A simple and sensitive flow injection chemiluminescence immunoassay for chloramphenicol based on gold nanoparticle‐loaded enzyme

A simple and ultrasensitive flow injection chemiluminescence competitive immunoassay based on gold nanoparticle‐loaded enzyme for the detection of chloramphenicol (CAP) residues in shrimp and honey has been developed. Due to their good biocompatibility and large specific surface area, carboxylic resin beads can be used as solid phase carriers to immobilize more coating antigens (Ag). In addition, gold nanoparticles could provide an effective matrix for loading more CAP antibody and horseradish peroxidase, which would effectively catalyze the system of luminol–p‐iodophenol (PIP)–H₂O₂. A competitive immunoassay strategy was used for detection of CAP, in which CAP in the sample would compete with the coating Ag for the limited antibodies, leading to a chemiluminescence (CL) signal decrease with increase in CAP concentration. A wide linear range 0.001–10 ng ml^?1 (R² = 0.9961) was obtained under optimized conditions, and the detection limit (3σ) was calculated to be 0.33 pg ml^?1. This method was also been successfully applied to determine CAP in shrimp and honey samples. The immunosensor proposed in this study not only has the advantages of high sensitivity, wider linear range, and satisfactory stability, but also expands the application of flow injection CL immunoassay in antibiotic detection.     

A new highly sensitive chemiluminescent assay of alkaline phosphatase using lucigenin and its application to enzyme immunoassay

The chemiluminescent reaction of lucigenin with various reducing sugars and reducing compounds has been studied. It was found that dihydroxyacetone gave the most intense chemiluminescence (CL). We have developed highly sensitive chemiluminescent methods for alkaline phosphatase (ALP) based on the production of dihydroxyacetone using NADP⁺ or glycerol-3-phosphate as substrate. The detection limits for ALP using each substrate were 1.25 × 10^?19 mol/assay and 2.5 × 10^?19 mol/assay, and the coefficient of variation (n = 7) was 2.8% and 3.7%, respectively. We have also applied the method using NADP⁺ as substrate in enzyme immunoassays (EIA) for cholecystokinin (CCK) and human chorionic gonadotropin (hCG). CCK-8 (octapeptide sulphated form of a carboxy terminal fragment of CCK) concentrations released from alimentary canal of rat were assayed using the chemiluminescent EIA (CLEIA) and a fluorimtric EIA (ALP label). The correlation between CCK-8 values obtained by these methods was y = 1.04x + 18.21, r = 0.946, n = 28. hCG values in serum and in urine were measured. The correlation between hCG values in serum samples obtained using the CLEIA and a time-resolved fluoroimmunoassay (TR-FIA), and in urine samples obtained using the CLEIA and the fluorimetric EIA using ALP were satisfactory. The correlations were y = 1.00x ? 0.04, r = 0.997 (n = 51) and y = 1.00x ? 0.03, r = 0.999 (n = 10), respectively.     

Chemiluminescence assay of lipase activity using a synthetic substrate as proenhancer for luminol chemiluminescence reaction.

A novel chemiluminescence (CL) assay method for lipase (triacylglycerol lipase, E.C.3.1.1.3) activity was developed by using the lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI) as a substrate. The method is based on the enhanced CL reaction of luminol-hydrogen peroxide-horseradish peroxidase (HRP) with HDI that is liberated from the substrate by enzymatic hydrolysis. To simplify the assay procedure, both the hydrolysis of the substrate and the enhanced CL reaction were performed in the same reaction mixture. Lipases from Candida cylindracea and porcine pancreas were successfully determined with the detection limits (blank signal + 3 SD) of 0.05 and 50.0 mU/tube, respectively. The method is simple and rapid, permitting the completion of single assay within 5 min. The reproducibilities obtained with replicate assays were relative standard deviations (RSDs) of <=> 4.7% for within-day and <=> 6.0% for between-day assays.     

An enhanced chemiluminescent enzyme immunoassay for serum carcinoembryonic antigen based on a modification of a commercial kit

A conventional colorimetric peroxidase end-point (ortho-phenylenediamine substrate), used in an enzyme immunoassay for carcinoembryonic antigen, employing plastic beads as solid support, has been replaced by a much faster (30 seconds versus 30 minutes) enhanced chemiluminescent assay for the peroxidase label. Para-iodophenol was used to enhance the light emission from the peroxidase catalysed chemiluminescent reaction between luminol and hydrogen peroxide. Values for precision and carcinoembryonic antigen concentration obtained with the chemiluminescent and colorimetric versions of the immunoassay on 62 serum specimens were in good agreement.     

Development of a high‐throughput,indirect antibody immobilization format chemiluminescence enzyme immunoassay (CLEIA) for the determination of progesterone in human serum

A high‐throughput and simple chemiluminescence (CL) enzyme immunoassay (CLEIA) for the determination of progesterone (P) in human serum was developed, with the highly sensitive 4‐methoxy‐4‐(3‐phosphatephenyl)‐spiro‐(1,2‐dioxetane‐3,2′‐adamantane) (AMPPD)–alkaline phosphatase (ALP) system as the CL detection system. The results showed that the indirect immobilization of rabbit anti‐progesterone polyclonal antibody (RAPA) through secondary antibody exhibited apparent advantages over direct coating in terms of antibody saving and improvement of the coating stability and uniformity. The direct analysis of P in human serum without extraction was realized by using 8‐anilino‐1‐naphthalenesulphonic acid (ANS) to displace P from its binding proteins. The effect of several relevant parameters of the immunoreaction were examined and optimized. Compared with some commercial progesterone kits, the presented CLEIA has higher sensitivity with detection limitation as low as 0.06 ng/mL. The recoveries were 95.9–101%. The coefficient of variation was <8.4% and 9.9% for intra‐ and inter‐assay precision, respectively. This method has been successfully applied to the evaluation of P in human serum. Copyright © 2008 John Wiley & Sons, Ltd.     

An investigation on the catalytic mechanism of enhanced chemiluminescence: Immunochemical applications of this reaction

The mechanism of peroxidase-catalysed oxidation of luminol by H₂O₂ was studied. The stopped-flow technique was used to measure the rate constants for the reactions between the oxidized forms of peroxidase with luminol and the following substrates: p-iodophenol, p-bromophenol, p-clorophenol, o-iodophenol, m-iodophenol, luciferin, and 2-iodo-6-hydroxybenzothiazole. The correlation between kinetic parameters and the degree of enhancement was established. The effect of charged synthetic polymers and specific antibodies on the peroxidase activity in the enhanced chemiluminescent reaction. Novel homogenous methods of luminescent immunoassay (LIA) for (1) antibodies to insulin, (2) insulin and (3) antibodies to trinitrophenyl group are proposed on the basis of regulatory facilities of the enhanced chemiluminescent reaction. Based on the enhanced chemiluminescent reaction a peroxidase flow-injection assay was developed and successfuly tested in the flow-injection enzyme immunoassays for human IgG and for thyroxin (T4). The immunoassay proposed has a detection limit of 10^?9M for IgG and 10^?11M for T4, the overall time of the assay being 5–15 min.     

A magnetic particles-based chemiluminescence enzyme immunoassay for rapid detection of ovalbumin

Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles–chemiluminescence enzyme immunoassay (MPs–CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol–H₂O₂ chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs–CLEIA method had a linear range from 0.31 to 100 ng/ml with a detection limit of 0.24 ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA.     

Enhanced chemiluminescence: A sensitive analytical system for detection of sweet potato peroxidase

3-(10'-Phenothiazinyl)propane-1-sulfonate (SPTZ) was shown to be a potent enhancer of anionic sweet potato peroxidase (aSPP)-induced chemiluminescence. The optimal conditions for aSPP-catalyzed oxidation of luminol were investigated by varying the concentrations of luminol, hydrogen peroxide, Tris, and SPTZ as well as the pH values of the reaction mixture. Addition of 4-morpholinopyridine (MORP) to the reaction mixture markedly increased the light intensity. Using SPTZ and MORP together enhanced the effect 265 times. The lower detection limit (LDL) of SPP was 0.09 pM, approximately in 10 times lower than that for the cationic isozyme c of horseradish peroxidase/4-iodophenol system. It was shown that aSPP in the presence of SPTZ produced a longer lasting chemiluminescent signal.     

Chemiluminescent enzyme immunoassay for alpha-fetoprotein using β-D-galactosidase as label and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside as substrate

We developed a sensitive chemiluminescent sandwich-type enzyme immunoassay (CLEIA) of alpha-fetoprotein (AFP) using b?-D -galactosidase (b?-gal) as a label and 5-bromo-4-chloro-3-indolyl-b?-D -galactopyranoside as a substrate. The CL-EIA for AFP was performed using two monoclonal antibodies, one antibody is labelled with b?-gal, the other is coated onto the inside surface of a polystyrene tube. The detection limit for AFP was 0.5 ng/mL, equivalent to 10 pg/assay tube. The coefficient of variation for within and between assay imprecision were 2.0%?4.9% (n = 10) and 4.4%?9.8% (n = 5), respectively. AFP values in serum determined by this method correlated well with those obtained by radioimmunoassay (n = 26, r = 0.99). This sensitive AFP assay can be performed within 4 h and can be used as a routine assay in clinical diagnosis.     

Development of a highly sensitive chemiluminescent assay for hydrogen peroxide under neutral conditions using acridinium ester and its application to an enzyme immunoassay

We developed a highly sensitive chemiluminescent (CL) assay for hydrogen peroxide using 10‐methyl‐9‐(phenoxycarbonyl) acridinium fluorosulfonate (PMAC) that produced chemiluminescence under neutral conditions and applied it to an enzyme immunoassay (EIA). One picomole of hydrogen peroxide could be detected using the optimized PMAC‐CL method and 6.2 × 10^‐20 mol β‐d ‐galactosidase (β‐gal) could be detected by combining an indoxyl derivative substrate and the proposed PMAC‐CL method. This highly sensitive CL β‐gal assay was applied to an EIA for thyroid‐stimulating hormone (TSH) using β‐gal as a label enzyme; 0.02–100.0 μU/mL TSH in human serum could be assayed directly and with high reproducibility. Copyright © 2013 John Wiley & Sons, Ltd.     

Highly sensitive chemiluminescence immunoassay on chitosan membrane modified paper platform using TiO2 nanoparticles/multiwalled carbon nanotubes as label

A highly sensitive chemiluminescence (CL) immunoassay was incorporated into a low‐cost microfluidic paper‐based analytical device (μ‐PAD) to fabricate a facile paper‐based CL immunodevice (denoted as μ‐PCLI). This μ‐PCLI was constructed by covalently immobilizing capture antibody on a chitosan membrane modified μ‐PADs, which was developed by simple wax printing methodology. TiO₂ nanoparticles coated multiwalled carbon nanotubes (TiO₂/MWCNTs) were synthesized as an amplification catalyst tag to label signal antibody (Ab₂). After sandwich‐type immunoreactions, the TiO₂/MWCNTs were captured on the surface of μ‐PADs to catalyze the luminol‐p‐iodophenol‐H₂O₂ CL system, which produced an enhanced CL emission. Using prostate‐specific antigen as a model analyte, the approach provided a good linear response range from 0.001 to 20 ng/mL with a low detection limit of 0.8 pg/mL under optimal conditions. This μ‐PCLI showed good reproducibility, selectivity and stability. The assay results of prostate‐specific antigen in clinical serum samples were in good agreement with that obtained by commercially used electrochemiluminescence methods at the Cancer Research Center of Shandong Tumor Hospital (Jinan, Shandong Province, China). This μ‐PCLI could be very useful to realize highly sensitive, qualitative point‐of‐care testing in developing or developed countries. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a rapid and high‐performance chemiluminescence immunoassay based on magnetic particles for protein S100B in human serum

Protein S100B is a clinically useful non‐invasive biomarker for brain cell damage. A rapid chemiluminescence immunoassay (CLIA) for S100B in human serum has been developed. Fluorescein isothiocyanate (FITC) and N‐(aminobutyl)‐N‐(ethylisoluminol) (ABEI) are used to label two different monoclonal antibodies of anti‐S100B. Protein S100B in serum combines with labeled antibodies and can form a sandwiched immunoreaction. A simplified separation procedure based on the use of magnetic particles (MPs) that were coated with anti‐FITC antibody is performed to remove the unwanted materials. After adding the substrate solution, the relative light unit (RLU) of ABEI is measured and is found to be directly proportional to the concentration of S100B in serum. The relevant variables involved in the CLIA signals are optimized and the parameters of the proposed method are evaluated. The results demonstrate that the method is linear to 25 ng/mL S100B with a detection limit of 0.02 ng/mL. The coefficient of variation (CV) is < 5% and < 6% for intra‐ and interassay precision, respectively. The average recoveries are between 97 and 107%. The linearity–dilution effect produces a linear correlation coefficient of 0.9988. Compared with the commercial kit, the proposed method shows a correlation of 0.9897. The proposed method displays acceptable performance for quantification of S100B and is appropriate for use in clinical diagnosis. Copyright © 2013 John Wiley & Sons, Ltd.     

Chemiluminescence sandwich enzyme immunoassay for determination of human granulocyte colony stimulating factor (G-CSF)

A chemiluminescence sandwich enzyme immunoassay, using a glucose oxidase (GO) label, was developed for detecting attomole amounts of human granulocyte colony stimulating factor (G-CSF). Purified goat F(ab′)₂ immobilized on a bead and purified goat Fab′ labelled with GO were selected in combination with a chemiluminescent detection system comprising luminol and ferricyanide. The detection limits for G-CSF were 4amol/assay (1 pg/mL) in buffer solution and 10 amol/assay (2.5 pg/mL) in human serum. Coefficients of variation within assay and between assay ranged from 5.5% to 7.8% and from 3.4% to 16.0%, respectively. The G-CSF content of serum from normal healthy individuals was measurable using this method. G-CSF in 24 normal human sera showed a mean value of 19.3 pg/mL and ranged from 3.6 to 83.0 pg/mL.     

Development of a sensitive flow‐injection–chemiluminescence detection method for the determination of laevodopa

A simple chemiluminometric method using flow injection has been developed for the determination of laevodopa, based on its sensitizing effect on the weak chemiluminescence (CL) reaction between Na₂SO₃ and acidic KMnO₄. Under optimum experimental conditions, the CL intensity was linearly related to the concentration of laevodopa from 3.4 × 10^–8 to 2.4 × 10^–5 mol/L and the detection limit was 1.1 × 10^–8 mol/L (s:n = 3). The relative standard deviation (RSD) of the proposed method calculated from 20 replicate injection of 3 × 10^–7 mol/L laevodopa was 3.3%. The correlation coefficient was 0.997. The method was successfully applied to the determination of laevodopa in commercial pharmaceutical formulations and spiked urine samples. Copyright © 2008 John Wiley & Sons, Ltd.     

Levels of indole-3-acetic acid in intact and decapitated coleoptiles as determined by a specific and highly sensitive solid-phase enzyme immunoassay

A specific solid-phase enzyme immunoassay for the detection of as little as 3–4 pg of indole-3-acetic acid (IAA) is described. The assay involves minimal procedural efforts and requires only standard laboratory equipment. Up to 50 samples in triplicate, processed simultaneously, can be assayed and evaluated in 2.5 h. As little as 1 mg oat coleoptile tissue is sufficient for a quantitative IAA analysis and little or no extract purification is necessary. Using this assay, levels of IAA have been determined in coleoptiles of maize and oat. The distribution of IAA within single coleoptiles was quantitated and the production of IAA during the regeneration of the physiological tip in Avena coleoptiles was investigated. The changes in levels of IAA and other major phytohormones were quantitated during the growth of oat coleoptiles.Abbreviations ABA abscisic acid - BHT butylated hydroxytoluene - BSA bovine serum albumin - IAA indole-3-acetic acid - TBS Trishydroxymethylaminomethane buffered saline Part 21 in the series Use of Immunoassay in Plant Science     

Development and clinical application of a bioluminescence enzyme immunoassay for oxytocin

The development of a highly sensitive analytical method for oxytocin could be useful in the diagnosis and treatment of autistic spectrum disorder. We previously developed a colorimetric enzyme immunoassay (EIA) for plasma oxytocin measurement. In this study, we developed a method to measure oxytocin concentrations using a higher sensitivity bioluminescent EIA. Biotinylated oxytocin bridged with five lysine residues was used in a competitive format. The standard curve range for oxytocin was 1.0 to 1000 pg/assay. In addition, there was good correlation between the colorimetric and bioluminescent immunoassays in terms of measured oxytocin concentration (r = 0.9665, n = 48). The bioluminescent EIA for plasma oxytocin was more rapid and provided higher sensitivity than the colorimetric immunoassay, making it suitable for clinical application.     

Flow‐injection chemiluminescence and electrogenerated chemiluminescence determination of escitalopram oxalate in tablet form

Rapid, simple and highly sensitive flow‐injection (FI) chemiluminescence (CL) and flow‐injection electrogenerated chemiluminescence (ECL) methods were developed for the determination of escitalopram oxalate (ESC), a selective serotonin reuptake inhibitor used as an antidepressant drug. The CL method was based on the CL reaction of ESC with acidic cerium(IV) and tris(2,2'‐bipyridyl)ruthenium(II) (Ru). Various experimental parameters affecting CL intensity were carefully studied and optimised. The method enabled the determination of 0.001‐50 µg/mL of ESC in bulk form with a correlation coefficient r = 0.9999. The limit of detection (LOD) was 0.01 ng/mL (S/N = 3). The ECL method was based on the ECL reaction of Ru with the drug in an acidic medium, permitting the determination of ESC in the range of 0.00001‐70 µg/mL with r = 0.9999 and LOD of 1 x 10^‐4 ng/mL. The proposed methods were applied to the determination of ESC in commercial tablets. The results were compared statistically with those obtained from a published method using t‐ and F‐tests. Copyright © 2012 John Wiley & Sons, Ltd.     

Highly sensitive chemiluminescence determination of tenoxicam using a cerium(IV)–sodium hyposulphite system in micellar medium

A simple, rapid and precise flow‐injection–chemiluminescence (FI–CL) method is presented for the determination of tenoxicam in pharmaceutical preparations and biological samples. The method is based on the weak chemiluminescence signal arising from the reaction of cerium(IV) in a nitric acid medium with sodium hyposulphite being significantly increased by tenoxicam in the presence of sodium dodecyl benzene sulphonate. Several experimental parameters affecting the CL reaction were examined and optimized systematically. Under the optimum conditions, the CL intensity was proportional to the concentration of tenoxicam in the range 7.0 × 10^–11–5.0 × 10^–8 g/mL. The detection limit was 2.3 × 10^–11 g/mL tenoxicam and the relative standard deviation (RSD) was 2.1% for 1.0 × 10^–9 g/mL tenoxicam solution (n = 11). The proposed method was applied to the determination of tenoxicam in pharmaceutical preparations, serum and human urine, with satisfactory results. The possible mechanism of the chemiluminescence reaction is also briefly discussed. Copyright © 2011 John Wiley & Sons, Ltd.     

Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N‐acetyl‐seryl‐aspartyl‐lysyl‐proline

N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is hydrolyzed by the N‐terminal active site of angiotensin converting enzyme and partially eliminated in urine, its plasma level is a result of a complex balance between its production, hydrolysis by ACE, and renal elimination. In this study, we attempted to establish an enzyme immunoassay (EIA) for quantifying AcSDKP‐like immunoreactive substance (IS), which is applicable for monitoring plasma AcSDKP levels in healthy subjects and patients with chronic renal failure. Using β‐ d ‐galactosidase‐labeled Gly‐γAbu‐SDKP as a marker antigen, an anti‐rabbit IgG‐coated immunoplate as a bound/free separator and 4‐methylumbelliferyl‐β‐ d ‐galactopyranoside as a fluorogenic substrate, a highly sensitive and specific EIA was developed for the quantification of AcSDKP‐IS in human plasma. The lower limit of quantification was 0.32 fmol/well, and the sharp inhibition competitive EIA calibration curve obtained was linear between 8.0 and 513 fmol/ml. This EIA was so sensitive that only 10 µl plasma sample was required for a single assay. The coefficients of variation (reproducibility) for human plasma concentrations of 0.2 and 2.1 pmol/ml were 7.2 and 7.7%, respectively, for inter‐assay and 13.3 and 7.8% for intra‐assay comparisons. Plasma AcSDKP‐IS level was significantly higher in patients with chronic renal failure (0.92 ± 0.39 pmol/ml) compared with healthy subjects (0.29 ± 0.07 pmol/ml). These results suggest that our EIA may be useful to evaluate plasma AcSDKP level as a biomarker in various patients. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.