PDF《CLONE 61016.11》

MONOCLONAL ANTI-EOTAXIN-2 (MPIF-2), HUMAN CLONE 61016.

11 Purified Mouse Immunoglobulin Product Number E

5768 Product Description Monoclonal Anti-Eotaxin-2 (mouse IgG1) is developed in mouse using purified recombinant human eotaxin-2, expressed in E. coli as immunogen. The antibody is purified from the IgG fraction of ascites fluid using protein G affinity chromatography. Monoclonal Anti-Eotaxin-2 (MPIF-2) recognizes recombinant human eotaxin-2 by various immuno- chemical techniques including immunoblotting, ELISA, and neutralization. When used as a capture antibody in human eotaxin-2 sandwich ELISAs, this antibody exhibits less than 0.03 % cross-reactivity with recombinant human eotaxin, MIP-1α, and MCP-3. Eotaxin-2, also named MPIF -2, Ckβ6, and SCYA24, is a member of the CC chemokine family, based on the presence of the CC motif and homology with other known CC chemokines. 1,2 Eotaxin-2 cDNA

2 encodes a

119 amino acid residue precursor protein with a

26 amino acid residue signal peptide that is cleaved to generate a mature protein predicted to contain

93 amino acid residues with an N-glycosylation site. Mature human eotaxin-2 has a predicted molecular mass of approximately 10.6 kDa. Compared to other CC chemokines, eotaxin-2 exhibits

40 %,

42 %, and

39 % amino acid identity to MCP-3, MIP-1α, and eotaxin, respectively. Human CC chemokine eotaxin-2 maps to chromosome 7q11.23.

3 Both eotaxin and eotaxin-2 activate and attract eosinophils and basophils. A receptor for human eotaxin has been identified and found to be the third numbered receptor in the C-C chemokine subfamily of receptors (CCR-3).

4 On eosinophils, the effects of eotaxin-2 is inhibited by an anti-CCR-3 antibody and cross-desensitized by eotaxin and MCP -4, suggesting that all three CC chemokines act through CCR-3. 2,5 Eotaxin-2 mRNA is weakly expressed in activated monocytes and T lymphocytes. Recombinant eotaxin-2 induces chemotaxis of eosinophils, basophils, and resting T lymphocytes but not monocytes and activated T lymphocytes. Eotaxin-2 inhibits colony formation in myleloid progenitor cells.

1 Reagents Monoclonal Anti-Eotaxin-2 is supplied as

500 ?g of antiserum lyophilized from a 0.2 ?m filtered solution in phosphate buffered saline (PBS). Preparation Instructions To one vial of lyophilized powder, add

1 ml of sterile phosphate-buffered saline (PBS) to produce a 0.5 mg/ml stock solution of antibody. Storage/Stability Prior to reconstitution, store at ?20 °C. Reconstituted product may be stored at

2 ° to

8 °C for at least one month. For prolonged storage, freeze in working aliquots at ?20 °C. Avoid repeated freezing and thawing. Product Profile Monoclonal Anti-Eotaxin-2 has the ability to neutralize the biological activity of recombinant human eotaxin-2. To measure this activity, recombinant human eotaxin-2 is incubated with various concentrations of the antibody for

30 minutes at room temperature in a

96 well microplate. Following this preincubation period,

35 ?l of the cytokine-antibody solution (containing recombinant human eotaxin-2 at a final concentration of 0.1 ?g/ml and antibody at concentrations from 0.01 to

100 ?g/ml) is transferred to the lower compartment of a

96 well chemotaxis chamber. The chemotaxis chamber is then assembled using a PVP-free polycarbonate filter (5 micron pore size) and

2 x

10 6 cells/ well (CCR-3 transfected Y3 cells) are added to the top chamber. After incubation for

3 hours at

37 °C in a

5 % CO2 humidified incubator, the chamber is disassembled and the cells that have migrated through to the lower chamber are transferred to a working plate and stained using MTT. Absorbance is read on a microplate reader. The exact concentration of antibody required to neutralize recombinant human eotaxin activity is dependent on the cytokine concentration, cell type, growth conditions, and the type of activity. The Neutralization Dose50 (ND50) for the antibody is defined as that concentration required to yield one-half maximal inhibition of the eotaxin-2 activity on a responsive cell line, when eotaxin-2 is present at a concentration just high enough to elicit a maximum response. The Neutralization Dose50 (ND50) for this antibody is approximately