Harmonization of SARS-CoV-2 antigen immunoassays:are they measuring the same“thing”?

Background:This study was planned to assess the accuracy and comparability of two commercially available,laboratory-based SARS-CoV-2(severe acute respiratory syndrome)antigen(Ag)immunoassays.Methods:We studied a cohort of subjects with acute SARS-CoV-2 infection,from whom a nasopharyngeal swab was taken and tested with a molecular assay(Altona Diagnostics RealStar SARS-CoV-2 RT-PCR Kit)and two laboratory-based,fully automated SARS-CoV-2 Ag immunoassays(Fujirebio Lumipulse G SARS-CoV-2 Ag and Roche Elecsys SARS-CoV-2 Ag).Results:The final population consisted in 93 subjects testing positive for SARS-CoV-2 RNA,34 with cycle threshold(Ct)values<29.5.The results of the two SARS-CoV-2 Ag immunoassays were significantly intercorrelated(r=0.77;P<0.001)in the entire cohort,though such correlation considerably improved in patients with high viral load(cycle threshold values<29.5:r=0.96;P<0.001).The accuracy for identifying samples with high viral load was excellent for both Lumipulse G SARS-CoV-2 Ag(AUC,0.99;P<0.001)and Elecsys SARS-CoV-2 Ag(AUC,0.99;P<0.001),with best cut-offs of 2.03 ng/mL for Lumipulse G SARS-CoV-2 Ag(1.00 sensitivity and 0.88 specificity)and 0.70 COI for Elecsys SARS-CoV-2 Ag(1.00 sensitivity and 0.80 specificity),respectively.Conclusion:The results of this study provide valuable support to usability of fully-automated,rapid,high throughput and accurate SARS-CoV-2 Ag immunoassays for complementing molecular assays.

Recent advances in immunoassays and biosensors for mycotoxins detection in feedstuffs and foods

Mycotoxins are secondary metabolites produced by fungus.Many mycotoxin species are highly toxic and are frequently found in cereals and feedstuffs.So,powerful detection methods are vital and effective ways to prevent feed contamination.Traditional detection methods can no longer meet the needs of massive,real-time,simple,and fast mycotoxin monitoring.Rapid detection methods based on advanced material and sensor technology are the future trend.In this review,we highlight recent progress of mycotoxin rapid detection strategies in feedstuffs and foods,especially for simultaneous multiplex mycotoxin determination.Immunoassays,biosensors,and the prominent roles of nanomaterials are introduced.The principles of different types of recognition and signal transduction are explained,and the merits and pitfalls of these methods are compared.Furthermore,limitations and challenges of existing rapid sensing strategies and perspectives of future research are discussed.

In situ enzyme immunoassays with tissue smears to study horizontal transmission of BMNV in penaeid shrimp and in beach animals

An enzyme immunoassay technique is developed for detecting the baculoviral midgut gland necrosis virus (BMNV) in penaeid shrimp and beach anieds. An antiserum was obtained from rabbits immunized with a BMNV preparation Purified from Penaeus japonicus. γ-globulin was isolated and labelled with horseradish peroxidase (HRP). This reagent was used to detect the virus in penaeid shrimp experimentally infected with the BMNV preparation, in cultured penaeid shrimp and in several beach animals. The technique is specific, simple and sensitive and can be developed into a practical method for the detection of penaeid shrimp viruses. Among 128 tissue samples from 11beach animals, 77 samples gave positive reactions with the enzyme-labelled immunoglobulin against BMNV from P.japonicus. These animals could possibly constitute a horizontal transmission pathway for cultured penaeid shrimp viruses.

Research Advances on Immunoassays for Phthalic Acid Esters

Phthalates( PAEs) compounds are a class of widely used environmental hormone plasticizers. Their harm to the environment and human health has caused a lot of attention. The detection of such substances,especially the simple and rapid detection,is of great significance for the protection of environmental food safety and consumer health. At present,a series of rapid immunoassay techniques have been applied to PAE detection with advantages of simple and fast operation,low cost,high sensitivity,high specificity and high throughput,achieving the goal of screening a large number of samples and making up for the shortcomings of chromatographic detection which uses expensive equipment with cumbersome operation,requires professional and technical personnel,and cannot achieve the target of screening a large number of samples. This paper introduced the research progress of PAE antibodies and their rapid immunoassay technology,and performed evaluation,in order to provide guidance and help for the development and application of PAE rapid detection technology.

The Effects of Sample Matrices on Immunoassays to Detect Microcystin-LR in Water

Immunoassays are widely used biochemical techniques to detect microcystins in environmental samples. The use of immunoassays for the detection of microcystins is vulnerable to matrix components and other interferents. This study is an evaluation of the effects of interfering substances commonly found in drinking and ambient water samples using commercially-available immunoassay kits for microcystin toxins. The microplate and strip test immunoassay formats were tested in the study. For the microplate ELISA, the following were found to inhibit microcystin-LR (MC-LR) detection: 250 μg/mL Ca2+ or Mg2+, 0.01% ascorbic acid, 0.1% EDTA chelating agent, 0.05 M glycine-HCl, pH 3. The following exhibited no effect: sodium chloride (NaCl, 1% to 4%) and sodium thiosulfate (0.001% and 0.01%), 0.01 to 0.1 M phosphate buffers (PB), pH 7 and 0.067 M PB at pH 5, 6, 7 and 8. Overall, up to 50 μg/mL of standard and reference natural organic matter (NOM) from various sources did not interfere in the assay system (without MC-LR) but diminished the detection of MC-LR at varying degrees. This is the first study evaluating standard and reference humic and fulvic acids from various sources in immunoassays for microcystins. The strip test also showed variable effects on MC-LR detection in the presence of NOM. This assay format was also sensitive to varying pHs and ionic strengths. MC-LR binding was inhibited at low pH (0.05 M glycine-HCl, pH 3), whereas, 0.067 M PB with pH 6, 7 and 8 can yield false positive results. Lower ionic strength of 0.01 M PB, pH 7 showed no interference in MC-LR binding whereas higher ionic strengths can interfere with MC-LR detection. NaCl at 3% and 4% can interfere with the analysis giving false positive results. Mg2+ at 50 and 250 μg/mL showed no effect on the analysis while the same concentration of Ca2+ can yield false positive results. The performance in marine, brackish and hard waters should be tested given the potential sensitivity to salinity. Results of this study may assist in the further refinement of existing assays and the development of practical antibody-based methods to clean-up samples and detect cyanotoxins in water.

Application of Green Fluorescent Protein in Immunoassays

Green fluorescent protein (GFP) is a protein that emits green fluorescence when exposed to a radiation of ultraviolet wavelength range, even without the addition of substrate and cofactor. Because of such characteristics, the usage of GFP is widespread in both in vivo and in vitro applications. In addition, recent advances in biotechnology have enabled GFP to be expressed in various hosts, including bacteria, yeast, plants, animals, and even living-cells, for multiple purposes. Currently, GFP is a subject of great interest in the analytical sciences, especially in immunoassays for qualitative and quantitative analyses, when it is fused with an antibody because of the high sensitivity of GFP and antigen-binding specificity of antibodies. Recently the fluobody, which is a fusion protein of GFP with single-chain variable fragment antibody (scFv), has become a useful tool in various fields. We review here the applications of GFP as fluobodies in immunoassays.

Nanomaterial Labels in Lateral Flow Immunoassays for Point-of-Care-Testing

Lateral flow immunoassays(LFIAs) have been developed rapidly in recent years and used in a wide range of application at point-of-care-testing(POCT),where small biomolecules can be conveniently examined on a test strip.Compared with other biochemical detection methods such as ELISA(enzyme linked immunosorbent assay) or mass spectrometry method,LFIAs have the advantages of low cost,easy operation and short time-consuming.However,it suffers from low sensitivity since conventional LFIA can only realize qualitative detection based on colorimetric signals.With the increasing demand for more accurate and sensitive determination,novel nanomaterials have been used as labels in LFIAs due to their unique advantages in physical and chemical properties.Colloidal gold,fluorescent nano particles,SERSactive nanomaterials,magnetic nanoparticles and carbon nanomaterials are utilized in LFIAs to produce different kinds of signals for quantitative or semi-quantitative detection.This review paper first gives a description of the LFIA principles,and then focuses on the state-of-the-art nanomaterial labelling technology in LFIAs.At last,the conclusion and outlook are given to inspire exploration of more advanced nanomaterials for the development of future LFIAs.

On-site rapid detection of multiple pesticide residues in tea leaves by lateral flow immunoassay

The application of pesticides (mostly insecticides and fungicides) during the tea-planting process will undoubtedly increase the dietary risk associated with drinking tea. Thus, it is necessary to ascertain whether pesticide residues in tea products exceed the maximum residue limits. However, the complex matrices present in tea samples comprise a major challenge in the analytical detection of pesticide residues. In this study, nine types of lateral flow immunochromatographic strips (LFICSs) were developed to detect the pesticides of interest (fenpropathrin, chlorpyrifos, imidacloprid, thiamethoxam, acetamiprid, carbendazim, chlorothalonil, pyraclostrobin, and iprodione). To reduce the interference of tea substrates on the assay sensitivity, the pretreatment conditions for tea samples, including the extraction solvent, extraction time, and purification agent, were optimized for the simultaneous detection of these pesticides. The entire testing procedure (including pretreatment and detection) could be completed within 30 min. The detected results of authentic tea samples were confirmed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), which suggest that the LFICS coupled with sample rapid pretreatment can be used for on-site rapid screening of the target pesticide in tea products prior to their market release.

Establishment of Double-antigen Sandwich Time-resolved Fluorescence Immunoassay for Detection of Pest des Petits Ruminants Virus

[Objectives]This study was conducted to explore rapid and large-scale screening and detection of peste des petits ruminants(PPR),so as to provide important technical means for prevention,control and purification of PPR.[Methods]Soluble N protein and NH fusion protein were successfully obtained in an Escherichia coli expression system by optimizing E.coli codon and expression conditions.Furthermore,based on purified soluble N protein and NH fusion protein,a double-antigen sandwich time-resolved fluorescence immunoassay method for detection of peste des petits ruminants virus(PPRV)was established.[Results]The method has high sensitivity and specificity and can specifically detect the antibody against PPRV in sheep serum,and it has no cross reaction with other related diseases.The method was used to detect 292 clinical samples,and compared with French IDVET competition ELISA kit.The coincidence rates of positive samples and negative samples from the two kinds of test kits were 92.47%and 97.26%,respectively,and the overall coincidence rate was 94.86%.The intra-group and inter-group coefficients of variation in the repeatability test were less than 10%.[Conclusions]Compared with the traditional ELISA method,the double-antigen sandwich time-resolved fluorescence immunoassay for detection of PPRV has equivalent sensitivity and specificity,and simple and rapid operation,and thus high application and popularization value.

3,6-Bis-β-Dicarbonylsubstituted Carbazoles Bearing N-Spacers and Their Eu(III) Complexes as Immunofluorescent Labelling Agents

New reagents for immunofluorescence analysis of carbazole series containing fluorinated β-dicarbonyl fragments and carboxylic substituent groups separated by spacers of different lengths from the light-gathering carbazole scaffold have been developed. The markers in complex with Eu3+ ions possess stability in the aqueous phase, intense and prolonged luminescence (τ 550 - 570 μs) with characteristic emission maxima in the region of 615 nm and excitation wavelengths in the region of 380 - 390 nm, which distinguishes them from most of the analogs used. In the study of marker conjugation with streptavidin, a reagent containing 4 - 5 europium labeling complexes based on spacer-containing carbazole tetraketone was obtained. The marker-doped silicate nanoparticles exhibit intense and long-lived luminescence in the characteristic region.

Recent progress in the application of microfluidic systems and gold nanoparticles in immunoassays

Immunoassays are useful for many bioassays. Many new techniques and materials are introduced into the immunoassay to improve the efficiency. This paper reviews recent progress in the application of microfluidic systems and gold nanoparticles in immunoassay. The micro/nano technologies and materials can offer good sensitivity, fast detection, cost-effectiveness and easy signal readout. In particular, the miniaturization of microfluidics and colorimetric assays based on gold nanoparticles have dramatically improved the efficiency of immunoassays.

Point-of-Care Immunoassays with Tunable Detection Range for Detecting Infection in Intensive Care Unit

One of the difficulties of a multiplexed analytical assay is matching the concentration range of different markers.Here,the authors develop an automatic,multiplexed point-of-care(POC)immunoassay that allows detecting C-reactive protein(CRP),procalcitonin(PCT),and interleukin 6(IL-6)in 50μL serum samples with limits of detection 1.87μg/mL,0.17 ng/mL,and 49.75 pg/mL,respectively.The authors use electrospun fibers and surface chemistry of gold nanoparticles(AuNPs)to adjust the detection range of different biomarkers.The authors have proposed some new diagnostic indicators(mScore and mScorePlus)that combine the results of CRP,PCT,IL-6,and complete blood counts(CBCs).The authors also tracked changes in CRP,PCT,and IL-6 of patients in an intensive care unit(ICU).The authors find that mScore and mScorePlus have advantages in improving the diagnostic accuracy and providing more analytical information.mScore and mScorePlus are effective tools to detect infections,differentiate bacterial and viral infections,and monitor disease.Integrating multiple markers into one straightforward parameter is an effective method for analytical applications.The authors believe that this method provides a general way for chemists to develop increasingly accurate detection methods and indicators.

An anti-passivation ink for the preparation of electrodes for use in electrochemical immunoassays

p-Nitrophenylphosphate （PNPP） is usually employed as the substrate for enzyme-linked immunosorbent assays, p-Nitrophenol （PNP）, the product of PNPP, with the catalyst alkaline phosphatase （ALP）, will passivate an electrode, which limits applications in electrochemical analysis. A novel anti-passivation ink used in the preparation of a graphene/ionic liquid/chitosan composited （rGO/IL/Ghi） electrode is proposed to solve the problem. The anti-passivation electrode was fabricated by directly writing the graphene-ionic liquid-chitosan composite on a single-side conductive gold strip. A glassy carbon electrode, a screen-printed electrode, and a graphene-chitosan composite-modified screen-printed electrode were investigated for comparison. Scanning electron microscopy was used to characterize the surface structure of the four different electrodes and cyclic voltammetry was carried out to compare their performance. The results showed that the rGO/IL/Ghi electrode had the best performance according to its low peak potential and large peak current. Amperometdc responses of the different electrodes to PNP proved that only the rGO/IL/Chi electrode was capable of anti-passivation. The detection of cardiac troponin I was used as a test example for electrochemical immunoassay. Differential pulse voltammetry was performed to detect cardiac troponin I and obtain a calibration curve. The limit of detection was 0.05 ng/ml.

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 6x 105-fold,reaching down to 33.9fg mL^(-1)(-1.4fM) 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.

Biofunctionalized semiconductor quantum dots for virus detection

Virus is a kind of microorganism and possesses simple structure and contains one nucleic acid,which must be replicated using the host cell system.It causes large-scale infectious diseases and poses serious threats to the health,social well-being,and economic conditions of millions of people worldwide.Therefore,there is an urgent need to develop novel strategies for accurate diagnosis of virus infection to prevent disease transmission.Quantum dots(QDs)are typical fluorescence nanomaterials with high quantum yield,broad absorbance range,narrow and size-dependent emission,and good stability.QDs-based nanotechnology has been found to be effective method with rapid response,easy operation,high sensitivity,and good specificity,and has been widely applied for the detection of different viruses.However,until now,no systematic and critical review has been published on this important research area.Hence,in this review,we aim to provide a comprehensive coverage of various QDs-based virus detection methods.The fundamental investigations have been reviewed,including information related to the synthesis and biofunctionalization of QDs,QDs-based viral nucleic acid detection strategies,and QDs-based immunoassays.The challenges and perspectives regarding the potential application of QDs for virus detection is also discussed.

Quantification of soluble epoxide hydrolase inhibitors in experimental and clinical samples using the nanobody-based ELISA

To ensure proper dosage of a drug,analytical quantification of it in biofluid is necessary.Liquid chromatography mass spectrometry(LC-MS)is the conventional method of choice as it permits accurate identification and quantification.However,it requires expensive instrumentation and is not appropriate for bedside use.Using soluble epoxide hydrolase(sEH)inhibitors(EC5026 and TPPU)as examples,we report development of a nanobody-based enzyme-linked immunosorbent assay(ELISA)for such small molecules and its use to accurately quantify the drug chemicals in human samples.Under optimized conditions,two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL,respectively,and two order of magnitude linear ranges with high precision and accuracy.The assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate EC5026.In addition,the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory potency.The results indicate that nanobody-based ELISA methods can efficiently analyze drug like compounds.These methods could be easily implemented by the bedside,in the field in remote areas or in veterinary practice.This work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and therapy.Attributes of nanobody based pharmaceutical assays are discussed.

Development and pathogenicity analysis of full-length infectious cDNA clones of citrus yellow mottle-associated virus in citrus plants

Citrus yellow mottle-associated virus(CiYMaV)belonging to the subgenus Mandarivirus within the genus Potexvirus,was first identified in 2018 from Pakistan(CiYMaV-PK),where it is endemic in several regions.Here,three full-length cDNA clones(pCiYMaV-FL-1,pCiYMaV-FL-18,and pCiYMaV-FL-22)corresponding to the genomic RNA of CiYMaV were constructed and then agroinfiltrated on Chandler pummelo(Citrus grandis)seedlings using the vacuum infiltration method.All the inoculated plants developed severe vein yellowing,leaf mottling,and dwarfing symptoms by 40 days post-infiltration(dpi).The results of a direct tissue blot immunoassay and reverse transcription polymerase chain reaction detection showed 94.7–100%infection rates of pCiYMaV-FL at 60 dpi.Despite there being no observed difference among the three clones in the severity of symptom,pCiYMaV-FL-22 showed the highest accumulation levels of viral RNA and coat proteins.Moreover,pCiYMaV-FL-22 successfully infected seven other citrus varieties and induced symptoms in five of them.Transmission electron microscopy identified the presence of filamentous virus particles in extracts from systemic leaves of the plants infected with pCiYMaV-FL-22 at 6-months post-infiltration.Taken together,the results indicate that Koch's postulates were fulfilled for CiYMaV in citrus plants.This is the first report of full-length infectious cDNA clones of CiYMaV,and thus,the data provide a basis for further study of the molecular mechanisms of virus infection and the development of a viral vector to express foreign genes in citrus plants.

Current testing strategies for hepatitis C virus infection in blood donors and the way forward

Screening tests for blood donations are based upon sensitivity, cost-effectiveness and their suitability for high-throughput testing. Enzyme immunoassay (EIAs) for hepatitis C virus (HCV) antibodies were the initial screening tests introduced. The &#x0201d;first generation&#x0201c; antibody EIAs detected seroconversion after unduly long infectious window period. Improved HCV antibody assays still had an infectious window period around 66 d. HCV core antigen EIAs shortened the window period considerably, but high costs did not lead to widespread acceptance. A fourth-generation HCV antigen and antibody assay (combination EIA) is more convenient as two infectious markers of HCV are detected in the same assay. Molecular testing for HCV-RNA utilizing nucleic acid amplification technology (NAT) is the most sensitive assay and shortens the window period to only 4 d. Implementation of NAT in many developed countries around the world has resulted in dramatic reductions in transfusion transmissible HCV and relative risk is now &#x0003c; 1 per million donations. However, HCV serology still continues to be retained as some donations are serology positive but NAT negative. In resource constrained countries HCV screening is highly variable, depending upon infrastructure, trained manpower and financial resource. Rapid tests which do not require instrumentation and are simple to perform are used in many small and remotely located blood centres. The sensitivity as compared to EIAs is less and wherever feasible HCV antibody EIAs are most frequently used screening assays. Efforts have been made to implement combined antigen-antibody assays and even NAT in some of these countries.

核酸检测技术在长春地区血液筛查的初步应用

目的进一步提高血液安全性,评估在长春地区开展核酸检测(NAT)的必要性和可行性。方法在国产和进口2种酶联免疫法(enzyme immunoassay,EIA)检测的基础上进行NAT分析。结果对2次EIA筛查合格的41615份血样进行NAT检测,其中乙型肝炎病毒核酸(HBVDNA)3份阳性、艾滋病毒核酸(HIVRNA)1份阳性,漏检率分别为0.07‰和0.02‰;未发现丙型肝炎病毒核酸(HCVRNA)阳性。结论经2次EIA法检测都合格的血液,并非绝对安全,还存在漏检现象。因此,在献血者筛查中应尽快开展NAT技术以缩短病毒检测的"窗口期",进一步提高血液安全性。

Recent Developments in SARS-CoV-2 Neutralizing Antibody Detection Methods

The ongoing Coronavirus disease 19 pandemic has likely changed the world in ways not seen in the past.Neutralizing antibody(NAb)assays play an important role in the management of the severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)outbreak.Using these tools,we can assess the presence and duration of antibody-mediated protection in naturally infected individuals,screen convalescent plasma preparations for donation,test the efficacy of immunotherapy,and analyze NAb titers and persistence after vaccination to predict vaccine-induced protective effects.This review briefly summarizes the various methods used for the detection of SARS-CoV-2 NAbs and compares their advantages and disadvantages to facilitate their development and clinical application.