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

The tick-borne protozoan Babesia microti 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 B. microti antibody testing. A fully automated prototype B. microti antibody test was developed on the Architect instrument. The specificity was determined to be 99.98% in volunteer blood donors (n = 28,740) from areas considered to have low endemicity for B. microti The sensitivity of the prototype test was studied in experimentally infected macaques; a total of 128 samples were detected as positive whereas 125 were detected as positive with an indirect fluorescent antibody (IFA) test; additionally, 83 (89.2%) of the PCR-positive samples were detected in contrast to 81 (87.1%) using an IFA test. All PCR-positive samples that tested negative in the prototype antibody test were preseroconversion period samples. Following seroconversion, periods of intermittent parasitemia occurred; 17 PCR-negative samples drawn in between PCR-positive bleed dates tested positive both by the prototype test (robust reactivity) and IFA test (marginal reactivity) prior to the administration of therapeutic drugs, indicating that the PCR test failed to detect samples from persistently infected macaques. The prototype assay detected 56 of 58 (96.6%) human subjects diagnosed with clinical babesiosis by both PCR and IFA testing. Overall, the prototype anti-Babesia assay provides a highly sensitive and specific test for the diagnosis of B. microti infection. While PCR is preferred for detection of window-period parasitemia, antibody tests detect infected subjects during periods of low-level parasitemia.

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. Analysis of different antibody lots using analytical hydrophobic interaction chromatography (HIC) uncovered multiple peaks of varying size. Electrospray ionization mass spectrometry (ESI-MS) indicated that the antibody contained a cysteinylation post-translational modification in complementarity-determining region (CDR) 3 of the antibody light chain. Fractionation of the antibody by HIC followed by ESI-MS and Biacore showed that the different peaks were antibody containing zero, one, or two cysteinylation modifications, and that the modification interferes with the ability of the modified antibody arm to bind antigen. Molecular modeling of the modified region shows that this oxidation of an unpaired cysteine in the antibody CDR would block a potential antigen binding pocket, suggesting an inhibition mechanism.

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). The improved method is simple to implement, robust, accurate, and (most important) precise.

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. The MRM-based MS assay using a stable internal standard that improved the selectivity, specificity, and sensitivity of the protein quantitation. Also, plasma albumin depletion and affinity enrichment of TnI by anti-TnI mAb-coated microparticles reduced the sample complexity, enhanced the dynamic range, and further improved the detecting sensitivity of the targeted protein in the biological matrix. Therefore, quantitation of TnI, a low abundant protein in human plasma, has demonstrated the applicability of the targeted protein quantitation strategy through its epitope peptide determined by epitope mapping method.

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. In order to maintain the integrity of these modifications, extensive studies were conducted on an implemented system suitability standard protein with specifically defined parameters indicating either sufficient or poor separation performance.

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. A direct and simple method applying a fluorescent protein stain that is compatible with subsequent detection by antibody or lectin recognition factors along with common image adjustment software is examined. The utility of this blot dual-mode development method for direct protein recognition and quantification in one- and two-dimensional electrophoresis is demonstrated for bioanalytical objectives where replicate experiments are challenged by sample complexity.

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. We modified the traditional dot-blot assay to serve as an identity test for monoclonal antibodies to carbamazepine, sirolimus, tacrolimus, cyclosporine, cortisol, methotrexate, and gentamicin. The primary obstacle was the immobilization of these organic compounds on nitrocellulose as nitrocellulose is also soluble in most of the organic solvents in which the compounds are soluble. We evaluated different membranes, solvents, and chemical forms of these organic compounds to overcome this challenge. A number of incubation and washing solutions were also investigated. By varying the chemical form, concentration, and incubation conditions, a set of effective and reproducible identity tests were developed for these monoclonal antibodies.

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. This study demonstrated that 7-9% of mutant (C87S) recombinant NGAL was N-glycosylated and no O-glycosylation was detected. The NGAL sequence was confirmed by nanoLC/MS/MS following in gel and in solution trypsin digestion, and the N-glycosylation site was localized by MS/MS. Six different mutant recombinant NGAL samples (samples A-F) were analyzed in this study; however, these samples demonstrated two different glycan patterns. Forty-one N-glycans were detected in sample A and the more abundant N-glycans were unsialylated. Forty-three N-glycans were detected in sample F and the more abundant N-glycans were sialylated. Each of the other four samples (B-E) had a similar N-glycan pattern as sample F.