A rapid enhanced chemiluminescent immunoassay for allergen-specific IgE antibodies

An enhanced chemiluminescent immunoassay is described for the measurement of allergen-specific IgE antibodies. The assay is demonstrated for pollen from four grass species. A comparison was made between results obtained by this method and those obtained by the radioallergosorbent (RAST) procedure; a high degree of correlation (r = 0.95) was found for D. glomerata specific IgE. The assay is rapid and can be carried out in under 1 hour. The advantages of the luminescent assay as compared with the RAST procedure are discussed.     

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.     

Enhanced chemiluminescent immunoassay for aldosterone

A solid phase immunoassay for aldosterone using enhanced chemiluminescent detection has been developed. Monoclonal antibodies against aldosterone were used for the immune reaction and compared with polyclonal antibodies. Uniform Protein A coated polystyrene tubes were used as solid phase for the monoclonal antibody and second (anti-rabbit) antibody coated tubes for the polyclonal antibody. Horseradish peroxidase was covalently linked to aldosterone as enzyme label. Optimum conditions were established for the generation and measurement of the luminescent reactions using luminol, p-iodophenol as enhancer and hydrogen peroxide. The advantages of this assay are the high sensitivity with a detection limit of 100fg/tube, the prolonged luminescence signal with a simplification of the measurement (simpler detectors, external start pipetting) and the short measure time with the possibility of repeated measurement. The coefficients of variation were 4.2%–7.3% in the concentration range 140–1180 pmol/l. The assay showed a significant correlation (r = 0.91) with the ELISA. The aldosterone concentrations in plasma and saliva of patients with Conn's syndrome were significantly increased, and in patients with Addison's disease were found near the detection limit.     

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.     

A rapid and sensitive 384‐well microtitre format chemiluminescent enzyme immunoassay for 19‐nortestosterone

We developed a competitive chemiluminescent (CL) enzyme immunoassay for rapid, sensitive analysis of 19‐nortestosterone (19‐NT) in bovine urine. Anti‐19‐NT polyclonal antibodies were raised in rabbits using a 19‐NT‐hemisuccinate derivative conjugated with ovalbumin; the derivative was also conjugated with horseradish peroxidase (HRP) as a label. Antibodies were immobilized on 384‐well black polystyrene microtitre plates and HRP‐labelled 19‐NT activity was measured using an efficient chemiluminescent substrate (SuperSignal® ELISA Femto) after 3 min incubation. Emitted light was recorded using a conventional, photomultiplier‐tube‐based microtitre plate reader or a sensitive back‐illuminated, cooled CCD camera. The developed method fulfils all the requirements of precision (intra‐ and inter‐assay CV < 10%) and accuracy (mean recovery 94–112%), with a detection limit of 0.03 ppb (1.1 × 10^?9 mol/L) in a urine matrix. Chemiluminescence enhances detectability of the HRP‐labelled tracer (thus lowering the limit of detection with respect to colorimetry) and reduces analysis time. The 384‐well microtitre plate cuts the sample/reagent volume (20 µL), a five‐fold reduction with respect to the conventional 96‐well microtitre plate. The developed method is suitable for high‐throughput screening of 19‐NT in urine samples, with reduced costs as compared with conventional colorimetric enzyme immunoassays. Copyright © 2003 John Wiley & Sons, Ltd.     

Incorporation of enhanced chemiluminescent reactions into fully automated enzyme immunoassays

Enhanced chemiluminescent quantitation of horseradish peroxidase labels has been incorporated into assays performed on the ES 600 automatic immunoassay analyser. The rapid end-point produced excellent dose-response curves and potential savings in total assay times of up to 40%.     

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.     

The development of Luminomaster™, a fully automated chemiluminescent enzyme immunoassay system

We have developed a fully automated discrete chemiluminescent heterogeneous enzyme immunoassay system called Luminomaster?. The characteristics of this analyser are: 120 test/h throughput, 14 test menu, wide dynamic range, automated sample dilution, automatic retest, communication with a host central processing unit (CPU) and connection with sample transfer system.     

A chemiluminescent metalloimmunoassay based on copper‐enhanced gold nanoparticles for quantification of human growth hormone

A chemiluminescence (CL) immunoassay was developed to determine human growth hormone (hGH) based on copper‐enhanced gold nanoparticles. In this method, gold nanoparticles were deposited on polystyrene wells for adsorption of human growth antibodies as well as catalyst for reducing of copper ions from the copper enhancer solution. The reduction of copper ions was prevented where the gold nanoparticles were covered by the antibody–antigen immunocomplex. The deposited copper on Au nanoparticles was then dissolved in HNO₃ solution and quantified using the CL method. The CL intensity response was logarithmically dependent on the hGH concentrations over the range 0.2–50 ng/mL, with a detection limit (3σ) of 0.036 ng/mL. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate

In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancers was developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP‐C) in the presence of 3‐(10'‐phenothiazinyl) propane‐1‐sulfonate (SPTZ) and 4‐morpholinopyridine (MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33 pg/well, which is lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail can significantly increase the light output of HRP‐catalyzed ECR, which can be translated into a corresponding improvement in sensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR of N‐azoles as secondary enhancer was also presented. Copyright © 2013 John Wiley & Sons, Ltd.     

猪O型口蹄疫病毒抗体化学发光酶联免疫检测方法的建立

表达并纯化猪O型口蹄疫病毒(FMDV)VP1重组蛋白作为检测抗原,建立了一种快速检测猪O型口蹄疫病毒抗体的化学发光酶联免疫(CLEIA)检测方法。建立的VP1-CLEIA方法特异性为100%,板内变异系数在1.10%–6.70%之间,板间变异系数在0.66%–4.80%之间,具有较好的特异性和重复性,且灵敏度高于ELISA方法。通过对山东、辽宁、河北地区采集的250份临床血清的检测表明,该方法与间接ELISA试剂盒的符合率为93.50%,与液相阻断ELISA试剂盒的符合率为94.00%,表明本次建立的VP1-CLEIA检测方法可以用于猪O型FMDV感染或疫苗免疫后抗体水平检测。     

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.     

An enhanced chemiluminescence enzyme immunoassay for serum progesterone

A competitive enhanced luminescent enzyme immunoassay for serum progesterone is described, which is based on a 11 alpha-hydroxyprogesterone 11-hemisuccinyl-horseradish peroxidase conjugate and a black polystyrene microtitre plate sensitised with anti-progesterone IgG. Bound label was determined using a mixture of 4-iodophenol, luminol and peroxide, and the light emitted from the wells of the plate quantitated using a luminescent plate reader. The assay was sensitive (detection limit 0.5 pg), precise (CV 2.7 - 9.0% in the concentration range 4.3-67.7 nM) and showed good correlation (r = 0.99) with a conventional radioimmunoassay.     

Multicenter comparison of rapid lateral flow stool antigen immunoassay and stool antigen enzyme immunoassay for the diagnosis of Helicobacter pylori infection in children

BACKGROUND AND AIM: The stool antigen enzyme immunoassay (EIA) methods are widely used for diagnosing Helicobacter pylori infection. Recently, a novel, rapid stool antigen test, the lateral flow immunoassay (LFI) method, has been developed. The primary purpose of this study was to compare the EIA method with the LFI method for the diagnosis of H. pylori infection in children. MATERIALS AND METHODS: Stool specimens from children being evaluated for H. pylori infection were also examined using the LFI (ImmunoCard STAT! HpSA) and EIA methods (Premier Platinum HpSA). The sensitivity, specificity and accuracy of the test were based on the 13C-labeled urea breath test. RESULTS: One hundred and eighty-two children and adolescents, 3-17 years of age (mean 9.2 years), were studied. In addition, 29 patients who received eradication therapy were re-evaluated 2 or 3 months post-treatment. The 13C-labeled urea breath test was positive in 64 patients (35.2%). The sensitivity, specificity and accuracy of the LFI method were 90.6% (95% CI = 80.7-96.5%), 95.8% (92.1-99.4%), and 94.0% (90.5-97.4%), respectively and for the EIA method, sensitivity, specificity and accuracy were 96.8% (95% CI, 89.0-99.6%) and 99.2% (97.5-100%), and 98.3% (96.5-100%), respectively. There were no significant differences in results among the age groups 3-5, 6-10 and 11-17 years. As for the assessment of H. pylori eradication, the results of the LFI and EIA methods agreed with those of 13C-urea breath test in 27/29 and 29/29 patients, respectively. CONCLUSIONS: The LFI stool antigen method showed a good sensitivity, specificity and accuracy for diagnosing H. pylori infection in children. This novel method may be useful in clinical practice as an office-based test because it is rapid, reliable and easy to perform.     

Ultrasensitive chemiluminescent immunoassay of Salmonella with silver enhancement of nanogold labels

In this paper, silver enhancement of nanogold labels coupled with chemiluminescence detection was developed for ultrasensitive immunoassay of Salmonella based upon antigen–antibody immunoreaction. Polyclonal rabbit anti‐Salmonella sp. antibodies (pAb) were employed to establish the analytical protocol. The pAb coated onto ELISA microwell plates and Au nanoparticles (Au NPs) conjugated pAb capture target Salmonella to form a sandwich‐type complex. Silver then was in situ deposited around the Au NPs core and resulted in the signal amplification. In consequence, silver was dissolved to form Ag⁺ and a sensitive chemiluminescence based on the Ag⁺–K₂S₂O₈–Mn²⁺–luminol system was coupled for further signal amplification. Under the optimized conditions, the chemiluminescent intensity is proportional to target Salmonella over the range of 5–1038 cfu mL^?1 with a detection limit of 5 cfu mL^?1. The relative standard deviation for 11 measurements of about 50–100 cfu/mL target Salmonella is 4.7%. The proposed method was successfully applied to measure Salmonella in food samples and the results are identical to those of the offical standard method of China. These offer us a more powerful tool for ultrasensitive assay of foodborne pathogens. Copyright © 2010 John Wiley & Son, Ltd.     

A versatile enzyme immunoassay for the determination of progestogens in feces and serum

The ability of zoos to monitor the reproductive status of their animals can vastly improve the effectiveness of husbandry/management practices, and noninvasive methods such as fecal steroid analysis are the easiest to apply in a zoo setting. Furthermore, enzyme immunoassay (EIA) is preferred to radioimmunoassay (RIA) as the method of quantifying hormones because EIAs do not involve the use, storage, and disposal of radioactive materials. However, progesterone is excreted in the feces as predominantly unconjugated metabolites (progestogens) and, until recently, antibodies able to cross‐react with a variety of progestogens were used primarily in RIAs. An EIA using a broad‐spectrum progestogen antibody is described and applied to serum and/or fecal samples from female African elephants, black rhinoceros, white rhinoceros, okapi, and hippopotami. The clear progestogen profiles generated in these species suggest that the described EIA would be as versatile as the RIA using the same antibody and could be a practical and economical alternative to RIAs for monitoring gonadal function via progestogen analysis in zoo species. Zoo Biol 20:227–236, 2001. © 2001 Wiley‐Liss, Inc.     

Chemiluminescence enzyme immunoassay (CLEIA) for the determination of chloramphenicol residues in aquatic tissues.

A competitive indirect chemiluminescence enzyme immunoassay (ic-CLEIA) for chloramphenicol (CAP) residues in shrimp has been developed. After optimization (incubation time, concentration of Tween-20, concentration of PBS and pH), the method gave a limit of detection of 0.01 ng/mL and a detection range of 0.03-23.7 ng/mL, with an ED(50) of 0.47 ng/mL. The method has been validated on spiked shrimp samples in terms of precision (intra- and interassay coefficient variations of less than 10% and 15%, respectively) and accuracy (mean recovery 95-123%). The assay performance is better than the ELISA method which is widely used to detect chloramphenicol and indicates that the CLEIA method can be used to test aquatic samples instead of ELISA.     

Characterization of a variant of carcinoembryonic antigen (CEA) using monoclonal and polyclonal anti-CEA antibodies

A variant of the carcinoembryonic antigen (CEA) with lower molecular weight than a CEA reference preparation has been separated from CEA. Using a polyclonal, spleen absorbed anti-CEA antiserum, the variant crossreacts with reference CEA in immunodiffusion. The CEA-activity of the variant has been demonstrated using an enzyme-immunoassay with monoclonal CEA specific antibodies. There is sufficient immunological evidence that this variant is a distinct antigen different from the crossreactive antigens described so far. The reactivity of the polyclonal anti-CEA antiserum with the CEA variant was abolished by absorption against the immobilized variant.     

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.