Testing different antibodies recognising against the same target in parallel and observing any common patterns of antibody reactivity can significantly strengthen confidence in the validation data generated. The use of such a TA enabled the reactivity of the NAT105 antibody to be tested using immunohistochemical labelling on a single glass slide. This specially designed TA contained lymph node, thymus, spleen and tonsil (positive control tissues known to express PD-1) and kidney, heart, brain, placenta (negative control tissues lacking PD-1 protein). Tissue Microarray (TA) technology was used to prepare a paraffin embedded block containing the cores from several deliberately chosen formalin-fixed paraffin-embedded normal tissue samples. (B) PRDM1α nuclei staining in the cell lines correlated with the pattern of protein expression found by Western blotting.Įxample 3 Validation studies of the anti PD-1 (clone name NAT105) monoclonal antibody using a tissue microarray. The negative control Burkitt´s lymphoma cell line, RAJI, expressed neither PRDM1α nor PRDM1β proteins. The myeloma cell lines, SK-MM-2, KARPAS-620, NCI-H929, OPM-2 and LP-1, also expressed the 80 kDa PRDM1β protein. All the myeloma cell lines show a 97 kDa band, corresponding to the PRDM1α protein. (A) Western blot analysis of PRDM1 expression in total protein extracts from multiple myeloma and RAJI cell lines. The RAJI (Burkitt′s lymphoma) cell line was used as a negative control.įigure legend Validation of the monoclonal antibody ROS for the PRDM1 protein using a combination of Western blotting and IHC. Several human myeloma cell lines expected to express the PRDM1α protein were tested with antibody ROS using a combination of Western blotting and immunohistochemistry (IHC). D) shows antibody LT19 labelling 19% of peripheral blood lymphocytes, a population consistent with the numbers of B-cell lymphocytes in blood.Įxample 2 Validation of the anti PRDM1 (clone name ROS) monoclonal antibody using myeloma cell lines. No labelling was observed of the JURKAT and U937 cell lines. Of the cell lines, only the RAJI Burkitt’s lymphoma derived B-cell line was stained by this monoclonal antibody. The percentage of positive cells and mean fluorescence intensity (MFI) are indicated. Negative control staining versus reactivity with the mAbs is shown in each histogram. Cells were incubated with antibody LT19 followed by anti-mouse labelled with PE. The reactivity of the antibody was also tested on normal peripheral blood lymphocytes.įigure legend Validation studies of a CD19 monoclonal antibody LT19 with flow cytometry using A) the Raji (Burkitt’s lymphoma cell), B) the JURKAT (T cell lymphoma) C) U937 (monocytic) cell lines and D) normal PBLs. RAJI was used as a positive control while the JURKAT and U937 cell represented the negative control. Cells or tissues over-expressing or lacking mRNA encoding the relevant antigens should be used as controls in this situation.Įxample 1 Validation of a monoclonal antibody (clone name LT19) raised against CD19 in cells known to express the endogenous target protein.Ī collection of several lymphoma or leukaemia-derived cell lines of different lineages (T, B cells and myeloid cells) was stained with the mAb LT19 using flow cytometry. Protein expression data may not, however, be available for many target antigens. Although the use of cell lines can be a valuable indicator of the expression of the target protein, the use of positive and negative control tissue(s) is essential for the full validation of the antibody and the assessment of non-specific binding ( Example 3). Cell lines that endogenously express lack the protein of interest are widely used as controls in antibody validation ( Example 1 and Example 2). What can you use as a positive and negative control?Ī. If possible, you should confirm the expression of the required target molecule in your sample using more than one assay. You should always use several positive and negative controls at the same time. Your negative control should consist of tissues or cells where your target protein is known to be absent. Your positive control should confirm that your target antigen is expressed on the relevant cells and tissues. Positive and negative controls for antibody validation
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |