Robert C. Boguslaski

[37] Monitoring specific protein-binding reactions with chemiluminescence

Publisher Summary This chapter discusses the monitoring of specific protein-binding reactions with chemiluminescence. Aminophthalhydrazides, aminonaphthylhydrazides, and their ligand conjugates can be measured at picomolar concentrations by chemiluminescence. Competitive protein-binding assays monitored by chemiluminescence can measure some ligands at concentrations present in biological materials. Light production by the biotin-isoluminol conjugate is enhanced about 10-fold when the biotin moiety is bound to avidin. This enhancement provided a means for monitoring competitive-binding assays for biotin without separation of the protein-bound and free forms. Among the oxidation systems tested, the H 2 O 2 -microperoxidase and the H 2 O 2 -hematin systems provide the most sensitive assays for the chemiluminescent compounds. The sensitivity of competitive-binding assays is determined in part by the lower limit for detection of the label. The lower limits for detection of these compounds using several oxidation systems are presented in the chapter along with their use in protein-binding assays.

Specific protein-binding reactions monitored by enzymatic hydrolysis of ligand-fluorescent dye conjugates

Abstract Specific protein-binding reactions were monitored utilizing ligand-fluorescent dye conjugates which were substrates for porcine esterase. Biotin was coupled directly to umbelliferone through an ester bond, while 2,4-dinitrophenyl derivatives were conjugated to fluorescein via an ester linkage. Hydrolysis of these nonfluorescent esters with an esterase yielded fluorescent products, and the reaction rate was related quantitatively to the conjugate concentration. When the conjugated ligands were bound to their specific proteins (avidin or antibody to the dinitrophenyl residue), they were inactive as substrates. This assay allowed the measurement of unbound conjugate without separation from protein-bound conjugate. The inactivation of the conjugated substrates was relieved by the addition of free (unconjugated) ligand in competitive binding reactions. In addition to measuring free ligand levels, this assay was used to detect specific binding proteins, e.g., during the purification of antibody to the dinitrophenyl residue.

A method for monitoring specific binding reactions with cofactor labeled ligands.

Abstract Specific binding reactions were monitored by a new enzymatic method which employs cofactor labeled ligands. The ligands biotin and 2,4-dinitrofluorobenzene were coupled covalently to a derivative of NAD, nicotinamide 6-(2-aminoethylamino) purine dinucleotide (AENAD) to provide cofactor conjugates which were active with several dehydrogenases. The conjugates were cycled in reactions employing lactic dehydrogenase and diaphorase and cycling rates were determined by spectrophotometric measurement of a reaction product, reduced thiazolyl blue. The cycling of biotinyl-AENAD was inhibited specifically by avidin, a biotin-binding protein. This inhibition was reversed by biotin in competitive binding reactions. Similarly, the cycling rate of 2,4-dinitrophenyl-AENAD was inhibited by antibody to the 2,4-dinitrophenyl residue and 2,4-dinitrophenyl-6-aminocaproate reversed this inhibition. Thus, the activity of a cofactor coupled to a ligand is modified by a binding protein specific for the ligand. This phenomenon provides a versatile method for conducting specific binding assays without separation of free and bound labeled ligand.

Specific binding reactions monitored with ligand-cofactor conjugates and bacterial luciferase

Abstract A unique rapid method for assaying specific binding reactions with ligand-cofactor conjugates and a bioluminescent reaction is described. Biotin and 2,4-dinitrofluorobenzene were coupled covalently to the free amino residue of nicotinamide 6-(2-aminoethylamino) purine dinucleotide (AENAD) to produce the enzymatically active conjugates biotinyl-AENAD and DNP-AENAD. After reduction with alcohol dehydrogenase and ethanol these two conjugates were measured quantitatively by means of light produced in a bioluminescent reaction employing luciferase from Photobacterium fisheri. Light production by biotinyl-AENADH and DNP-AENADH was inhibited by the specific binding proteins avidin and antibody to DNP, respectively. The counter ligands, biotin and DNP-6-aminocaproate, reversed the inhibition by the respective binding proteins in competitive binding reactions. Thus, specific binding reactions can be assayed rapidly without separation of free and bound labeled ligands.

Immunoassay for serum thyroxine monitored by chemiluminescence.

An immunoassay for thyroxine (T4) monitored by chemiluminescence was evaluated with clinical serums. A thyroxine-label conjugate (T4-L) and serum samples were applied sequentially to alkaline Sephadex G-25 columns which adsorbed the thyroxine species. Other serum components and potential interferents were washed from the column with barbital buffer. Subsequently addition of antibody initiated the binding reaction. After 1 h incubation, the antibody was eluted from the column with a barbital buffer wash. The bound T4-L in the eluate was oxidized in a chemiluminescent detection reaction. The peak light intensity, attained in about 1 sec, was related to T4 concentration by means of standards. The intra-assay precision of the chemiluminescence immunoassay was +/-5% (C.V.). Statistical comparison of T4 levels determined for 28 serums by this method and a reference assay was acceptable (y = 0.95x + 5.9, r = 0.98, Sy square root of y . 100 = 13.1%).

Specific protein-binding reactions monitored with ligand-ATP conjugates and firefly luciferase

Abstract A new method was developed to monitor specific protein binding reactions with an ATP-labeled ligand and firefly luciferase. The ligand, 2,4-dinitrobenzene, was covalently coupled to four ATP derivatives and three of these conjugates were measured quantitatively at nanomolar levels with firefly luciferase. Incubation of the conjugates with antibody to the 2,4-dinitrophenyl residue diminished the peak light intensities produced in the bioluminescent assay, whereas incubation with immunoglobulin from a nonimmunized rabbit did not affect light production. Therefore, the antibody-bound ligand-ATP conjugates were inactive in the bioluminescent assay and levels of unbound conjugate could be measured in the presence of the bound form. The firefly luciferase was used to monitor competitive binding reactions between the antibody, the conjugates, and N(2,4-dinitrophenyl)-β-alanine.

Homogeneous substrate-labeled fluorescent immunoassay for IgG in human serum

A homogeneous substrate-labeled fluorescent immunoassay for IgG has been developed. Purified IgG was covalently labeled with 6-(7-beta-galactosylcoumarin-3-carboxamido)-hexylamine to form a stable conjugate, GU-IgG. The galactosyl residue was hydrolyzed from GU-IgG by beta-galactosidase and the progress of the hydrolysis was monitored by the increase in fluorescence emission at 450 nm with excitation at 400 nm. Antibody to IgG diminished the activity of GU-IgG as a substrate for beta-galactosidase. Competitive binding immunoassays were conducted by allowing added IgG and GU-IgG to compete for a limited number of antibody binding sites. Hence, the fluorescence produced by enzymic hydrolysis increased with the level of added IgG. This method provides a simple and reliable immunoassay for IgG and is applicable to other proteins.

A colorimetric immunoassay based on an enzyme inhibitor method

A competitive binding immunoassay was developed using an enzyme inhibitor for labeling the analyte. Thyroxine was labeled by covalent coupling of the alpha-amino group to the gamma-carboxyl of the glutamyl residue of methotrexate. This thyroxine-methotrexate conjugate was a potent inhibitor of dihydrofolate reductase. When antibody was bound to the thyroxine moiety, the inhibitor was inactivated. Thus, a competitive binding immunoassay for thyroxine was demonstrated based on colorimetric measurement of dihydrofolate reductase activity.

[12] ATP-Labeled ligands and firefly luciferase for monitoring specific protein-binding reactions

Publisher Summary This chapter discusses binding reactions employing radiolabeled ligands that are used extensively for measurement of low levels of ligands and binding proteins in biological materials. Bioluminescent emission from firefly luciferase can be influenced by materials in the sample being assayed. Many biological fluids, such as serum or plasma, might contain endogenous adenosine triphosphate (ATP) that can interfere with the measurement of ligand–ATP conjugates. However, these fluids often contain enzymes that degrade ATP rapidly and the endogenous cofactor can be eliminated by incubation of the sample prior to the binding reaction. The degradation of ligand–ATP conjugates by the endogenous enzymes can be inhibited by including EDTA in the buffer for the binding reaction. Ten millimolar EDTA in the sample being assayed does not influence bioluminescence.

Multilayer film immunoassay

The workload in clinical laboratories has increased over the years because increasing numbers of specimens are being tested and new analytes discovered. As assay methods for important analytes have become available, systems for simplification and automation of the measurements have been developed. At the same time, there has been a need for convenient and robust assays that can be carried out in doctors’ offices and other decentralised settings by personnel with little formal technical training.