A5037448040- Monoclonal and Polyclonal Antibodies Research
- T-cell and B-cell Immunology
- Immune Cell Function and Interaction
- Glycosylation and Glycoproteins Research
- Viral Infectious Diseases and Gene Expression in Insects
- Protein purification and stability
- Blood groups and transfusion
- Immunotherapy and Immune Responses
- Influenza Virus Research Studies
- Immunodeficiency and Autoimmune Disorders
- Chemical Synthesis and Analysis
- Bacteriophages and microbial interactions
- Biosimilars and Bioanalytical Methods
- HIV Research and Treatment
- Advanced Proteomics Techniques and Applications
- Transgenic Plants and Applications
- Adenosine and Purinergic Signaling
- Immune Response and Inflammation
- Respiratory viral infections research
- Cytomegalovirus and herpesvirus research
- CAR-T cell therapy research
- Cell Adhesion Molecules Research
- Diabetes and associated disorders
- Cellular transport and secretion
- Advanced Biosensing Techniques and Applications
The University of Texas at Austin
2010-2016
Sequence analysis of immunoglobulin (Ig) heavy and light chain transcripts can refine categorization B cell subpopulations shed on the selective forces that act during immune responses or dysregulation, such as autoimmunity, allergy, malignancy. High-throughput sequencing yields Ig transcript collections unprecedented size. The authoritative web-based IMGT/HighV-QUEST program is capable analyzing large provides annotated output files to describe many key properties transcripts. However,...
Significance Most vaccines confer immunity by eliciting long-term production of antibodies that bind to and neutralize the vaccine antigen. Remarkably, very little is known regarding identities, sequence diversity, relative concentrations, or binding functionalities mAbs comprise serum repertoire elicited vaccination. Here, we have delineated constituent human IgG after vaccination examined their relationship antibody V gene encoded circulating B cells. The results detail molecular...
We have developed and validated a methodology for determining the antibody composition of polyclonal serum response after immunization. Pepsin-digested IgGs were subjected to standard antigen-affinity chromatography, resulting elution, wash, flow-through fractions analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification individual monoclonal antibodies required generation database IgG variable gene (V-gene) sequences constructed NextGen sequencing...
Abstract Obtaining full-length antibody heavy- and light-chain variable regions from individual B cells at scale remains a challenging problem. Here we use high-throughput single-cell B-cell receptor sequencing (scBCR-seq) to obtain accurately paired in massively parallel fashion. We sequenced more than 250,000 rat, mouse human repertoires characterize their lineages expansion. In addition, immunized rats with chicken ovalbumin profiled antigen-reactive lymph nodes of animals. The scBCR-seq...
Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of hematopoiesis. In particular, NOD-scid-IL2Rγnull engrafted have been shown to reasonable levels T and B cell repopulation can mount T-cell dependent responses; however, antigen-specific B-cell responses this model are generally poor. We explored whether developmental defects immunoglobulin gene repertoire might be partly responsible for low level antibody model. Roche 454 sequencing was used...
Rabbits have been used extensively as a model system for the elucidation of mechanism immunoglobulin diversification and production antibodies. We employed Next Generation Sequencing to analyze Ig germline V J gene usage, CDR3 length amino acid composition, conversion frequencies within functional (transcribed) IgG repertoire New Zealand white rabbit (Oryctolagus cuniculus). Several previously unannotated heavy chain variable (VH) light (VL) elements were deduced bioinformatically using...
The vast initial diversity of the antibody repertoire is generated centrally by means a complex series V (D) J gene rearrangement events, variation in site segment joining, and TdT catalyzed N- region addition. Although great, close inspection has revealed distinct unique characteristics repertoires expressed different B cell developmental subsets. In order to illustrate our approach analysis, we present an in-depth comparison usage, hydrophobicity, length, DH reading frame, amino acid usage...
Monoclonal antibodies (mAbs) have become a major class of protein therapeutics that target spectrum diseases ranging from cancers to infectious diseases. Similar any molecule, mAbs are susceptible chemical modifications during the manufacturing process, long-term storage, and in vivo circulation can impair their potency. One such modification is oxidation methionine residues. Chemical occur complementarity-determining regions (CDRs) lead abrogation antigen binding reduce drug's potency...
The immunoglobulin lambda (IGL) repertoires from two unrelated human blood samples, three NOD-scid-IL2Rγnull mice engrafted with hematopoietic stem cells and pairs of monozygotic twin samples were determined by Roche 454 sequencing to generate a total about 700 000 IGL sequences. We applied bioinformatic analysis examine wherein, surprisingly, ⩾20% CDR-L3 peptide sequences 'public' (shared across individuals); moreover, full-length protein (VJ recombinants) also present in the public domain....
An enzyme engineering technology involving yeast endoplasmic reticulum (ER) sequestration screening (YESS) has been recently developed. Here, a new method is established, in which the YESS platform combined with NextGen sequencing (NGS) to enable comprehensive survey of protease specificity. In this approach, combinatorial substrate library targeted ER and transported through secretory pathway, interacting any protease(s) residing ER. Multicolor FACS used isolate cells labeled...
Since the invention of Hybridoma technology by Milstein and Köhler in 1975, its application has greatly advanced antibody discovery process. The enables both functional screening long-term archival immortalized monoclonal producing B cells. Despite dependable cryopreservation for hybridoma cells, practicality storage been outpaced recent progress robotics automations, which routine identification thousands antigen specific clones. Such throughput increase imposes two nascent challenges...
Abstract Antibody variable domain sequence diversity is generated by recombination of germline segments. The third complementarity-determining region the heavy chain (CDR H3) highest and formed joining V H , D J segments combined with random nucleotide trimming additions between these We show that CDR H3 junctional segment length distributions are biased in human antibody repertoires as a function L utilization. Most biases apparent naive antigen experienced B cell compartments but not...
The proactive generation of anti-idiotypic antibodies (anti-IDs) against therapeutic with desirable properties is an important step in pre-clinical and clinical assay development supporting their bioanalytical programs. Here, we describe a robust platform to generate anti-IDs using rabbit single B cell sorting-culture cloning technology by immunizing rabbits drug Fab fragment sorting complementarity determining regions (CDRs) specific cells designed framework control as negative gate exclude...
Antibodies are fundamental effectors of humoral immunity, and have become a highly successful class therapeutics. There is increasing evidence that antibodies utilize transient homotypic interactions to enhance function, elucidation such can provide insights into their biology new opportunities for optimization as drugs. Yet the transitory nature weak makes them difficult investigate. Capitalizing on rich structural data high conservation, we characterized all ways antibody fragment...
Significance Weak transient interactions are fundamental to immune responses, enabling avidity-driven triggers for pathogen neutralization and cellular regulation. In contrast obligate binding that can be directly investigated structurally, the low or transitory abundance of weak make them difficult identify characterize. This study leverages receptor agonism systems sensitive oligomerization investigate homotypic interfaces between antibody Fab regions. Our results show self-association...
ABSTRACT Using high-throughput single-cell B-cell receptor sequencing (scBCR-seq) we obtained accurately paired full-length heavy- and light-chain variable domains from thousands of individual B cells in a massively parallel fashion. We sequenced more than 250,000 rat, mouse human repertoires to characterize their lineages expansion. In addition, immunized rats with chicken ovalbumin profiled antigen-reactive lymph nodes animals. The scBCR-seq data recovered 81% (n = 56/69) identified...
In the process of generating stable monoclonal antibody (mAb) producing cell lines, reagents such as methotrexate (MTX) or methionine sulfoximine (MSX) are often used. However, using selection reagent(s) increases possibility having higher occurrence sequence variants in expressed molecules due to effects MTX MSX on de novo nucleotide synthesis. Since inhibits glutamine synthase (GS) and results both amino acid nucleoside starvation, it is questioned whether supplementing nucleosides into...
Abstract Genetic and environmental cues shape the evolution of B cell Ig repertoire. Activation-induced cytidine deaminase (AID) is essential to generating diversity through isotype class switching somatic mutations, which then directly influence clonal selection. Impaired development in AID-knockout mice has made it difficult study diversification an aging Therefore, this report, we used a novel inducible mouse model discovered that deleting AID adult caused spontaneous germinal center...
Abstract Antibody variable domain sequence diversity is generated by recombination of germline segments. The third complementarity-determining region the heavy chain (CDR H3) highest and formed joining V H , D J segments combined with random nucleotide trimming additions between these We show that CDR H3 length distribution biased in human antibody repertoires as a function L segment utilization. Most biases are apparent naïve B cell compartment, significant bias towards shorter sequences...
Abstract Antibodies are fundamental effectors of humoral immunity, and have become a highly successful class therapeutics. There is increasing evidence that antibodies utilize transient homotypic interactions to enhance function, elucidation such can provide insights into their biology new opportunities for optimization as drugs. Yet the transitory nature weak makes them difficult investigate. Capitalizing on rich structural data high conservation, we characterized all ways antibody Fab...