Sensitive Immunoassay Method Research Articles

Establishment of a high-sensitivity time-resolved fluorescence immunoassay with PLA2R-IgG1 antibody and its clinical application in idiopathic membranous nephropathy prognosis

IntroductionThe objective of this study was to develop a highly sensitive time-resolved fluorescence immunoassay (TRFIA) method to detect phospholipase A2 receptor (PLA2R)-IgG1 antibodies and evaluate its clinical relevance in predicting the prognosis of individuals with idiopathic membranous nephropathy (IMN). Materials and methodsA three-step indirect TRFIA method was established using a PLA2R antigen-coated microtiter plate to capture PLA2R-IgG antibodies, followed by detection using mouse anti-human IgG1 and Eu3+-labeled goat anti-mouse IgG antibodies. This method was applied to the initial serum of 56 patients with PLA2R-IMN to investigate the clinical value of PLA2R-IgG1 antibody levels in predicting IMN prognosis. ResultsThe detection range of PLA2R-IgG1-TRFIA was 0.85–300RU/mL, with intra-assay precision of 3.54–5.93 % and inter-assay precision of 4.39–9.36 %. Recoveries were 101.77–108.04 %. A PLA2R-IgG1 level above 2.21RU/mL indicated PLA2R-IMN. At initial diagnosis, the median PLA2R-IgG level was 51.24RU/mL in the remission group and 93.27RU/mL in the non-remission group. The median PLA2R-IgG1 level was 603.32RU/mL in the non-remission group, which was 4.29 times higher than that in the remission group (140.67RU/mL). PLA2R-IgG1 levels (P = 0.001) more effectively distinguished between remission and non-remission groups compared with PLA2R-IgG levels (P = 0.094). ConclusionsThe first quantitative TRFIA for PLA2R-IgG1 was established, showing greater clinical value in predicting IMN prognosis, compared to that for PLA2R-IgG levels.

Direct competitive immunoassay method for sensitive detection of the histamine in foods based on a MI-Cu-GMP nanozyme marker and molecularly imprinted biomimetic antibody.

Histamine may lead to low blood pressure, skin flushing and edema when it accumulates in large amounts in the body. Therefore, establishing sensitive methods for the detection of histamine in foods is extremely important to ensure food safety and human health. The MI-Cu-GMP NPs (2-methylimidazole-copper-guanosine monophosphate nanozymes) with high laccase-like activity were synthesized. Using the prepared molecular imprinted membrane as biomimetic antibody and MI-Cu-GMP NPs as marker, a sensitive direct competitive biomimetic enzyme-linked immunoassay (BELISA) method for rapid detection of the histamine in foods was developed. Under optimal conditions, the limit of detection (LOD, IC15) and sensitivity (IC50) of the BELISA method for histamine was 0.05 mg L-1 and 1.22 mg L-1, respectively. The liquor samples spiked with histamine was detected by the BELISA method with satisfactory recoveries ranging from 90.00% to 116.00%. Further, the level of histamine in three samples (cooking wine, rice vinegar and soy sauce) was tested by the BELISA and high-performance liquid chromatography (HPLC), with no significant difference found between the two methods. Given the advantages, the established BELISA method is expected to provide practical guidance for the monitoring of histamine in food and provides a foundation for the detection of other food hazards. © 2024 Society of Chemical Industry.

Development of an immunoassay based on a specific antibody for the detection of diphenyl ether herbicide fomesafen

Fomesafen belongs to the diphenyl ether herbicide, and is widely used in the control of broadleaf weeds in crop fields due to its high efficiency and good selectivity. The residual of fomesafen in soil has a toxic effect on subsequent sensitive crops and the microbial community structure because of its long residual period. Therefore, an efficient method for detecting fomesafen is critical to guide the correct and reasonable use of this herbicide. Rapid and sensitive immunoassay methods for fomesafen is unavailable due to the lack of specific antibody. In this study, a specific antibody for fomesafen was generated based on rational design of haptens and a sensitive immunoassay method was established. The half maximal inhibitory concentration (IC50) of the immunoassay was 39 ng/mL with a linear range (IC10–90) of 1.92–779.8 ng/mL. In addition, the developed assay had a good correlation with the standard UPLC-MS/MS both in the spike-recovery studies and in the detection of real soil samples. Overall, the developed indirect competitive enzyme immunoassay reported here is important for detecting and quantifying fomesafen contamination in soil and other environmental samples with good sensitivity and high reproducibility.

Ultrasensitive detection of salivary SARS-CoV-2 IgG antibodies in individuals with natural and COVID-19 vaccine-induced immunity

We assessed the feasibility of a highly sensitive immunoassay method based on single molecule array (Simoa) technology to detect IgG and IgA antibodies against SARS-CoV-2 spike protein receptor binding domain (RBD) in saliva from individuals with natural or vaccine-induced COVID-19 immunity. The performance of the method was compared to a laboratory-developed SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay (ELISA). Paired serum and saliva specimens were collected from individuals (n = 40) prior to and 2 weeks after receiving an initial prime COVID-19 vaccine dose (Pfizer/BioNTech BNT162b2 or Moderna mRNA-1273). Saliva was collected using a commercially available collection device (OraSure Inc.) and SARS-CoV-2 RBD IgG antibodies were measured by an indirect ELISA using concentrated saliva samples and a Simoa immunoassay using unconcentrated saliva samples. The IgG results were compared with paired serum specimens that were analyzed for total RBD antibodies using the ELISA method. The analytical sensitivity of the saliva-based Simoa immunoassay was five orders of magnitude higher than the ELISA assay: 0.24 pg/mL compared to 15 ng/mL. The diagnostic sensitivity of the saliva ELISA method was 90% (95% CI 76.3–97.2%) compared to 91.7% (95% CI 77.5–98.2%) for the Simoa immunoassay without total IgG-normalization and 100% (95% CI 90.3–100%) for the Simoa immunoassay after total IgG-normalization when compared to the serum ELISA assay. When analyzed using the SARS-CoV-2 RBD IgG antibody ELISA, the average relative increase in antibody index (AI) between the saliva of the post- and pre-vaccinated individuals was 8.7 (AIpost/pre). An average relative increase of 431 pg/mL was observed when the unconcentrated saliva specimens were analyzed using the Simoa immunoassay (SARS-CoV-2 RBD IgGpost/pre). These findings support the suitability of concentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG antibodies via ELISA, and unconcentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG and IgA using an ultrasensitive Simoa immunoassay.

Competitive fluorescence immunoassay for the rapid qualitative screening and accurate quantitative analysis of ketamine.

In this paper, a sensitive and specific competitive fluorescence immunoassay (CFIA) method was developed for the qualitative and quantitative analysis of ketamine (KET). A novel competitive model in which ketamine hapten (KET-BSA), coated on microporous plates, competed with ketamine antigen (KET-Ag) in actual samples to bind fluorescein isothiocyanate-labeled antibody (KET-Ab) could be used for rapid and indirect quantitative analysis of KET in human urine, blood, or sewage. In the CFIA method, KET concentration in the sample negatively correlated with the detected fluorescence intensity. The linear correlation coefficient of the competitive quantitative equation was 0.992, the linear range was 0.01-0.5 μg mL-1, and the limit of detection (LOD) was 0.1 pg mL-1. The specificity results showed that the cross-reaction rate of norketamine was less than 10%. Recoveries of spiked samples at low, medium, and high concentrations ranged from 96% to 117%. The CFIA method and classical gas chromatography-tandem mass spectrometry (GC-MS/MS) were used to detect the actual samples simultaneously. The relative deviation of the quantitative results was less than 10%. The LOD value of KET by CFIA was four orders of magnitude lower than that by GC-MS/MS. Additionally, CFIA had great advantages over GC-MS/MS in terms of sample pretreatment and economic investment. In conclusion, this study provided a targeting detection platform for KET, which achieved a rapid, portable, and sensitive analysis of trace KET in various materials.

Development of Time-Resolved Fluorescence Immunochromatographic Assays for Simultaneously Detecting Tylosin and Tilmicosin in Milk in Group-Screening Manner.

Tylosin and tilmicosin (T&T) residues in livestock products have received extensive attention from consumers. Time-resolved fluorescence immunochromatographic assay (TRFICA), as a fast, efficient and sensitive immunoassay method, has played an increasingly important role in the food safety field. Therefore, herein a quantitative and visual TRFICA was established for simultaneously detecting T&T in milk in a group-screening manner. Under the optimal conditions, the standard curve range of developed TRFICA based on the T&T was 1.87~7.47 ng/mL, and the half-maximal inhibition concentrations (IC50) were 4.06 ng/mL and 3.74 ng/mL, respectively. The limits of detection (LOD) of the TRFICA method were from 1.72 ng/mL to 1.39 ng/mL, and the visual cut-off values were 31.25 ng/mL and 62.50 ng/mL for T&T in milk, respectively. Moreover, the stability experiments showed that the strips could be stored at 4 °C for more than 6 months, the total detection time was less than 13 min, and the cross-reactivities (CRs) with related compounds were less than 0.1%, which concluded that the developed TRFICA method could be used in real milk sample detection.

Design of Hapten Synthesis and Antibody Characterization of G-group Aflatoxins

Food and feed contamination with Aflatoxins pose a serious threat to human health and animal husbandry development and has caused widespread concern, among them, G-group Aflatoxins as the main pollutant has attracted more and more attention. In order to establish a rapid, sensitive, specific and efficient immunoassay method for G-group aflatoxins, this study aimed to designed to synthesize 3 immunogens and coating antigens and identified by UV and SDS-PAGE. Then used to immunize Balb/c mice with prepared of three immunogen the titers were determined by indirect ELISA and the sensitivity was determined by competitive indirect ELISA (icELISA), the specificity was assessed by the cross-reaction test (CR). The results of UV and SDS-PAGE showed that the three immunogens and the corresponding coated antigens were successfully synthesized and the best one was SA method among three synthesis methods of G-group AF artificial antigen and its conjugation ratio of AFG1 to BSA was about 5.64∶1. The immune efficacy of SA method was the best, its AFG1 pAb had high titers of 1∶(6.4×103) by indirect ELISA, a good sensitivity with the 50 % inhibition concentration(IC50) of 13.6 μg/kg－1 to AFG1 by blocking ELISA and a high CR to AFG2 of 82.19 %, it showed high specificity for other aflatoxins. The experimental results not only obtained the ideal G group aflatoxin antibody, but also established a substance and technology foundation for G group aflatoxin immunization methods, and can be referenced in the similar tests.

A sensitive biomimetic enzyme-linked immunoassay method based on Au@Pt@Au composite nanozyme label and molecularly imprinted biomimetic antibody for histamine detection

ABSTRACTIn this paper, a sensitive biomimetic enzyme-linked immunoassay (BELISA) method based on an Au@Pt@Au composite nanozyme label was developed for histamine (HA) detection. A molecular imprinted polymer prepared directly on the 96-well plate served as a biomimetic antibody. A BELISA method was developed for HA standard solutions at concentrations ranging from 0.01 to 100.00 mg/L. Under optimal conditions, the novel BELISA method had a low limit of detection (0.069 mg/L) and high sensitivity (7.20 mg/L). The HA spiked in yellow rice wine and liqueur samples were detected by this BELISA method with the recoveries ranging from 84.28% to 108.82%. There was no significant difference of the results between the BELISA method and HPLC for the detection of HA in the red and white grape wine samples.

A highly sensitive immunofluorescence sensor based on bicolor upconversion and magnetic separation for simultaneous detection of fumonisin B1 and zearalenone.

Mycotoxins cause significant harm to human health, so it is imperative to develop a highly sensitive and easy-to-operate method for the detection of mycotoxins. Herein, a fluorescence-based magnetic separation immunoassay for simultaneous detection of mycotoxins fumonisin B1 and zearalenone is established. The method employed high fluorescent upconversion-nanoparticles(UCNPs) conjugated with biotinylated antigens as upconversion fluoroscent probes. Magnetic nanoparticles(MNPs) immobilized with monoclonal antibodies are used as immune-capture probes. Highly sensitive detection of FB1 and ZEN was achieved based on the luminescence properties of UCNPs and the separation effects of MNPs. The results showed a robust linear correlation between the enhanced fluorescence emission intensity and the logarithmic concentrations of FB1 and ZEN under the optimal conditions (R2(FB1) = 0.9965, R2(ZEN) = 0.9976), and the linear ranges were 0.05-5 ng mL-1. Furthermore, the limits of detection (LOD) were 0.016 ng mL-1 for FB1 and 0.012 ng mL-1 for ZEN. The standard addition method was used to determine the content of FB1 and ZEN in the samples to evaluate the accuracy of the process. The average recoveries were 89.48% to 113.69% and 85.97% to 113.82%, respectively. Compared with the other five mycotoxins, this method had high selectivity. It is expected that the multi-component simultaneous detection can be further realized by using the multicolor labeling characteristics of UCNPs.

Research Progress of Green Immunoassay for Mycotoxins

Mycotoxin pollution not only causes huge economic losses, but also seriously threatens the health of humans and animals. Immunoassay methods are widely used in the rapid detection of mycotoxins, but the method requires the use of toxin standards and artificial antigens.The artificial antigen synthesis is not only difficult to prepare, but also requires toxin standards as raw materials. Toxin standards are expensive and harmful to the health of producers and operators, which restricts the application and popularization of immunoanalytic methods in mycotoxin detection. If the toxic antigens and standards involving in the immune reaction is substituted with the non-toxic antigen, a green immunology test method can be established to overcome the above deficiencies. Establishing a green, fast, simple and highly sensitive immunoassay method to detect these toxins has become a hot topic in recent years. This paper reviews the application and prospect of green immunoanalysis methods based on anti-unique antibody and phage display techniques inmycotoxins.

A Calibration Curve Implanted Enzyme-Linked Immunosorbent Assay for Simultaneously Quantitative Determination of Multiplex Mycotoxins in Cereal Samples, Soybean and Peanut.

In this study, a rapid and sensitive immunoassay method has been established based on calibration curve implanted enzyme-linked immunosorbent assay (C-ELISA) for the simultaneously quantitative determination of aflatoxin B1, deoxynivalenol and zearalenone in cereal samples, soybean and peanut. The C-ELISA avoids using the standard substances during the detection. The principle of the C-ELISA is to implant the optimized standard curve data into the matched analysis software which can make data processing more convenient and faster. The implanted calibration curve software was programmed with C plus plus. In the new immunoassay system for aflatoxin B1, deoxynivalenol and zearalenone, their linear detection ranges were from 0.03~0.81, 1.00~27.00 and 5.00~135.00 ng/g, respectively. Recovery rates from spiked samples ranged from 85% to 110% with the intra-assay coefficients of variation under 5%. Compared with HPLC method, the new method showed consistence in all the observed contents of the three mycotoxins in real samples. The new method can rapidly and reliably high throughput simultaneously screen for multiplex mycotoxins.

Polydopamine coated zirconium metal-organic frameworks-based immunochromatographic assay for highly sensitive detection of deoxynivalenol

Conventional immunochromatographic assays (ICAs) based on gold nanoparticles (GNPs) suffer from the disadvantage of low sensitivity. In this work, a highly sensitive ICA based on polydopamine coated zirconium metal-organic frameworks labeled antibodies (ZrPA-Ab) as a novel probe was developed for visual determination of deoxynivalenol (DON). The ZrPA was synthesized via an oxidative self-polymerized assembly (OPMA) strategy using porphyrin functionalized zirconium metal-organic frameworks (Zr-MOFs, MOF-525) and polydopamine (PDA). The Abs could directly attach to the ZrPA surface owing to the large specific surface area, excellent water-stability and bio-compatibility of the ZrPA, on this basis, a sensitive, precise and reliable immunoassay method can be developed for rapid and selective detection of DON. Under optimized conditions, a non-linear calibration curve was obtained in the range of 0–50 ng/mL DON concentration with an IC50 of 1.22 ng/mL, and the visual detection limit was 0.18 ng/mL, which was about 8-times more sensitive than that of the conventional GNPs-based ICA. Finally, the proposed ZrPA-ICA was successfully applied for the detection of DON in pig hind legs meat, green bean, maize and millet samples, revealing the feasible and reliable application of this biosensor in different food matrices. Thus, this work broadens the possibilities for the use of MOFs as a novel labeling carrier in immunoassays.

Perovskite Nanocrystal Fluorescence-Linked Immunosorbent Assay Methodology for Sensitive Point-of-Care Biological Test

Summary Quantitative fluorescence immunoassay is a vital and convenient method in point-of-care (POC) testing for food safety and clinical healthcare. However, current marking materials, from organic luciferin to quantum dots, still lack the requirement of increasing need for multiple targets of interest because of their too broad emission. Here, a new label material, perovskite nanocrystals (Pe-NCs), with a narrower photoluminescence spectrum is reported, and a sensitive and quantitative immunoassay method named the perovskite nanocrystal fluorescence-linked immunosorbent assay is preliminarily demonstrated. The key point of realizing water-dispersed and stable Pe-NCs was addressed by functionalizing with hydroxyl groups. The fluorescent probes (perovskite antibody) were constructed via electrostatic adsorption with narrow full-width at half-maximum (∼18 nm), laying the foundation for future study of multi-target detection. Both indirect competitive immunity and sandwich immunoassay were successfully demonstrated. This work provides a possible label material for POC tests and may open up a promising avenue of next-generation immunoassay.

Alkaline Hydrolysis Behavior of Metal-Organic Frameworks NH2-MIL-53(Al) Employed for Sensitive Immunoassay via Releasing Fluorescent Molecules.

Nanosized metal-organic frameworks (MOFs) NH2-MIL-53(Al) were synthesized from 2-aminoterephthalic acid (NH2·H2BDC) and AlCl3 by a facile hydrothermal method. The synthesized MOFs displayed good stability and a uniform particle size in a netural medium and were hydrolyzed in alkaline medium to release a large amount of fluorescent ligand NH2·H2BDC. Therefore, they can act as large-capability nanovehicles to load signal molecules for investigating various biorecognition events. In this work, based on the alkaline hydrolysis behavior of MOFs NH2-MIL-53(Al), a sensitive immunoassay method was developed for the detection of aflatoxin B1 (AFB1) by employing them as fluorescent signal probes. With a competitive immunoassay mode on microplate, AFB1 can be detected within a linear range of 0.05-25 ng mL-1. The method was successfully employed to detect AFB1 spiked in Job tears, Polygala tenuifolia and with acceptable recovery values of 83.00-114.00%. The detection results for moldy Fructus xanthii displayed an acceptable agreement with those from the high-performance liquid chromatography method, with relative errors of -14.21 to 3.49%. With the merits of high sensitivity, facile manipulation, and ideal reliability, the approach can also be extended to other areas such as aptasensor and receptor-binding assay.

Sensitive and portable electrochemical immunoassay for lipoprotein-associated phospholipase A2 using BSA-doped CaCO3 nanospheres to regulate pH readout

A simple and sensitive electrochemical immunoassay method was developed for the quantitative monitoring of lipoprotein-associated phospholipase A2 (Lp-PLA2) on a low-cost microtiter plate by using a portable handheld pH meter.

A voltammetric immunoassay for the carcinoembryonic antigen using a self-assembled magnetic nanocomposite.

The authors describe a voltammetric immunoassay for the carcinoembryonic antigen (CEA). It is based on the use of a self-assembled magnetic nanocomposite as multifunctional signal amplification platform. The core of the nanocomposite consists of Fe3O4 microspheres, and the shell of zirconium hexacyanoferrate loaded with gold nanoparticles (AuNPs@ZrHCF@Fe3O4). The material was synthesized by an electrostatic self-assembly process which is caused by the strong interaction between cyano groups and AuNPs. The surface of the Fe3O4 microspheres was functionalized with amino groups to facilitate the immobilization of ZrHCF which acts as an electron mediator. The nanocomposite was placed on a glassy carbon electrode which then displays noteworthy electrocatalytic activity toward the reduction of hydrogen peroxide (H2O2). The AuNPs serve as a support for the immobilization of antibodies by the interaction between AuNPs and amino groups on antibodies to construct a covalent Au-N bond. This facilitates electron transfer on the electrode surface using H2O2 as the electrochemical probe. Square wave voltammetry (measured typically at +0.2V vs. SCE) was carried out to record the electrochemical behavior. Under the optimal conditions, a response is linear in the 0.5pg·mL-1 to 50ng·mL-1 CEA concentration range, and the detection limit is as low as 0.15pg·mL-1 (S/N= 3). The method is selective, highly stable and acceptably reproducible. Graphical abstract A self-assembly magnetic nanocomposite for voltammetric immunoassay of CEA. GCE glassy carbon electrode; Au NPs gold nanoparticles; ZrHCF zirconium hexacyanoferrate; CEA carcinoembryonic antigen; Anti-CEA CEA antibody; BSA bovine serum albumin; SWV square wave voltammetry. A high sensitive voltammetric immunoassay method has been used for detecting CEA, It is based on a self-assembled magnetic nanocomposite (Au NPs@ZrHCF@Fe3O4) as multifunctional signal amplification platform.

A simple and sensitive immunoassay method for the detection of low-molecular-weight proteins and neuropeptides

BACKGROUND: Low-molecular-weight proteins and peptides perform numerous regulatory roles in biological systems and could be useful as biomarkers. Hence, qualitative and quantitative measurement of peptide levels has become increasingly important. AIM: This present study was aimed at developing a simple and sensitive dot immunoblot assay (DIA) by modifying existing protocols. MATERIALS AND METHODS: Using polyclonal antibody generated against FMRFamide peptide, at a dilution ratio of 1:10,000 in buffer, as a primary antibody, alkaline phosphatase-conjugated goat anti-rabbit antibody as secondary antibody and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate development system, peptides were dotted on 0.2μm nitrocellulose membrane in volumes of 1μl, 0.5μl and 0.2μl. RESULTS: Results show immunoreactivity of FMRFamide antibody to small quantities of dotted neuropeptide as low as 0.2ng and 33.4pM. CONCLUSION: This indicates that this protocol is sensitive with the additional advantage of simplicity, speed, small sample volume, low cost, and production of nonhazardous waste. This DIA protocol could be useful in resource-poor settings and laboratories with low budgets.

Rapid and Highly Sensitive Non-Competitive Immunoassay for Specific Detection of Nodularin.

Nodularin (NOD) is a cyclic penta-peptide hepatotoxin mainly produced by Nodularia spumigena, reported from the brackish water bodies of various parts of the world. It can accumulate in the food chain and, for safety reasons, levels of NOD not only in water bodies but also in food matrices are of interest. Here, we report on a non-competitive immunoassay for the specific detection of NOD. A phage display technique was utilized to interrogate a synthetic antibody phage library for binders recognizing NOD bound to an anti-ADDA (3-Amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E),6(E)-dienoic acid) monoclonal antibody (Mab). One of the obtained immunocomplex binders, designated SA32C11, showed very high specificity towards nodularin-R (NOD-R) over to the tested 10 different microcystins (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF, -LW, -LA, -WR). It was expressed in Escherichia coli as a single chain antibody fragment (scFv) fusion protein and used to establish a time-resolved fluorometry-based assay in combination with the anti-ADDA Mab. The detection limit (blank + 3SD) of the immunoassay, with a total assay time of 1 h 10 min, is 0.03 µg/L of NOD-R. This represents the most sensitive immunoassay method for the specific detection of NOD reported so far. The assay was tested for its performance to detect NOD using spiked (0.1 to 3 µg/L of NOD-R) water samples including brackish sea and coastal water and the recovery ranged from 79 to 127%. Furthermore, a panel of environmental samples, including water from different sources, fish and other marine tissue specimens, were analyzed for NOD using the assay. The assay has potential as a rapid screening tool for the analysis of a large number of water samples for the presence of NOD. It can also find applications in the analysis of the bioaccumulation of NOD in marine organisms and in the food chain.

Copper chromogenic reaction based colorimetric immunoassay for rapid and sensitive detection of a tumor biomarker

A new colorimetric immunoassay method was developed for the rapid and sensitive detection of a tumor biomarker of carcinoembryonic antigen (CEA) by combination of a magnetic bead (MB)-based sandwich immunoassay and a copper chromogenic reaction. The magnetic immunoassay platform was constructed through the covalent immobilization of the capture antibody on the surface of carboxylated magnetic beads. After immuno-recognition of CEA, signal antibody-functionalized copper oxide nanoparticle (CuO NP) probes were applied for sandwich immunoreaction to form an immunocomplex. The CuO NP labels quantitatively captured onto the immunocomplex were then dissolved in acid solution to release high-content copper ions. Based on the coordination of these ions with the newly synthesized chromogenic agent of 1,2-diphenyl-2-(2-(pyridin-2-yl)hydrazono)ethanone, a red complex was produced for the colorimetric signal readout, resulting in the successful construction of a sensitive immunoassay method for CEA detection. Under the optimum conditions, this method showed a wide linear range over three orders of magnitude and a low detection limit of 26 pg/mL. Besides, this method showed excellent performance with low cost, rapid and convenient operation as well as satisfactory reproducibility, stability and accuracy, thus providing great potentials for practical applications.

A novel microchip electrophoresis-based chemiluminescence immunoassay for the detection of alpha-fetoprotein in human serum

A sensitive immunoassay method based on microchip electrophoresis chemiluminescence (MCE-CL) detection technology was developed for the detection of tumor marker alpha-fetoprotein (AFP). This method adopts the non-competitive immunoassay mode, and was conducted after AFP reacted with excessive horseradish peroxidase (HRP) labeled monoclonal antibody. The extreme pH value was adopted in the electrophoresis buffer solution. The use of brij 35 as an additive of electrophoresis buffer increased dramatically the resolution (Rs) and the reproducibility of the analysis. Under the optimized experimental conditions, effective separation of the immune complex Ag-Ab* and free Ab* was achieved within 60s. The peak height of the immune complex Ag-Ab* was taken as quantification of AFP. Good linearity was observed within AFP concentrations ranging from 10ng/mL to 150ng/mL, and the detection limit was found to be 7.2ng/mL (1.0×10−10M). The present method was successfully applied for the detection of AFP in human serum from both healthy and cancer patients, and the AFP levels in the both were found be in the range of 16.5–23.4ng/mL and 416.2–825.4ng/mL, respectively.