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Major histocompatibility complex
Published in Gabriel Virella, Medical Immunology, 2019
Ellen Klohe, Janardan P. Pandey
An astonishing number of alleles have been identified at several HLA loci. Alleles are designated by a number that follows the relevant locus (e.g., HLA-A1, HLA-B8, or HLA-DR17). In fact, these are the most polymorphic structural gene loci in the entire human genome. Alleles may differ from each other by a single nucleotide or multiple nucleotides. Nucleotide substitutions may be either synchronous (no change to the amino acid in the encoded protein) or nonsynchronous (a different amino acid in the encoded protein). Many nonsynchronous substitutions are clustered in particular stretches of nucleotides in the gene sequence. This suggests that these differences did not arise by chance but rather were the result of natural selection. As of December 2017, 4,081 HLA-A, 4,950 HLA-B, and 3,685 HLA-C alleles have been identified. Many class II loci are also polymorphic, with 4,802 alleles currently identified. The IPD-IMGT/HLA Database is the repository for HLA DNA sequences and approved alleles and is located at https://www.ebi.ac.uk/ipd/imgt/hla/.
Preexisting antibodies targeting SARS-CoV-2 S2 cross-react with commensal gut bacteria and impact COVID-19 vaccine induced immunity
Published in Gut Microbes, 2022
Liqiu Jia, Shufeng Weng, Jing Wu, Xiangxiang Tian, Yifan Zhang, Xuyang Wang, Jing Wang, Dongmei Yan, Wanhai Wang, Fang Fang, Zhaoqin Zhu, Chao Qiu, Wenhong Zhang, Ying Xu, Yanmin Wan
The V(D)J genes of P144 reactive monoclonal antibodies were sequenced by AZENTA life science. Briefly, total RNA was extracted from hybridoma cells using Trizol reagent (Cat# R4801-02, Invitrogen, USA). 5` RACE was performed with SMARTer RACE cDNA Amplification Kit (Cat# 634923, Clontech, USA), total RNA input was 500–2000 ng. V(D)J genes of heavy and light chains were amplified by PCR. The PCR products were purified through gel extraction using a QIAquick Gel Extraction Kit (Cat# 28704, Qiagen, USA). NGS libraries were constructed by using VAHTS Universal DNA Library Prep Kit for Illumina (Cat# ND607, Vazyme, China). The qualified libraries were sequenced on the Illumina Miseq 2 × 300 platform (Illumina, San Diego, CA, USA). Raw fastq files were first subject to quality assessment. Adapters and bases with poor quality scores (Q value lower than 20) were removed using Trimmomatic (v0.36) to generate clean data (trimmed data). Pandaseq (2.10) was used to merge pair-end read. Merged sequences were processed by IgBLAST software to identify the V(D)J sequences. The reference sequences were obtained in IMGT database (IMGT, https://www.imgt.org/).
Impact of IgG subclass on molecular properties of monoclonal antibodies
Published in mAbs, 2021
Yu Tang, Paul Cain, Victor Anguiano, James J. Shih, Qing Chai, Yiqing Feng
Homology models of Fab region and Fc portion are built by the Molecular Operating Environment (MOE2019) (Chemical Computing Group, Montreal, Canada), based on crystal structures of Fab obtained in house and closest matching templates. Specifically, structure modeling of Fv-B, and Fv-C is based on crystal structures obtained in house. Fv-A homology model is built using MOE with the top scoring homology model among 5 models selected for further analysis. Solvent-accessible surface area, surface exposure (%) for each residue, and net charge and hydrophobicity score for each CDR were calculated based on the modeled structure using the protein properties analysis module (MOE2019). Specifically, hydrophobicity and net charge of each CDR segment are computed and reported in supplemental material. IMGT definition is applied to CDR segment and standard calculation by MOE protein descriptor calculation was used, which include pro_patch_cdr related calculation, Fv-CSP, and parsed score for each CDR segment. In addition, electrostatic potential calculation, spatial aggregation propensity (SAP), and spatial-charge map (SCM) were computed according to the method in the BIOVIA Discovery Studio 2020 software (San Diego, CA).
Therapeutic blockade of CXCR2 rapidly clears inflammation in arthritis and atopic dermatitis models: demonstration with surrogate and humanized antibodies
Published in mAbs, 2020
Md Jahangir Alam, Liang Xie, Caroline Ang, Farnaz Fahimi, Stephen B. Willingham, Andrew J. Kueh, Marco J. Herold, Charles R. Mackay, Remy Robert
Total cellular RNA was extracted from 5 × 106 hybridoma cells using TRIzol reagent (Invitrogen) and poly-A+ RNA was isolated from total cellular RNA using the Oligotex mRNA mini kit (Qiagen, Vic., Australia). Single-stranded (ss) cDNA was produced by reverse-transcription of RNA using the Omniscript reverse transcriptase kit (Qiagen). Reactions contained 500 ng of poly-A+ RNA and 10 pmol of isotype-specific anti-sense oligonucleotide primer (CH1-γ and CL-κ cDNA). A poly-G tail was appended to the 3ʹ-end of ss-cDNA using terminal transferase (NEB, MA, USA). Double-stranded cDNA was generated by PCR using Vent polymerase (NEB) with the poly-C anchor and CH-γ or CL-κ specific primers. The VH and VL genes were sequenced and analyzed by the IMGT® databases (the international ImMunoGeneTics information system®, http://www.imgt.org).