Antibody mAb33 from Transduction Laboratories detects human CD95L in ELISA but not in immunoblots

Abstract

The expression and function of CD95 ligand (CD95L, FasL, Apo-1L) in normal and pathological immune responses are subject of extensive studies. The analysis of CD95L protein expression relies on antibodies specific for CD95L. Testing CD95L expression by immunoblotting using the monoclonal antibody 33 from Transduction Laboratories (Lexington, KY, USA) raised against CD95L we found CD95L protein in many different cell types even if CD95L transcripts were undetectable by RT ± PCR. Because of these contradictory results we further examined mAb33 specificity using a panel of different antibodies including mAb33 for binding to CD95L. Our immunoblotting experiments showed that mAb33 most probably detects a protein expressed by many different cell types but not CD95L. 1 Referring to the experiments performed by us and by others, 1 ± 3 Herr et al 4 (this issue) reinvestigated the specificity of mAb33 using human embryonic kidney carcinoma cell stably transfected with CD95L. Testing the mAb33 specificity by comparison to two other CD95L specific antibodies (NOK-1 5 and G247-4) they found that under reducing conditions (immunoblots) mAb33 binds to full-length CD95L. Since these results are different from our data, we repeated our previous experiments using our original and a new batch of mAb33 and several other CD95L specific antibodies as controls. As shown below, these new tests confirmed our earlier experiments with the only exception, that mAb33 was found to detect native CD95L protein in ELISA.At first we compared by immunoblotting the binding specificities of the old and the new batch of mAb33 to CD95L expressed by transiently transfected 293T cells ( Figure 1A). To include also potential effects of tags attached to CD95L two different types of expression vectors were used encoding either human CD95L or a modified form carrying an N-terminal FLAG-tag fused directly in front of the first methionine residue of the cytoplasmic N-terminus. This tagged version of CD95L differs from the recombinant soluble form used by Herr et al 4 as the FLAG-tag used here is separated by 100 amino acid residues from the extracellular domain carrying the antigenic epitopes. As expected, under reducing conditions both forms of CD95L were stained in immunoblots with the antibodies G247-4 (Pharmingen, mouse IgG1) and MIKE-2 (Alexis, rat IgG), the FLAG specific antibody M2 (Sigma) only stained the tagged form and mock-transfected controls showed no staining ( Figure 1A, a). Both batches of mAb33, however, stained a 37 kD signal in untransfected and transfected 293T cells ( Figure 1A, b) but not the human endothelial cell lysate that is provided by Transduction Laboratories as a positive control. Although it was used in 2 ± 4-fold excess, this control lysate was also not stained by the CD95L specific antibody G247-4 ( Figure 1A, c). Low-level CD95L expression in mock-transfected 293T cells was excluded by CD95L specific RT ± PCR using two different primer combinations and total RNA from 293T cells. Samples from human peripheral blood cells or...

CD95 ligand (CD95L) immunohistochemistry: a critical study on 12 antibodies

“…7 Finally, in 2D electrophoresis, mAb 33 detected a protein with different mobility than the CD95L specific signal detected by G247-4 and a polyclonal rabbit anti-CD95L antibody, PE62. 9 In our present study, mAb33 did not only stain CD95L-transfected and control cells to a similar extent, but also strongly labeled tonsillar epithelial cells in tissue sections which lacked CD95L mRNA by in situ hybridization. Even though there may be differences in binding properties under reducing and non-reducing conditions, 9,13 our findings support the assumption by Fiedler and co-workers 9 that mAb 33 most probably detects a protein different from CD95L.…”

“…Specificity of the monoclonal CD95L antibody 33 has first been doubted when CD95L was found in normal thyrocytes in Western blots using mAb 33 although they lacked detectable CD95L mRNA in both RT ± PCR and nuclease protection assays. 6 Fiedler et al 7,9 reported that, in immunoblots, mAb 33 detected an identical 37 kDa signal in both CD95L-transfected and untransfected 293T cells while, using mAb G247-4, only transfected cells showed a CD95L-specific signal. Moreover, mAb 33 did not recognize immunoprecipitated CD95L from transfected 293T cells, but a 37 kDa protein in the supernatant of the immunoprecipitate.…”

Lack of FasL Expression in Cultured Human Retinal Pigment Epithelial Cells

“…This confirms the findings reported by Rezai et al [6], Farrokh-Siar et al [7] and Ferguson and Griffith [1] using cultured human and murine RPE cells. It contradicts the results of a recent study by Jiang et al [13] where cultured human RPE cells p (6) [5], and that the specificity of the anti-FasL antibody (C-33) applied in immunoblotting has been put in question [14][15][16]. Additionally, the RT-PCR reported by Jiang et al [13] lacks a positive control.…”

Cell death in the colonic epithelium during inflammatory bowel diseases

“…54 • Finally, a great deal of confusion was caused by a number of reports that used antibodies of questionable CD95L specificity. [59][60][61] Although a recent editorial suggested that certain anti-CD95L antibodies should be removed from the market, 59 such antibodies have been some of the most widely used reagents for immunohistochemical detection of CD95L.…”