One-step Immunoassay Research Articles

Analysis of Biochemical and Inflammatory Markers for Predicting COVID-19 Severity: Insights From a Tertiary Healthcare Institution of Eastern India.

Background Coronavirus disease-19 (COVID-19) patients often deteriorate rapidly based on viral infection-related inflammation and the subsequent cytokine storm. The clinical symptoms were found to be inconsistent with laboratory findings. There is a need to develop biochemical severity score to closely monitor COVID-19 patients. Methods This study was conducted in the department of biochemistry at All India Institute of Medical Sciences (AIIMS) Bhubaneswar in collaboration with the intensive care unit. Laboratory data of 7,395 patients diagnosed with COVID-19 during the first three waves of the pandemic were analyzed. The serum high sensitivity high-sensitivity C-reactive protein (hs-CRP, immuno-turbidity method), lactate dehydrogenase (LDH, modified Wacker et al. method), and liver enzymes (kinetic-UV method) were estimated by fully automated chemistry analyzer. Serum ferritin and interleukin-6 (IL-6) were measured by one-step immunoassay using chemiluminescence technology. Three models were used in logistic regression to check for the predictive potential of biochemical parameters, and a COVID-19 biochemical severity score was calculated using a non-linear regression algorithm. Results The receiver operating characteristic curve found age, urea, uric acid, CRP, ferritin, IL6, and LDH with the highest odds of predicting ICU admission for COVID-19 patients. COVID-19 biochemical severity scores higher than 0.775were highly predictive (odds ratio of 5.925) of ICU admission (AUC=0.740, p<0.001) as compared to any other individual parameter. For the validation, 30% of the total dataset was used as testing data (n=2095) with a sensitivity of 68.3%, specificity of 74.5%, and odds ratio of 6.304. Conclusion Age, urea, uric acid, ferritin, IL6, LDH, and CRP-based predictive probability algorithm calculating COVID-19 severity was found to be highly predictive of ICU admission for COVID-19 patients.

Au-X (X=Pt/Ru)-Decorated Magnetic Nanocubes as Bifunctional Nanozyme Labels in Colorimetric, Magnetically-Enhanced, One-Step Sandwich CRP Immunoassay

Au-X (X=Pt/Ru)-Decorated Magnetic Nanocubes as Bifunctional Nanozyme Labels in Colorimetric, Magnetically-Enhanced, One-Step Sandwich CRP Immunoassay

One-step immunoassay based on filtration for detection of food poisoning-related bacteria

A one-step immunoassay based on filtration was presented, which used microbeads for target analyte detection and filters with appropriate pore sizes to distinguish the complexity of target analyte and microbeads. For effective bacterial detection, the microbead size and the filter's pore size must be optimized. The optimal concentrations of the enzyme (urease) and antibody were determined at the maximum absorbance change, that is, the maximum pH change. The pH change was measured using a field-effect transistor (FET). The correlation between pH change and threshold voltage was estimated to be 21.7 mV/pH, and the correlation between pH change and the source-drain current was estimated to be −379 nA/pH. For the one-step immunoassay, antibodies against target bacteria were isolated from horse serum by filtration, and these antibodies were estimated to have a sufficiently high specificity to overcome cross-reactivity among five types of food poisoning-related bacteria: Escherichia coli O157, Salmonella typhimurium, Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus. Finally, the FET-based one-step immunoassay was demonstrated for five types of food poisoning-related bacteria in human serum.

Label-Free Fluorescence Quantitative Detection Platform on Plasmonic Silica Photonic Crystal Microsphere Array.

We have demonstrated the proof-of-concept of a label-free fluorescence quantitative detection platform based on gold nanoparticle (AuNP) enhancement intrinsic fluorescence of protein on the silica photonic crystal microsphere (SPCM) array. The label-free one-step competitive fluorescence immunoassay protocol has been proposed on the surface of the SPCM. Aflatoxin B1 (AFB1) as a model molecule was detected by the newly established method. AFB1-bovine serum albumin and monoclonal antibodies (Abs) of anti-AFB1 have been immobilized on the surfaces of SPCMs and AuNPs, respectively. AuNPs remarkably enhanced the intrinsic fluorescence of artificial antigens on the surface of the SPCM at near UV excitation. The simulation of electric field distribution showed that the maximum value of the near-field enhancement |E/E0| of the SPCM with AuNPs could reach 20. The label-free fluorescence enhancement effect comes from the synergistic effects of photonic crystal effect and AuNP plasmon effect. Such a label-free fluorescence detection method can provide a linear detection range from 0.1 to 10 ng/mL with a limit of detection of 0.025 ng/mL and good specificity for AFB1. The recovery rates in the spiked cereal samples were measured in the range of 84.07 ± 5.71%-101.02 ± 5.13%, which were consistent with that of the traditional enzyme linked immunosorbent assay method. The label-free detection platform displays great application potential in biology, medicine, agriculture, food industry, chemical industry, energy source, and environmental protection.

One-step immunoassay based on switching peptides for analyzing ochratoxin A in wines

A one-step immunoassay is presented for the detection of ochratoxin A (OTA) using an antibody complex with switching peptides. Because the switching peptides (fluorescence-labeled) were able to bind the frame region of antibodies (IgGs), they were dissociated from antibodies immediately when target analytes were bound to the binding pockets of antibodies. From the fluorescence signal measurements of switching peptides, a quantitative analysis of target analytes, via a one-step immunoassay without any washing steps, could be performed. As the first step, the binding constant (KD) of OTA to the antibodies was estimated under the continuous flow conditions of a surface plasmon resonance biosensor. Then, the optimal switching peptide, among four types of switching peptides, and the reaction condition for complex formation with the switching peptide were determined for the one-step immunoassay for OTA analysis. Additionally, the selectivity test of one-step immunoassay for OTA was carried out in comparison with phenylalanine and zearalenone. For the application to the one-step immunoassay to detect OTA in wines, two types of sample pre-treatment methods were compared: (1) a liquid extraction was carried out using chloroform as a solvent with subsequent resuspension in phosphate-buffered saline (total analysis time < 1 h); (2) direct dilution of the wine sample (total analysis time < 0.5 h). Finally, the direct dilution method was found to be effective for the one-step immunoassay based on the switching peptide assay for OTA in wines with a markedly improved total analysis time (< 0.5 h). Additionally, the assay results were compared with commercial lateral flow immunoassay.

Phosphate-triggered ratiometric multicolor immunosensor based on nanobody-alkaline phosphatase fusion protein for sensitive detection of fenitrothion

Multicolor immunosensor is a promising tool for on-site rapid analysis by naked eye without bulky instruments. Herein, a multicolor immunosensor employed a specific nanobody-alkaline phosphatase fusion protein (VHHjd8-ALP) for biosensing of fenitrothion was developed. After one-step indirect competitive immunoassay, the binding VHHjd8-ALP in microplate catalyzed ascorbyl-2-phosphate (AAP) into ascorbic acid, which propose dual readout including multicolor ratiometric visible immunoassay (MRVIA) and multicolor ratiometric fluoroimmunoassay (MRFIA). For MRVIA, K3[Fe(CN)6] and Fe3+ was employed for multicolor generation. The substrate AAP can chelate with Fe3+ to form orange AAP-Fe3+ complex. When ALP catalyzed AAP into ascorbic acid, the latter can reduced K3[Fe(CN)6] into K4[Fe(CN)6], which reacted with Fe3+ to produced Prussian blue via self-assembly. A visible color transition from orange to blue against fenitrothion was obtained. For MRFIA, the generated ascorbic acid etched MnO2 nanosheet to control the concentration of Na2SO3, which can convert 9-anthraldehyde into sulfuretted anthracene accompanied by a fluorescent color transition from green to blue. These two methods showed visible multicolor transition against fenitrothion for naked-eye analysis. The recovery and blind samples test verified good accuracy and practicability, which can be used for rapid screening of fenitrothion by a naked-eye semiquantitative analysis mode.

Different patterns of exosomal α-synuclein between Parkinson's disease and probable rapid eye movement sleep behavior disorder.

The insidious onset of Parkinson's disease (PD) makes early diagnosis difficult. Notably, idiopathic rapid eye movement sleep behavior disorder (iRBD) was reported as a prodrome of PD, which may represent a breakthrough for the early diagnosis of PD. However, currently there is no reliable biomarker for PD diagnosis. Considering that α-synuclein (α-Syn) and neuroinflammation are known to develop prior to the onset of clinical symptoms in PD, it was hypothesized that plasma total exosomal α-Syn (t-exo α-Syn), neural-derived exosomal α-Syn (n-exo α-Syn) and exosomal apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) may be potential biomarkers of PD. In this study, 78 PD patients, 153 probable iRBD patients (pRBD) and 63 healthy controls (HCs) were recruited. α-Syn concentrations were measured using a one-step paramagnetic particle-based chemiluminescence immunoassay, and ASC levels were measured using the Ella system. It was found that t-exo α-Syn was significantly increased in the PD group compared to the pRBD and HC groups (p &lt; 0.0001), whilst n-exo α-Syn levels were significantly increased in both the PD and pRBD groups compared to HCs (p &lt; 0.0001). Furthermore, although no difference was found in ASC levels between the PD and pRBD groups, there was a positive correlation between ASC and α-Syn in exosomes. Our results suggest that both t-exo α-Syn and n-exo α-Syn were elevated in the PD group, whilst only n-exo α-Syn was elevated in the pRBD group. Additionally, the adaptor protein of inflammasome ASC is correlated with α-Syn and may facilitate synucleinopathy.

One-Step Homogeneous Immunoassay for the Detection of Influenza Virus Using Switching Peptide and Graphene Quencher.

One-step homogeneous immunoassay was developed for detecting influenza viruses A and B (Inf-A and Inf-B) using the switching peptide H2. As the fluorescence-labeled switching peptide dissociated from the binding pocket of detection antibodies, the fluorescence signal could be directly generated by the binding of Inf-A and Inf-B without washing (i.e., one-step immunoassay). For the one-step homogeneous immunoassay with detection antibodies in solution, graphene was labeled with the antibodies as a fluorescence quencher. To test the feasibility of the homogeneous one-step immunoassay, the stability of the antibody complex with the switching peptide was evaluated under different pH and salt conditions. The one-step homogeneous immunoassay with switching peptide was conducted using influenza virus antigens in phosphate-buffered saline and real samples with inactivated Inf-A and Inf-B spiked in serum. Finally, the one-step homogeneous immunoassay results were compared with those of commercially available lateral flow immunoassays.

Homogeneous One-Step Immunoassay Based on Switching Peptides for Detection of the Influenza Virus.

In this study, a homogeneous one-step immunoassay based on switching peptides is presented for the detection of influenza viruses A and B (Inf-A and Inf-B, respectively). The one-step immunoassay represents an immunoassay method that does not involve any washing steps, only treatment of the sample. In this method, fluorescence-labeled switching peptides quantitatively dissociate from the antigen-binding site of immunoglobulin G (IgG). In particular, the one-step immunoassay based on soluble detection antibodies with switching peptides is called a homogeneous one-step immunoassay. The immunoassay developed uses switching peptides labeled with two types of fluorescence dyes (FAM and TAMRA) and detection antibodies labeled with two types of fluorescence quenchers (TQ2 for FAM and TQ3 for TAMRA). The optimal switching peptides for the detection of Inf-A and Inf-B have been selected as L1-peptide and H2-peptide. The interactions between the four kinds of switching peptides and IgG have been analyzed using computational docking simulation and SPR biosensor. The location of labeling for the fluorescence quenchers has been determined based on the distance between the fluorescence dyes of the switching peptides and the fluorescence quenchers, calculated on the basis of the efficiency of fluorescence quenching, using the Förster equation. To demonstrate the feasibility of the one-step immunoassay, binding constants (KD) have been calculated for detection antibodies against Inf-A and Inf-B with target antigens (Inf-A and Inf-B) and switching peptides (L1- and H2-peptides), using an isotherm model. The immunoassay has been demonstrated to be feasible using antigens as well as real samples of Inf-A and Inf-B with a critical cycle number (Ct). The immunoassay has also been compared to other commercially available rapid test kits for Inf-A and Inf-B and found to be far more sensitive for detection of Inf-A and Inf-B over the entire detection range.

One-step homogeneous micro-orifice resistance immunoassay for detection of chlorpyrifos in orange samples

In this work, a one-step homogeneous micro-orifice resistance immunoassay has been proposed for chlorpyrifos detection by integrating functionalized polystyrene (PS) microsphere probes with particle counting technology. The particle counter is highly sensitive and accurate for detecting the state of PS microspheres, where the particles of different states exhibit significant differences in resistance. The state of the functionalized PS microspheres is altered from dispersed to aggregated during the antigen-antibody recognition. Based on the degree of aggregation of the functionalized PS microsphere probes, chlorpyrifos can be quantitatively detected through the competitive immune response between PS antibodies and PS complete antigens. This one-step homogeneous micro-orifice resistance immunoassay simplified the procedures and greatly increased the sensitivity of detection, which has been successfully applied to detect chlorpyrifos in orange samples within 0.5 h, with the detection limit of 0.058 ng/mL.

Time-resolved fluorescence immunoassay (TRFIA) for the simultaneous detection of hs-CRP and lipoprotein(a) in serum.

Elevated serum high-sensitivity C-reactive protein (hs-CRP) and lipoprotein(a) (Lp(a)) levels are associated with the development of native coronary atherosclerosis. We aimed to establish a new method for the simultaneous detection of hs-CRP and Lp(a) to predict the development of atherosclerosis. A one-step time-resolved fluorescence immunoassay (TRFIA) with europium(III) (Eu3+ ) or samarium(III) (Sm3+ ) labels was established, and the performance of this TRFIA (in terms of sensitivity, specificity, accuracy, and cutoff values) was evaluated using clinical serum samples and compared with those of registered kits. The sensitivity was 0.052μg/ml for hs-CRP and 0.64μg/ml for Lp(a). The intra-assay and inter-assay cross-reactivities (CVs) were very low, ranging from 2.05% to 4.67% for hs-CRP and from 2.42% to 6.43% for Lp(a). The CVs were very low (<0.34% and <2.65%, respectively) with five interferents. Additionally, there was a high Pearson coefficient between the present TRFIA method and the registered kits (R2 = 0.9967 and 0.9906, respectively). These data indicate that this study developed a TRFIA method that can be used for the quantitative detection of hs-CRP and Lp(a) in serum with high sensitivity, specificity, and accuracy. This TRFIA provides a new method for predicting the development of atherosclerosis.

One-step immunoassay for food allergens based on screened mimotopes from autodisplayed FV-antibody library

One-step immunoassay detects a target analyte simply by mixing a sample with a reagent solution without any washing steps. Herein, we present a one-step immunoassay that uses a peptide mimicking a target analyte (mimotope). The key idea of this strategy is that the mimotopes are screened from an autodisplayed FV-antibody library using monoclonal antibodies against target analytes. The monoclonal antibodies are bound to fluorescence-labeled mimotopes, which are quantitatively released into the solution when the target analytes are bound to the monoclonal antibodies. Thus, the target analyte is detected without any washing steps. For the mimotope screening, an FV-antibody library was exhibited on the outer membrane of E. coli with a diversity of >106 clones/library using autodisplay technology. The targeted clones were screened from the autodisplayed FV-antibody library using magnetic beads with immobilized monoclonal antibodies against food allergens. The analysis of binding properties of a control strain with mutant FV -antibodies composed of only CDR1 and CDR2 demonstrated that the CDR3 regions of the screened FV-antibodies showed binding affinity to food allergens. The CDR3 regions were synthesized into peptides as mimotopes for the corresponding food allergens (mackerel, peanuts, and pig fat). One-step immunoassays for food allergens were demonstrated using mimotopes against mackerel, peanut, and pig fat without any washing steps in solution without immobilization of antibodies to a solid support.

Electrochemical One-Step Immunoassay Based on Switching Peptides and Pyrolyzed Carbon Electrodes.

Switching peptides were designed to bind reversibly to the binding pocket of antibodies (IgG) by interacting with frame regions (FRs). These peptides can be quantitatively released when antigens bind to IgG. As FRs have conserved amino acid sequences, switching peptides can be used as antibodies for different antigens and different source animals. In this study, an electrochemical one-step immunoassay was conducted using switching peptides labeled with ferrocene for the quantitative measurement of analytes. For the effective amperometry of the switching peptides labeled with ferrocene, a pyrolyzed carbon electrode was prepared by pyrolysis of the parylene-C film. The feasibility of the pyrolyzed carbon electrode for the electrochemical one-step immunoassay was determined by analyzing its electrochemical properties, such as its low double-layer capacitance (Cdl), high electron transfer rate (kapp), and wide electrochemical window. In addition, the factors influencing the amperometry of switching peptides labeled with ferrocene were analyzed according to the hydrodynamic radius, the number of intrahydrogen bonds, dipole moments, and diffusion coefficients. Finally, the applicability of the electrochemical one-step immunoassay for the medical diagnosis of the human hepatitis B surface antigen (hHBsAg) was assessed.

One-step immunoassay for the detection of food-poisoning related bacteria using a switching peptide.

A one-step immunoassay was developed for five types of food-poisoning-related bacteria using a switching peptide and antibodies isolated from unimmunized horse serum. The one-step immunoassay involves mixing samples and reagents in a homogeneous solution without any washing steps. In this work, a one-step immunoassay configuration was developed using isolated antibodies labelled with an organic fluorescence quencher and a switching-peptide labelled with a fluorescent dye. The fluorescence-labelled switching-peptide was bound to the antigen-binding site of the isolated antibodies before binding to the bacteria (no fluorescence signal), and the switching-peptide dissociated from the antibodies as soon as they bound to the bacteria (fluorescence signal turns on). By quantifying the generated fluorescence signal, the one-step immunoassay presented here allows microbial detection without any washing step.

Machine-Learning-Assisted Microfluidic Nanoplasmonic Digital Immunoassay for Cytokine Storm Profiling in COVID-19 Patients.

Cytokine storm, known as an exaggerated hyperactive immune response characterized by elevated release of cytokines, has been described as a feature associated with life-threatening complications in COVID-19 patients. A critical evaluation of a cytokine storm and its mechanistic linkage to COVID-19 requires innovative immunoassay technology capable of rapid, sensitive, selective detection of multiple cytokines across a wide dynamic range at high-throughput. In this study, we report a machine-learning-assisted microfluidic nanoplasmonic digital immunoassay to meet the rising demand for cytokine storm monitoring in COVID-19 patients. Specifically, the assay was carried out using a facile one-step sandwich immunoassay format with three notable features: (i) a microfluidic microarray patterning technique for high-throughput, multiantibody-arrayed biosensing chip fabrication; (ii) an ultrasensitive nanoplasmonic digital imaging technology utilizing 100 nm silver nanocubes (AgNCs) for signal transduction; (iii) a rapid and accurate machine-learning-based image processing method for digital signal analysis. The developed immunoassay allows simultaneous detection of six cytokines in a single run with wide working ranges of 1–10,000 pg mL–1 and ultralow detection limits down to 0.46–1.36 pg mL–1 using a minimum of 3 μL serum samples. The whole chip can afford a 6-plex assay of 8 different samples with 6 repeats in each sample for a total of 288 sensing spots in less than 100 min. The image processing method enhanced by convolutional neural network (CNN) dramatically shortens the processing time ∼6,000 fold with a much simpler procedure while maintaining high statistical accuracy compared to the conventional manual counting approach. The immunoassay was validated by the gold-standard enzyme-linked immunosorbent assay (ELISA) and utilized for serum cytokine profiling of COVID-19 positive patients. Our results demonstrate the nanoplasmonic digital immunoassay as a promising practical tool for comprehensive characterization of cytokine storm in patients that holds great promise as an intelligent immunoassay for next generation immune monitoring.

Study of Effect of Whitening Creams on Serum Basal Cortisol level In Egyptian Females

Abstract Objective The aim of the present study is to evaluate the relationship between practice of whitening creams (Topical Corticosteroids) and serum cortisol level in a sample of healthy females at Ain- shams University Teaching hospital. Background Topical corticosteroids (TCs) are widely used as depigmenting agents alone or in combination with other fairness creams. Secondary adrenocortical insufficiency occurs due to deficiency of corticotropin (adrenocorticotropic hormone) [ACTH] in the pituitary gland. With low levels of corticotropin, cortex layer in the adrenal gland is not sufficiently stimulated to release adequate amount of cortisol. Excessive glucocorticoid therapy is the primary cause of adrenocortical insufficiency. Methods we recruited 45 subjects practice whitening creams (TCs) for three months or more; matched with a healthy control group consists of 45 participants. Early morning basal serum cortisol level (8:00 am) done by one-step competitive immunoassay using Beckman Coulter device. Cortisol ELISA Kit provided by CALBIOTECH, Inc (Co_103S). Results Early morning serum cortisol level was statistically insignificant among subjects practice whitening creams in combination with TCs vs. healthy controls (p value 0.307). However, there were 7 out of 45 participants in the study group (15.6%) had low serum cortisol level (&amp;lt;5ug/dl) with highly statistically significant difference regarding whitening creams usage methods. In comparison and data analysis between subjects of the study group regarding practice duration, used quantity, application frequency, and method of exposure, there was high statistically significant difference between subjects with normal cortisol level vs. subjects with low cortisol level (p value &amp;lt;0.001). There were, no significant difference found regarding BMI, and arterial blood pressure between study group and controls. Conclusion Whitening creams abuse especially high potency TCs is a common phenomenon that may induce several local and systemic complications including adrenal gland insufficiency.

Monolithic integration of nanorod arrays on microfluidic chips for fast and sensitive one-step immunoassays

Here, we present integrated nanorod arrays on microfluidic chips for fast and sensitive flow-through immunoassays of physiologically relevant macromolecules. Dense arrays of Au nanorods are easily fabricated through one-step oblique angle deposition, which eliminates the requirement of advanced lithography methods. We report the utility of this plasmonic structure to improve the detection limit of the cardiac troponin I (cTnI) assay by over 6 × 105-fold, reaching down to 33.9 fg mL−1 (~1.4 fM), compared with an identical assay on glass substrates. Through monolithic integration with microfluidic elements, the device enables a flow-through assay for quantitative detection of cTnI in the serum with a detection sensitivity of 6.9 pg mL−1 (~0.3 pM) in <6 min, which was 4000 times lower than conventional glass devices. This ultrasensitive detection arises from the large surface area for antibody conjugation and metal-enhanced fluorescent signals through plasmonic nanostructures. Moreover, due to the parallel arrangement of flow paths, simultaneous detection of multiple cancer biomarkers, including prostate-specific antigen and carcinoembryonic antigen, has been fulfilled with increased signal-to-background ratios. Given the high performance of this assay, together with its simple fabrication process that is compatible with standard mass manufacturing techniques, we expect that the prepared integrated nanorod device can bring on-site point-of-care diagnosis closer to reality.

Application of an AlphaLISA method for rapid sensitive detection of African swine fever virus in porcine serum.

Infection with African swine fever virus (ASFV) causes an acute and highly lethal hemorrhagic disease that has been responsible for huge economic losses in China. To exactly detect the antigen of ASFV, we established a rapid, no-wash, one-step sandwich-type immunoassay based on the amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) using two monoclonal antibodies (mAbs) M-5 and M-6 against ASFV p72. ASFV p72 in samples was captured by biotinylated mAb M-5 connected to the donor bead surface via streptavidin and "sandwiched" by mAb M-6 which was coated onto the acceptor bead. Efficacy and sensitivity trials revealed that the AlphaLISA could detect ≥0.78 ng/ml of purified p72 and with a linear range of 0.78-100 ng/ml. The AlphaLISA was specific for ASFV and did not cross-react with other common pathogenic porcine viruses. Compared with RealPCR ASFV DNA test and ASFV antigen detection kit, the sensitivity of the AlphaLISA evaluated in 60 porcine serum samples was 93% and 100%, respectively. The specificity was 100% and 91.7%, respectively. This study presents a good laboratory diagnostic tool for sensitive and efficient detection of ASFV in porcine serum. KEY POINTS: • MAbs M-5 and M-6 recognized various epitopes of ASFV p72. • The established ASFV p72 AlphaLISA showed well specificity, high sensitivity, and satisfied correlation coefficient.

A quantitative detection of Cardiotrophin-1 in chronic heart failure by chemiluminescence immunoassay.

BackgroundCardiotrophin‐1 (CT‐1) is a cytokine that could induce cardiomyocytes hypertrophy and dysfunction. Plasma CT‐1 might serve as a cardiac biomarker both in diagnosis, staging, and prognostic assessment of heart failure.MethodsIn this study, a one‐step paramagnetic particles‐based chemiluminescence immunoassay (MPs‐CILA) for rapid and sensitive detection of plasma CT‐1 was established. Plasma samples were directly incubated with biotin‐labeled anti‐CT‐1 antibody (bio‐Ab) and acridine ester labeled anti‐CT‐1 antibody (AE‐Ab) to form sandwiched complex. The sandwiched CT‐1 was then captured by streptavidin modified paramagnetic particles (MPs‐SA) for rapid separation and signal generation.ResultsThe proposed MPs‐CLIA presents a laudable linear relationship ranging from 7.8 pg/mL to 200 ng/mL with a detection limit of 1.0 pg/mL. The recoveries of spiked human plasma samples at low (10pg/mL), medium (100 pg/mL), and high (800 pg/mL) levels of CT‐1 were 96%, 104%, and 110% respectively. The intra‐analysis coefficient variation (CVs) of the 3 samples was 8.92%, 6.69%, and 3.54%, respectively. And the inter‐analysis coefficient variation (CVs) was 9.25%, 10.9%, and 4.3%, respectively. These results strongly indicate high sensitivity, wide linear range, acceptable precision, and applicable reproducibility of the proposed method to detect plasma level of CT‐1. Finally, Plasma CT‐1 from 140 subjects with or without chronic heart failure was analyzed to assess the clinical application of MPs‐CILA.ConclusionsNoteworthily, the MPs‐CLIA method is highly automated such that it is suitable for high‐throughput detection of CT‐1 in clinical inspection.

One-step, wash-free, bead-based immunoassay employing bound-free phase detection

We present a simple and fast one-step heterogeneous immunoassay, with performance characteristics that can enable easy and versatile adaptation to miniaturized, automated point-of-care systems. This novel analytical method uses magnetic and fluorescent beads as capture and detection agents respectively. Its main feature is the measurement of the fluorescent signal in the bound-free phase for (semi-)quantitative detection of analytes. Thus, no washing is required and the workflow consists only of sample and reagent supply, incubation, separation and detection. The immunoassay concept is demonstrated with C-reactive protein (CRP), a systemic inflammation marker. CRP in only 5 μL of undiluted serum was measured in the range 20–140 mg L−1 (includes clinically relevant cut-off values). The limit of detection (LOD) was 22.1 ± 6.3 mg L−1 (incubation 15 min). A CRP certified reference material was measured on five different days. Intra- and inter-assay coefficients of variation were 4.6 ± 1.9% and 5.6% respectively. To demonstrate the compatibility of the assay concept with additional matrices and concentration ranges, three oral inflammation markers, namely matrix metalloproteinases 8 and 9 (MMP-8, MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1), were measured in saliva in the ranges 0.47–30 ng mL−1 for MMP-8 and MMP-9, and 0.69–44 ng mL−1 for TIMP-1. LODs were 0.24 ng mL−1, 0.38 ng mL−1 and 0.39 ng mL−1 respectively (incubation 20 min). Multiplexing capacity of the assay concept was also shown with these markers. The demonstrated excellent reproducibility of the results, combined with the versatility and low complexity of the introduced immunoassay concept, make it an attractive candidate for applied analytical chemistry and automated point-of-care testing.