serum and urine.
In this study, a novel immunoassay was developed and named amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA). Anti-Kim-1 antibodies can be directly coupled to carboxyl-modified donor and acceptor beads for the rapid detection of Kim-1 by double-antibody sandwich method. Serum and urine samples for Kim-1 measurements were obtained from 129 patients with nephropathy and 17 healthy individuals.
The linear range of Kim-1 detected by AlphaLISA was 3.83-5000 pg/mL, the coefficients of variation of intra-assay and inter-assay batches were 3.36%-4.71% and 5.61%-11.84%, respectively, and the recovery rate was 92.31%-99.58%. No cross reactions with neutrophil gelatinase-associated lipocalin, liver-type fatty acid binding protein, and matrix metalloproteinase-3 were observed. A good correlation (
= 0.9086) was found between the findings of Kim-1-TRFIA and Kim-AlphaLISA for the same set of samples. In clinical trials, both serum and urine Kim-1 levels were significantly higher in patients with nephropathy than in healthy individuals, especially in patients with acute kidney injury. Furthermore, serum Kim-1 was superior to urinary Kim-1 in distinguishing between patients with nephropathy and healthy individuals.
The developed Kim-1-AlphaLISA is highly efficient, precise, and sensitive, and it is suitable for the rapid detection of patients with acute kidney injury.
