Dataset Information

A Refined Protocol for Identifying Citrulline-specific Monoclonal Antibodies from Single Human B Cells from Rheumatoid Arthritis Patient Material.

ABSTRACT: We describe here a detailed, refined protocol for the generation of citrulline-specific monoclonal antibodies from single human B cells from rheumatoid arthritis (RA) patients. This protocol provides a detailed guide of the procedure starting from single B cells of your choice and followed by amplification of the variable region of immunoglobulin genes by RT-PCR, subsequent immunoglobulin gene cloning, recombinant IgG1 monoclonal antibody (mAb) production and quality controls. The produced mAbs can be used for further studies including reactivity towards candidate antigens and functionality both in vitro and in vivo. This protocol can be used to generate antigen-specific mAbs from B cells derived from different tissues and compartments, including peripheral blood, synovial fluid, digested biopsies, bone marrow aspirations, and bronchoalveolar lavage fluid. Notably, although examples are given on how to identify citrulline-specific autoantibodies the general methods can also be applied to other reactivities.

SUBMITTER: Amara K

PROVIDER: S-EPMC7854072 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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A Refined Protocol for Identifying Citrulline-specific Monoclonal Antibodies from Single Human B Cells from Rheumatoid Arthritis Patient Material.

Amara Khaled K Israelsson Lena L Stålesen Ragnhild R Sahlström Peter P Steen Johanna J Malmström Vivianne V Grönwall Caroline C

Bio-protocol 20190905 17

We describe here a detailed, refined protocol for the generation of citrulline-specific monoclonal antibodies from single human B cells from rheumatoid arthritis (RA) patients. This protocol provides a detailed guide of the procedure starting from single B cells of your choice and followed by amplification of the variable region of immunoglobulin genes by RT-PCR, subsequent immunoglobulin gene cloning, recombinant IgG1 monoclonal antibody (mAb) production and quality controls. The produced mAbs ...[more]

PMID: 33654849

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Project description:BackgroundDifferential diagnosis among psoriatic arthritis (PsA) and seronegative rheumatoid arthritis (Abneg RA) can be challenging particularly in the clinical setting of peripheral phenotype and autoantibodies seronegativity. The aim of the study was to identify synovial tissue (ST) biomarkers differentially expressed in PsA and Abneg RA and test their predictive value of therapeutic response.MethodsThirty-four PsA patients [12 DMARD naive and 22 non-responder to methotrexate (MTX-IR)] with peripheral joint involvement and 55 Abneg RA (27 DMARD naive and 28 MTX-IR) underwent US-guided ST biopsy and immunohistochemistry (IHC) for CD68+, CD3+, CD20+, CD21+, CD117+, and CD138+ cells. After study entry, each DMARD-naive patient started MTX therapy and was followed in an outpatient setting for at least 6 months to define the achievement of Minimal Disease Activity (PsA) and DAS remission (Abneg RA) status respectively. Each IR-MTX patient was treated according to EULAR recommendations.ResultsAt study entry, IHC analysis revealed that PsA patients had comparable levels of lining and sublining CD68+ and sublining CD21+, CD20+, and CD3+ cells than Abneg RA, despite the therapeutic regimen. Moreover, regardless of the therapeutic scheme, PsA patients showed higher IHC score of CD117+ cells (p = 0.0004 and p = 0.0005 for naive and MTX-IR patients respectively) compared to Abneg RA patients. Conversely, Abneg RA patients showed higher IHC score of CD138+ cells, irrespective to the therapeutic scheme (p = 0.04 and p = 0.002 for naive and MTX-IR patients respectively). Analyzing the response rate to the therapeutic scheme, naive PsA patients reaching MDA status at 6 months follow-up, showed, at the study entry, lower IHC score of CD3+ cells compared to PsA patients not reaching this outcome (p = 0.02); conversely, naive Abneg RA patients reaching DAS remission status at 6 months follow-up, showed, at the study entry, lower IHC score of sublining CD68+ cells compared to Abneg RA patients not reaching this outcome (p < 0.001).ConclusionsCD117+ and CD138+ cells are differentially distributed among PsA and Abneg RA. Histological analysis of ST may help to solve the clinical overlap between the two diseases and provides prognostic data about the therapy success.

Dataset Information

A Refined Protocol for Identifying Citrulline-specific Monoclonal Antibodies from Single Human B Cells from Rheumatoid Arthritis Patient Material.

Publications

A Refined Protocol for Identifying Citrulline-specific Monoclonal Antibodies from Single Human B Cells from Rheumatoid Arthritis Patient Material.

Similar Datasets

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