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.

Chemiluminescence enzyme immunoassay based on magnetic nanoparticles for detection of hepatocellular carcinoma marker glypican-3

Glypican-3 （GPC3） is reported as a great promising tumor marker for hepatocellular carcinoma （HCC） diagnosis. Highly sensitive and accurate analysis of serum GPC3 （sGPC3）, in combination with or instead of traditional HCC marker alpha-fetoprotein （AFP）, is essential for early diagnosis of I-ICC. Biomaterial-functionalized magnetic particles have been utilized as solid supports with good biological compatibility for sensitive immunoassay. Here, the magnetic nanoparticles （MnPs） and magnetic microparticles （MmPs） with carboxyl groups were further modified with streptavidin, and applied for the development of chemiluminescence enzyme immunoassay （CLEIA）. After comparing between MnPs- and MmPs-based CLEIA, MnPs-based CLEIA was proved to be a better method with less assay time, greater sensitivity, better linearity and longer chemiluminescence platform. MnPs-based CLEIA was applied for detection of sGPC3 in normal liver, hepatocirrhosis, secondary liver cancer and HCC serum samples. The results indicated that sGPC3 was effective in diagnosis of HCC with high performance.

Immunoassay of chemical contaminants in milk:A review

The detection of chemical contaminants is critical to ensure dairy safety. These contaminants include veterinary medicines, antibiotics, pesticides, heavy metals, mycotoxins, and persistent organic pollutants （POPs）. Immunoassays have recently been used to detect contaminants in milk because of their simple operation, high speed, and low cost. This article describes the latest developments in the most important component of immunoassays--antibodies, and then reviews the four major substrates used for immunoassays （i.e., microplates, membranes, gels, and chips） as well as their use in the detection of milk contaminants. The paper concludes with prospects for further aDDlications of these immunoassavs.

A New Hapten for Immunoassay of Aldicarb

A new hapten, aldicarb oxime succinic ester （AOSE）, was synthesized for immunoassay of aldicarb. It was conjugated to proteins by active ester method. Polyclonal antibody was raised against AOSE-BSA （bovine serum albumin） conjugate. Enzyme-linked immunosorbent assays （ELISAs） showed that this antiserum had high affinity to aldicarb and can be used for sensitive and selective immunoassay of aldicarb.

Hierarchical Nanogold Labels to Improve the Sensitivity of Lateral Flow Immunoassay

Lateral flow immunoassay(LFIA) is a widely used express method and offers advantages such as a short analysis time, simplicity of testing and result evaluation.However, an LFIA based on gold nanospheres lacks the desired sensitivity, thereby limiting its wide applications.In this study, spherical nanogold labels along with new types of nanogold labels such as gold nanopopcorns and nanostars were prepared, characterized, and applied for LFIA of model protein antigen procalcitonin. It was found that the label with a structure close to spherical provided more uniform distribution of specific antibodies on its surface, indicative of its suitability for this type of analysis.LFIA using gold nanopopcorns as a label allowed procalcitonin detection over a linear range of 0.5–10 ng mL^(-1) with the limit of detection of 0.1 ng mL^(-1), which was fivefold higher than the sensitivity of the assay with gold nanospheres. Another approach to improve the sensitivity of the assay included the silver enhancement method,which was used to compare the amplification of LFIA for procalcitonin detection. The sensitivity of procalcitonin determination by this method was 10 times better the sensitivity of the conventional LFIA with gold nanosphere as a label. The proposed approach of LFIA based on gold nanopopcorns improved the detection sensitivity without additional steps and prevented the increased consumption of specific reagents(antibodies).

Characterization of Hapten-Protein Conjugates for Immunoassay of Polycyclic Aromatic Hydrocarbons(PAHs)

Preparation and characterization of the hapten-protein conjugates are fundamental to developing environmental immunoassays. As a hapten, 1-pyrenebutyric acid（PBA） was conjugated to the carrier protein of bovine serum albumin（BSA） or ovalbumin（OVA） by active ester method. Infrared spectra（IR） showed that PBA-BSA and PBA-OVA conjugates were successfully prepared. The number of the haptens conjugated to the carrier protein was determined by ultraviolet spectra（UV） and matrix-assisted laser desorption ionization time-of-flight mass spectrometry（MALDI-TOF-MS）. The calculated average binding ratios of PBA/BSA and PBA/OVA were 18：1 and 10：1 by UV, and 31：1 and 22：1 by MALDI-TOF-MS, respectively. Although there was a discrepancy between the results determined by the two methods, both of them were useful for the characterization of the hapten-protein conjugates. The antibody was produced against the antigen of PBA-BSA, and the affinity was tested by the double agar diffusion method The conjugates and the antibody could be used for developing a sensitive and selective immunoassay of polycyclic aromatic hydrocarbons（PAHs）.

Comparison of chemiluminescence enzyme immunoassay based on magnetic microparticles with traditional colorimetric ELISA for the detection of serum α-fetoprotein

A chemiluminescence enzyme immunoassay based on magnetic microparticles (MmPs-CLEIA) was developed to evaluate serum a-fetoprotein (AFP) in parallel with traditional colorimetric enzyme-linked immunosorbent assay (ELISA).A systematic comparison between the MmPs-CLEIA and colorimetric ELISA concluded that the MPs-CLEIA exhibited fewer dosages of immunoreagents,less total assay time,and better linearity,recovery,precision,sensitivity and validity.AFP was detected in forty human serum samples by the proposed MPs-CLEIA and ELISA,and the results were compared with commercial electrochemiluminescence immunoassay (ECLIA) kit.The correlation coefficient between MPs-CLEIA and ELISA was obtained with R 2 0.6703;however,the correlation between MPs-CLEIA and ECLIA (R 2 0.9582) was obviously better than that between colorimetric ELISA and ECLIA (R 2 0.6866).

The clinical value of enzyme-multiplied immunoassay technique monitoring the plasma concentrations of cyclosporine A after renal transplantation

The feasibility and the clinical value of the enzyme-multiplied immunoassay technique （EMIT） monitoring of blood concentrations of cyclosporine A （CsA） in patients treated with CsA were investigated after kidney transplantation. The validation method was performed to the EMIT determination of CsA blood concentration, the CsA whole blood trough concentrations （Co） of patients in different time periods after renal transplantation were monitored, and combined with the clinical complications, the statistical results were analyzed and compared. EMIT was precise, accurate and stable, also with a high quality control. The mean postoperative blood concentration of CsA was as follows： 〈1 month, （281.4± 57.9）ng/mL; 2 - 3 months, （264.5 ± 41.2） ng/mL; 4 - 5 months, （236.4 ± 38.9） ng/mL; 6 - 12 months, （206.5± 32.6）ng/mL; 〉12 months, （185.6± 28.1）ng/mL. The toxic reaction rate of CsA blood concentration within the recommended therapeutic concentration was 14.1%, significantly lower than that of the none-recommended dose group （37.2%） （P〈0.05）; the transplantation rejection rate was 4.4%, significantly lower than that of the none- recommended dose group （22.5%） （P〈0.05）. Using EMIT to monitor the blood concentration of CsA as the routine laboratory method is feasible, and is able to reduce the CsA toxicity and rejection significantly, leading to achieving the desired therapeutic effect.

Development of Lateral-flow Immunoassay for WSSV with Polyclonal Antibodies Raised against Recombinant VP (19+28) Fusion Protein

我们为 WSSV 开发了敏感、快速的侧面流动的免疫分析(LFIA ) ，用象指示物的胶体的黄金。熔化蛋白质， VP (19+28 ) ，在 E 被表示。coli，净化并且过去常准备 polyclonal 抗体。净化的 anti-VP (19+28 ) IgG 与胶体的黄金被结合。Unconjugated anti-VP (19+28 ) IgG 和山羊反兔子 IgG 在硝化纤维素膜上被使不能调动。在汇编以后，虾样品的三个组(在每个组的 5 个单个动物) 被测试它包括了健康、垂死、死了的虾。为每个组，三不同纸巾(身体蜜汁，鳃和 hepatopancreas ) 同时被测试。在平行，所有样品也为比较用 PCR 被分析。从测试的 45 件样品，当 15 作为否定被分类时， 30 作为积极被检测。LFIA 的结果相关，那些由 PCR 分析获得了，显示这二个察觉方法有在在这初步的研究测试的样品的有限数字的一样的功效。关键词侧面流动的免疫分析 - 白点症候群病毒(WSSV )- VP19 - VP28 CLC 数字 S945.

Capillary Electrophoretic Immunoassay with Laser-induced Fluorescence Detection for Interferon-gamma

Capillary electrophoretic immunoassay with laser-induced fluorescence detection for recombinant human interferon-gamma (IFN-g) was established. The limits of detection for three forms of IFN-g are 6.9 ng/L, 5.7 ng/L and 5.0 ng/L, respectively.

The Development of A Fluorescence Polarization Immunoassay for Aflatoxin Detection

A fluorescence polarization immunoassay （FPIA） was developed for the analysis ofaflatoxins （AFs） using an anti-aflatoxin B1 （AFB1） monoclonal antibody and a novel fluorescein-labeled AFB1 tracer. The FPIA showed an IC50 value of 23.33 ng/mL with a limit of detection of 13.12 ng/mL for AFB1. The cross-reactivities of AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 with the antibody were 100%, 65.7%, 143%, 23.5%, 111.4%, and 2%, respectively. The group-specificity of anti-AFB1mAb indicated that the FPIA could potentially be used in a screening method for the detection of total AFs, albeit not AFG2 and AFM2. The total time required for analyzing 96 samples in one microplate was less than 5 rain. This study demonstrates the potential usefulness of the FPIA as a rapid and simple technique for monitoring AFs.

A novel bead-based fluorescence immunoassay for aldosterone

Aldosterone quantification helps evaluate the rennin-angiotensin-aldosterone system. The new bead-based mul-tiplex platform has not been applied in aldosterone detection to achieve simultaneous measurements of multiple hormones. A new sensitive competitive bead immunoassay based on Luminex technology for detecting aldoster-one in small sample volumes was developed using two-antibody coupled beads and biotinylated aldosterone as tracer in combination with an extraction step. The assay was validated in human and mouse samples and exhibited a linear working range from 10 to 1,000 pg/mL. The assay was reproducible and precise with intra-assay coeffi-cient of variations （CVs） from 6.0% to 11.2%, inter-assay CVs from 8.0% to 13.0% and good recovery [（90-110）%] and linearity [（89-107）%]. Excellent correlation was found between this new assay and the reference method （r = 0.96, P 0.000,1）. The successful establishment of this assay provides high possibility for carrying out bead-based multiplex assay measuring aldosterone and other parameters simultaneously in one 50 μL sample so that the efficiency can be improved and precious samples can be saved.

Electrochemiluminescence immunoassay method underestimates cortisol suppression in ulcerative colitis patients treated with oral prednisone

AIM: To evaluate cortisolemia by using conventional electrochemiluminescence immunoassay (ECLIA) method compared to liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in active ulcerative colitis (UC) patients treated with oral prednisone (PD).

Precision medicine allergy immunoassay methods for assessing immunoglobulin E sensitization to aeroallergen molecules

Molecular-based allergy diagnosis for the in vitro assessment of a patient immunoglobulin E(IgE) sensitization profile at the molecular level uses allergen molecules(also referred to as allergen components), which may be well-defined, highly purified, natural allergen components or recombinant allergens. Modern immunoassay methods used for the detection of specific Ig E against aeroallergen components are either singleplex(such as the fluorescence enzyme immunoassay with capsulated cellulose polymer solid-phase coupled allergens, the enzyme-enhanced chemiluminescence immunoassay and the reversed enzyme allergosorbent test, with liquid-phase allergens), multiparameter(such as the line blot immunoassay for defined partial allergen diagnostics with allergen components coating membrane strips) or multiplex(such as the microarraybased immunoassay on immuno solid-phase allergen chip, and the two new multiplex nanotechnology-based immunoassays: the patient-friendly allergen nanobead array, and the macroarray nanotechnology-based immunoassay used as a molecular allergy explorer). The precision medicine diagnostic work-up may be organized as an integrated "U-shape" approach, with a "top-down" approach(from symptoms to molecules) and a "bottomup" approach(from molecules to clinical implications), as needed in selected patients. The comprehensive and accurate Ig E sensitization molecular profiling, with identification of the relevant allergens, is indicated within the framework of a detailed patient's clinical history to distinguish genuine Ig E sensitization from sensitization due to cross-reactivity(especially in polysensitized patients), to assess unclear symptoms and unsatisfactory response to treatment, to reveal unexpected sensitizations, and to improve assessment of severity and risk aspects in some patients. Practical approaches, such as anamnesis molecular thinking, laboratory molecular thinking and postmolecular anamnesis, are sometimes applied. The component-resolved diagnosis of the specific IgE repertoire has a key impact on optimal decisions making for prophylactic and specific immunotherapeutic strategies tailored for the individual patient.

Determination of theophylline concentration in serum by chemiluminescent immunoassay

Objective: This study aimed to establish chemiluminescent immunoassay (CLIA) for quantitative determination of theophylline levels in human serum. Methods: To measure the concentration of theophylline (n=122) and evaluate the assay.Results: The linear range of the CLIA method was 0.51～40 mg/L (Y=1.02X+0.44, r=0.995). The intra and inter CV (coefficient variance) of CLIA were 3.20% and 3.57%, respectively. The average recovery rate was 102.3%. This method was free from interference by brilirubin (＜200 μmol/L), hemoglobin (＜10 g/L), and triglycerides (＜15 mmol/L). Conclusion: This method is simple, convenient and precise for clinical pharmacokinetics study oftheophylline.

Electrochemical Determination of Cortisol by Capillary Electrophoretic Enzyme Immunoassay

An electrochemical method for detection of cortisol based on capillary electrophoretic enzyme immunoassay has been developed. A limit of detection of 1.7?0-9 mol/L was obtained.

Determination of Thyroxine with Capillary Electrophoretic Enzyme Immunoassay

A Capillary electrophortic enzyme linked immunoassay with electrochemical detection (CE-EIA-ED) has been developed. The method can be used to determine thyroxine with a limit of 3.8×10-9 mol/L.

Lateral Flow Immunoassay for Quantitative Detection of Ractopamine in Swine Urine

A strip reader based lateral flow immunoassay （LFIA） was established for the rapid and quantitative detection of ractopamine （RAC） in swine urine. The ratio of the optical densities （ODs） of the test line （AT） to that of the control line （Ac） was used to effectively minimize interference among strips and sample variations. The linear range for the quantitative detection of RAC was 0.2 ng/mL to 3.5 ng/mL with a median inhibitory concentration （IC50） of 0.59＋0.06 ng/mL. The limit of detection （LOD） of the LFIA was 0.13 ng/mL. The intra-assay recovery rates were 92.97%, 97.25%, and 107.41%, whereas the inter-assay rates were 80.07%, 108.17%, and 93.7%, respectively.