Homogeneous Immunoassay Research Articles

The Use of Inosine 5′-Monophosphate Dehydrogenase (IMPDH) in the Development of a New Liquid Homogeneous Enzyme Immunoassay Technology

The objective of the present study was the development of a quantitative liquid homogeneous immunoassay specific for theophylline based on the specific uncompetitive inhibition of inosine 5′-monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). This was accomplished by covalent coupling of theophylline to MPA to form a theophylline-MPA conjugate (Fig. 1⇓ ). IMPDH (EC 1.1.1.205) (1) catalyzes the NAD-dependent oxidation of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP). The enzyme follows an ordered Bi-Bi reaction sequence of substrate and cofactor binding and product release. In the first step, IMP binds to IMPDH, followed by the binding of the cofactor NAD. After IMP oxidation, the reduced cofactor, NADH, is released from IMPDH, followed by the release of XMP. To monitor the reaction, the rate of NADH formation is measured at 340 nm. Uncompetitive inhibition occurs when MPA combines with the IMPDH-XMP complex at the active site of the enzyme to form IMPDH-XMP-MPA complex, which is unable to release XMP. IMPDH inhibition depends only on the concentration of MPA because of the uncompetitive nature of inhibition by MPA. Thus, the greater the concentration of MPA inhibitor, the greater the inhibition of the enzyme. An uncompetitive inhibitor of IMPDH inhibits by binding at the active site of the enzyme and does not compete with IMP …

A POLARIZATION FLUORESCENCE IMMUNOASSAY FOR THE HERBICIDE PROPANIL

A rapid, homogeneous polarization fluorescence immunoassay (PFIA) has been developed for herbicide propanil. Polyclonal antiserum were raised in rabbits using 3,4-dichloroaniline coupled to succinylated bovine serum albumin (BSA) as immunogen. The various haptens and fluorescein-labeled tracers were synthesized to achieve high-sensitivity PFIA. The method can detect propanil concentrations in the range 1–100 ng/mL in 50 μL of sample. Analysis of one sample takes less then 1 min. In order to achieve better sensitivity a preconcentration step is possible and has been done for the low concentration samples. The use of SPE was suitable to enrich the sample, allowing measuring water samples at the limit required at EC Directive (permitted residue level in water: 0.1 ng/ml). The specificity of the PFIA for propanil was investigated using chloranilines, phenylureas, and other pesticides. Some cross-reactivity was found for closely related phenylureas (13–18%), but the major metabolite 3,4-dichloroaniline had negligible cross-reactivity (0.1%). SPE-PFIA and SPME-GC-MS showed a good correlation, when testing propanil in drinking water.

DEVELOPMENT OF A HOMOGENEOUS PHOSPHORESCENT IMMUNOASSAY FOR THE DETECTION OF POLYCHLORINATED DIBENZO-p-DIOXINS

A homogeneous immunoassay for dioxins was developed using a phosphorescent label for detection. A dioxin derivative was conjugated to Pt-coproporphyrin. In the assay, when the antibodies against dioxin (DD3) were bound to the phosphorescent conjugate, the signal from Pt-coproporphyrin was quenched. The interaction between antibody and the conjugate was studied by time-resolved luminescence spectroscopy. As concentrations of dioxin standards increased from 39 pg/well to 2.5 ng/well the lifetime of the phosphorescence of the shortlived component increased from 25.6 microseconds to 58.9 microseconds. This increase in half-life was associated with a dose dependent quenching of the phosphorescence. The inhibition obtained is similar to that for a reported enzyme-based immunoassay, but the data were obtained in minutes instead of hours.

Effects of the ionic environment, charge, and particle surface chemistry for enhancing a latex homogeneous immunoassay of C-reactive protein.

The role of the solution environment for a light-scattering, latex-particle-enhanced, homogeneous immunoassay of C-reactive protein (CRP) has been investigated in order to assess and optimize the immunoagglutination response. Latex particles of 50-170-nm sizes were covalently coupled with an IgG polyclonal antibody and subjected to an extensive optimization regime. This consisted of conditions responsible, in different degrees, for the principal attractive/repulsive forces affecting both colloidal stability and the antibody/antigen interaction: particle size, antibody concentration, ionic strength and species, pH, and amino acid chemistry of the particle surface. Careful control of these parameters was found to be necessary to achieve the desired effects of balancing high colloidal stability in the absence of antigen but promoting a rapid, sensitive, and dose-dependent agglutination with pathological serum samples. In addition, the estimation of fundamental properties governing intermolecular interaction (i.e. the "Hamaker" constant and critical coagulation concentration) was attempted to order to investigate a simple, practical means of defining a colloidal/immunoassay system under "real conditions" as well as "real time". It is concluded that because each antibody system is unique, a similar optimization should be performed in diagnostic immunoassays of this type to maximize their clinical utility.

A new homogeneous enzyme immunoassay for thyroxine using glycogen phosphorylase b–thyroxine conjugates

Background: Measurement of serum thyroxine (T 4) concentration is important for diagnosis of thyroid gland diseases. We developed a practical homogeneous enzyme immunoassay for thyroxine analysis in unextracted sera. Methods: A thyroxine derivative conjugated to a reactive sulfhydryl group of glycogen phosphorylase b (GPb). Conjugation caused inhibition of enzyme activity and the enzyme conjugate was re-activated upon the binding of a polyclonal anti-T 4 antibody. Antibody-activation was blocked by the presence of free T 4. Results: Conjugation affected the allosteric character of the enzyme and the K m for the allosteric activator AMP was increased 28 times, while anti-T 4 antibody partially reversed this effect. The optimum concentration ratio of enzyme conjugate to anti-T 4 antibody was determined, and T 4 was measured with desired sensitivity and accuracy in the range between 10 and 240 μg/l. Furosemide was used to inhibit the interaction of thyroxine with serum T 4-binding sites. Human serum T 4 values obtained by this method correlated well with those obtained by a radioimmunoassay ( y=1.9+1.0 x, r=0.97, N=72). Conclusions: Chemical modification of glycogen phosphorylase b with a T 4 derivative led to the development of a simple homogenous enzyme immunoassay for T 4 analysis with the desired sensitivity and accuracy.

Homogeneous Enzyme Immunoassay for Triiodothyronine in Serum

The concentration of triiodothyronine (T3) in human serum is extremely low and can be determined only by very sensitive methods. We developed a homogeneous enzyme immunoassay for T3 analysis in unextracted serum. A T(3) derivative was conjugated to the -SH groups of glycogen phosphorylase b (GPb) from rabbit muscle. Conjugation caused inhibition of enzyme activity, and the enzyme conjugate was reactivated upon binding of anti-T3 antibody. Activation was blocked by the presence of non-antibody-bound T3; this was the basis for the development of the homogeneous enzyme immunoassay for T3 by determining GPb activity fluorometrically. We used furosemide to block the interaction of T3 with serum proteins with T3-binding sites, avoiding any serum treatment step. T3 was measured in the range 0.3-8 microg/L. T3 values obtained by this assay correlated well with those obtained by a RIA (y = 0.97x - 0.07 microg/L; r = 0.96; n = 92). Within- and between-run imprecision (CV) was 5-9% for normal and high concentrations and 16-20% for low concentrations. Chemical modification of GPb with a T3 derivative allows the development of a simple homogeneous enzyme immunoassay for T3 in unextracted serum.

Nonseparation Binding/Immunoassays Using Polycation-Sensitive Membrane Electrode Detection

Two novel homogeneous potentiometric immunoassay formats suitable for the detection of small molecules are described based on polycation-sensitive membrane electrode detection. One approach involves the use of synthetic polycationic-analyte conjugates (polycation labeled analyte) and a limited amount of binding protein sites (antibodies, etc.) to modulate the electrochemical response toward the conjugate. Upon conjugate binding to large binding proteins/antibodies in solution, the polycation label cannot be efficiently extracted into the transduction membrane of the polycation-sensitive device, greatly diminishing the observed EMF response. In the presence of analyte molecules, competitive binding frees up more of the conjugate, and an increase in EMF response is observed in proportion to the concentration of analyte present. With a 15 min reaction incubation period and 3 min electrochemical response time, the assay is demonstrated to function effectively for measurement of theophylline (model analyte) at sub-micromolar levels without any discrete washing or separation steps. The second approach is based on the use of trypsin as an enzymatic label in a homogeneous enzyme binding/immunoassay scheme. Trypsin-biotin conjugates (biotin used as model analyte) are shown to be inhibited nearly 100 % by avidin, but catalytic activity is regained as free biotin is present at increasing levels in the sample solution. The activity of trypsin can be conveniently monitored via polycation-sensitive membrane electrode detection by following the decrease in the EMF response to the polycationic protein protamine, as it is cleaved into smaller fragments by the enzyme. This new electrochemical homogeneous enzyme binding/immunoassay method is shown to respond to the analyte at ≥10 nM levels, again without need for washing or separation steps.

Demonstration of a Homogeneous Noncompetitive Immunoassay Based on Bioluminescence Resonance Energy Transfer

We describe a noncompetitive homogeneous bioluminescent immunoassay based on the antigen-dependent reassociation of antibody variable domains (open sandwich bioluminescent immunoassay, OS-BLIA). The reassociation of two chimeric proteins, an antibody heavy-chain fragment (VH)–Renilla luciferase (Rluc) and an antibody light-chain fragment (VL)–enhanced yellow fluorescent protein (EYFP), was monitored by a bioluminescence resonance energy transfer (BRET) between the two. Upon simple mixing of the reagents with the sample, an antigen-dependent increase in BRET was observed with a measurable concentration range of 0.1∼10 μg/ml antigen hen egg lysozyme. Compared with our comparable assays based on fluorescence resonance energy transfer (FRET), a 10-fold improvement in the sensitivity was attained, probably due to a reduction in reagent concentration.

Liposome Immune Lysis Assay of Antibodies by Liposomes Encapsulating Coenzyme .BETA.-NAD+.

Liposome immune lysis assay (LILA), which depends on immune lysis of artificial phospholipid vesicles by the activation of complement, is a homogeneous immunoassay method and much better than ELISA in respects of simplicity and rapidity in measurements. Fluorescent agents have been used as markers encapsulated in liposomes, but they need an expensive apparatus for detection. In this work, we measure the concentration of polyclonal and monoclonal antibodies by LILA by use of antigen-bound liposomes encapsulating coenzyme β-NAD+. Conjugated redox reactions involving β-NAD+ can accumulate a colored material and attain a high sensitivity similar to fluorescent agents in detection of released marker. We also analyze the characteristics of immune lysis of liposomes in LILA depending on the equilibrium relationship between antigen and antibody. The marker release depends on the concentration of the antibodies, and the decrease in the concentration of liposomes raises the sensitivity. The marker release is proportional to the concentration of antigen-antibody complex formed on liposomes. Polyclonal antibody can sensitively be detected even at a lower average association constant than monoclonal antibody.

Ultrasensitive magnetic biosensor for homogeneous immunoassay.

A technique is described for specific, sensitive, quantitative, and rapid detection of biological targets by using superparamagnetic nanoparticles and a "microscope" based on a high-transition temperature dc superconducting quantum interference device (SQUID). In this technique, a mylar film to which the targets have been bound is placed on the microscope. The film, at room temperature and atmospheric pressure, is typically 40 micrometer from the SQUID, which is at 77 K in a vacuum. A suspension of magnetic nanoparticles carrying antibodies directed against the target is added to the mixture in the well, and 1-s pulses of magnetic field are applied parallel to the SQUID. In the presence of this aligning field the nanoparticles develop a net magnetization, which relaxes when the field is turned off. Unbound nanoparticles relax rapidly by Brownian rotation and contribute no measurable signal. Nanoparticles that are bound to the target on the film are immobilized and undergo Néel relaxation, producing a slowly decaying magnetic flux, which is detected by the SQUID. The ability to distinguish between bound and unbound labels allows one to run homogeneous assays, which do not require separation and removal of unbound magnetic particles. The technique has been demonstrated with a model system of liposomes carrying the FLAG epitope. The SQUID microscope requires no more than (5 +/- 2) x 10(4) magnetic nanoparticles to register a reproducible signal.

Rapid Homogeneous Immunoassay for Human Ferritin in the Cobas Mira Using Colloidal Gold as the Reporter Reagent

Serum ferritin concentrations, with some exceptions (1)(2)(3), reflect iron stores (4)(5). Ferritin assays must have a broad dynamic range because the serum concentrations can be 1 mg/L in some types of malignancies (7). Radio- and enzyme immunoassays have been used routinely (8), but rapid, automated latex agglutination immunoassays have been developed and validated (9)(10)(11). These methods have drawbacks (12), of which the disturbance of colloidal stability by nonspecific bridging processes particularly should be avoided (13). In our quest for new reporter reagents (14)(15), our attention was drawn to colloidal gold as a potential substitute for latex in particle-enhanced agglutination immunoassays. Colloidal gold was used by Leuvering et al. (16) in sol particle immunoassays (SPIAs) for several serum or urine analytes. Unfortunately, the technique was prone to interference when undiluted serum samples were used (17). Recently, we showed (18)(19) that the change in visible absorbance at 600 nm ( A 600) observed when colloidal gold particles coated with an antibody interact with the antigen results not only from agglutination but also from subtle changes in the refractive index at the particle surface [surface plasmon resonance effect (SPR)]. Thus, the unidentified random interferences noted by Leuvering et al. (16) could result from interactions between nonspecific reacting sites on the antibody molecule (distinct from the binding site) and several serum components (distinct from the analyte). According to this model, these interactions were transduced as a photometric signal by the SPR effect of gold (19)(20), which adds to the signal produced by the agglutination. With this in mind, we optimized the buffer to be used in the SPIA with colloidal gold and carefully selected the antibodies to be used for their lack of SPR …

Size Exclusion Redox-Labeled Immunoassay (SERI): A New Format for Homogeneous Amperometric Creatinine Determination

A homogeneous amperometric immunoassay for creatinine has been developed by using anti-creatinine antibodies, redox-labeled creatinine and a glassy carbon electrode covered with a semipermeable cellulose membrane with 20 kD cutoff. Creatinine from the sample competes with redox-labeled creatinine for the antigen binding sites of the antibody. Unbound conjugate passes through the membrane and is indicated at the electrode whereas antibody bound conjugate is size excluded. For redox labeling of creatinine a new labeling substance, 2-acetamido-3-chloro-1,4-naphthoquinone (AcClNQ), was used, which can be indicated electrochemically at an interference free working potential of – 200 mV (vs. Ag/AgCl). With the developed size exclusion redox-labeled immunoassay (SERI) creatinine can be determined within a range from 10 ng/mL to 100 µg/mL (0.09–900 µM).

Fluorescence Polarization Immunoassay: Detection of Antibody to Brucella abortus

Fluorescence polarization immunoassay (FPA) is a homogeneous immunoassay useful for rapid and accurate detection of antibody or antigen. The principle of the assay is that a fluorescent dye (attached to an antigen or an antibody fragment) can be excited by plane-polarized light at the appropriate wavelength. As a rule, a small molecule rotates faster when in solution than a larger molecule. The rotation rate may be assessed by measuring light intensity in the vertical and horizontal planes. Generally, the time it takes a molecule to rotate through a given angle is an indication of its size. When a small molecule that rotates rapidly is bound to a larger molecule, the rotation rate is decreased and this decrease is measured. Because it is a primary antigen–antibody interaction, the rate of reaction is very rapid and usually a result may be obtained in minutes. This technology was applied to the detection of antibody to Brucella abortus in serum and milk, providing for the first time a rapid primary binding assay that is cost effective for use in the field.

Fluorescence Polarization Competition Immunoassay for Tyrosine Kinases

To increase the sensitivity and throughput of protein tyrosine kinase (PTK), simple, homogeneous, nonradioactive, direct and indirect fluorescence polarization (FP) protein tyrosine kinase immunoassays have been developed that are compatible with high-throughput and ultrahigh-throughput screening for developing drugs. In the direct method, a fluorescinylated peptide substrate is incubated with the kinase, ATP, and antiphosphotyrosine antibody. The phosphorylated peptide product is immunocomplexed with the antiphosphotyrosine antibody, resulting in an increase in the polarization signal. Since the direct method can be used only with a peptide substrate and requires large amounts of antiphosphotyrosine antibody, a modified indirect method, wherein a phosphorylated peptide or protein produced by kinase reaction will compete with a fluorescent phosphopeptide used as a tracer for immunocomplex formation with phosphotyrosine antibody, was developed. In this format kinase activity will result in loss of the polarization signal. Both the direct and indirect FP-PTK immunoassays have been compared with a more commonly used 32PO4 transfer assay and validated using lymphoid T-cell protein tyrosine kinase (Lck). In both assays, Lck activity showed a similar dependence on ATP, Lck enzyme, and peptide substrate concentration, comparable to the 32PO4 transfer assay. Inhibition by staurosporine and the Lck inhibitor 4-amino-5-(methylphenyl)-7-(tert-butyl)pyrazolo[3,4-d]pyrimidine in these two FP assays was similar to that obtained in the 32PO4 transfer assay. The advantages of these FP-PTK assays over the other kinase assays, besides high sensitivity, are use of inexpensive nonisotropic substrate; environmental safety; homogeneous nature of FP kinase assays that are done in the same tube (or in a well of 96- or 384-well microtiter plates), without separation, precipitation, or washing; and increase of throughput.

A Novel and Robust Homogeneous Fluorescence-Based Assay Using Nanoparticles for Pharmaceutical Screening and Diagnostics

We have established a new type of homogeneous immunoassay based on nanoparticles (nanoparticle immunoassay, or NPIA) being analyzed using fluorescence intensity distribution analysis (FIDA). This method allows the characterization of single fluorescently labeled molecules or particles with respect to their molecular brightness and concentration. Upon binding of conjugates to molecules coupled to the nanoparticle surface, the brightness of the complex scales with the number of bound conjugates. The complexes can then be distinguished accurately from free conjugate and concentrations of free and bound molecules can be determined reliably. In this study we present various examples of NPIAs where capture antibodies were linked to the nanoparticles, which were either artificial beads or bacteria. Two assay formats have been developed; first, direct labeling of the conjugate was used to quantitate free antigen through competition experiments, and second, an antigen-directed antibody was labeled to establish an assay similar to a sandwich ELISA setup. The major advantages of a NPIA are the robustness and high signal-to-noise ratio at short measurement times, as demonstrated with a miniaturized experiment in a Nanocarrier™ holding a volume of 1 μl/well. In addition to the good data quality, NPIAs are straightforward to perform because they require no washing steps. NPIAs open new dimensions for high throughput pharmaceutical screening and diagnostics. Assay development times can be reduced significantly because of a simple toolbox principle that is applicable to most types of assays.

Fluorolabeling of antibody variable domains with green fluorescent protein variants: application to an energy transfer-based homogeneous immunoassay.

A site-specific and efficient fluorolabeling of antibody variable regions with green fluorescent protein (GFP) variants and its application to an energy transfer-based homogeneous fluoroimmunoassay (open sandwich FIA) were attempted. Two chimeric proteins, Trx-V(H)-EBFP and Trx-V(L)-EGFP, consisting of V(H) and V(L) fragments of anti-hen egg lysozyme (HEL) antibody HyHEL-10 and two GFP color variants, EBFP and EGFP, respectively, were designed to be expressed in cytoplasm of trxB - mutant Escherichia coli as fusions with thioredoxin from E.coli The mixture of two proteins could be purified with HEL-affinity chromatography, retaining sufficient intrinsic fluorescence and binding activity to HEL. A significant increase in fluorescence resonance energy transfer (FRET) dependent on HEL concentration was observed, indicating the reassociation of the V(H) and V(L) domains of these chimeric proteins due to co-existing antigen. With this open sandwich FIA, an HEL concentration of 1-100 microg/ml could be non-competitively determined. The assay could be performed in a microplate format and took only a few minutes to obtain a sufficient signal after simple mixing of the chimeric proteins with samples. This represents the first demonstration that the FRET between GFP variants is applicable to homogeneous immunoassay.

Measurement of nine human milk proteins by nephelometric immunoassays: application to the determination of mature milk protein profile

Objectives: Microparticle-enhanced nephelometric immunoassays for six human milk proteins (β-casein, κ-casein, α-lactalbumin, serum albumin, lactoferrin, and lysozyme) and conventional immunonephelometry assays for immunoglobulin A, C3, and C4 complement proteins were developed and characterized. Design and methods: Microparticle-enhanced nephelometric immunoassays are competitive assays based on the nephelometric quantification of the inhibition of microparticle-protein conjugates immunoagglutination by the proteins to be assayed. Results: High precision (CVs ranged from 1% to 14% in within- and between-assays) and recovery (linear recovery in dilution-overloading assay) ensure a reliable determination of the main human milk proteins by single-step homogeneous nephelometric immunoassays, accurate over wide ranges of concentration. These immunoassays were easily applied to a large number of mature human milk samples (between 373 and 503 according to the proteins tested). Conclusions: The immunoassays developed could be applied to the fast determination of human milk protein profile usable for nursery milk bank and fortification.

Immunoanalytical techniques for pesticide monitoring based on fluorescence detection.

In the field of environmental analysis there is still great potential for development and application of immunoanalytical techniques (IT). Heterogeneous and homogeneous immunoassays (IA), flow-injection immunoanalysis (FIIA) and immunosensors (IS) with different detection principles have been developed. In this review we focus on fluorescence methods for pesticide monitoring published since 1992. These techniques offer a high degree of selectivity and, in principle, sensitivity. Restrictions on the limits of detection (LOD) due to background signals are minimized by development of solid-phase separation systems, new fluorescent probes, and new instrumentation.

Coupling immunoassays with chromatographic separation techniques.

Coupling immunoassays with HPLC separation techniques is becoming increasingly useful in the analysis of biological and nonbiological samples of both large and small molecules. This is because it provides both sensitivity and selectivity for molecular analysis at relatively low cost, low maintenance and with excellent potential for automation. This paper reviews application of this hyphenated approach both in the pre-column immunoextraction and post-column immunodetection modes. Systems in which immunoassays are interfaced to chromatographic separations in order to separate bound and free fractions of the immunoassay will not be included since these systems do not provide the enhanced selectivity common to hyphenated systems. Post-column immunodetection is based on various immunoassay formats such as direct detection, one-site, competitive and sandwich immunoassays. Homogeneous immunodetectors are more convenient than heterogeneous immunodectors since there are no separation and column regeneration steps involved in homogeneous immunoassays. On the other hand, heterogeneous immunoassays are generally more sensitive than homogeneous immunoassays since interfering substances are removed prior to immunodetection. Advantages and limitations for the various approaches will be discussed.

New Donor−Acceptor Pair for Fluorescent Immunoassays by Energy Transfer

A novel Förster donor-acceptor dye pair for an immunoassay based on resonance energy transfer (RET) is characterized with respect to its photophysical properties. As donor and acceptor, we chose the long-wavelength excitable cyanine dyes Cy5 and Cy5.5, respectively. Due to the perfect spectral overlap, an exceptionally high R(0) value of 68.7 A is obtained in solution. For biochemical applications, antibodies (IgG) are labeled with Cy5, while a tracer for competitive binding is synthesized by labeling bovine serum albumin (BSA) with an analyte derivative and Cy5.5. Binding the dyes to proteins at a low dye/protein ratio increases the fluorescence lifetimes and quantum yields, leading to an enhanced R(0) value of 85.2 A. At higher dye/protein ratios, the formation of nonfluorescent dimeric species causes a decrease in the fluorescence lifetime and quantum yield due to RET from monomeric dyes to dimers within one protein molecule. The Förster distances could be calculated using the dimer absorption spectra to 83.9 and 83.6 A for Cy5 and Cy5.5, respectively. Upon binding of the Cy5-labeled IgG to the tracer, efficient quenching of Cy5 fluorescence is observed. Steady-state and time-resolved measurements reveal that approximately 50% of the quenching results in Förster-type RET, while the residual quenching effect is caused by static quenching processes. The applicability of this dye pair is demonstrated in a homogeneous competitive immunoassay for the pesticide simazine.