A sensitive bioluminescent immunoassay for dinitrophenol and trinitrotoluene

A bioluminescent immunoassay for measuring dinitrophenol and trinitrotoluene (TNT) has been developed. The DNP and TNT were covalently linked to firefly luciferase, resulting in a conjugate containing 1 mol of DNP or trinitrophenyl (TNP) per mole of luciferase. The conjugate retained 90% of its original catalytic activity. When the conjugate was incubated with immobillzed anti-TNT or anti-DNP and varying concentrations of free TNT or DNP-leucine, the amount of conjugate bound was inversely proportional to the concentration of the free compound. Using this procedure it is possible to detect 2.5 pmol of DNP-leucine and 1.0 pmol of TNT. If the TNP or DNP is linked to glucose-6-phosphate dehydrogenase instead of luciferase, much lower quantities of antigen can be detected. This is due to the fact that this enzyme has a large turnover number so that amplification is possible. The NADH produced is measured using immobilized bacterial NADH:FMN oxidoreductase and luciferase. With this procedure, 10 amol (10^?17 mol) of antigen can be measured. These procedures should be suitable for measuring any antigen.     

Electrochemical luminescence-based homogeneous immunoassay

Aromatic hydrocarbon such as pyrene capable of generating electrochemical luminescence was employed as a label of immunoassay. Pyrene labeled antigen generated luminescence upon electrolytic reduction, while the luminescence decreased remarkably in the presence of antibody. The labeled antigen (constant) and free antigen were competitively reacted to the constant amount of antibody. The luminescence was correlated to the antigen concentration as little as 10(-6)M antigen. The proposed method is a very unique immunochemical technique which requires no BF separation.     

Giant unilamellar vesicles containing Rhodamine 6G as a marker for immunoassay of bovine serum albumin and lipocalin-2

Functionalized giant unilamellar vesicles (GUVs) containing a fluorescence dye Rhodamine 6G is proposed as a marker in sandwich-type immunoassay for bovine serum albumin (BSA) and lipocalin-2 (LCN2). The GUVs were prepared by the electroformation method and functionalized with anti-BSA antibody and anti-LCN2 antibody, respectively. The purification of antibody-modified GUVs was achieved by conventional centrifugation and a washing step in a flow system. To antigen on an antibody slip, antibody-modified GUVs were added as a marker and incubated. After wash-out of excess reagents and lysis of the bound GUVs with Triton X-100, the fluorescence image was captured. The fluorometric immunoassays for BSA and LCN2 exhibited lower detection limits of 4 and 80 fg ml⁻¹, respectively.     

An immunoassay method for quantitative detection of proteins using single antibodies

A new immunoassay method called specific analyte labeling and recapture assay (SALRA) to quantitatively measure protein abundance was developed, and the assay conditions were optimized. The key features of this method include labeling the antigen bound to the capture antibody, eluting the labeled antigen, and recapturing it by the same capture antibody on the detection plate. The reporter molecules on the labeled antigen provide a convenient and reliable means for signal detection. We demonstrated that the dose-response curve of SALRA was comparable to that of sandwich enzyme-linked immunosorbent assay (ELISA) and better than that of the antigen direct labeling method. In addition, multiple proteins can be measured simultaneously by SALRA. Using the SALRA method, the detection limit for most of the cytokines tested was approximately 0.01 ng/ml. Further SALRA tests on interleukin 6 (IL-6) showed the linear dose-response was 3.3 to 0.01 ng/ml, the accuracy of the test was 71 to 91%, the intraassay variation was 3.6 to 7.4%, and the interassay variation was 3.8 to 10.0%. The applications of SALRA include quantitatively measuring proteins for which there are no ELISA tools available and providing a new platform for protein microarrays.     

An enzyme immunoassay for plasma betamethasone

A sensitive enzyme immunoassay for plasma betamethasone was developed using betamethasone-3-(O-carboxymethyl)oxime-β-D-galactosidase conjugate as a labelled antigen and 4-methylumbelliferyl-β-D-galactoside as a fluorescence substrate. The performances of the enzyme immunoassay were compared with that of a radioimmunoassay using ³H-betamethasone and the same antiserum. The minimal detectable level for the enzyme immunoassay was 0.15 pg/tube or 0.15 ng/ml of plasma, which was remarkably more sensitive than the radioimmunoassay level of 10 pg/tube or 2 ng/ml of plasma. The specificity was sufficient, in particular, the cross reactivity of cortisol was 0.008%. However, the precision of the enzyme immunoassay was inferior to that of the radioimmunoassay.     

Enzyme-linked immunoassay (ELISA) for connective tissue components

Enzyme-linked immunoadsorbant assays have been developed for types I, II, III, and IV collagen and for laminin and fibronectin. These assays offer a specific, sensitive, and convenient method for the measurement of various connective tissue components either separately or simultaneously. Plastic microtiter wells are coated with purified antigen, then antibodies to the antigen are allowed to bind to the insolubilized antigen in each well. The amount of bound antibody is determined by incubation with a second antibody which is covalently linked to alkaline phosphatase or horseradish peroxidase. The amount of bound enzyme is assayed after adding an appropriate substrate. The level of protein in a sample is estimated from its ability to inhibit the binding of the first antibody to the antigen-coated well. Specificity of the assay depends on the purity of the adsorbed antigen and allows for highly specific tests. Under optimum conditions the sensitivity of the assay is determined by the K_B of the antibody and allows 10–20 ng of a specific type of collagen or laminin and 1 ng fibronectin to be detected.     

Antigen Binding Cells in Embryonic Chicken Bursa and Thymus

THE autoradiographic detection of the binding of ¹²⁵I-labelled antigen to a proportion of normal lymphocytes is now well established and characterized^1–4. Such cells are thought to possess patches of immunoglobulin surface receptors which combine specifically with an appropriate antigen⁵. Evidence for the specificity of the antigen binding includes the capacity of specific anti-L and anti-µ chain antisera to block the receptor sites⁶ and the demonstration that at least the more heavily labelled antigen binding lymphocytes in mouse spleen are immunologically competent⁷.     

Development of electron spin resonance radical immunoassay for measurement of airborne orchard grass (<Emphasis Type="Italic">Dactylis glomerata</Emphasis>) pollen antigens

We have developed a highly sensitive method for the measurement of airborne orchard grass (Dactylis glomerata: Dac g) pollen antigens using an electron spin resonance (ESR) radical immunoassay. In this immunoassay, the lowest detectable level of Dac g antigen in a sample is 0.1 arbitrary unit; the amount of Dac g antigen in single pollen grains was found to be as 1.84 units. Thus, Dac g antigens can be detected in amounts of 1/20th of that contained in the grain. This immunoassay enables early detection of grass pollen antigens. Such information may be useful for patients with grass pollinosis, especially for those who show symptoms when only low levels of the pollen antigens are present in air. In this study, minor amounts of Dac g antigen (cross-reactive antigens) were detected in late March, after which the levels gradually increased. The levels were detected to be 10 units/m³ until the middle of May and then increased after blooming of orchard grass. High levels were maintained until the middle of June. Some patients who suffer from grass pollinosis show symptoms in late April and early May, when the airborne Dac g antigen levels were found to be 5–10 units/m³.     

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.     

Elimination of I131-labelled heterologous antigen from the blood of chickens after total body X-irradiation

A study was made on the effect of ionizing radiation upon the rate of elimination of I¹³¹. labelled human serum albumin (HSA I¹³¹) from the blood and upon antibody formation in chickens irradited with 1,2 00R(i.e.with LD₅₀) and injected with antigen 30 min, 6 days or 14 days after irradiation. The elimination curve from unirradiated control birds followed the typical three-phase pattern. The effect of irradiation was most marked with chickens injected with antigen 6 days after irradiation, resulting in an extension of the second phase with practically no third phase at all. Exposure to irradiation 30 min prior to antigen administration resulted in an extension of the second phase by 2 days as compared to the controls, with the onset of the third phase occurring on day 7. Irradiation 14 days prior to antigen administration resulted in an extension of the second phase by 1 day as compared to the controls, with the onset of the third phase occurring on day 6. Elimination of HSA I¹³¹ in the second phase was more rapid than that of I¹³¹-labelled chicken serum albumin (CSA I¹³¹) no matter whether the chickens were irradiated or not. This suggests that the capacity of specific antigen uptake is not affected by irradiation. Antigen elimination curves from control irradiated groups given CSA I¹³¹ followed the same pattern as that found in control unirradiated birds injected with homologous antigen.     

Comparison of stool antigen immunoassay and serology for screening for Helicobacter pylori infection in intellectually disabled children

Diagnosis of active Helicobacter pylori infection in intellectually disabled (ID) children is problematic because they are unable to cooperate with performance of invasive tests. In this study, the non‐invasive methods of measuring serum IgG antibody concentrations and performing stool antigen tests were used to screen for H. pylori infection in ID children. Eighty‐seven children with intellectual disabilities were studied. The amount of serum IgG antibody against H. pylori was measured by the ELISA method. Stool samples were examined using an amplified IDEIA HpStAR kit. To assess categorical variables, X², Fisher's exact and Kappa tests were used. The stool antigen tests showed that 93.1% of the children had H. pylori antigen and the serology test that 85.1% of children were positive for H. pylori IgG antibodies. Agreement between results of H. pylori stool antigen (HpSA) testing and IgG antibody serology was 82.8%; however, according to the kappa measure of agreement this agreement is not statistically significant (value, 0.128; P = 0.19). Discordant results were observed for 15 children (17.2%): 11 (12.6%) who were positive on HpSA test but negative by serology and 4 (4.6%) who were IgG seropositive but had negative HpSA tests. This study showed a notably higher rate of H. pylori infection in ID children than has been reported by others for non‐ID children from the same geographical area. The HpSA test is a valid method for primary screening for H. pylori infection in ID children; it detects the specific antigens shed during active infections and has less cross‐reactivity than serological tests that detect antibodies. HpSA is a sensitive non‐invasive method for detecting infection in ID children and may serve as an accurate alternative to serology.     

A label-free visual immunoassay on solid support with silver nanoparticles as plasmon resonance scattering indicator

By taking silver nanoparticles (Ag-NPs) as plasmon resonance scattering (PRS) indicator considering that Ag-NPs have strong plasmon resonance light scattering signals corresponding to their plasmon resonance absorption (PRA), we propose a label-free visual immunoassay on the solid support of glass slides. Our investigations showed that Ag-NPs could be adsorbed on the surface of glass slides where immunoreactions between a previously immobilized antigen and its antibody have occurred if the glass slides were immersed in an Ag-NP suspension whose pH value has been carefully adjusted. The optimal pH of the Ag-NP suspension depends on the nature of previously immobilized antigen and its antibody. It was found that the adsorption of negative-charged Ag-NPs on the surface of glass slides depends only on the content of antibody under optimal conditions. With a common spectrofluorometer to measure the PRS signals of the Ag-NPs adsorbed on the surface, we could detect antibody in the range of 10 to 160 ng ml⁻¹. If a white light-emitting diode (LED) torch is employed to illuminate the glass slides, we can make visual detection of the antibody by the naked eye.     

Autoradiographic immunoassay (ARIA): a rapid technique for the semiquantitative mass screening of haptens.

A semi-quantitative radioimmunoassay was developed which allows the mass screening of more than 10⁴ samples per day per person. The assay is performed in multiwell test plates and employs charcoal separation of antibody-bound and free antigen fractions. The radioactivity of the charcoal phase is transformed into fluorescent light which exposes a sheet of X-ray film. By this autoradiographic immunoassay (ARIA) technique, both visual and quantitative evaluation of the tests can be accomplished. The applicability of this new method is demonstrated for two haptens, digoxin and nicotine. Correlation coefficients between charcoal radioactivity and film density were found to exceed 0.95. Measuring ranges extend from 0.2 to 50 ng (digoxin) and from 5 to 500 ng (nicotine). Interassay variabilities are about twice as high for ARIA as for radioimmunoassays. By proper choice of the sample dilution used for assays, the screening for high or low antigen concentrations is possible.     

Bioelectrochemical immunoassay for human chorionic gonadotrophin in serum using an electrode-immobilised capture antibody

An amperometric technique for the quantification of an enzyme immunoassay which utilises a capture antibody covalently attached to a carbon electrode is described. The electrode is used both to separate the assay and to monitor the activity of the bound enzyme label. A ‘two-site’ immunometric assay with monoclonal antibodies directed against human chorionic gonadotrophin (HCG) was used as the model system. The activity of the enzyme bound to the electrode is determined electrochemically by the use of an electron transfer mediator (dimethylaminomethyl ferrocene) permitting rapid quantification of the analyte without the need for a separate incubation step to measure enzyme activity. The sensitivity of the assay is 9mIU HCG ml⁻¹ in serum (1st International Reference Preparation). The correlation between the amperometric measurement of serum HCG and data for an immunoradiometric assay was r = 0·988. The assay is rapid requiring a total assay time of 20 min per sample, which includes 15 min for antibody—antigen binding.     

Quantitative determination of factor VIII antigen with an enzyme immunoassay

We describe an enzyme-immunoassay for the determination of factor VIII antigen. After representation of the isolation of proteins the enzyme-immunoassay is presented. The principle of the method is the following: Test plasma is mixed with rabbit antibody in excess and incubated at 37 degrees C. The incubation mixture is added to polystyrene tubes, which are coated with human factor VIII. The rabbit antibody is available to adhere to factor VIII coating the tube and can be detected with an enzyme-labeled antibody to rabbit IgG. This method is sensitive to 7.8 . 10(-3) U/ml factor VIII antigen; the variation coefficient is 10.9%.     

Sandwich microgravimetric immunoassay: sensitive and specific detection of low molecular weight analytes using piezoelectric quartz crystal

A multi-channel sandwich microgravimetric immunoassay (sMIA), using the quartz crystal microbalance (QCM) principle, has been developed to quantify low molecular weight substances in standard solutions. An antigen is sandwiched between two antigen-specific antibodies: the first antibody is coated on the quartz crystal surface and the second antibody is used for the detection of analyte. The concentration of low molecular weight antigen (insulin was used in this study, M _r6000 Da) was correlated with the shift of resonant frequency of QCM system before and after second antibody binding to insulin. The developed assay is highly specific showing low cross-reactivity, and is sensitive to approx. 1 ng insulin ml^–1 with a linear response for insulin from 10 g ml^–1 to 10 ng ml^–1 in standard solutions. The technique may also be applied for the detection of other small biomolecules.     

Localization of the 7H6 Antigen at Tight Junctions Correlates with the Paracellular Barrier Function of MDCK Cells

An important function of the tight junction is to act as a selective barrier to ions and small molecules, although no molecule responsible for the barrier function has been identified. Here we report evidence that the localization of the 7H6 tight junction-associated antigen identified in our laboratory at tight junctions correlates with the barrier function of MDCK cells. MDCK cells in a confluent monolayer possessed a polarized morphology, having an apical plasma membrane and a basolateral membrane, which is separated from the former by tight junctions. MDCK cells expressed both ZO-1 and 7H6 antigen at tight junctions, which maintain a tight barrier as determined by resistance to lanthanum permeation and high transepithelial electrical resistance (TER, 1500 ohm-cm²). The 7H6 antigen disappeared as tight junctions became permeable to lanthanum with a decrease in TER (below 100 ohm-cm²) due to treatment with metabolic inhibitors (10 μm antimycin A and 10 mM 2-deoxyglucose) for 30 min, while leaving ZO-1 at the cell border. The 7H6 antigen appeared at tight junctions again as TER recovered to a high level (1500 ohm-cm²) within 3 h after withdrawal of metabolic inhibitors. In addition, we found that 7H6 antigen is a phosphorylated protein and that phosphorylation is closely related to the localization of 7H6 antigen in the area of tight junctions.     

Electrochemical enzyme immunoassay for detection of toxic substances.

Sensors that provide reliable, rapid measurement of toxic substances are needed to solve significant human health and safety problems. We developed a new biosensor design that combines the advantages of immunoassay with electrochemical response. We established that this enzyme-linked immunosensor measures toxic substances in biological samples. The biosensor consists of two major elements: (1) an electrical conducting layer having immobilized enzyme, polyclonal or monoclonal antibodies, and other necessary reagents, and (2) the electronic components used in the signal readout. The result is an amperometric immunoassay based on coupling the immunochemical reaction to the enzyme electrode response by using a soluble, electrochemically active mediator. The specific question addressed was: Does the system's immunochemical detection reliably respond at sufficiently low analyte concentrations? We present our results in these areas: (1) enzyme immobilization on colloidal gold; (2) colloidal gold-enzyme deposition on the electrode surface; (3) mediator-antigen conjugate synthesis; (4) antibody incorporation at the electrode surface; (5) bioelectrode characterization and optimization; and (6) immunosensor demonstration to detect antigen. Sensors that employ immunochemical detection will have broad applicability to detect/diagnose toxic substances in biological samples such as blood and urine and in environmental samples such as wastewater and drinking water.     

Characierization of monoclonal antibodies specific for isopentenyl adenosine derivatives occurring in transfer RNA

A family of isopentenyl adenosine derivatives are naturally occurring components of transfer RNA and are involved in several different functional roles in the cell. To facilitate the study of the biochemistry of these modified nucleosides we have raised monoclonal antibodies to N⁶-(Δ²-isopentenyl)adenosine and N⁶-(4-hydroxy-3-methyl-but-2-enyl)adenosine. The antibodies show considerable specificity and three characteristic types are distinguishable. The first type have the hydroxylated derivative as the preferred antigen, the second type have isopentenyl adenosine as the preferred antigen and a third type show a specificity for all isopentenyl-containing derivatives.