Performance Evaluation of a Prototype Architect Antibody Assay for Babesia microti.

The tick-borne protozoan is responsible for more than 200 cases of transfusion-transmitted babesiosis (TTB) infection in the United States that have occurred over the last 30 years. Measures to mitigate the risk of TTB include nucleic acid testing (NAT) and antibody testing. A fully automated prototype antibody test was developed on the Architect instrument.

Cysteinylation of a monoclonal antibody leads to its inactivation.

Post-translational modifications can have a signification effect on antibody stability. A comprehensive approach is often required to best understand the underlying reasons the modification affects the antibody's potency or aggregation state. Monoclonal antibody 001 displayed significant variation in terms of potency, as defined by surface plasmon resonance testing (Biacore), from lot to lot independent of any observable aggregation or degradation, suggesting that a post-translational modification could be driving this variability.

A precise spectrophotometric method for measuring sodium dodecyl sulfate concentration.

Sodium dodecyl sulfate (SDS) is used to denature and solubilize proteins, especially membrane and other hydrophobic proteins. A quantitative method to determine the concentration of SDS using the dye Stains-All is known. However, this method lacks the accuracy and reproducibility necessary for use with protein solutions where SDS concentration is a critical factor, so we modified this method after examining multiple parameters (solvent, pH, buffers, and light exposure).

Epitope mapping and targeted quantitation of the cardiac biomarker troponin by SID-MRM mass spectrometry.

The absolute quantitation of the targeted protein using MS provides a promising method to evaluate/verify biomarkers used in clinical diagnostics. In this study, a cardiac biomarker, troponin I (TnI), was used as a model protein for method development. The epitope peptide of TnI was characterized by epitope excision followed with LC/MS/MS method and acted as the surrogate peptide for the targeted protein quantitation.

CIEF method optimization: development of robust and reproducible protein reagent characterization in the clinical immunodiagnostic industry.

Several method parameters have been refined for application of CIEF methods to provide optimal capillary robustness and performance longevity while maintaining desired analytical output for the ever increasing characterization scrutiny of protein reagents used in clinical assay formulations. Demonstrated here are significant modifications to the existing protocols in order to attain a robust, reproducible method that achieves as much as a 20-fold increase in the number of consecutive runs before capillary degradation. Not only is it a concern for the rudimentary analysis of acidic and basic components of the isoform profile for monoclonal antibodies, but a comprehensive identification of each individual isoform to obtain a characteristic fingerprint is necessary for minor distinguishable properties between multiple proteins in unambiguous identification.

Utility of a direct dual-mode development analysis on blotted protein mixtures.

Classic blotting methods remain a commonly applied approach to specific protein identification in gel electrophoresis of complex mixtures despite the inherent difficulty in band or spot matching due to significant variability of protein migration or localization in replicate blotting experiments. A direct application of both protein stain and protein blotting on a single membrane significantly reduces the complexity of the experiment and provides increased confidence of signal matching. Digital alignment of images acquired from both total protein stain and blotting development modes on a single membrane allows unambiguous spot or band assignments in these experiments as well as retention of quantitative information acquired from both modes of signal generation.

Structural characterization of glycoprotein NGAL, an early predictive biomarker for acute kidney injury.

Neutrophil gelatinase-associated lipocalin (NGAL) is a promising new renal biomarker that can reduce the time to diagnose acute kidney injury (AKI). There is little information available about complex glycans on NGAL. Detailed structural characterization of NGAL is necessary to understand the structural variability of NGAL used as a standard in the NGAL immunoassay.

Development of a dot-blot assay for screening monoclonal antibodies to low-molecular-mass drugs.

Dot-blot is a versatile and simple analysis to perform. We adapted this method as a simple identity test for monoclonal antibodies to a number of small compounds: three transplant drugs, an anticonvulsant, a steroid, an anticancer drug, and an antibiotic. Immunology-based identity tests using low-molecular-mass organic compounds have historically been a challenge to develop.

Linker-mediated modulation of the chemiluminescent signal from N(10)-(3-Sulfopropyl)-N-sulfonylacridinium-9-carboxamide tracers.

Four chemiluminescent N-sulfonylacridinium-9-carboxamide active esters (17-20) were prepared from the corresponding acids and coupled to both of the aminated phenobarbital (13) and N-(6-aminohexyl)phenytoin (16) haptens. The level of signal produced by chemiluminescent N-sulfonylacridinium-9-carboxamide phenobarbital and phenytoin tracers in a solid-phase immunoassay format was found to be modulated by at least 20-fold by the judicious choice of the reactive acridinium-hapten linking group.

Resin-supported labeling reagents.

Resin-supported fluorescein, coumarin, acridinium, and biotin active esters were prepared from a new N-hydroxysuccinimidyl resin in high yield. The active esters were used to prepare representative conjugates with estriol, thyroxine, phenytoin, and desipramine haptens without need for purification beyond removal of the spent resin.

Tracermer signal generators: an arborescent approach to the incorporation of multiple chemiluminescent labels.

The synthesis, conjugation, and chemiluminescent evaluation of zero, first, and second order acridinium-based Tracermer signal generators are described. Members of this family of labels have potential use as tracers in diagnostic assays and are structurally similar to arborol dendrimers. Tracermer-BSA conjugates showed up to a sixfold increase in light emission compared to the normal acridinium label.

Preparation of succinimidyl and pentafluorophenyl active esters of 5- and 6-carboxyfluorescein.

A mixture of 5- and 6-carboxyfluorescein was activated with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride in the presence of either N-hydroxysuccinimide or pentafluorophenol to give the corresponding succinimidyl and pentafluorophenyl esters. The regioisomeric mixtures were separated to give the 5- and 6-succinimidyl and pentafluorophenyl active esters in > 98% purity.

Quantitative determination of E- and Z-doxepin and E- and Z-desmethyldoxepin by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method was developed for the quantitative, simultaneous determination of the following four compounds in serum: E-doxepin, Z-doxepin, E-desmethyldoxepin, and Z-desmethyldoxepin. A 3-microns analytical silica column (6 x 100 mm) was employed with the mobile phase 0.025 M phosphate:acetonitrile:n-nonylamine (80:20:1).

Immunoassay reagents for psychoactive drugs. Part 5. Quantitative determination of imipramine and desipramine by fluorescence polarization immunoassay.

Two methods for the quantitative determination of imipramine (IMI) and desipramine (DMI) by fluorescence polarization immunoassay (FPIA) are described. One immunoassay allows for the accurate quantification of imipramine in the presence of desipramine, while the other allows for the accurate quantification of desipramine in the presence of imipramine.

Characterization of protein-hapten conjugates. 1. Matrix-assisted laser desorption ionization mass spectrometry of immuno BSA-hapten conjugates and comparison with other characterization methods.

Several different low molecular weight haptens were conjugated to BSA to produce immunogens useful for antibody development. The extent of BSA modification due to covalent attachment of hapten was estimated by matrix-assisted laser desorption ionization mass spectrometry. The average number of hapten incorporated to immunogen was determined from the difference in the measured molecular weights of the conjugate from nonmodified BSA.

Immunoassay reagents for thyroid testing. 1. Synthesis of thyroxine conjugates.

Immunoreagents were designed to improve the performance of a commercial fluorescent polarization immunoassay for thyroxine. The thyroxine immunogen was prepared by selective coupling of N-acetyl-L-thyroxine to BSA via an aminocaproic acid spacer arm. The fluorescent tracer was prepared by a multistep reaction sequence which relied on extensive use of orthogonal protecting groups.

Immunoassay reagents for psychoactive drugs. Part 4. Quantitative determination of amitriptyline and nortriptyline by fluorescence polarization immunoassay.

Methods for the quantitative determination of amitriptyline and nortriptyline by fluorescence polarization immunoassay (FPIA) is described. One immunoassay allows for the accurate quantification of amitriptyline in the presence of nortriptyline while the second immunoassay allows for the accurate quantification of nortriptyline in the presence of amitriptyline.

Immunoassay reagents for psychoactive drugs. Part 3. Removal of phenothiazine interferences in the quantification of tricyclic antidepressants.

Phenothiazines and their metabolites are known to interfere in the quantification of tricyclic antidepressants (TCAs). A method for selective chemical modification of phenothiazines by chloramine-T in the presence of TCAs is described. This method allows for accurate quantification of the TCA analyte in a serum sample without interference from the modified phenothiazine.

Immunoassay reagents for psychoactive drugs. II. The method for the development of antibodies specific to imipramine and desipramine.

Imipramine and desipramine were structurally modified by the attachment of spacer arms to the aromatic ring which were subsequently attached to bovine serum albumin. This approach utilized novel spacer arms and conjugation methods. This method yielded antisera with excellent selectivity and good titers.

Immunoassay reagents for psychoactive drugs. I. The method for the development of antibodies specific to amitriptyline and nortriptyline.

Amitriptyline and nortriptyline were structurally modified by the attachment of spacer arms to the aromatic ring which were subsequently attached to bovine serum albumin (BSA). Rabbits inoculated with these conjugates yielded polyclonal antisera with high selectivity and good titers. This approach required novel spacer arms and new conjugation methods.

Structure of (+/-)-(1R*,6S*)-1-benzyloxy-8,11,11-trimethyl-6-phenylthiobicyclo [5.3.1 ]undec-7-en-3-one.

C27H32O2S, Mr = 420.61, monoclinic, P2(1)/c, a = 8.4756(13), b = 14.

Structure of (+/-)-(1S*,3S*)-3-hydroxy-8,11,11-trimethyl-9-oxobicyclo[5.3.1]undec-7- en-1-yl benzoate.

C21H26O4, Mr = 342.43, monoclinic, P2(1)/n, a = 7.211 (2), b = 19.

Structure of (+/-)-(1R*,4S*,6S*)-1-benzyloxy-4,8,11,11-tetramethyl-6- phenylthio-bicyclo[5.3.1]undec-7-en-3-one.

C28H34O2S, Mr = 434.64, triclinic, P1, a = 8.9868 (7), b = 11.