Article

Screening of anti-human leukocyte monoclonal antibodies for reactivity with equine leukocyte

Department of Clinical Sciences, Colorado State University, Fort Collins, Colorado, United States
Veterinary Immunology and Immunopathology (Impact Factor: 1.54). 10/2007; 119(1-2):63-80. DOI: 10.1016/j.vetimm.2007.06.034
Source: PubMed

ABSTRACT

Three hundred and seventy-nine monoclonal antibodies (mAbs) against various human CD molecules supplied to the HLDA8 animal homologues section (including four isotype controls) were analysed for cross-reactivity with equine leukocytes. First, flow cytometric identification of positively reacting mAbs was performed in one laboratory. Thereafter, a second round of flow cytometric evaluation was performed, involving three laboratories participating in the study. The first test-round indicated 17 mAbs as potentially positive. After the second round of flow cytometric analysis, 14 mAbs remained (directed against CD2, CD11a, CD18, CD44, CD45, CD49d, CD91, CD163 and CD172) where cross-reactivity was anticipated based on similarities between the human and equine staining pattern. Additionally, there was 1 mAb with weak likely positive reactivity, 12 mAbs with positive staining, which likely do not reflect valuable data, 5 mAbs with clear alternate expression pattern from that expected from humans, 5 mAbs with a questionable staining pattern itself, i.e. that was variable between the three labs, 32 mAbs with weak-positive expression and alternate staining pattern, and 279 negative mAbs (including the four isotype controls) were detected. In 31 cases, more appropriate target cells, such as thymocytes or stem cells, were not available for the screening. The results underline the value of this "cross-reactivity" approach for equine immunology. However, as only a few mAbs against leukocyte surface antigens reacted positively (approximately 4% of the mAbs submitted), the analysis of further anti-human mAbs and directed efforts to develop species-specific anti-CD mAb are still required.

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    • "Evaluation of cross-species reactivity of commercially available antibodies, particularly CD antigens, has been largely performed in equine tissues prepared for whole-cell analysis like flow-cytometry (Johne et al., 1997;Mérant et al., 2003;Terio et al., 2003;Kunisch et al., 2004;Ibrahim et al., 2007) or on fresh or frozen specimens (Bilzer et al., 1995;Zeng et al., 1996;Lemos et al., 2008;Härtig et al., 2009). Large screenings of non-equine derived antibodies have often resulted in limited identification of equine reactive reagents (Ibrahim et al., 2007;Schnabel et al., 2013;Szabo & Gulya, 2013). Of the 26 antibodies in this study, six antibodies successfully reacted with FFPE equine tissues. "
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    • "Greater than 90% of the analyzed PBMC were CD172a+, indicating that they were monocytes/macrophages (data not shown). Although the anti-CD172a mAb also identifies equine granulocytes [40], the ficoll hypaque PBMC isolation protocol (which removes the majority of granulocytes) together with the lack of internal complexity in the labeled cells (data not shown), provided further evidence that they were monocytes/macrophages. Overall, these data confirmed the SCID phenotype and indicated that the establishment of T. equi infection in SCID1 and SCID2 did not involve B or T lymphocytes. "
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    • "According to Borjesson and Peroni (2011), the capacity to characterize equine MSCs is hampered by the limited availability of commercial antibodies and by variability in cross-reactions . Ibrahim et al. (2007) showed that of 379 human CD antibodies, only 14 (4%) showed cross-reactivity with equine leukocytes. Alternatively, De Schauwer et al. (2011) suggested the use of a panel of cell surface markers for equine MSCs. "
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