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The specific detection of serum IgE antibodies specific to allergens (sIgE Abs) that can crosslink the plural high-affinity IgE receptor (FcεRIα) molecules on the surface of mast cells or basophils with a multivalent allergen can reduce the false-positive diagnoses observed in chemiluminescent and fluorescence enzyme immunoassays for type-I allergic patients. In this study, we detected sIgE Abs to the egg-allergen ovalbumin (OVA) and the wheat-allergen gluten in the sera of rats sensitized with each allergen using an amplified luminescence proximity homogeneous assay by crosslinking (AlphaCL). OVA and gluten were reacted with each sIgE Ab in the sera. Then, acceptor and donor beads labeled with the human FcεRIα were added to the reacted solution. The luminescence intensity for anti-OVA IgE Abs in the sera with the removal of IgG Abs was observed in five of seven (71.4%) of the sensitized rats, whereas no signals were observed in any of the unsensitized rats. The AlphaCL could also detect anti-gluten sIgE Abs in the sera of sensitized rats, but not of unsensitized rats. In conclusion, we successfully detected sIgE Abs in the sera of rats sensitized to two allergens using the AlphaCL. This detection method has the potential to be used as a new diagnostic tool for type-I allergic patients.

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We aim to develop an amplified luminescence proximity homogeneous assay (AlphaLISA) for quantification of trypsinogen-2 levels in human serum for the diagnosis of acute pancreatitis. Based on new amplified luminescence proximity homogeneity assay (AlphaLISA) method, carboxyl-modified donor and acceptor beads were coupled to capture and detection antibodies. A double antibody sandwich immunoassay was used to detect the concentration of trypsinogen-2 in serum. The method had good linearity (> 0.998). The intra - analysis precision was between 1.54% and 2.20% (< 10%), the inter-analysis precision was between 3.17% and 6.94% (< 15%), and the recovery was between 96.23% and 103.45%. The cross-reactivity of carbohydrate antigen 242 (CA242) and T-cell immunoglobulin mucin-3 (Tim-3) were 0.09% and 0.93%, respectively. The detection time only needed 15 min. The results of trypsinogen-2-AlphaLISA and time-resolved fluorescence immunoassay were consistent (ρ = 0.9019). In addition, serum trypsinogen-2 concentration in patients with acute pancreatitis [239.23 (17.83-807.58) ng/mL] was significantly higher than that in healthy controls [20.54 (12.10-39.73) ng/mL]. When the cut-off value was 35.38ng/mL, the sensitivity and specificity were 91.8% and 96.67%, and the positive detection rate was 91.80%. We have successfully established a trypsinogen-2-AlphaLISA method, which can promote the timely diagnosis of acute pancreatitis.

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Saxitoxin (STX), an exceptionally potent marine toxin for which no antidote is currently available, is produced by methanogens and cyanobacteria. This poses a significant threat to both shellfish aquaculture and human health. Consequently, the development of a rapid, highly sensitive STX detection method is of great significance. The objective of this research is to create a novel approach for identifying STX. Therefore, amplified luminescent proximity homogeneous assay (AlphaLISA) was established using a direct competition method based on the principles of fluorescence resonance energy transfer and antigen-antibody specific binding. This method is sensitive, rapid, performed without washing, easy to operate, and can detect 8-128 ng/mL of STX in only 10 min. The limit of detection achieved by this method is as low as 4.29 ng/mL with coefficients of variation for the intra-batch and inter-batch analyses ranging from 2.61% to 3.63% and from 7.67% to 8.30%, respectively. In conclusion, our study successfully establishes a simple yet sensitive, rapid, and accurate AlphaLISA method for the detection of STX which holds great potential in advancing research on marine biotoxins.

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A homogeneous assay was developed to evaluate ligands that target the membrane progesterone receptor alpha (mPRα) of goldfish. This was achieved by employing graphene quantum dots (GQDs), a type of semiconductor nanoparticle conjugated to the goldfish mPRα. When progesterone-BSA-fluorescein isothiocyanate (P4-BSA-FITC) was combined with the other agents, fluorescence was observed through Förster resonance energy transfer (FRET). However, this fluorescence was quenched by binding between the ligand and receptor. This established method demonstrated the ligand selectivity of the mPRα protein. Then, the methylotrophic yeast Pichia pastoris was used to express the goldfish mPRα (GmPRα) protein. The recombinant purified GmPRα protein was coupled with graphene quantum dots (GQDs) to generate GQD-conjugated goldfish mPRα (GQD-GmPRα). Fluorescence at a wavelength of 520 nm was observed through FRET upon the combination of P4-BSA-FITC and subsequent activation by ultraviolet (UV) light. Adding free P4 to the reaction mixture resulted in a decrease in fluorescence intensity at a wavelength of 520 nm. The fluorescence was reduced by the administration of GmPRα ligands but not by steroids that do not interact with GmPRα. The findings indicated that the interaction between the ligand and receptor led to the formation of a complex involving GQD-GmPRα and P4-BSA-FITC. The interaction between the compounds and GQD-GmPRα was additionally validated by a binding experiment that employed the radiolabeled natural ligand [3H]-17α,20ß-dihydroxy-4-pregnen-3-one. We established a ligand-binding assay for the fish membrane progesterone receptor that is applicable for screening compounds.

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Fat mass and obesity-associated protein (FTO) is a crucial eraser of RNA N6- methyladenosine (m6A) modification, and abnormal FTO expression level is implicated in pathogenesis of numerous cancers. Herein, we demonstrate the construction of a label-free fluorescent biosensor for homogeneous detection of m6A eraser FTO in breast cancer tissues. When FTO is present, it specifically erases the methyl group in m6A, inducing the cleavage of demethylated DNA by endonuclease DpnII and the generation of a single-stranded DNA (ssDNA) with a 3'-hydroxyl group. Subsequently, terminal deoxynucleotidyl transferase (TdT) promotes the incorporation of dTTPs into the ssDNA to obtain a long polythymidine (T) DNA sequence. The resultant long poly (T) DNA sequence can act as a template to trigger hyperbranched strand displacement amplification (HSDA), yielding numerous DNA fragments that may be stained by SYBR Gold to produce an enhanced fluorescence signal. This biosensor processes ultrahigh sensitivity with a detection limit of 1.65 × 10-10 mg/mL (2.6 fM), and it can detect the FTO activity in a single MCF-7 cell. Moreover, this biosensor can screen the FTO inhibitors, evaluate enzyme kinetic parameters, and discriminate the FTO expression levels in the tissues of breast cancer patients and healthy persons.

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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.

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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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Remnant lipoproteins (RLs), which are typically present at high concentrations in patients with type 2 diabetes mellitus (T2DM), are associated with cardiovascular disease (CVD). Although an RL cholesterol homogeneous assay (RemL-C) is available for the measurement of RL concentrations, there have been no studies of the relationship between RemL-C and clinical parameters in T2DM. Therefore, we evaluated the relationships between RemL-C and CVD-related parameters in patients with T2DM. We performed a cross-sectional study of 169 patients with T2DM who were hospitalized at Kumamoto University Hospital. Compared with those with low RemL-C, those with higher RemL-C had higher fasting plasma glucose, homeostasis model assessment for insulin resistance (HOMA-R), total cholesterol, triglyceride, small dense low-density lipoprotein cholesterol (sdLDL-C), and urinary albumin-creatinine ratio; and lower high-density lipoprotein cholesterol, adiponectin, and ankle brachial pressure index (ABI). Multivariate logistic regression analysis showed that sdLDL-C and ABI were significantly and independently associated with high RemL-C. Although LDL-C was lower in participants with CVD, there was no difference in RemL-C between participants with or without CVD. Thus, RemL-C may represent a useful index of lipid and glucose metabolism, and that may be a marker of peripheral atherosclerotic disease (PAD) in male patients with T2DM.

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The general electrochemical biosensors for telomerase detection require the immobilization of primers on the electrode surface for telomeric extension and hybridization reactions. However, immobilization of primers may suffer from the challenges of hindrance effect and configuration freedom, thus reducing the extension and hybridization efficiency. Herein, we developed a sensitive electrochemical biosensor for telomerase detection by integration of homogeneous extension and hybridization reactions and surface-tethered detection. In the presence of telomerase, the biotinylated primer (bio-primer) was efficiently elongated with telomeric repeats of (TTAGGG)n at the 3' end in solution. Then, the extension product (bio-DNA) was hybridized with the signal probe DNA modified on the surface of ferrocene (Fc)-capped gold nanoparticle (AuNP). The bio-DNA/DNA/Fc-AuNP hybrids were then tethered by streptavidin-modified electrodes through the specific avidin-biotin interactions, thus producing strong electrochemical signals from the oxidation of Fc tags. The biosensor was successfully used to determine telomerase in HeLa cells and monitor the inhibition efficiency of inhibitor. A wide linear range for the detection of telomerase extracted from HeLa cells was attained. This method has great potential in clinical diagnosis and anti-cancer drug development, and should be beneficial for the fabrication of novel biosensors by integration of homogeneous catalysis and hybridization reactions.

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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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AIM: To compare LDL-C concentrations using the Friedewald formula, the Martin-Hopkins formula, a direct assay and polyacrylamide gradient gel electrophoresis (PGGE) to the reference standard density gradient ultracentrifugation in patients with Familial Dysbetalipoproteinemia (FD) patients. We also compared non-HDL-cholesterol concentrations by two methods. METHODS: For this study data from 28 patients with genetically confirmed FD from the placebo arm of the EVOLVE-FD trial were used. Four different methods for determining LDL-C were compared with ultracentrifugation. Non-HDL-C was measured with standard assays and compared to ultracentrifugation. Correlation coefficients and Bland-Altman plots were used to compare the methods. RESULTS: Mean age of the 28 FD patients was 62 ± 9 years, 43 % were female and 93 % had an ɛ2ɛ2 genotype. LDL-C determined by Friedewald (R2 = 0.62, p <0.01), Martin-Hopkins (R2 = 0.50, p = 0.01) and the direct assay (R2 = 0.41, p = 0.03) correlated with density gradient ultracentrifugation. However, Bland-Altman plots showed considerable over- or underestimation by the four methods compared to ultracentrifugation. Non-HDL-C showed good correlation and agreement. CONCLUSION: In patients with FD, all four methods investigated over- or underestimated LDL-C concentrations compared with ultracentrifugation. In contrast, standard non-HDL-C assays performed well, emphasizing the use of non-HDL-C in patients with FD.

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Background: 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. Methods: 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. Results: 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 (R 2 = 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. Conclusion: 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.

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Electrochemical biosensors generally require the immobilization of recognition elements or capture probes on the electrode surface. This may limit their practical applications due to the complex operation procedure and low repeatability and stability. Magnetically assisted biosensors show remarkable advantages in separation and pre-concentration of targets from complex biological samples. More importantly, magnetically assisted sensing systems show high throughput since the magnetic materials can be produced and preserved on a large scale. In this work, we summarized the design of electrochemical biosensors involving magnetic materials as the platforms for recognition reaction and target conversion. The recognition reactions usually include antigen-antibody, DNA hybridization, and aptamer-target interactions. By conjugating an electroactive probe to biomolecules attached to magnetic materials, the complexes can be accumulated near to an electrode surface with the aid of external magnet field, producing an easily measurable redox current. The redox current can be further enhanced by enzymes, nanomaterials, DNA assemblies, and thermal-cycle or isothermal amplification. In magnetically assisted assays, the magnetic substrates are removed by a magnet after the target conversion, and the signal can be monitored through stimuli-response release of signal reporters, enzymatic production of electroactive species, or target-induced generation of messenger DNA.

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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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BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

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Rotavirus is the main pathogen causing acute viral gastroenteritis. Accurate and rapid diagnosis of rotavirus infection is important to determine appropriate treatment, prevention of unnecessary antibiotics use and control of infection spread. In this study, we established a rapid, accurate, and sensitive amplified luminescent proximity homogeneous assay linked immunosorbent assay (AlphaLISA) for detecting rotavirus and evaluated its efficacy in human stool samples. Our results demonstrated that the sensitivity of AlphaLISA (5-8) significantly exceeded that of the immunochromatographic assay (ICA, 5-4) for rotavirus antigen detection. The intra-assay and inter-assay coefficients of variation were 2.99-3.85% and 5.27-6.51%, respectively. Furthermore, AlphaLISA was specific for rotavirus and did not cross-react with other common diarrhea viruses. AlphaLISA and real-time reverse transcription polymerase chain reaction (RT-qPCR, which is considered a gold standard for detecting diarrhea viruses) tests showed consistent results on 235 stool samples, with an overall consistency rate of 97.87% and a kappa value of 0.894 (P < 0.001). The overall consistency rate of ICA compared with RT-qPCR was 95.74%. AlphaLISA showed better consistency with RT-qPCR than the routinely used ICA for rotavirus detection in stool samples. The AlphaLISA method can be used in clinical practice for the rapid, accurate, and sensitive detection of rotavirus infection.

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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.

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Protein sensors based on allosteric enzymes responding to target binding with rapid changes in enzymatic activity are potential tools for homogeneous assays. However, a high signal-to-noise ratio (S/N) is difficult to achieve in their construction. A high S/N is critical to discriminate signals from the background, a phenomenon that might largely vary among serum samples from different individuals. Herein, based on the modularized luciferase NanoLuc, we designed a novel biosensor called NanoSwitch. This sensor allows direct detection of antibodies in 1 µl serum in 45 min without washing steps. In the detection of Flag and HA antibodies, NanoSwitches respond to antibodies with S/N ratios of 33-fold and 42-fold, respectively. Further, we constructed a NanoSwitch for detecting SARS-CoV-2-specific antibodies, which showed over 200-fold S/N in serum samples. High S/N was achieved by a new working model, combining the turn-off of the sensor with human serum albumin and turn-on with a specific antibody. Also, we constructed NanoSwitches for detecting antibodies against the core protein of hepatitis C virus (HCV) and gp41 of the human immunodeficiency virus (HIV). Interestingly, these sensors demonstrated a high S/N and good performance in the assays of clinical samples; this was partly attributed to the combination of off-and-on models. In summary, we provide a novel type of protein sensor and a working model that potentially guides new sensor design with better performance.

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Homogeneous assays for determining the concentration of small molecules in biological fluids are of importance for monitoring blood levels of critical drugs in patients. We have developed a strand displacement competition assay for the drugs dabigatran, methotrexate, and linezolid, which allows detection and determination of the concentration of the drugs in plasma; however, a surprising kinetic behavior of the assay was observed with an initial rapid change in apparent FRET values. We found that protein-induced fluorescent enhancement or quenching (PIFE/Q) caused the initial change in fluorescence within the first minute after addition of protein, which could be exploited to construct assays for concentration determination within minutes in the low nanomolar range in plasma. A kinetic model for the assay was established, and when taking the new finding into account, the in silico simulations were in good agreement with the experimentally observed results. Utilizing these findings, a simpler assay was constructed for detection of dabigatran, which allowed for detection within minutes without any time dependencies.

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BACKGROUND: Autoantibodies (AAbs) against immunoglobulin E (IgE) antibodies (Abs) and their high-affinity receptor alpha subunits (FcεRIα) are key factors in the elicitation of type IIb autoimmune chronic spontaneous urticaria (type IIb aiCSU). In this study, we aimed to develop a new method to detect functional anti-FcεRIα and anti-IgE AAbs, which can crosslink the plural FcεRІα molecules and IgE Abs on the surface of mast cells and basophils, in sera from aiCSU patients using the amplified luminescence proximity homogeneous assay (Alpha). METHODS: Sera were obtained from 14 aiCSU patients, as diagnosed by recurrent chronic spontaneous urticaria episodes and positive results for the autologous serum skin test and/or histamine release test (HRT). The AAbs to FcεRIα and IgE Abs were determined in sera from aiCSU patients using enzyme-linked immunosorbent assay (ELISA) and Alpha by cross-linking (AlphaCL) of IgE Abs and/or FcεRІα. RESULTS: Serum anti-FcεRIα and anti-IgE AAb levels were not significantly different between aiCSU patients and healthy subjects in ELISA. Anti-FcεRIα AAbs were detected in 10 of 14 aiCSU patients who displayed positive (5/5) and negative (5/9) results in the HRT for anti-FcεRIα AAbs by AlphaCL, whereas no signals were observed in healthy subjects. Additionally, anti-IgE AAbs were detected in two of four aiCSU patients who displayed positive results in the HRT for anti-IgE AAbs. CONCLUSIONS: A new assay method using AlphaCL can detect anti-FcεRIα and anti-IgE AAbs with FcεRIα- and IgE-crosslinking abilities in sera from aiCSU patients. This simple and practical assay method may be available as a diagnostic tool for urticaria patients.

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