Monoclonal Antibody Designated T2G1 Reacts With Human Fibrin β-Chain but Not With the Corresponding Chain From Mouse Fibrin
To the Editor:
This communication concerns data presented by Tabrizi and coworkers,1 wherein the authors used antibody T2G1 to recognize mouse fibrin β-chain. There is some confusion as to the antibody, since the authors link T2G1 to a different antibody (designated 59D8) used in a separate study.2 Antibody T2G1 was prepared by our group,3 and to the best of our knowledge, no one has ever shown it to be reactive with mouse fibrin.
At about the time we identified T2G1, Hui and coworkers4 described several antibodies, including 59D8, that reacted with human fibrin. To evaluate such monoclonals as clot imaging agents, we compared T2G1 and 59D8 and found that both were specific for the new NH2-terminus of fibrin β-chain.5 Matsueda and Margolies6 found that 59D8 also bound dog but not bovine, ovine, or porcine fibrins and speculated that Leu5 in the fibrin β-chain was the structural determinant underlying species specificity. A more recent study showed that antibody 59D8 also reacts with mouse fibrin β-chain.2 In contrast, we had observed that plastic-coated human3 as well as dog and rhesus monkey fibrinogen—after in situ activation with thrombin—bound T2G1, whereas mouse fibrinogen, treated in the same fashion, did not (B.J.K. et al, unpublished data, 1993).
The results of Weiler-Guettler and coworkers2 prompted us to reexamine the reactivity of antibody T2G1 with mouse fibrin. The Figure⇓ shows that T2G1 reacts not only with fibrin β-chain made from pure human fibrinogen or clotted plasma but also with several faster-moving degradation products present in both samples. No reactivity was obtained with T2G1 and human fibrinogen Bβ-chain or with any chain constituting the mouse plasma clot.
Our concern deals with the use of antibody T2G1 to detect mouse fibrin. Regarding histopathology (see Figure 3 in Tabrizi et al1 ), the major difficulty is that the authors most likely used a secondary anti-mouse antibody, which would not only detect T2G1 but would also react with any mouse immunoglobulin found in tissues. These authors state that brain sections were fixed in formalin. We have previously shown that human fibrin–containing pathological specimens fixed in formalin react poorly, if at all, with antibody T2G1.7 Concerning the standard curve (see Figure 4 in Tabrizi et al1 ), the authors show a section of membrane that must not only contain closely migrating fibrin β- and γ-chains but also may have some amount of H-chain derived from mouse antibody present as a contaminant in the commercial fibrinogen they used. Because the authors did not identify their secondary antibody, it may be that they obtained a positive signal with mouse H-chain. These authors also stated that the fibrin standard curve is linear between 0.1 and 3 μg/0.1 mL. However, in their Figure 4, the lowest dose shows little if any signal. In our experience, a membrane with a similar range of human fibrin—blotted with enzyme-labeled T2G1 and visualized via chemiluminescent detection—would be overexposed.
Based on the points raised in this communication, we think that Tabrizi and coworkers1 should provide needed clarification and also address our concerns on the data obtained with antibody T2G1.
Tabrizi P, Wang L, Seeds N, McComb JG, Yamada S, Griffin JH, Carmeliet P, Weiss MH, Zlokovic BV. Tissue plasminogen activator (tPA) deficiency exacerbates cerebrovascular fibrin deposition and brain injury in a murine stroke model: studies in tPA-deficient mice and wild-type mice on a matched genetic background. Arterioscler Thromb Vasc Biol.. 1999;19:2801–2806.
Hui KY, Haber E, Matsueda GR. Monoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not fibrinogen. Science.. 1983;222:1129–1132.
Kudryk BJ, Grossman ZD, McAfee JG, Rosebrough SF. Monoclonal antibodies as probes for fibrin(ogen) proteolysis. In: Chatal J-F, ed. Monoclonal Antibodies in Immunoscintigraphy. Boca Raton, FL: CRC Press; 1989:365–398.
Dr Kudryk expressed his concerns to us initially by e-mail indicating that he “used both T2G1 and 59D8 [anti-human fibrin] antibodies side by side, and in [his] hands, neither antibody cross-reacts with mouse fibrin.” We responded that in our hands, three different anti-human fibrin antibodies, including EA3 and T2G1 from Accurate Chemical and A008 from Dako, cross-react with mouse fibrin. We also mentioned that another group reported that anti-human fibrin antibody [59D8] cross-reacts with mouse fibrin.R1
From Dr Kudryk’s letter, it is encouraging to us that he is now able to demonstrate that anti-human fibrin antibody 59D8 cross-reacts with mouse fibrin. This is an important advance, as only a few months ago he was very much concerned about this antibody as well.
Dr Kudryk is concerned that our Western blot analysis does not detect fibrin bands but the H-chain of mouse IgG with the secondary antibody. As indicated in our methods,R2 we use only primary T2G1 antibody labeled with horseradish peroxidase and the enhanced chemiluminescent system. We do not use secondary antibody, and Dr Kudryk’s suggestion is unrealistic. In addition to high sensitivity towards mouse fibrin, this antibody also detects rat fibrin on Western blots, though with somewhat lower affinity when compared with A008 Dako anti-human fibrin/fibrinogen antibody (our unpublished observations, 1999).
Regarding immunocytochemical analysis, Dr Kudryk indicates that in his hands, “human fibrin-containing pathologic specimens fixed in formalin react poorly with T2G1 antibody.” Because this antibody is anti-human, it should react with human fibrin chain. We would recommend using the antigen retrieval techniques described by Shi et al,R3 which work very well in our hands, or a signal amplification system such as fluorophore tyramide that reacts well with antibodies tagged with horseradish peroxidase.
In response to the additional concern of Dr Kudryk that in our immunocytochemical analysis “secondary antibody will react with any mouse immunoglobulin that may be present in tissue along with fibrin,” we indicated in the methods of our articleR2 that “routine control sections included deletion of primary antibody, deletion of secondary antibody, and use of an irrelevant antibody.” All of these negative controls exclude the possibility that the reaction product is due to the recognition of any mouse IgG by the secondary antibody. Furthermore, we included two control negative stainings with primary and secondary antibodies in Figure 3.R2 First, Figure 3C shows negative staining in the contralateral hemisphere of a tPA−/− mouse that does not contain any reaction product, in contrast to significant vascular staining for fibrin in ischemic infarcted hemisphere in the same animal (Figures 3A, 3B, 3D, and 3E). Second, Figures 3G and 3H show mostly negative staining for fibrin in tPA+/+ mice or weakly positive staining in the ischemic hemisphere studied under the same experimental conditions. The difference in fibrin deposition between tPA−/− and tPA+/+ mice after postischemic occlusion is therefore very specific, and as indicated by quantitative Western blot analysis in section IV (level of the optic chiasm), this difference is 8.2-fold (Figure 4).R2
Thus, in summary, several anti-human fibrin or fibrin/fibrinogen antibodies cross-react with corresponding mouse fibrin. Of course, cross-species reactivity of antibodies is highly influenced by experimental conditions.
Weiler-Guettler H, Christie PD, Beeler DL, Healy AM, Hancock WW, Rayburn H, Edelberg JM, Rosenberg RD. A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state. J Clin Invest.. 1998;101:1983–1991.
Tabrizi P, Wang L, Seeds N, McComb JG, Yamada S, Griffin JH, Carmeliet P, Weiss MH, Zlokovic BV. Tissue plasminogen activator (tPA) deficiency exacerbates cerebrovascular fibrin deposition and brain injury in a murine stroke model. Arteioscler Thromb Vasc Biol.. 1999;19:2801–2806.
Shi SR, Cote RJ, Taylor CR. Antigen retrieval immunohistochemistry: past, present and future. J Histochem Cytochem.. 1997;45:327–343.