University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Journal of Immunological Methods

  • Publisher: Elsevier
  • FAD Code: JIME
  • CODEN: JIMMBG
  • ISSN: 0022-1759
  • Abbreviation: J. Immunol. Methods
  • DOI Prefix: 10.1016/j.jim,10.1016/0022-1759
  • Language: English
  • Comments: Fulltext from 1971 V1

Citations 13

"Flow-injection Chemiluminescent Immunoassay For α-fetoprotein Based On Epoxysilane Modified Glass Microbeads"
J. Immunol. Methods 2006 Volume 312, Issue 1-2 Pages 61-67
Zhifeng Fu, Chen Hao, Xiaoqing Fei and Huangxian Ju

Abstract: A flow-injection chemiluminescent immunoassay system based on a novel transparent immunoaffinity reactor is proposed for the quantitation of α-fetoprotein. The reactor prepared with α-fetoprotein immobilized epoxysilane modified glass microbeads was used as an immunosensor for chemiluminescent detection. With a non-competitive immunoassay format, the proposed immunosensor system is a low cost, flexible and rapid assay for α-fetoprotein. After an off-line incubation of the analyte α-fetoprotein with horseradish peroxidase-labeled α-fetoprotein antibody as enzyme tracer, the mixture was injected into the reactor, which led to trapping of the free enzyme tracer by the reactor. The trapped enzyme tracer was detected by the p-iodophenol-luminol-H2O2 chemiluminescence system. Under optimal conditions, the decrease in chemiluminescence intensity was proportional to the α-fetoprotein concentration in the range of 5.0-100 ng/ml with a detection limit of 2.7 ng/ml at a signal/noise ratio of 3. The immunosensor system showed an acceptable reproducibility and stability. Clinical serum samples were assayed with this method and the results were in acceptable agreement with those obtained from immunoradiometric assay.

"Flow Injection Immunoassay For Carcinoembryonic Antigen Combined With Time-resolved Fluorometric Detection"
J. Immunol. Methods 2005 Volume 305, Issue 2 Pages 120-127
Feng Yan, Jiannong Zhou, Jiehua Lin, Huangxian Ju and Xiaoya Hu

Abstract: Time-resolved fluorescence has been developed for immunoassay to obtain higher sensitivity than usual immunoassays. In this paper, a simple, sensitive and specific method was developed for immunoassay of serum carcinoembryonic antigen (CEA) by combining time-resolved fluoroimmunoassay (TRFIA) and flow injection analysis. Based on a sandwich immunoassay format, a monoclonal antibody immobilized immunoaffinity column inserted in a flow system was used for immunoreactions. The cleaved solution was collected after the reaction between the immunocomplex in the immunoaffinity column and the enhancement solution that was used to cleave the Eu-labels from the immunocomplex, and then detected by time-resolved fluorescence. Serum CEA could be detected in the linear range from 2.5 to 100 ng/ml with a correlation coefficient of 0.997 and the detection limit of 1.0 ng/ml. Twenty human serum samples detected by this method were in good agreement with the results obtained by the electrochemiluminescence immunoassay. This method could be further developed for fast practical clinical detection of serum CEA levels. © 2005 Elsevier B.V. All rights reserved.

"Chemiluminescent Immunosensor For CA19-9 Based On Antigen Immobilization On A Cross-linked Chitosan Membrane"
J. Immunol. Methods 2004 Volume 291, Issue 1-2 Pages 165-174
Jiehua Lin, Feng Yan, Xiaoya Hu and Huangxian Ju

Abstract: A novel chemiluminescent immunosensor for carbohydrate antigen 19-9 (CA19-9) based on the immobilization of CA19-9 on the cross-linked chitosan membrane was developed. The different membranes were characterized by atomic force microscopy (AFM) and infrared spectrum, respectively. Based on a noncompetitive immunoassay format, this proposed chemiluminescent immunosensor enabled a low-cost, flexible and rapid determination for CA19-9 in combination with flow injection analysis (FIA). After an off-line incubation of the analyte CA19-9 with horseradish peroxidase (HRP)-labeled anti-CA19-9, the mixture was injected into the immunosensor, which led to the trapping of free HRP-labeled anti-CA19-9 by the immobilized antigen in the immunosensor. The trapped HRP-labeled antibody was detected by chemiluminescence due to its catalytic activity following the reaction of luminol and H2O2. Under optimal conditions, the decreased chemiluminescent signal of the immunosensor was proportional to the CA19-9 concentration in the range of 2.0-25 U/ml with a detection limit of 1.0 U/ml. The immunosensor showed an acceptable accuracy and good reproducibility. The results of 20 human serum samples detected by this method were in acceptable agreement with those obtained by immunoradiometric assay. The proposed immunosensor provided a new promising tool for practical clinical detection of the serum CA19-9 level.

"Online Flow Displacement Immunoassay For Fatty Acid-binding Protein"
J. Immunol. Methods 1998 Volume 217, Issue 1-2 Pages 103-111
Wilhelmina A. Kapteina, b, e, Jakob Korfb, Shong Chenga, Michael Yangd, Jan F. C. Glatzc and Reinhard Renneberga,*

Abstract: In standard displacement flow immunoassays the analyte in the sample creates an active dissociation of labelled antigens (or antigen homologues) from an antigen binding site of an immobilized antibody, after which the labelled substance is measured downstream. Such systems have been described for molecules up to 1 kDa. In this study, we demonstrate displacement in a flow system for the detection of a small protein, cytoplasmic heart-type fatty acid-binding protein (15 kDa), a plasma marker for myocardial injury. The displacement system uses an inverse set-up: enzyme labelled monoclonal antibodies are associated to immobilized antigen, and are displaced by analyte in the sample. The system permits detection of both physiological (2-12 µg l-1) and pathological concentrations (12-2000 µg l-1) of fatty acid-binding protein in an on-line flow system.
Protein Immunoassay

"Design Of Non-competitive Flow Injection Enzyme Immunoassays For Determination Of Haptens: Application To Digoxigenin"
J. Immunol. Methods 1997 Volume 208, Issue 2 Pages 159-168
Ulf Lövgren*, Karin Kronkvist, Birthe Bäckström, Lars-Erik Edholm and Gillis Johansson

Abstract: A theoretical model immunoradiometric assay (IRMA) was adapted to provide a non-competitive flow injection enzyme immunoassay for haptens and used as a guide in studying the effects of different parameters on the sensitivity, precision and dynamic range of the assay. As well as the concentration of the antibody-enzyme conjugate, the affinity constant, the run time through the affinity column, the homogeneity of the antibody population and purity of the antibody-enzyme conjugate were all shown to be important parameters in the optimization of the assay. The findings were used to design an optimized enzyme flow injection immunoassay for the model compound digoxigenin in standard solutions. A linear calibration curve was established in the range 0.38- 7.7 fmol of digoxigenin, resulting in a precision of 14.8% RSD at 1 fmol and 3.7% RSD at 7.7 fmol. Antibody fragments reacting with digoxigenin and labelled with alkaline phosphatase, (Fab-AP) were used to convert 4-methyl umbelliferyl phosphate to a fluorescent product measured downstream. The sample throughput was 15 h-1 and over 60 injections were possible before regenerating the affinity column.
Digoxigenin Haptens Radioimmunoassay Optimization

"Liposomes And Immunoassays"
J. Immunol. Methods 1997 Volume 204, Issue 2 Pages 105-133
H. A. H. Rongen*, A. Bult and W. P. van Bennekom

Abstract: Various aspects of the application of liposomes as a label in immunoassays are reviewed. Methods for the preparation of liposomes, from the basic film method to the more advanced dehydration-rehydration method, are discussed. Furthermore, the markers used in liposome labels, as well as the methods to conjugate liposomes to antigens or antibodies, are summarized. Liposome immunoassays are applied as homogeneous or heterogeneous assays. Homogeneous assays often rely on the lytic activity of complement on antibody-associated liposomes. Another group of homogeneous assays utilizes the inhibitory action of antibodies on the activity of conjugates of mellitin (a bee venom protein) with a hapten. Free mellitin conjugates are able to lyse liposomes effectively. Heterogeneous liposome immunoassays, performed either competitively or non-competitively, resemble more closely standard enzyme linked immunosorbent assays, with the enzyme being replaced by a liposome label. Washing steps are used to separate antigen-specifically bound liposomes from unbound liposomes. All bound liposomes are lysed with a detergent, giving an instantaneous amplification. Flow injection liposome immunoassays and liposome immunosensors are also described as examples of other possible immunoassay formats.
Sensor Immunoassay Review Liposomes

"Assessment Of Affinity Constants By Rapid Solid Phase Detection Of Equilibrium Binding In A Flow System"
J. Immunol. Methods 1997 Volume 201, Issue 2 Pages 189-206
Jacob Piehler*, Andreas Brecht, Thomas Giersch, Bertold Hock and Günter Gauglitz

Abstract: We present a method for the determination of affinity constants based on equilibrium binding between an analyte and an antibody in liquid phase by a heterogeneous phase detection scheme. Equilibrium concentration of free antibody binding sites was probed kinetically by direct optical detection of specific binding to an immobilized analyte derivative. The additional binding signal due to dissociation of the analyte-antibody complex during detection was minimised by the use of fast flow-through conditions. The concentration of free antibody binding sites was titrated by adding increasing analyte concentrations. The affinity constant was derived from the titration curve by a non- linear least square fit of a model function. The affinity of monoclonal triazine antibodies to several s-triazine pesticides and a relevant metabolite was investigated. Kinetic determination of equilibrium concentration of free binding sites was carried out by reflectometric interference spectroscopy (RIfS) using flow injection analysis. The capabilities of the model were investigated using different analyte- antibody pairs and various antibody concentrations. Both bivalent IgG and monovalent Fab fragments were used to compare different binding models. The applied model corresponds well to the titration curves for affinity constants of 10(7) M-1 and higher. For lower affinity constants significant deviations due to dissociation of the analyte- antibody complex during detection were observed.
Antibodies Spectroscopy Kinetic Immobilized analyte Complexation Interferences Solid phase detection

"Competitive Flow Injection Enzyme Immunoassay For Steroids Using A Post-column Reaction Technique"
J. Immunol. Methods 1997 Volume 200, Issue 1-2 Pages 145-153
Karin Kronkvist, Ulf Lövgren*, Johan Svenson, Lars-Erik Edholm and Gillis Johansson

Abstract: Two types of flexible, sensitive and rapid competitive flow injection enzyme immunoassay were developed and evaluated for their potential use in the bioanalysis of steroids. Instead of the more typical approach where the signal is generated from antibody-bound hapten-enzyme conjugate, the non-bound fraction passing through the affinity column was allowed to react with an enzyme substrate from a merging channel in a post-column reaction system. The enzyme product (p-aminophenol or 4-methyl umbelliferol) was amperometrically or fluorometrically detected. Several parameters known to affect signal generation in the immunoassay were evaluated, including flow rate through the affinity column and through the reaction coil, the length of the reaction coil and of the affinity column. In the pre-incubation approach, where samples were mixed with enzyme conjugate and antibodies before injection, a sample throughput as high as 20 h-1 was possible. The signal precision was about 1% (RSD) for cortisol (0.6-80 pmol) and 2% (RSD) for budesonide (0.02-12.5 pmol). In the displacement assay for cortisol, enzyme-labelled analyte was displaced from the affinity column when the sample was injected into the flow. A standard curve was obtained with a signal precision of 4-20% for 12.5-1250 pmol injected. The same instrumental set-up was used in both types of immunoassay, and thus a highly flexible system was obtained. A simple replacement of the affinity column from protein G in the pre-incubation approach to a column containing primary antibodies in the displacement assay was needed.
Steroids Amperometry LC Immunoassay Fluorescence Post-column derivatization Optimization Column

"Highly Sensitive Amperometric Enzyme Immunoassay For α-fetoprotein In Human Serum"
J. Immunol. Methods 1996 Volume 193, Issue 1 Pages 51-62
Leopoldo Della Ciana*, Giuliana Bernacca, Concetta De Nitti and Anilla Massaglia

Abstract: A sandwich amperometric enzyme immunoassay with flow injection for α-fetoprotein in human serum has been developed with alkaline phosphatase as the enzyme label. p-Hydroxyphenyl phosphate was used as the substrate for alkaline phosphatase. The hydrolysis product, hydroquinone, was detected by oxidative amperometry in a flow injection system. The amperometric wall jet detector was fitted with a glassy carbon working electrode held at 350 mV vs. Ag/AgCl. The detection limit of hydroquinone in 30 mM borate buffer pH 9.5 was 1.2 x 10^-10 M (linearity range: 10^-9 - 5.12 x 10^-6 M. A detection limit for free alkaline phosphatase of 1.2 x 10^-15 M (linearity range: 10^-15 - 10^-13 M), or about 36 000 molecules, was observed (same borate buffer and incubations of 10 min at 25°C). These conditions were maintained for the amperometric α-fetoprotein immunoassay. For comparison purposes, a photometric detection system was set up, with p-nitrophenyl phosphate as enzyme substrate and the same pair of antibodies and incubation conditions. The detection limit for α-fetoprotein obtained by amperometry, 0.07 ng/ml (linearity range = 5-500 ng/ml), was 14 times lower than by photometry. The amperometric enzyme immunoassay correlates well with a commercial colorimetric immunoassay (r = 0.986, slope = 0.967, n = 240).
α-Fetoprotein Serum Human Immunoassay Amperometry Electrode Method comparison Enzyme

"New Immunoassay Technique Using Antibody Immobilized On A Membrane And A Flow Cuvette As Reaction Vessel"
J. Immunol. Methods 1993 Volume 157, Issue 1-2 Pages 11-17
B. M. Gorovits, A. P. Osipov* and A. M. Egorov

Abstract: Immunoenzymatic detection systems have been developed using human IgG as a model antigen. A membrane with covalently immobilized specific antibodies was placed into a specially constructed ultranarrow flow cuvette and solutions containing the antigen and antibody-peroxidase conjugate were then successively passed through the flow capillary cell. After washing, the membrane was placed into the substrate solution and the intensity of developed color on the membrane was recorded visually or by a reflection spectrophotometer. The lower detection limit was about 5 x 10^-11 M and the overall analysis time was 10 min. Photoimmobilization was used to immobilize the antibody and thereby permitting control of the protein surface concentration on the membrane as well as the dimensions and shape of the activated region.
Immunoglobulin G Immunoassay Spectrophotometry Immobilized antibody Photochemistry Membrane

"Latex Immunoassay Of Transferrin In Urine"
J. Immunol. Methods 1991 Volume 144, Issue 1 Pages 49-55
A. Bernard*, K. S. Chia and R. Lauwerys

Abstract: A fully automated assay based on latex particle agglutination is described. The assay is carried out in a continuous-flow system (as described by Bernard and Lauwerys (Clin. Chem., 1983, 29, 1007) which was modified to include a Tecan 505 manipulator which automatically dilutes urine samples and standards. Urine was incubated for 30 min at 50°C with latex particles adsorbed with anti-transferrin antibody (prep. described); residual unagglutinated particles were quantified with an optical particle counter. The assay ranged from 0.5 to 10 µg L-1 of transferrin. Intra- and inter-assay coefficient of variation were from 2.1 to 11.8% and the average recovery was 102.5%. The latex immunoassay of transferrin was adapted to give a turbidimetric reading; agglutination was quantified by measuring the decrease in absorbance at 360 nm (full details given).
Transferrin Urine Immunoassay Spectrophotometry Turbidimetry Automation Dilution Heated reaction Latex

"Continuous-flow Immunoassay For Rapid And Sensitive Detection Of Small Molecules"
J. Immunol. Methods 1990 Volume 135, Issue 1-2 Pages 191-197
Anne W. Kusterbeck, Gregory A. Wemhoff, Paul T. Charles, Doyle A. Yeager, Reinhard Bredehorst, Carl-Wilhelm Vogel and Frances S. Ligler,*

Abstract: The assay is based on the binding of labelled antigen to an immobilized antibody with subsequent displacement of the labelled antigen when antigen is present in the buffer flow. The hapten 2,4-dinitrophenol (DNP) as DNP-lysine was used as model antigen with either 125I-labelled DNP-insulin or fluorescein-labelled DNP-insulin as labelled antigen. The radiolabelled antigen was used to establish assay conditions. Anti-DNP antibody was immobilized on tresyl chloride-activated Sepharose 4B and, after immobilization, the available antigen-binding sites were occupied with labelled antigen and the gel was packed into 6-mm i.d. columns. Borate-buffered saline (pH 8.2) was continuously flushed through the column. Sample (200 µL) was injected into the buffer flow and the displaced labelled antigen to the effluent was monitored fluorimetrically at 515 nm (excitation at 492 nm) or by collection of fractions followed by liquid scintillation counting. The calibration graphs were rectilinear from 570 to 4600 nM-DNP and down to 140 nM could be detected using a 200 µL column.
Immunoassay Buffer Column Calibration

"Use Of Phycobiliproteins As Fluorescent Labels In Immunoassay"
J. Immunol. Methods 1986 Volume 92, Issue 1 Pages 1-13
Mel N. Kronick

Abstract: The fluorescent characteristics of phycoerythrins, phycocyanins and allophycocyanins, their coupling to proteins or haptens and applications of the conjugates in immunoassay methods, cell labelling and flow cytometry are reviewed. (67 references).
Fluorescence Flow cytometry Immunoassay Review