Homogeneous Immunoassay Research Articles

BSA‐Gold Nanoclusters Enhanced Clusterization‐Triggered–Emission of Nonconventional Luminophores‐Hydrogels in Dilute Aqueous Solution

AbstractThe application of nonconventional nonaromatic luminophores‐based hydrogels (NLHs) in bioanalysis is severely limited due to their luminescence in concentrated solution or solid state. Herein, a nonconventional NLH (B‐Au@PDMAA) with high signal‐to‐noise ratio and stability in dilute solutions is designed by enhancing nonaromatic luminophores intermolecular aggregation through bovine serum albumin‐gold nanoclusters (B‐Au) confined space interaction. Importantly, the established NLHs could be rapidly polymerized in situ on magnetic beads (MB) surface in dilute solutions, and the fluorescence intensity after polymerization is 5.63‐fold higher than that of direct polymerization. Moreover, proposed B‐Au@PDMAA@MB constructed homogeneous immunoassay platform is demonstrated to be highly sensitive for detecting hepatitis B surface antigen with a sensitivity of 3.39 ng mL−1, which is 28.6‐fold lower than that of B‐Au@PDMAA based immunoassay and comparable to the sensitivity of the chemiluminescence immunoassay. This work presents a new strategy for the synthesis of NLHs with high brightness and stability in dilute solutions, and opens new avenues for the construction of novel homogeneous immunoassay platforms.

Establishment of a microspheres-based homogeneous fluorescence immunoassay for the rapid detection of cardiac troponin I.

Myocardial infarction occurs rapidly, and thus the rapid detection of cTnI levels is the key to its diagnosis. Most current assays take 10-30 min. In this study, we developed a method for accurately measuring cardiac troponin I (cTnI) levels in human sera with amplified luminescence neighborhood homogeneous assay (AlphaLISA). The method involves coupling two cTnI antibodies targeting different epitopes to the surface of carboxylated donor and acceptor beads. The final signal values were detected by the double-antibody sandwich method, and the best reaction conditions were obtained by optimizing the experimental conditions. The sensitivity, specificity, accuracy, and precision of the method were evaluated. Results showed that the method requires only 3 min to produce the results, the detection sensitivity is 27.06 ng L-1, and the measurement range is 34.56-62 500 ng L-1. cTnI-AlphaLISA has an intra-assay precision of 2.18-4.57% (<10%) and an inter-assay precision of 5.60-6.95% (<10%). The relative recovery rates are within reasonable limits. In addition, the serum assay results of the method were compared with chemiluminescence immunoassay, and the results are in agreement with one another (ρ = 0.8803; P < 0.0001). The method is expected to be developed as a routine method, but further studies and evaluations are needed.

Homogeneous fluorescence immunoassay based on AuNPs quenching dendritic silica assembled with multicolor QDs for the simultaneous determination of four mycotoxins in cereals

In this work, a novel homogeneous fluorescence immunoassay for the simultaneous detection of ochratoxin A (OTA), aflatoxin B1 (AFB1), fumonisins B1 (FB1), and zearalenone (ZEN) in cereal samples is established. Here, amino groups and thiol groups are grafted onto the surface of dendritic mesoporous silica nanoparticles (DMSNs) to improve loading rate and fluorescence preservation rate of quantum dots (QDs), enabling enrichment and amplification of fluorescence signals. In this test system, the fluorescence of the signal probe (DMSNs@QDs@Ab) is quenched by AuNPs, and then the presence of the targets makes the fluorescence of the signal probes recover. The correlation between the recovered fluorescence value and mycotoxin concentration is applied to achieve quantitative detecting mycotoxins. This assay displays good linear correlation in range of 0.01–100 μg L-1, with the limit of detection (LOD) of 0.0001 μg L-1 for OTA, 0.0008 μg L-1 for AFB1, 0.001 μg L-1 for FB1, and 0.0006 μg L-1 for ZEN, respectively. The proposed assay is applied to detect multiple mycotoxins in contaminated cereal samples and the test results is confirmed by liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Avail of silica with high loading capacity for assembling multicolor QDs to achieve simultaneous multi-signal output, a simple and efficient homogeneous fluorescence immunoassay is proposed for the first time. This multiplex test strategy provides a good idea for simultaneous, rapid, and sensitive detection of multiple hazardous substances in food and environment samples.

Quenchbodies That Enable One-Pot Detection of Antigens: A Structural Perspective.

Quenchbody (Q-body) is a unique, reagentless, fluorescent antibody whose fluorescent intensity increases in an antigen-concentration-dependent manner. Q-body-based homogeneous immunoassay is superior to conventional immunoassays as it does not require multiple immobilization, reaction, and washing steps. In fact, simply mixing the Q-body and the sample containing the antigen enables the detection of the target antigen. To date, various Q-bodies have been developed to detect biomarkers of interest, including haptens, peptides, proteins, and cells. This review sought to describe the principle of Q-body-based immunoassay and the use of Q-body for various immunoassays. In particular, the Q-bodies were classified from a structural perspective to provide useful information for designing Q-bodies with an appropriate objective.

An LF-NMR homogeneous immunoassay for Vibrio parahaemolyticus based on superparamagnetic 2D nanomaterials

In this work, a sensitive low field nuclear magnetic resonance (LF-NMR) homogeneous immunoassay, also called magnetic resonance switch (MRSw) sensor, for Vibrio parahaemolyticus (VP) was developed. Superparamagnetic 2D nanomaterial was designed and used as the magnetic probe of MRSw sensor. It was GO@SPIONs&Ab, a composite nanomaterial with many superparamagnetic Fe3O4 nanoparticles (SPIONs) providing a magnetic signal and VP antibody (Ab) specifically recognizing the target VP evenly distributed on the surface of GO. The presence of VP controllably changed the aggregation state of the probe, eliminating the uncertainty of MRSw sensor type, and thus then achieving a regular variation of transverse relaxation time T2 and ensuing quantitative detection of VP. Triple signal enhancement of the MRSw sensor was obtained due to the application of the designed 2D probe, by increasing the number of SPIONs, improving the magnetic intensity and susceptibility, and forming a synergistic effect. Under optimized experimental conditions, VP could be detected with satisfied sensitivity, selectivity, precision, accuracy, and stability, even in turbid real samples. LOQ for VP was 10 CFU/mL. This detection principle is widely applicable, providing an idea for the construction of highly sensitive MRSw sensors.

B-330 Homogeneous Enzyme Immunoassay for Hydrocodone

Abstract Background Hydrocodone is a semi-synthetic derivative of codeine and produces opioid-like effects similar to morphine. Hydrocodone (Trade names: Vicodin, Lortab, Hycodan, Vicoprofen) is one of the most frequently prescribed opioids in the U.S. as an antitussive (cough suppressant) and narcotic analgesic agent for the treatment of moderate to severe pain. Its ease of prescription and opioid-like effects has led to widespread drug diversion and abuse. The two commercially available homogeneous immunoassays for the detection of hydrocodone in urine at a cutoff of 300 ng/mL suffer undesirable cross-reactivity to oxycodone and morphine at concentrations below 25 000 ng/mL. ARK Diagnostics has developed the ARK Hydrocodone Assay to detect hydrocodone at a cutoff of 300 ng/mL with no cross-reactivity to oxycodone, morphine, and codeine at concentrations below 100 000 ng/mL. Methods The ARK™ Hydrocodone Assay is a liquid stable homogeneous enzyme immunoassay, consisting of two reagents, with a cutoff of 300 ng/mL and semi-quantitative range up to 800 ng/mL. The performance of this assay was evaluated on the Beckman Coulter AU680 Automated Clinical Chemistry Analyzer. Precision, spiked recovery, specificity, Histogram Overlap Analysis of ±25% controls and the cutoff, and method comparison with LC-MS/MS were evaluated. Results In semi-quantitative mode, total precision ranged from 3.9% to 9.9%CV. Spiked recovery of hydrocodone ranged from 86.2% (720 ng/mL) to 102.9% (240 ng/mL). The active metabolite, hydromorphone, showed an approximate equivalence to the 300 ng/mL hydrocodone cutoff at 299 ng/mL (100.3% cross-reactivity). Histogram overlap analysis showed no overlap between cutoff and control levels. Method correlation with LC-MS/MS using authentic urine samples showed an excellent agreement with a specificity of 100% and sensitivity of 96.9% (98 positives and 128 negatives). Conclusion The ARKTM Hydrocodone Assay measures hydrocodone and its metabolite hydromorphone in human urine with acceptable performance. The assay is sensitive, rapid, and applicable to a wide range of clinical chemistry analyzers.

B-334 How to Prevent the Next Wave in Overdose Crisis?—Detect Designer Benzodiazepines Using CEDIA and DRI Assays

Abstract Background Benzodiazepines are a class of drugs used for relieving symptoms of anxiety, insomnia, agitation, muscle spasms, and alcohol withdrawal. Benzodiazepines are CNS depressants enhancing the effect of GABA neurotransmitter at GABA receptors and they are well-known for drug abuse potential and drug dependence.Designer benzodiazepines are new psychoactive substances emerging in the past two decades. Designer benzodiazepines are often taken by individuals who use hallucinogenic and stimulant drugs. The drug abuse can lead to respiratory depression, coma, or death. There exists a need to develop a new homogeneous enzyme immunoassay. Existing commercial immunoassay antibodies may also cross-react with designer benzodiazepines which have chemical structure closely related to classic therapeutic benzodiazepines. Herein, we evaluated designer benzodiazepines cross-reactivity using DRI Benzodiazepines and CEDIA Benzodiazepines assays and demonstrated the potential of detecting designer benzodiazepines in urines. Methods DRI assay is based on competition between drug labeled with glucose-6-phosphate dehydrogenase (G6PDH) and free drug in the urine sample for a fixed amount of antibody binding sites. The enzyme activity is determined spectrophotometrically at 340 nm by measuring its ability to convert NAD to NADH. CEDIA technology is based on the bacterial enzyme β-galactosidase which has been genetically engineered into two inactive fragments, Enzyme Acceptor (EA) and Enzyme Donor (ED). The enzyme activity is then determined spectrophotometrically at 570 nm. Designer Benzodiazepines tested in this study are 3-Hydroxyphenazepam, Adinazolam, Bromazolam, Clonazolam, Cloniprazepam, Deschloroetizolam, Diclazepam, Etizolam, Flubromazepam, Flubromazolam, Flunitrazolam, Meclonazepam, N-Desmethylflunitrazepam, Nifoxipam, Nimetazepam, Nitrazolam, and Pyrazolam. Compounds were spiked into drug free urine and cross-reactivity was evaluated using DRI® Benzodiazepine Assay and CEDIA® Benzodiazepine Assays on the Beckman Coulter AU680. Results 14 out of 17 (82%) tested designer benzodiazepines showed high cross-reactivity (&amp;gt;100%) in DRI benzodiazepine assay. 12 out of 17 (71%) tested designer benzodiazepines showed high cross-reactivity (&amp;gt;100%) in CEDIA assays with 200 ng/mL cutoff and 300 ng/mL cutoff. Compared to NPS-benzodiazepines trend data, this study demonstrated that DRI and CEDIA benzodiazepine assays can detect more than 50% (10 out of 19) designer benzodiazepines over 2020 Q1 to 2022 Q4 in the United States. Conclusion DRI and CEDIA Benzodiazepine Assays showed high cross reactivity of most seventeen designer benzodiazepines. The results provide potential tools for detect emerging designer benzodiazepines. Note: This was preliminary data that has not been reviewed by any regulatory authorities/cleared by FDA.

B-331 Homogeneous Enzyme Immunoassay for the Quantitative Determination of Methotrexate

Abstract Background Methotrexate (MTX), a classical antifolate, can be safely administered over a wide dose range as maintenance chemotherapy for acute lymphoblastic leukemia and treatment of nononcologic diseases including rheumatoid arthritis or psoriasis. When combined with leucovorin (LV) rescue, high-dose MTX (HDMTX; doses of 1000–33 000 mg/m2) is usually administered as a prolonged i.v. infusion for a variety of cancers, including acute lymphoblastic leukemia, lymphoma, osteosarcoma, breast cancer, and head and neck cancer. HDMTX can be safely administered to patients with normal renal function by vigorously hydrating and alkalinizing the patient to enhance the solubility of MTX in urine. Serum levels may reach 1000 μmol/L or more. Pharmacokinetically guided LV rescue by monitoring MTX serum levels is required to prevent potentially lethal MTX toxicity. Ability to measure MTX accurately at 0.050 μmol/L enables clinical determination of non-toxic status Methods The ARK™ Methotrexate II Assay is a homogeneous enzyme immunoassay for the quantitative determination of methotrexate in human serum or plasma on automated clinical chemistry analyzers. The assay was evaluated on the Beckman AU680 analyzer. The assay consists of two reagents, six-level calibrators, and six-level (0.070, 0.400, 0.800, 5.0, 50.0, 500.0 μmol/L) quality controls. Performance of the assay was determined by assessing precision, limit of quantitation, linearity, endogenous substances interference, high sample dilution (1:10 serial dilutions), on-board auto dilution (at 1:10 and 1:50), cross-reactivity, and method comparison. Results Within-lab precision (%CV) for control levels were 3.00% (0.070 µmol/L), 1.40% (0.400 µmol/L), 2.05% (0.800 µmol/L), 1.58% (5.0 µmol/L), 2.30% (50.0 µmol/L), and 1.62% (500.0 µmol/L). Limit of Detection and Quantitation: LoD was 0.004 µmol/L and LoQ was 0.030 µmol/L (4.87% CV, 113.6% analytical recovery). The ARK Methotrexate II Assay showed less than 8% deviation from linearity over the range 0.030 to 1.300 µmol/L. Endogenous substances did not interfere with measurement of MTX at the levels tested. High sample manual dilution (2.0, 20.0, 200.0, 1200.0, 5.0, 50.0, and 500.0 µmol/L) and on-board auto dilution (1:10 and 1:50) results were within 10% of nominal values. Cross-reactivity to compounds (potentially co-administered drugs, folate derivatives, and compounds of similar structure) tested was &amp;lt;10% interference. The major metabolite, 7-hydroxy MTX, at 50 µmol/L produced less than 10% interference for MTX levels of 0.050 and 0.500 µmol/L. Passing Bablok analysis of the results for 90 patient specimens of ARK MTX II Assay compared to liquid chromatography with tandem mass spectrometry (LC-MS/MS) was 1.03 + 0.00 (r2 = 0.98). Conclusion The ARK™ Methotrexate Assay II quantitated methotrexate accurately and precisely in serum and plasma. Performance on the Beckman AU680 automated clinical chemistry analyzer demonstrated excellent precision, recovery, and clinical accuracy vs LC-MS/MS.

The Cross Reactivity of Cannabinoid Analogs (Delta-8-THC, Delta-10-THC and CBD), their metabolites, and Chiral Carboxy HHC metabolites in Urine of Six Commercially Available Homogeneous Immunoassays.

There has been an exponential surge in the presence and use of cannabinoids since the federal legalization of hemp (Agricultural Improvement Act of 2018). This growth is attributed to Delta-9-tetrahydrocannabinol (Delta-9-THC) and cannabidiol (CBD), the most abundant phytocannabinoid components of cannabis and hemp, respectively, but with many other emerging THC analogs. Structurally, these analogs are similar to Delta-9-THC, yet very little information is available about their potency and even less information is available regarding their detectability using commercially available cannabinoid screening kits. Due to their structural similarity, current cannabinoid homogeneous immunoassay screening methods may be able to detect these emerging cannabinoid analogs and their metabolites. Six urine immunoassay kits (Abbott Cannabinoids - Abbott Diagnostics, LZI Cannabinoids (cTHC) Enzyme Immunoassay - Lin-Zhi International, DRI® Cannabinoid Assay and CEDIA™ THC - Thermo Fisher Scientific, ONLINE DAT Cannabinoid II - Roche Diagnostics, and Syva EMIT®II Plus - Siemens Healthineers) were evaluated at two different cutoff concentrations: 50ng/mL and 20 or 25ng/mL, assay dependent. The analysis was performed on an Abbott Architect Plus c4000 (Abbott Diagnostics). Delta-8-THC, CBD, olivetol and their major metabolites, and Delta-10-THC and HHC carboxylic acid chiral analogs were evaluated. The cross reactivity was evaluated by preparing each analyte at 20, 50, 100, and 1000ng/mL in urine. Analytes that did not cross react at 1000ng/mL for a cutoff were considered not detectable. If detected, the lowest concentration was used as the decision point to determine the precision at the immunoassay's cutoff. The six commercially available urine cannabinoid homogeneous immunoassay screening kits cross reacted with the following analogs: Delta-8-THC, 11-OH-Delta-8-THC, 11-COOH-Delta-8-THC, 6-OH-CBD, 7-OH-CBD, all Delta-10-THC and HHC carboxylic acid chiral analogs, and olivetol with varying selectivity depending on the screening kit and cutoff concentration. The kits did not cross react with the following analogs: CBD, 7-COOH-CBD, Abnormal CBD, CBDA-A, and olivetolic acid.

Biological variation of serum thyrotropin and thyroid hormones concentrations determined at 8-week intervals for 1 year in clinically healthy cats.

Cats commonly develop thyroid disease but little is known about the long-term biological variability of serum thyroid hormone and thyrotropin (thyroid-stimulating hormone; TSH) concentrations. We aimed to determine the long-term biological variation of thyroid hormones and TSH in clinically healthy cats. A prospective, observational study was carried out. Serum samples for analysis of total thyroxine (T4, by radioimmunoassay [RIA] and homogenous enzyme immunoassay [EIA]), triiodothyronine (T3 ), free T4 (by dialysis), and TSH were obtained every 8 weeks for 1 year from 15 healthy cats, then frozen until single-batch analysis. Coefficients of variation (CV) within individual cats ( ) and among individual cats ( ), as well as the variation between duplicates (ie, analytical variation [ ]) were determined with restricted maximum likelihood estimation. The indices of individuality (IoI) and reference change values (RCVs) for each hormone were calculated. Some thyroid hormones showed similar (total T4 by EIA) or greater (TSH) interindividual relative to intraindividual variation resulting in intermediate to high IoI, consistent with previous studies evaluating the biological variation of these hormones weekly for 5-6 weeks. By contrast, total T4 (by RIA) and free T4 had a low IoI. Total T3 had a high ratio of to ; therefore, interindividual variation could not be distinguished from analytical variation. No seasonal variability in the hormones could be demonstrated. Clinicians might improve the diagnosis of feline thyroid disease by establishing baseline concentrations for analytes with intermediate-high IoI (total T4, TSH) for individual cats and applying RCVs to subsequent measurements.

Two strategies for rapid and sensitive detection of procalcitonin using carbon dots-encapsulated nanocapsule and magnetic carbon dots as coupled labels

Procalcitonin (PCT) is a crucial biomarker for diagnosing bacterial infections in clinical settings. Here, a biosensing system utilizing a fluorescence amplification system of nanocapsules and magnetic carbon dots was developed. The system incorporated two PCT sensing strategies: highly sensitive immunomagnetic separation and rapid homogeneous immunoassay. In these strategies, nanocapsules modified with antibodies function as immunosensors and energy transmitters, while magnetic carbon dots modified with antibodies serve as capture probes and immunosensors. The immunomagnetic separation analysis strategy utilized immunonanocapsules as sensors and immunomagnetic carbon dots as capture probes. This strategy demonstrated the capability to achieve ultrasensitive trace detection of PCT within the range of 1–1000 pg/mL, following normal immunoreactivity and magnetic separation. The limit of detection to 0.3 pg/mL (serum) and 0.33 pg/mL (plasma). Via the fluorescence resonance energy transfer principle, the homogeneous immunoassay strategy used immunonanocapsules as energy transmitters and immunomagnetic carbon dots as sensors. This enabled direct and rapid detection of PCT in a wide range of 0–100 ng/mL in diluted serum, with a limit of detection of 0.41 ng/mL. Both strategies demonstrated high recoveries (93.5%-104.6% and 98.4%-103.8%) and exhibited reliability in achieving accurate quantitative PCT detection. Such a novel analytical approach to PCT may broaden the potential uses of carbon dots-based immunofluorescent sensing. Furthermore, it was direct implications for ultrasensitive and rapid point-of-care detection of various serum biomarkers in clinical settings.

Freeze-drying of few microliters of antibody formulations to implement 384-wells homogeneous instant assays

Enzyme-linked immunosorbent assay protocols have traditionally complex workflows with several intensive wash steps. Analytical tools with both shorter time-to-result and hands-on-time using smaller sample and assays reagents volumes are now investigated. In this context, fluorescence resonance energy transfer (FRET)-based assays are emerging as one of the most promising analytical tools in high-throughput screening (HTS). These immunoassays allow fast quantification of antigens at the nano-gram level in a final assay volume of only a few μL. We used a homogeneous time-resolved FRET (called HTRF) assay to develop a freeze-dried screening and ready-to-use format with only one rehydration step called “instant assay”.To assure optimal performance of the developed homogeneous instant assay, we investigated the critical quality attributes by studying the functionality and stability of the critical reagents and fluorophores. The cyclic adenosine 3′-5′-monophosphate (cAMP) was selected as the antigen target. We tested various formulations (with different buffers, sugars, bulking reagents, surfactants and co-solvants) combined with a slow freezing and the use of an aluminium plate holder during the freeze-drying of few microliter of bioreagents.The optimized freeze-drying procedure permits to preserve more than 70% of Ab recognition properties. The developed off-the-shelf homogeneous FRET immunoassay allows direct and fast quantification of cAMP at a nanogram level.

Establishment of a homogeneous chemiluminescence immunoassay for cat dander sIgE antibody detection

Background In vitro specific IgE (sIgE) testing has become an important tool for the diagnosis of IgE-mediated allergic diseases. Current methods used to detect allergen sIgE are time consuming and/or expensive. Therefore, a new method was developed for rapid quantitative detection of cat dander-sIgE antibody based on homogeneous chemiluminescence immunoassay. Methods Selection of chemibeads with different chemical groups, and the best Light-initiated chemiluminescence assay (LiCA) analytical mode for cat dander-sIgE detection. To validate and eliminate the interference of IgE on the detection of cat dander-sIgE, concentration of biotinylated anti-human IgE antibody was optimized. For quantification of cat dander-sIgE, a calibration curve was established, and the performance of the assay was evaluated according to clinical guidelines. Results Indirect LiCA is the best mode of analysis and biotinylated anti-human IgE antibody at a dilution ratio of 1:250 minimizes IgE interference. The coefficient of variation of the developed LiCA was 1.49% to 4.66%, with an intermediate precision of 6.90% to 8.21%. The LoB, LoD, and LoQ of the assay were 0.023 kUA/L, 0.056 kUA/L and 0.185 kUA/L. The coefficient of correlation (r) between LiCA and ImmounoCAP was 0.9478. Conclusions A cat dander-sIgE quantitation assay based on homogeneous chemiluminescence immunoassay was established, which could be a new reliable analytical tool for the determination of cat dander-sIgE.

A Fluorescence Cross-Correlation-Spectroscopy-Based Immunoassay for Rapid, Selective, and Accurate Protein Sizing in Human Plasma, Applied to the von Willebrand Factor.

Multimeric abnormalities in plasma von Willebrand factor (VWF) cause bleeding or clotting disorders. Electrophoretic analysis of multimers is used to detect such abnormalities but is qualitative, slow, and difficult to standardize. Fluorescence correlation spectroscopy (FCS) is a good alternative but is affected by low selectivity and concentration bias. Here, we report the development of a homogeneous immunoassay based on dual-color fluorescence cross-correlation spectroscopy (FCCS) that overcomes these challenges. By performing a mild denaturation treatment followed by reacting with polyclonal antibodies, the concentration bias was drastically reduced. The use of a dual antibody assay improved selectivity. Diffusion times of immunolabeled VWF were measured with FCCS and standardized relative to calibrator measurements. The assay measures size changes in VWF using 1 μL of plasma and less than 10 ng of antibody per measurement and was validated over a 16-fold range of VWF antigen concentration (VWF:Ag), with a sensitivity of VWF:Ag 0.8%. Concentration bias and imprecision were less than 10%. Measurements were unaffected by hemolytic, icteric, or lipemic interference. Strong correlations were obtained with reference densitometric readouts (0.97 for calibrators, 0.85 for clinical samples), and significant differences were found between normal (n = 10), type 2A (n = 5), and type 2B (n = 5) von Willebrand's disease and acquired thrombotic thrombocytopenic purpura (n = 10) samples (p < 0.01). This FCCS based immunoassay accurately and selectively determines changes in the multimeric status of plasma VWF and may be used as a simpler, faster, and a standardizable alternative for multimer analysis, following further clinical validation in larger cohorts.

Differential T Cell Responsiveness to Proliferative Stimuli Assessed with a Novel, Homogeneous Ki-67 Immunoassay

Abstract Proliferative capacity is an important component of T cell fitness and a relevant parameter for characterization of CAR T cells and T cell activating agents. Ki-67 protein is a well-validated marker of cell proliferation. We used Lumit™ technology to develop a homogeneous, luminescent immunoassay for determination of intracellular Ki-67 levels. Purified CD8 +T cells from different human donors were plated in 96-well format, then treated with a panel of T cell activators +/− IL-2, including αCD3/αCD28, αCD3/αCD28/αCD2, phytohemagglutinin, concanavalin A, and a cocktail of PMA and ionomycin. An optimized lytic reagent was added to control and treated cells, followed by two anti-Ki-67 monoclonal antibodies labeled with SmBiT and LgBiT subunits, respectively. Upon coincident binding of these labeled antibodies to Ki-67 protein molecules, the resultant proximity of SmBiT and LgBiT reconstitutes NanoBiT luciferase. Addition of Lumit Detection Reagent produces light proportional to the level of Ki-67 expression, with a total assay time of 2 h. It was determined that stimulation of Ki-67 levels varied markedly between T cell activators tested, with the maximum responses observed exceeding 13-fold. Notably, T cell responses varied significantly between donors, including with respect to the degree of IL-2 dependence. This assay system was compatible with same-well fluorescent determination of cell death. In parallel treated wells, Lumit cytokine immunoassays were used to assess concomitant increases in cytokine levels. This simple, no-transfer, no-wash immunoassay for quantitative determination of Ki-67 levels provides an improved workflow for rapid and efficient comparative assessments of T cell proliferative responses.

Homogeneous immunoassay for cyclopiazonic acid based upon mimotopes and upconversion-resonance energy transfer

Strains of Penicillium spp. are used for fungi-ripened cheeses and Aspergillus spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin α-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel epitope mimicking peptide, or mimotope, selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved, and the analysis of individual phage clones revealed four different mimotope peptide sequences. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, surface plasmon resonance binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion nanoparticles (UCNP, type NaYF4:Yb3+, Er3+) were used as energy donors and coated with streptavidin to anchor the synthetic biotinylated mimotope. Alexa Fluor 555, used as an energy acceptor, was conjugated to the anti-CPA antibody fragment. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg mL−1 (1.5 μg kg−1) and an IC50 value of 0.36 ng mL−1. No significant cross-reactivity was observed with other co-produced mycotoxins. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) as a reference method.

A Sensitive Fluorescence Polarization Immunoassay for the Rapid Detection of Okadaic Acid in Environmental Waters

In this study, a homogeneous fluorescence polarization immunoassay (FPIA) for the detection of hazardous aquatic toxin okadaic acid (OA) contaminating environmental waters was for the first time developed. A conjugate of the analyte with a fluorophore based on a fluorescein derivative (tracer) was synthesized, and its interaction with specific anti-OA monoclonal antibodies (MAbs) was tested. A MAbs–tracer pair demonstrated highly affine immune binding (KD = 0.8 nM). Under optimal conditions, the limit of OA detection in the FPIA was 0.08 ng/mL (0.1 nM), and the working range of detectable concentrations was 0.4–72.5 ng/mL (0.5–90 nM). The developed FPIA was approbated for the determination of OA in real matrices: river water and seawater samples. No matrix effect of water was observed; therefore, no sample preparation was required before analysis. Due to this factor, the entire analytical procedure took less than 10 min. Using a compact portable fluorescence polarization analyzer enables the on-site testing of water samples. The developed analysis is very fast, easy to operate, and sensitive and can be extended to the determination of other aquatic toxins or low-molecular-weight water or food contaminants.

A Robust Homogeneous Fluorescence Polarization Immunoassay for Rapid Determination of Erythromycin in Milk.

Erythromycin (ERY) is one of the most common macrolides applied in veterinary medicine to treat diseases or as a feed additive for animal growth promotion. Long-term irrational use of ERY could lead to residues in animal-derived food and the emergence of drug-resistant strains, posing potential threats to human health. In this study, a highly sensitive, specific, robust, and rapid fluorescence polarization immunoassay (FPIA) for the determination of ERY in milk has been described. Herein, to achieve high sensitivity, five tracers of ERY with different fluorescein structures were synthesized and paired with three monoclonal antibodies (mAbs). Under the optimized conditions, the combination of mAb 5B2 and tracer ERM-FITC achieved the lowest IC50 value in the FPIA with 7.39 μg/L for ERM. The established FPIA was used to detect ERY in milk, revealing a limit of detection (LOD) of 14.08 μg/L with recoveries of 96.08-107.77% and coefficients of variations (CVs) of 3.41-10.97%. The total detection time of the developed FPIA was less than 5 min from the addition of samples to the result readout. All the above results showed that the proposed FPIA in this study was a rapid, accurate, and simple method for the screening of ERY in milk samples.

Analytical Performance of the New Siemens Affinity Chrome-Mediated Immunoassay Everolimus Assay and Its Interchangeability With the Thermo Quantitative Microsphere System for Routine Therapeutic Drug Monitoring of Patients After Solid Organ Transplantation.

A new homogeneous affinity chrome-mediated immunoassay (ACMIA) "EVRO" from Siemens Healthcare was evaluated for therapeutic drug monitoring of everolimus (EVL) with automated sample pretreatment and compared with quantitative microsphere system (QMS) "EVER" from Thermo Fisher Scientific. Imprecision, inaccuracy, and limit of quantitation (LoQ) of ACMIA/EVRO were verified using both hemolysate quality control (QC) samples and pooled whole blood specimens. The interchangeability of methods and the agreement of results were analyzed using 72 specimens (from 38, 30, and 4 kidney, liver, and lung transplant recipients, respectively). Within-run imprecision ranged within %CV = 2.81-2.53 with pooled whole blood specimens and within %CV = 2.88-2.53 with QCs; total imprecision with QCs was within %CV = 2.14-1.51. Inaccuracy with value assigned QC was %△ = 5.36 at the 5.6 ng/mL level and %△ = 5.56 at the 11.7 ng/mL level. LoQ was 0.93 ng/mL (%CV = 10). Passing-Bablok regression showed a constant bias of 0.679 ng/mL (95% CI: 0.216-1.026) and a proportional bias of 1.326 (95% CI: 1.240-1.425). Bland-Altman analysis showed 5/72 (6.9%) paired differences exceeding the limits of agreement and 1/72 (1.4%) paired differences exceeding 1.96 SD to a combined bias of 39.9% after detrending. ACMIA/EVRO shows satisfactory analytical performances that comply with recommendations, but it does not fulfill requirements for interchangeability with QMS/EVER. Particularly, this new assay using sirolimus-specific antibody shows a sizable proportional bias versus the more specific comparator, which may be because of EVL metabolites. This is supported by the lack of agreement for individual differences in most samples collected at the peak concentration (C2). Therefore, further evidence is needed to support the transition of EVL level monitoring from QMS/EVER to ACMIA/EVRO without making extensive changes to both reference interval and patient's baseline.

Cluster-bomb type magnetic biosensor for ultrasensitive detection of Vibrio parahaemolyticus based on low field nuclear magnetic resonance.

Herein, a novel cluster-bomb type signal sensing and amplification strategy in low field nuclear magnetic resonance was proposed, and a magnetic biosensor for ultrasensitive homogeneous immunoassay of Vibrio parahaemolyticus (VP) was developed. The capture unit MGO@Ab was magnetic graphene oxide (MGO) immobilized by VP antibody (Ab) to capture VP. And, the signal unit PS@Gd-CQDs@Ab was polystyrene (PS) pellets covered by Ab to recognize VP and Gd-CQDs i.e. carbon quantum dots (CQDs) containing lots of magnetic signal labels Gd3+. In presence of VP, the immunocomplex signal unit-VP-capture unit could be formed and separated by magnetic force conveniently from the sample matrix. With the successive introduction of disulfide threitol and hydrochloric acid, signal units were cleaved and disintegrated, resulting in a homogeneous dispersion of Gd3+. Thus, cluster-bomb type dual signal amplification was achieved through increasing the amount and the dispersity of signal labels simultaneously. Under optimal experimental conditions, VP could be detected in the concentration range of 5-1.0×106CFU/mL, with a limit of quantitation (LOQ) 4CFU/mL. In addition, satisfactory selectivity, stability and reliability could be obtained. Therefore, this cluster-bomb type signal sensing and amplification strategy is powerful in designing magnetic biosensor and detecting pathogenic bacteria.