Isotype matched antibody

Manufactured by BioLegend

Sourced in United States

Isotype-matched antibody is a laboratory tool used to provide a negative control for immunoassays. It is an antibody that shares the same isotype as the primary antibody being used, but does not bind to the target antigen. This allows for the assessment of non-specific binding in the assay.

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5 protocols using isotype matched antibody

Phenotypic Analysis of Regulatory T Cells

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Cryopreserved PBMC were thawed and rested for 6 hours at 37°C,
5% CO₂ in RPMI 1640 medium (Lonza) containing 10%
FBS, 2mM glutamine, 100 IU/ml penicillin, and 100 µm/ml streptomycin.
1×10⁶ PBMC were stained with a marker to distinguish live
and dead cells (Invitrogen) followed by surface staining with the following
directly conjugated mAbs: anti-CD3, anti-CD4, anti-CD25 (clone 4E3, Miltenyi
Biotech), anti-CD45RA, and anti-HLA-DR for 25 minutes at room temperature. Cells
were then washed once with FACS wash buffer and fixed with 1× fix/perm
buffer (Tonbo Biosciences) for 60 minutes at room temperature, then washed again
with 1× perm buffer solution and incubated in this buffer solution for
10 minutes, washed again with 1× perm buffer solution, and incubated for
45 minutes with mAb to Foxp3 (clone 206D, Biolegend). An isotype-matched
antibody was used as a control (Biolegend). Cells were then washed with
1× perm buffer solution, followed by an additional wash with FACS wash
buffer, re-suspended in 1% formalin in PBS, and acquired by the LSR-II
flow cytometer.

Kalokhe A.S., Ibegbu C.C., Kaur S.P., Amara R.R., Kelley M.E., del Rio C, & Stephenson R. (2016). Intimate Partner Violence is Associated with Increased CD4+ T-Cell Activation Among HIV-Negative High-Risk Women. Pathogens & Immunity, 1(1), 193-213.

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Flow Cytometric Analysis of iRC Cells

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Flow cytometric analysis was performed by staining 1-2 × 10⁷ iRCs using an FACSCalibur flow cytometer (BD Biosciences, Japan) [55 (link)]. In brief, cells were stained with the mouse anti HLA-G FITC antibody for 30 minutes at 4 °C. Control aliquots were stained with an isotype-matched antibody (Biolegend, Cat#400110) to evaluate nonspecific binding to target cells. Intracellular staining was performed using the Cytofix/Cytoperm (BD Biosciences) as previously described [47 (link)]. The experiment was repeated three times. See Supplementary Table 2 for the antibodies used.

Sato M., Kawana K., Adachi K., Fujimoto A., Yoshida M., Nakamura H., Nishida H., Inoue T., Taguchi A., Ogishima J., Eguchi S., Yamashita A., Tomio K., Wada-Hiraike O., Oda K., Nagamatsu T., Osuga Y, & Fujii T. (2017). Regeneration of cervical reserve cell-like cells from human induced pluripotent stem cells (iPSCs): A new approach to finding targets for cervical cancer stem cell treatment. Oncotarget, 8(25), 40935-40945.

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Assessing TNFα Neutralization in T-cell Inhibition

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To assess the role of the TNFα cytokine in growth inhibition of CD4 + T cells induced by the anti-Ly-6A antibody, the biological activity of TNFα was neutralized with an anti-TNFα antibody (Clone MP6-XT22) (BioLegend, San Diego, CA, USA). Cultures with isotype-matched antibody (BioLegend, San Diego, CA, USA) served as controls for determining the specificity in these blocking assays.

Patel A.G., Moxham S, & Bamezai A.K. (2023). Ly-6A-Induced Growth Inhibition and Cell Death in a Transformed CD4⁺ T Cell Line: Role of Tumor Necrosis Factor-α. Archivum immunologiae et therapiae experimentalis, 71(1).

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Multiparametric Flow Cytometry of CLL Cells

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The CLL cells from all the blood samples were stained with optimal concentrations of antibody combinations and directed against the following surface antigens: CD183(CXCR3)-FITC, CD20-PE, CD5-PerCP-Cy5.5, CD38-Pe-Cy7, CD49d-APC, CD19-APC-Cy7, CD184 (CXCR4)-BV421 and HLA-DR-BV510 (all procured from BioLegend), as previously reported [5 (link),15 (link)]. Isotype-matched antibodies (BioLegend) were used as negative controls.
The determination of s-CLL and l-CLL cells was conducted using FSC data and a back-gating strategy. The analysis was performed using a BD FACSCanto II (Becton Dickinson) instrument, and data acquisition was performed using BD FACSDiva software (v.8.0.2; Becton Dickinson). Flow cytometry data were analysed using FlowJo v.X0.7 software (Tree Star, Inc., San Carlos, CA, USA). In all the experiments, a minimum of 10,000 events was counted. The results were expressed as a percentage and mean fluorescence intensity (MFI).

Manukyan G., Mikulkova Z., Turcsanyi P., Savara J., Trajerová M., Kubova Z., Papajik T, & Kriegova E. (2021). Towards a Better Characterisation of Leukemic Cells in Chronic Lymphocytic Leukaemia: Cell-Size Heterogeneity Reflects Their Activation Status and Migratory Abilities. Cancers, 13(19), 4922.

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PD-L1 Expression Analysis in Macrophages and Tumors

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Human monocyte-derived macrophages were treated with human FcR-blocking reagent (BioLegend) and then reacted with phycoerythrin-labeled or Alexa 488-labeled anti-human PD-L1 antibody (BioLegend) or isotype-matched control antibody (BioLegend). For analysis of murine subcutaneous tumor, anti-CD11b antibody, anti-PD-L1 antibody, and isotype-matched antibodies (BioLegend) were used. The stained cell samples were analyzed on a FACSverse (Becton Dickinson, Franklin Lake, NJ, USA) flow cytometer with FACSuite (Becton Dickinson) software.

Shinchi Y., Ishizuka S., Komohara Y., Matsubara E., Mito R., Pan C., Yoshii D., Yonemitsu K., Fujiwara Y., Ikeda K., Tamada K., Sakagami T, & Suzuki M. (2022). The expression of PD-1 ligand 1 on macrophages and its clinical impacts and mechanisms in lung adenocarcinoma. Cancer Immunology, Immunotherapy, 71(11), 2645-2661.

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