CXCL10 (IP-10)

 

Description

IFN-g-inducible protein 10 (IP-10, CXCL10), a chemokine secreted from cells stimulated with type I and II interferons (IFNs) and lipopolysaccharide (LPS,) is a chemoattractant for activated T cells. Expression of IP-10 is seen in many Th1-type inflammatory diseases, where it plays an important role in recruiting activated T cells into sites of tissue inflammation (Dufour, Dziejman et al. 2002). IP-10 has been recently identified as a sensitive biomarker for inflammatory conditions including among others, development and treatment of tuberculosis (Ruhwald, Bjerregaard-Andersen et al. 2009, Goosen, Cooper et al. 2015), acute respiratory infections (Hayney, Henriquez et al. 2017), Kawasaki disease (Ko, Kuo et al. 2015), viral infections (Quint, Donaldson et al. 2010), and cystic fibrosis exacerbation (Solomon, Frederick et al. 2013). There are no available immunological reagents for MAR IP-10.

 

Alignment

Protein alignment for human, rhesus macaque and marmoset IP-10:

The referenced media source is missing and needs to be re-embedded.

 

Protein alignment for marmoset, owl monkey, and squirrel monkey IP-10:

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References

  • Dufour, J. H., M. Dziejman, M. T. Liu, J. H. Leung, T. E. Lane and A. D. Luster (2002). "IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking." J Immunol 168(7): 3195-3204.
  • Goosen, W. J., D. Cooper, M. A. Miller, P. D. van Helden and S. D. Parsons (2015). "IP-10 Is a Sensitive Biomarker of Antigen Recognition in Whole-Blood Stimulation Assays Used for the Diagnosis of Mycobacterium bovis Infection in African Buffaloes (Syncerus caffer)." Clin Vaccine Immunol 22(8): 974-978.
  • Hayney, M. S., K. M. Henriquez, J. H. Barnet, T. Ewers, H. M. Champion, S. Flannery and B. Barrett (2017). "Serum IFN-gamma-induced protein 10 (IP-10) as a biomarker for severity of acute respiratory infection in healthy adults." J Clin Virol 90: 32-37.
  • Ko, T. M., H. C. Kuo, J. S. Chang, S. P. Chen, Y. M. Liu, H. W. Chen, F. J. Tsai, Y. C. Lee, C. H. Chen, J. Y. Wu and Y. T. Chen (2015). "CXCL10/IP-10 is a biomarker and mediator for Kawasaki disease." Circ Res 116(5): 876-883.
  • Quint, J. K., G. C. Donaldson, J. J. Goldring, R. Baghai-Ravary, J. R. Hurst and J. A. Wedzicha (2010). "Serum IP-10 as a biomarker of human rhinovirus infection at exacerbation of COPD." Chest 137(4): 812-822.
  • Ruhwald, M., M. Bjerregaard-Andersen, P. Rabna, J. Eugen-Olsen and P. Ravn (2009). "IP-10, MCP-1, MCP-2, MCP-3, and IL-1RA hold promise as biomarkers for infection with M. tuberculosis in a whole blood based T-cell assay." BMC Res Notes 2: 19.
  • Solomon, G. M., C. Frederick, S. Zhang, A. Gaggar, T. Harris, B. A. Woodworth, C. Steele and S. M. Rowe (2013). "IP-10 is a potential biomarker of cystic fibrosis acute pulmonary exacerbations." PLoS One 8(8): e72398.

 

Status

Anti-marmoset (MAR) IP-10 monoclonal antibodies were generated by immunizing mice with recombinant MAR IP-10 with a C-tag (MAR IP-10.C-tag) or with C-tag and the addition of the murine IFN- leader sequence at the N-terminus (mIFNg.MARIP-10.C-tag). Generated monoclonal antibodies (mAbs) were screened by ELISA.  total of 19 mAbs were processed and produced as pure and biotinylated forms, and ELISA assays were performed using all possible combinations; supernatants of marmosets PBMCs stimulated with SEB were used for detection of working pairs. Figure 1 shows some of the mAb pairs that detected both the recombinant MAR IP-10 immunizing antigen as well as IP-10 in the PBMC supernatants. 

ELISA screening of capture/detection mAb pairs specific for MAR IP-10
Figure 1. ELISA screening of capture/detection mAb pairs specific for MAR IP-10

Based on the results of the ELISA, mAbs CD6, CD7 and CD11 were covalently attached to LMX beads and an assay was also run using different mAbs for detection (Figure 2). The strongest signal originated when using CD11 as capture antibody and MTR148 as detection.

LMX assay for MAR IP-10 using different capture/detection mAb combinations.
Figure 2. LMX assay for MAR IP-10 using different capture/detection mAb combinations. Epitope map for all mAbs is shown on the right.

The combination of LMX beads coated with CD11 and biotinylated MTR148 was further tested for the generation of a STD curve with recombinant MAR IP-10 as well as testing plasma samples from MAR, SQM, and OLM challenged with LPS. The STD curve showed that the LMX assay is very sensitive and able to detect signals for MAR IP-10 below 10 pg/ml (Figure 3). Further assays will be run to determine the limit of detection.  

STD curve for MAR IP-10 in a LMX format.
Figure 3. STD curve for MAR IP-10 in a LMX format.

Testing plasma samples from animals challenged with LPS showed that the assay can detect IP-10 for marmosets, squirrel monkeys, and owl monkeys. After exposure to LPS IV, circulating levels of IP-10 increase and remain elevated for 4 to 6 hours, taking up to 24 hours before returning to pre-challenge levels. 

LMX assay for IP-10 using plasma samples from NWM challenged IV with LPS.
Figure 4. LMX assay for IP-10 using plasma samples from NWM challenged IV with LPS.

In summary, we have generated CD11 and MTR148 mAbs, which work in ELISA and LMX formats for detection of marmoset, squirrel monkey, and owl monkey IP-10. Production of these antibodies for distribution is underway.