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1.
J Immunol Methods ; 516: 113460, 2023 05.
Article in English | MEDLINE | ID: mdl-36967060

ABSTRACT

The performance of enzyme-linked immunoassays is directly dependent on the storage, handling, and long-term stability of the critical reagents used in the assay. Currently, antibody reagents are routinely stored as concentrated, multi-use, frozen aliquots. This practice results in material waste, adds complexity to laboratory workflows, and can compromise reagents via cross-contamination and freeze-thaw damage. While refrigeration or freezing can slow down many degradation processes, the freezing process itself can have damaging effects, including introduction of aggregation and microheterogeneity. To address these challenges, we evaluated the application of capillary-mediated vitrification (CMV) as a tool for storing antibody reagents in a thermostable, single-use format. CMV is a novel biopreservation method that enables vitrification of biological materials without freezing. Using an anti-human IgG-alkaline phosphatase conjugate as a model system, we prepared CMV-stabilized aliquots which were stored in a single-use format at temperatures ranging from 25 to 55 °C for up to 3 months. Each stabilized aliquot contained enough antibody to perform a single assay run. We evaluated the assay performance and functional stability of the CMV-stabilized reagents using a plate-based ELISA. Assays run using the CMV stabilized reagents exhibited good linearity and precision that was comparable to results obtained with a frozen control. Throughout the stability study, the maximum signal and EC50s observed for ELISAs run using CMV-stabilized reagents were generally consistent with those obtained using a frozen control. These results indicate that the CMV process has the potential to improve both reagent stability and long-term assay performance, while also reducing reagent waste and simplifying assay workflows.


Subject(s)
Cytomegalovirus Infections , Vitrification , Humans , Indicators and Reagents , Immunoassay/methods , Antigens , Immunoglobulin G
2.
AAPS J ; 24(4): 75, 2022 06 16.
Article in English | MEDLINE | ID: mdl-35710853

ABSTRACT

RNA is a fundamental tool for molecular and cellular biology research. The recent COVID-19 pandemic has proved it is also invaluable in vaccine development. However, the need for cold storage to maintain RNA integrity and the practical and economic burden associated with cold chain logistics highlight the need for new and improved preservation methods. We recently showed the use of capillary-mediated vitrification (CMV), as a tool for stabilizing temperature-sensitive enzymes. Here, we demonstrate the use of CMV as a method to preserve mRNA. The CMV process was performed by formulating a green fluorescent protein (GFP)-encoding mRNA with common excipients, applying the solution to a porous support, referred to as the scaffold, and drying the samples under vacuum for 30 min. The CMV preserved samples were stored at 55 °C for up to 100 days or 25 °C for 60 days and analyzed by electrophoresis and for transfection efficiency in a cell-based assay. The 55 °C-stressed mRNA exhibited comparable electrophoresis banding patterns and band intensity when compared to a frozen, liquid control. Additionally, the CMV stabilized mRNA maintained 97.5 ± 8.7% transfection efficiency after 77 days and 78.4 ± 3.9% after 100 days when stored 55 °C and analyzed using a cell-based assay in the CHO-K1 cell line. In contrast, a liquid control exhibited no bands on the electrophoresis gel and lost all transfection activity after being stored overnight at 55 °C. Likewise, after 60 days at 25 °C, the CMV-processed samples had full transfection activity while the activity of the liquid control was reduced to 40.1 ± 4.6%. In conclusion, CMV is a simple formulation method that significantly enhances the thermal stability of mRNA, requires minimal processing time, and could enable formulation of mRNA that can tolerate exposure to temperatures well above 25 °C during shipment and deployment in extreme environments.


Subject(s)
COVID-19 , Cytomegalovirus Infections , Animals , CHO Cells , COVID-19/prevention & control , Cricetinae , Freeze Drying , Humans , Pandemics , RNA, Messenger , Temperature , Vitrification
3.
J Pharm Sci ; 111(8): 2280-2287, 2022 08.
Article in English | MEDLINE | ID: mdl-35235844

ABSTRACT

Capillary-mediated vitrification (CMV) is a novel method for stabilizing biological molecules and complexes. CMV leverages capillary evaporation to enable rapid desiccation of aqueous solutions while avoiding both freezing and boiling. In the CMV process, an aqueous solution containing the biological material of interest and common excipients is applied to a solid, porous support, referred to as the scaffold, and desiccated under vacuum. The pores within the scaffold accelerate drying by increasing surface area while preventing boiling through the interaction of the vapor pressure, capillary forces, and viscous forces. The process, which can be completed in under an hour, produces an amorphous dried product with enhanced thermal stability. In this study, CMV is demonstrated using luciferase as a model system. Using a 30-minute drying time, residual moisture levels of <4% were achieved. CMV-stabilized luciferase maintained full activity when stored for up to 6 weeks at 25 °C and >70% activity after 6 weeks at temperatures up to 45 °C. The liquid formulated enzyme lost all activity after 1 day at 37 °C or 4 h at temperatures above 37 °C. The data presented in this report demonstrate that CMV is a promising alternative to traditional biopreservation methods.


Subject(s)
Cytomegalovirus Infections , Desiccation , Desiccation/methods , Drug Stability , Excipients , Freeze Drying/methods , Humans , Proteins , Vitrification
4.
AAPS J ; 22(6): 145, 2020 11 08.
Article in English | MEDLINE | ID: mdl-33161491

ABSTRACT

Monoclonal antibodies (mAbs) are widely used as critical reagents in analytical assays. While regulatory guidelines exist for stability monitoring of biopharmaceutical antibodies, they do not apply directly to the stability of mAbs used as assay reagent. We investigated alternative approaches to real-time stability monitoring of assay reagents. We compared functional (ELISA and cell-based) and biochemical (aggregation, deamidation) assay results using temperature-stressed mAb reagents. Data from both assay groups were compared for indications of antibody degradation. Arrhenius model kinetics was used to further extrapolate stability trends. Changes detected by traditionally monitored biochemical changes were not directly predictive of assay function. Instead, monitoring of reportable results was a closer indication of changes in assay performance related to mAb degradation. Using Arrhenius kinetic modeling, we combined forced degradation of individual reagents with reportable assay results to classify reagents into risk groups with associated re-evaluation and monitoring plans. This combined approach mitigates risk by monitoring each mAb reagent individually under stressed conditions while streamlining expiry assignment through simplified Arrhenius kinetics with only limited real-time stability data.


Subject(s)
Antibodies, Monoclonal/chemistry , Protein Denaturation , Proteolysis , Arabidopsis Proteins , Biological Assay/methods , Guidelines as Topic , Indicators and Reagents/chemistry , Indicators and Reagents/standards , Laboratories/standards , Models, Biological , Nuclear Proteins , Quality Control
5.
BMC Infect Dis ; 18(1): 613, 2018 Dec 04.
Article in English | MEDLINE | ID: mdl-30509199

ABSTRACT

BACKGROUND: Community-acquired pneumonia is a leading infectious cause of hospitalization. A few vaccines exist to prevent pneumococcal disease in adults, including a pneumococcal polysaccharide unconjugated vaccine and a protein conjugated polysaccharide vaccine. Previous studies on the human immune response to the unconjugated vaccine showed that the vaccine boosted the existing memory B cells. In the present study, we investigated the human B cell immune response following pneumococcal polysaccharide conjugate vaccination. METHODS: Plasmablast B cells from a pneumococcal polysaccharide conjugate vaccinee were isolated and cloned for analysis. In response to primary vaccination, identical sequences from the plasmablast-derived antibodies were identified from multiple B cells, demonstrating evidence of clonal expansion. We evaluated the binding specificity of these human monoclonal antibodies in immunoassays, and tested there in vitro function in a multiplexed opsonophagocytic assay (MOPA). To characterize the plasmablast B cell response to the pneumococcal conjugated vaccine, the germline usage and the variable region somatic hypermutations on these antibodies were analyzed. Furthermore, a serotype 4 polysaccharide-specific antibody was tested in an animal challenge study to explore the in vivo functional activity. RESULTS: The data suggests that the pneumococcal polysaccharide conjugate vaccine boosted memory B cell responses, likely derived from previous pneumococcal exposure. The majority of the plasmablast-derived antibodies contained higher numbers of variable region somatic hypermutations and evidence for selection, as demonstrated by replacement to silent ratio's (R/S) greater than 2.9 in the complementarity-determining regions (CDRs). In addition, we found that VH3/JH4 was the predominant germline sequence used in these polysaccharide-specific B cells. All of the tested antibodies demonstrated narrow polysaccharide specificity in ELISA binding, and demonstrated functional opsonophagocytic killing (OPK) activity in the MOPA assay. The in-vivo animal challenge study showed that the tested serotype 4 polysaccharide-specific antibody demonstrated a potent protective effect when administered prior to bacterial challenge. CONCLUSIONS: The findings on the pneumococcal polysaccharide conjugate vaccine responses from a vaccinated subject reported in this study are similar to previously published data on the pneumococcal polysaccharide unconjugated vaccine responses. In both vaccine regimens, the pre-existing human memory B cells were expanded after vaccination with preferential use of the germline VH3/JH4 genes.


Subject(s)
Antibodies, Monoclonal/isolation & purification , B-Lymphocytes/immunology , Immunologic Memory , Pneumococcal Infections/prevention & control , Pneumococcal Vaccines/therapeutic use , Somatic Hypermutation, Immunoglobulin , Adult , Animals , Antibodies, Bacterial/immunology , B-Lymphocytes/metabolism , Cells, Cultured , Female , Gene Rearrangement, B-Lymphocyte/genetics , Gene Rearrangement, B-Lymphocyte/immunology , Humans , Immunologic Memory/genetics , Immunologic Memory/immunology , Mice , Mice, Inbred C57BL , Pneumococcal Infections/genetics , Pneumococcal Infections/immunology , Pneumococcal Infections/microbiology , Pneumococcal Vaccines/immunology , Serogroup , Somatic Hypermutation, Immunoglobulin/genetics , Somatic Hypermutation, Immunoglobulin/immunology , Streptococcus pneumoniae/immunology , Vaccination , Vaccines, Conjugate/immunology , Vaccines, Conjugate/therapeutic use
6.
Bioanalysis ; 10(3): 163-180, 2018 Feb.
Article in English | MEDLINE | ID: mdl-29333863

ABSTRACT

Compared with biologics, vaccine potency assays represent a special challenge due to their unique compositions, multivalency, long life cycles and global distribution. Historically, vaccines were released using in vivo potency assays requiring immunization of dozens of animals. Modern vaccines use a variety of newer analytical tools including biochemical, cell-based and immunochemical methods to measure potency. The choice of analytics largely depends on the mechanism of action and ability to ensure lot-to-lot consistency. Live vaccines often require cell-based assays to ensure infectivity, whereas recombinant vaccine potency can be reliably monitored with immunoassays. Several case studies are presented to demonstrate the relationship between mechanism of action and potency assay. A high-level decision tree is presented to assist with assay selection.


Subject(s)
Biological Assay , Drug Evaluation, Preclinical/methods , Vaccine Potency , Vaccines, Attenuated/immunology , Vaccines, Inactivated/immunology , Vaccines, Subunit/immunology , Animals , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Hep G2 Cells , Humans , Immunogenicity, Vaccine , Mice , Vaccination , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/genetics , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/genetics , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/genetics , Vero Cells
7.
Vaccine ; 35(41): 5495-5502, 2017 10 04.
Article in English | MEDLINE | ID: mdl-28433327

ABSTRACT

Vaccine in vitro potency assays are vital regulatory tests that are used to confirm the presence and concentration of an antigen of interest in a form that directly or indirectly relates to protective activity in patients. Current assays come in many forms, but they almost exclusively use antibody reagents for selective detection of the target antigen. Antibodies provide specific recognition of vaccine antigens but also exhibit drawbacks such as stability limitations, cost, and lot-to-lot variation, which can make it challenging to maintain the reagent throughout the lifetime of the vaccine. We explored replacing antibodies with aptamers. Aptamers are macromolecules, such as nucleic acids, which can bind to their targets with high specificity and affinity, similar to that of antibodies. Some of the advantages of using aptamers over antibodies is that aptamers can be more stable, smaller, less expensive to produce, synthesized in vitro, and logistically easier to supply throughout the multi-decade lifespan of a commercial vaccine. We created modified DNA aptamers against the common vaccine carrier protein, CRM197. Several aptamers were discovered and one was chosen for further characterization. The binding kinetics of the aptamer revealed an off-rate 16-fold slower than anti-CRM197 antibodies used for comparison. The aptamers were more sensitive than available antibodies in some assay formats and comparable in others. The aptamer epitope was mapped to the receptor-binding domain of CRM197, a site adjacent to a known antibody binding site. These data address some key aspects for a path forward in replacing antibodies with aptamers for use as critical reagents in vaccine assays. We further highlight the possibility of using nucleic acid reagents to develop next generation potency assays.


Subject(s)
Antibodies/immunology , Aptamers, Nucleotide/immunology , Antigens/immunology , Bacterial Proteins/immunology , Biological Assay/methods , Humans , Protein Binding/immunology , Vaccine Potency , Vaccines/immunology
8.
Anal Chem ; 89(6): 3554-3561, 2017 03 21.
Article in English | MEDLINE | ID: mdl-28233502

ABSTRACT

Measuring vaccine potency is critical for vaccine release and is often accomplished using antibody-based ELISAs. Antibodies can be associated with significant drawbacks that are often overlooked including lot-to-lot variability, problems with cell-line maintenance, limited stability, high cost, and long discovery lead times. Here, we address many of these issues through the development of an aptamer, known as a slow off-rate modified DNA aptamer (SOMAmer), which targets a vaccine antigen in the human papillomavirus (HPV) vaccine Gardasil. The aptamer, termed HPV-07, was selected to bind the Type 16 virus-like-particle (VLP) formed by the self-assembling capsid protein L1. It is capable of binding with high sensitivity (EC50 of 0.1 to 0.4 µg/mL depending on assay format) while strongly discriminating against other VLP types. The aptamer competes for binding with the neutralizing antibody H16.V5, indicating at least partial recognition of a neutralizing and clinically relevant epitope. This makes it a useful reagent for measuring both potency and stability. When used in an ELISA format, the aptamer displays both high precision (intermediate precision of 6.3%) and a large linear range spanning from 25% to 200% of a typical formulation. To further exploit the advantages of aptamers, a simplified mix and read assay was also developed. This assay format offers significant time and resource reductions compared to a traditional ELISA. These results show aptamers are suitable reagents for biological potency assays, and we expect that their implementation could improve upon current assay formats.


Subject(s)
Antigens, Viral/immunology , Aptamers, Nucleotide/immunology , Epitopes/immunology , Human papillomavirus 16/immunology , Papillomavirus Vaccines/immunology , Antigen-Antibody Reactions , Aptamers, Nucleotide/chemical synthesis , Aptamers, Nucleotide/chemistry , Enzyme-Linked Immunosorbent Assay , Humans
9.
J Immunol Methods ; 442: 20-28, 2017 03.
Article in English | MEDLINE | ID: mdl-28034712

ABSTRACT

Dilutions are a common source of analytical error, both in terms of accuracy and precision, and a common source of analyst mistakes. When serial dilutions are used, errors compound, even when employing laboratory automation. Direct point dilutions instead of serial dilutions can reduce error but is often impractical as they require either large diluent volumes or very small sample volumes when performed with traditional liquid handling equipment. We evaluated preparation of dilution curves using a picoliter digital dispenser, the HP, Inc. / TECAN D300 which is capable of accurately delivering picoliter volumes directly into sample wells filled with assay diluent. Dilution linearity and variability of the direct dilutions were similar to or less than those generated with a traditional liquid handler as measured using a fluorophore assay and an ELISA used to measure vaccine potency. Minimum concentrations for detergent in the dispensed sample were identified but no correlation with detergent characteristics was observed. The tolerance to protein in the sample was evaluated as well with up to 5% BSA having no impact on dispense linearity and precision. We found the digital dispenser to reduce automation complexity while maintaining or improving assay performance in addition to facilitating complex plate lay-outs.


Subject(s)
Enzyme-Linked Immunosorbent Assay/instrumentation , High-Throughput Screening Assays/instrumentation , Vaccine Potency , Automation, Laboratory , Calibration , Detergents/chemistry , Enzyme-Linked Immunosorbent Assay/standards , Equipment Design , Fluorescent Dyes/chemistry , High-Throughput Screening Assays/standards , Miniaturization , Reference Standards , Reproducibility of Results , Spectrometry, Fluorescence
10.
Bioanalysis ; 8(14): 1451-64, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27314462

ABSTRACT

BACKGROUND: Dilution bias is a major cause of immunoassay variability due to the lack of an internal standard to determine the true versus the expected dilution value. METHODOLOGY: We used an internal control to measure dilution bias in an ELISA. Acridine-orange was added at the first dilution step and monitored throughout dilutions. Assay results were corrected using the fluorescent signal ratio between samples and reference. Acridine dilution correlated with analyte-specific assay measurements (R2 = 0.987). Correction of assay results with the measured dilution factor improved both accuracy and precision resulting in a reduction of >50% %CV reduction. CONCLUSION: Dilution correction can significantly improve accuracy and precision of immunoassays. Additional control strategies may further mitigate other sources of variability.


Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Acridine Orange/analysis , Enzyme-Linked Immunosorbent Assay/standards , Fluorescence , Fluorescent Dyes/analysis , Indicator Dilution Techniques
11.
Anal Bioanal Chem ; 408(15): 3969-79, 2016 Jun.
Article in English | MEDLINE | ID: mdl-27116421

ABSTRACT

Microtiter plate-based assays are a common tool in biochemical and analytical labs. Despite widespread use, results generated in microtiter plate-based assays are often impacted by positional bias, in which variability in raw signal measurements are not uniform in all regions of the plate. Since small positional effects can disproportionately affect assay results and the reliability of the data, an effective mitigation strategy is critical. Commonly used mitigation strategies include avoiding the use of outer regions of the plate, replicating treatments within and between plates, and randomizing placement of treatments within and between plates. These strategies often introduce complexity while only partially mitigating positional effects and significantly reducing assay throughput. To reduce positional bias more effectively, we developed a novel block-randomized plate layout. Unlike a completely randomized layout, the block randomization scheme coordinates placement of specific curve regions into pre-defined blocks on the plate based on key experimental findings and assumptions about the distribution of assay bias and variability. Using the block-randomized plate layout, we demonstrated a mean bias reduction of relative potency estimates from 6.3 to 1.1 % in a sandwich enzyme-linked immunosorbent assay (ELISA) used for vaccine release. In addition, imprecision in relative potency estimates decreased from 10.2 to 4.5 % CV. Using simulations, we also demonstrated the impact of assay bias on measurement confidence and its relation to replication strategies. We outlined the underlying concepts of the block randomization scheme to potentially apply to other microtiter-based assays.


Subject(s)
Enzyme-Linked Immunosorbent Assay/instrumentation , Vaccines/analysis , Enzyme-Linked Immunosorbent Assay/methods , Random Allocation
12.
Bioanalysis ; 5(20): 2531-45, 2013 Oct.
Article in English | MEDLINE | ID: mdl-24138626

ABSTRACT

BACKGROUND: As quality by design (QbD) for pharmaceutical product development is being expanded to include analytical methods, we applied QbD to the development of antigenicity assays measuring in vitro relative potency of a quadrivalent vaccine candidate. RESULTS: After establishing development targets together with customers, immunoassays were developed to meet objectives. Statistical design of experiments was used to optimize method parameters and establish a design space. Systematic risk analysis enabled identification of potential risks to method performance that were mitigated by investigating the available design space around risk factors and by establishing appropriate control strategies. CONCLUSION: We found QbD-based method development was a more efficient and systematic approach that could also potentially facilitate assay transfers and life cycle management.


Subject(s)
Antigens/analysis , Factor Analysis, Statistical , Fluorescent Antibody Technique, Direct/standards , Fluorescent Antibody Technique, Indirect/standards , Vaccines/analysis , Animals , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/isolation & purification , Antibody Specificity , Humans , Immunoglobulin G/blood , Immunoglobulin G/isolation & purification , Rabbits , Reproducibility of Results , Risk Assessment , Sensitivity and Specificity , Vaccines/immunology , Validation Studies as Topic
13.
Clin Vaccine Immunol ; 16(5): 739-48, 2009 May.
Article in English | MEDLINE | ID: mdl-19321693

ABSTRACT

A direct binding Luminex assay has been developed and validated for the detection of human immunoglobulin G (IgG) antibodies to the Staphylococcus aureus iron surface determinant B protein (IsdB) in serum following natural infection or immunization with investigational Saccharomyces cerevisiae-derived IsdB-based vaccines. To ensure that IsdB-specific IgG antibodies are measured following immunization with S. cerevisiae-derived IsdB, an Escherichia coli-produced IsdB antigen is used in the assay. The IsdB antigen is covalently conjugated to maleimide microspheres via an engineered carboxy-terminal cysteine residue. Antibody titers are determined in a direct binding format, where the phycoerythrin-labeled monoclonal antibody (HP6043) specific for IgG1 to IgG4 binds to human serum IgG antibodies. Fluorescent signal emitted from bound HP6043 is directly proportional to an individual's antibody levels. A pooled human reference serum from vaccinees with high titers to IsdB is used to generate a 12-point standard curve. The correlation of mean fluorescent intensity (MFI) units to microg/ml of IsdB-specific IgG is made by interpolating the MFI data through a four-parameter curve-fitting algorithm. The assay is sensitive to 1.06 microg/ml with a dynamic range of 2.1 to 10,625 microg/ml. The overall specificity of the assay is >96% and the linearity (parallelism) of the assay is -4% per 10-fold dilution. The total precision of the assay was 16.6% relative standard deviation across three different IsdB antigen lots, three different microsphere lots, two secondary antibody lots, and three different operators. The assay has proven useful for evaluating the immune response following the administration of different dosages and formulations of investigational IsdB-based vaccines.


Subject(s)
Antibodies, Bacterial/blood , Cation Transport Proteins/immunology , Immunoglobulin G/blood , Adult , Aged , Female , Humans , Immunoassay/methods , Male , Middle Aged , Recombinant Proteins/immunology , Sensitivity and Specificity , Serum/immunology , Staphylococcus aureus/immunology , Young Adult
14.
Hum Vaccin ; 2(4): 147-54, 2006.
Article in English | MEDLINE | ID: mdl-17012891

ABSTRACT

The thermostability of GARDASIL (Merck & Co., Inc, Whitehouse Station, NJ, USA), a developmental vaccine against human papillomavirus (HPV), was evaluated using an enzyme immunoassay, referred to as the in vitro relative potency (IVRP) assay and differential scanning calorimetry (DSC). Gardasil samples were stored at temperatures ranging from 4 to 42 degrees C and tested for IVRP at various time points. Extrapolation of the IVRP results indicates GARDASIL is extremely stable. The half-life of the vaccine is estimated to be 130 months or longer at temperatures up to 25 degrees C. At 37 degrees C, the half-life is predicted to be 18 months and at 42 degrees C, the half-life is predicted to be approximately three months. Differential scanning calorimetry (DSC) analysis was used to evaluate the process of protein denaturation during a rapid temperature increase (as opposed to long-term storage at a specific temperature). Differences were seen among the DSC profiles of the four HPV types tested. This indicates that small differences in the amino acid structure can have a significant effect on the intermolecular contacts that stabilize the L1 proteins and the VLP assembly. For the Gardasil samples evaluated here, DSC results demonstrated the relative overall structural stability of the VLPs, but were not predictive of the excellent long-term stability observed with the IVRP assay.


Subject(s)
Viral Vaccines/standards , Calorimetry, Differential Scanning , Half-Life , Hot Temperature , Human Papillomavirus Recombinant Vaccine Quadrivalent, Types 6, 11, 16, 18 , Papillomavirus Vaccines , Virion/physiology
15.
Carbohydr Res ; 337(4): 335-44, 2002 Feb 18.
Article in English | MEDLINE | ID: mdl-11841814

ABSTRACT

NMR spectroscopy can be used to characterize bacterial polysaccharides such as that of Streptococcus pneumoniae type 1 which is a component of the 23-valent pneumococcal vaccine in clinical use. This particular polysaccharide gives NMR spectra with wide lines apparently due to restricted molecular mobility and chain flexibility which leads to rapid dipolar T(2) relaxation limiting the possibility of detailed spectral analysis. Removal of O-acetyl groups found on approximately two thirds of the repeating subunits of pneumococcal type 1 capsule leads to narrower NMR lines facilitating a complete assignment of the 1H and 13C NMR spectra. Degradation of the polysaccharide by periodate oxidation followed by base treatment leads to an oligosaccharide fragment of approximately three repeating trisaccharide units. This oligosaccharide has narrow NMR lines and 1H and 13C assignments very similar to those of the O-deacetylated polysaccharide. In the native polysaccharide, O-acetyl groups are located on the 2- and 3-positions of the 4-linked galacturonic acid residue providing protection against periodate oxidation. Analysis of NOESY spectra combined with molecular modeling of the oligosaccharide shows that flexibility occurs in certain of the saccharide linkages.


Subject(s)
Polysaccharides, Bacterial/chemistry , Streptococcus pneumoniae/chemistry , Bacterial Vaccines/chemistry , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Oxidation-Reduction
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