Monoclonal antibody-based enzyme immunoassay for mercury(II) determination

A monoclonal antibody (K3C6) was developed against Hg(II) and applied in different enzyme immunoassay (EIA) formats to determine the test system with the highest sensitivity. A detection limit of 1. 0 microg/L Hg(II) could be achieved with a competitive format in contrast to a detection limit of 2.1 microg/L Hg(II) with a noncompetitive EIA. A competitive displacement EIA yielded the best detection limit of 0.4 microg/L Hg(II) and was well suited to measuring real samples. For this purpose different water samples were diluted at least 1:10 to avoid matrix effects and subsequently spiked with 1 microg/L HgCl(2). Recovery of the spiked samples was between 80 and 120%.     

Simultaneous competitive enzyme immunoassay for human chorionic gonadotropin.

A simultaneous competitive enzyme immunoassay (SICEIA) for hCG was developed using beta-D-galactosidase (beta-Gal) as a labelled enzyme and anti-hCG antibody coated sheep red blood cells (SRBC) as a solid phase. In this report, a new coupling agent, MCAE, was used to couple beta-Gal with hCG. The sensitivity was improved to the degree of 2.5 mIU/ml, equal to that of RIA. The present procedure was safer and rapider than RIA. The value of hCG in urine by our procedure had good correlation with that by RIA.     

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.     

A rapid electrochemical method for assimilation test of microorganisms

Summary A microbial electrode consisting of the immobilized microorganisms to be tested and an oxygen electrode was used to study the assimilation characteristics of microorganisms. When a sample solution containing a substrate was injected into the microbial sensor system, the current of the sensor markedly decreased with time if the microorganisms assimilated the substrate. On the other hand, no current decrease was observed if the microorganisms could not assimilate the substrate. Assimilation characteristics of various microorganisms such as molds, yeasts, bacteria, actinomycetes and activated sludges were tested with various substrates. The time required for a test was 30 min per substrate by the pulse method (sample injection period, 5 min). Good correlations were obtained between this electrochemical method and the conventional growth test. The fundamental differences between the two methods and the application of the electrochemical method are discussed.     

Capillary electrophoretic enzyme immunoassay with electrochemical detection for thyroxine

A capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) has been developed. In this method, antigen (Ag) competes with horseradish peroxidase (HRP)-labeled antigen (HRP-Ag) for a limited number of antibody (Ab) binding sites. The free HRP-Ag and the bound HRP-Ag-Ab complex are separated by capillary electrophoresis in a separation capillary. Then they catalyze the oxidation of their enzyme substrate 3,3',5,5'-tetramethylbenzide (TMB (reduced form)) with H(2)O(2) in a reaction capillary, which follows the separation capillary. The reaction product (TMB (oxidized form)) is amperometrically determined using a carbon fiber microdisk bundle electrode at the outlet of the reaction capillary. Due to the amplification of the enzyme, the concentration of TMB(Ox) is much higher than those of free HRP-Ag and the bound HRP-Ag-Ab complex. Therefore, the limit of detection (LOD) of CE-EIA-ED is very low. The method has been used to determine thyroxine in human serum. A concentration of LOD of 3.8 x 10(-9)mol/L, which corresponds to a mass LOD of 23.2 amol, was achieved.     

Development of a competitive enzyme immunoassay for factor VIII-related antigen

An enzyme immunoassay system basing on a competitive method has been developed to measure factor VIII related antigen (F. VIII R:Ag). A sufficient discrimination at low F. VIII R:Ag concentrations was gained. This method appears to be sensitive to 7,8 X 10(-3) U/ml F. VIII R:Ag showing an intraassay coefficient of variation (CV) of 0,11. In comparison to the commonly used Laurell electroimmunodiffusion assay for factor VIII significant less antisera per sample for the enzyme immunoassay technique is necessary.     

Direct enzyme immunoassay in microtitration plate and test strip format for the detection of saxitoxin in shellfish

Saxitoxin was coupled to horseradish peroxidase via a novel adaptation of the periodate reaction. Based on polyclonal antibodies against saxitoxin, this conjugate was used for the development of two formats of direct enzyme immunoassay (EIA)–a microtitration enzyme-linked immunosorbent assay (ELISA) and a test strip EIA. The detection of saxitoxin without instrumentation by visual evaluation of the test strip EIA is described. The detection limits for saxitoxin were 7 pg/ml (0·35 pg/assay) in the ELISA and 200 pg/ml in the test strip EIA using visual evaluation. Employing a simple procedure of sample preparation, both ELISA and test strip EIA were applied to the analysis of shellfish. The detection limits for saxitoxin in shellfish tissue of the ELISA and the test strip assay were 3 and 4 ng/g, respectively.     

A competitive microtitre plate enzyme immunoassay for plasma aldosterone using a monoclonal antibody

A competitive microtitre plate enzyme immunoassay for plasma aldosterone was developed using an immobilised aldosterone-bovine serum albumin conjugate and a monoclonal anti-aldosterone preparation, followed by the use of enzyme-labelled second antibodies to determine the degree of competition. The quantity of immobilised aldosterone-protein conjugate was adjusted to give optimum assay sensitivity with respect to the antibodies used. The lower limit of detection of aldosterone (55 fmol) was much less than that of an ELISA for aldosterone, using identical reagents but with an excess of immobilised aldosterone-protein conjugate, and up to 1400 fmol could be determined. Aldosterone levels in small amounts of male and female plasma could be assayed with good reproducibility.     

A rapid bioluminescent enzyme immunoassay (BLEIA) for the detection of Shiga toxin types 1 and 2.

In recent years, Escherichia coli O157: H7 has emerged as a global public health concern. Among the more important virulence characteristics of this strain is its ability to produce one or more Shiga toxins (Stx). Traditional culture-based methods for assay of enteric toxins in foods and clinical samples are relatively slow and results can be ambiguous. In this work, we established a toxin-detection system based on bioluminescent enzyme immunoassay (BLEIA) using a simple and inexpensive device. The system could detect both Shiga toxin types 1 and 2 individually within 150 min with a detection limit for each toxin at 5 pg/ml. In our study of previously characterized Shigatoxigenic and all non-Shigatoxigenic E. coli and other bacterial species, we found all Shigatoxigenic strains to be positive and non-Shigatoxigenic E. coli and other bacterial species to be negative. This assay was also used to detect Stxs in milk and supernatant fluids from minced chicken and beef. For clinical stool samples we noted a tendency for the system to give unexpectedly high background level. Our results suggest the feasibility of using BLEIA methodology for the simple, rapid and sensitive detection of toxins from culture supernatant, various foods and clinical samples.     

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.     

A simple and rapid immunochromatographic test strip for detection of white spot syndrome virus (WSSV) of shrimp

A simple strip-test kit for white spot syndrome virus (WSSV) detection was developed using monoclonal antibody W29 (against the VP28 structural protein) conjugated with colloidal gold as the detector antibody. A rabbit anti-recombinant VP28F118 (rVP28) protein antibody in combination with a W28 monoclonal antibody was used as the capture complex at the test line (T), and goat anti-mouse IgG antibody (GAM) was used as the capture antibody at the control line (C). For evidence, the ready-to-use strip was kept in a plastic case and stored in a desiccated plastic bag. A sample volume of 100 microl gill homogenate in application buffer was applied to the sample chamber at one end of the strip and allowed to flow by chromatography through the nitrocellulose membrane to the other end. In test samples containing WSSV, the virus bound to the monoclonal antibody conjugated with colloidal gold and the resulting complex was captured by the antibodies at T to give a reddish-purple band. Any unbound monoclonal antibody conjugated with colloidal gold moved across T to be captured by the GAM and formed a band at C. In samples without WSSV or with WSSV below the limit of detection of the kit, only the band at C was seen. This method was 4 times less sensitive than dot blotting, and about 2 000 000 times less sensitive than 1-step PCR. Nonetheless, it could be used to screen individual shrimp or pooled shrimp samples to confirm high levels of WSSV infection or WSSV disease outbreaks. The beneficial features of this kit are that simple, convenient and quick results can be obtained without the requirement of sophisticated tools or special skills.     

A convenient immunochromatographic test strip for rapid detection of Scylla serrata reovirus

<正>Dear Editor,Scylla serrata reovirus(SsRV),first discovered in China in 2004,is now known to be widespread in the mud crab(Scylla serrata)farming pond of China.(Chen et al.,2008).Since there are no effective treatments currently available for this viral pathogen,early diagnosis is the     

A rapid microassay for detecting hepatitis B surface antigen by dot enzyme immunoassay

This study describes a dot enzyme immunoassay (Dot-EIA) for detecting hepatitis B surface antigen (HBsAg). The results demonstrated that the detection level of this assay for HBsAg was 1.5 ng/ml; no false-positive or -negative readings were observed. Also, this Dot-EIA had some advantages over standard EIA: (1) antiserum could be directly and immediately bound on nitrocellulose paper set into microfiltration apparatus, (2) the paper could be easily washed under reduced pressure using a water aspirator, (3) all assay steps could be performed at room temperature within 2 h, (4) the well-defined brown spots could be evaluated by both visual observation and densitometric reading. The Dot-EIA reported here may be useful for rapid diagnosis and screening of HBsAg in serum.     

A rapid microassay for detecting hepatitis B surface antigen by dot enzyme immunoassay

Abstract This study describes a dot enzyme immunoassay (Dot-EIA) for detecting hepatitis B surface antigen (HBsAg). The results demonstrated that the detection level of this assay for HBsAg was 1.5 ng/ml; no false-positive or -negative readings were observed. Also, this Dot-EIA had some advantages over standard EIA: (1) antiserum could be directly and immediately bound on nitrocellulose paper set into microfiltration apparatus, (2) the paper could be easily washed under reduced pressure using a water aspirator, (3) all assay steps could be performed at room temperature within 2 h, (4) the well-defined brown spots could be evaluated by both visual observation and densitometric reading. The Dot-EIA reported here may be useful for rapid diagnosis and screening of HBsAg in serum.     

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.     

Determination of an antibody-antigen binding constant by enzyme immunoassay and a theory for analysis of competitive binding of two ligands to heterogeneous receptor

A method for determining antigen-antibody binding constants by using enzyme-labeled antigens has been developed. In the measurement, enzyme-labeled and unlabeled antigens (Ag* and Ag) were allowed to compete in binding to the antibody (Ab) under conditions where Ag* much less than Ab much less than Ag. The data were analyzed according to a new theory developed for the analysis of competitive binding of two ligands to a heterogeneous receptor. The theory indicates that the binding degree of a labeled ligand measured at various concentrations of the receptor can be used to prepare a standard curve relating the binding degree of the labeled ligand and the average of the concentrations of the free receptor components which are in binding equilibrium with another unlabeled ligand. For homogeneous receptors, the method gives usual binding constants for the unlabeled ligand, but for heterogeneous receptors, it gives a new type of average binding constant for the unlabeled ligand in which the contribution of each receptor component is amplified in proportion to its affinity against the labeled ligand. This average binding constant was named the "affinity-average binding constant." A rabbit anti-blasticidin S (BLS) antiserum analyzed by the present method using beta-galactosidase-labeled BLS as the labeled ligand was found to be fairly homogeneous with respect to the affinity and to have a binding constant of 1.48 +/- 0.24 (S.D.) X 10(8) M-1 for unlabeled BLS.     

A sensitive two-site enzyme immunoassay for human epidermal growth factor (urogastrone)

A sensitive enzyme immunoassay (EIA) was developed for human epidermal growth factor (hEGF) or urogastrone, which was isolated from human urine. Our EIA system is based on the sandwiching of an antigen between anti-hEGF IgG coated on a polystyrene tube and anti-hEGF antibody Fab'-linked beta-D-galactosidase (beta-D-galactosidase, EC 3.2.1.23). This method has the advantages that the procedures are simple and rapid and that the antibody Fab'-beta-D-galactosidase complex is more stable than radioisotope-labeled IgG. Purified hEGF is detectable at as low as 100 pg/ml, which is very sensitive compared to the radioimmuno-assays or radioreceptor assays already reported. Using this new EIA system, hEGF levels in human urine were examined. The values for normal males and females were 48.4 and 83.5 ng/mg creatinine, respectively, which shows that females excrete 1.7 times more hEGF than males.     

A quantitative enzyme immunoassay for miraculin in Richadella dulcifica (miracle fruit)

We have developed an enzyme-linked immunoabsorbent assay formiraculin, a glycoprotein which is capable of modifying sourtaste into sweet. Antiserum against purified miraculin was raisedin rabbits and the anti-miraculin in IgG was purified usinga Protein A-Cellulofine column and then conjugated with horseradishperoxidase. The established enzyme immunoassay method was ableto accurately quantitate pg amounts of miraculin in crude extractsof miracle fruit. The amounts of miraculin in miracle fruitincreased dramatically seven weeks after pollination and ateight weeks reached 102 µg/mg protein in the fruit.