Description
The NFLD.D1 monoclonal antibody is specific for the HLA-DR4 serogroup. This panspecific HLA-DR4 antibody binds to an epitope expressed in the extracellular beta-2 (β2) domain of all HLA-DRB1 beta chain variants encoded by HLA-DRB1*04 alleles. The epitope recognized by the NFLD.D1 antibody depends on both a leucine present at sequence position 180 and a threonine at position 181. HLA-DR antigens are heterodimers comprised of type I transmembrane glycoprotein alpha and beta subunits that contain two extracellular domains, transmembrane regions, and cytoplasmic tails. The ~34 kDa HLA-DR alpha (HLA-DRα) chain is encoded by HLA-DRA whereas the alternative ~28 kDa HLA-DR beta (HLA-DRβ) chains are encoded by one of the four different HLA-DRB loci (HLA-DRB1,3,4, and 5) that are located within the Human Leukocyte Antigen (HLA) Complex of chromosome 6. HLA-DR is variably expressed on antigen-presenting cells (APCs), such as dendritic cells, B lymphocytes, monocytes, macrophages, and thymic epithelial cells as well as by activated T cells, epithelial cells, inflammatory fibroblasts, and tumor cells. Polymorphisms in the HLA-DRβ chains allow for variability in peptide binding and the presentation of self and foreign peptide antigens to CD4+ T lymphocytes that generate and regulate adaptive immune responses. The NFLD.D1 antibody can be especially useful along with other HLA-DRβ allotype-specific antibodies in applications such as immunofluorescence and flow cytometry for analyzing allelic HLA-DRB expression patterns by individual cells within heterogeneous cell populations. Some HLA-DRB1*04 alleles or their aberrant expression patterns have been associated with susceptibility to diseases including rheumatoid arthritis.
BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to the dye under optimum conditions that minimize unconjugated dye and antibody.
1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
3. For U.S. patents that may apply, see bd.com/patents.
4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
5. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
6. The production process underwent stringent testing and validation to assure that it generates a high-quality conjugate with consistent performance and specific binding activity. However, verification testing has not been performed on all conjugate lots.
7. When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
8. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
9. An isotype control should be used at the same concentration as the antibody of interest.
10. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
11. Please observe the following precautions: We recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to protect exposure of conjugated reagents, including cells stained with those reagents, to any room illumination. Absorption of visible light can significantly affect the emission spectra and quantum yield of tandem fluorochrome conjugates.
12. Tandem fluorochromes contain both an energy donor and an energy acceptor. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from the donor may be observed. Additionally, multi-laser cytometers may directly excite both the donor and acceptor fluorochromes. Therefore, we recommend for every tandem conjugate, a matched individual single-stain control be acquired for generating a compensation or spectral unmixing matrix.
13. Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
