Anti-Human CD14 Recombinant Antibody (rMil2) (CAT#: TAB-1586CL)

Figure 1 The Ab concentration in cell culture supernatant (mg/ml; s) and the Ab production rate (pg/cell/day; d) during a fed-batch expression period of 12 d were determined with ELISA.

Data are given as mean and SEM for rMil2 (A; n = 12), r18D11 (B; n = 5) and raNIP (C; n = 2).

Lau, C., Gunnarsen, K. S., Høydahl, L. S., Andersen, J. T., Berntzen, G., Pharo, A.,... & Nielsen, E. W. (2013). Chimeric anti-CD14 IGG2/4 hybrid antibodies for therapeutic intervention in pig and human models of inflammation. The Journal of Immunology, 191(9), 4769-4777.

Figure 2 Whole porcine blood was incubated with 150 mg/ml FITC-conjugated Mil2 (FITC-Mil2) and increasing concentrations of unlabeled rMil2 (s), control IgG2/4 (eculizumab) (N), the original clone Mil2 (d) or mIgG2b isotype control (n).

Remaining FITC-Mil2 binding to the granulocyte population was recorded as median fluorescence intensity of using flow cytometry. Fluorescence intensity in presence of FITC-Mil2 alone was set to 100%.

Lau, C., Gunnarsen, K. S., Høydahl, L. S., Andersen, J. T., Berntzen, G., Pharo, A.,... & Nielsen, E. W. (2013). Chimeric anti-CD14 IGG2/4 hybrid antibodies for therapeutic intervention in pig and human models of inflammation. The Journal of Immunology, 191(9), 4769-4777.

Figure 3 Release of the proinflammatory cytokines IL-1b (A) and TNF (B) from porcine whole blood induced with 1 × 10⁵/ml E. coli strain LE392 in presence of increasing concentrations of rMil2 (s), Mil2 (d), control IgG2/4 (N), or mIgG2b (n). After 120 min incubation, plasma cytokine levels were determined with ELISA, and those measured in absence of any exogenous Ab were set to 100%.

Lau, C., Gunnarsen, K. S., Høydahl, L. S., Andersen, J. T., Berntzen, G., Pharo, A.,... & Nielsen, E. W. (2013). Chimeric anti-CD14 IGG2/4 hybrid antibodies for therapeutic intervention in pig and human models of inflammation. The Journal of Immunology, 191(9), 4769-4777.

Figure 4 Blood slides from samples containing 71.4 mg/ml rMil2 or Mil2 were stained with nuclear stain and investigated by light microscopy.

Pictures were taken at a 1000-fold magnification. WBCs, RBCs, and platelets (thrombocytes [TRCs) are indicated by arrows. The single asterisk in the picture for Mil2 indicates a platelet aggregate with surrounding leukocytes that was not observed in rMil2-treated blood.

Lau, C., Gunnarsen, K. S., Høydahl, L. S., Andersen, J. T., Berntzen, G., Pharo, A.,... & Nielsen, E. W. (2013). Chimeric anti-CD14 IGG2/4 hybrid antibodies for therapeutic intervention in pig and human models of inflammation. The Journal of Immunology, 191(9), 4769-4777.

Figure 5 Effect of rMil2 in combination with C5-inhibitor OmCI on the inflammatory response in porcine blood in vitro.

Porcine whole blood was incubated with E. coli in the presence of PBS (E. coli), the complement C5 inhibitor OmCI (0.32 mM), original anti-CD14 Ab Mil2 (25 mg/ml), recombinant anti-CD14 Ab rMil2 (25 mg/ml), the combination of OmCI and the two Mil2 Abs, or an isotype control Ab. Plasma was analyzed for cytokines. (A) TNF. (B) IL-1b. (C) IL-8. (D) TF expression on granulocytes was measured by flow cytometry and expressed as median fluorescence intensity (MFI). Results are shown as mean 6 SEM of four to eight experiments. # Statistical significance between combined inhibition versus the two single inhibitions (for detailed p values, see Results). *Statistical significance as compared with E. coli alone (left column). *p,0.05, **p, 0.01, ***p, 0.001.

Lau, C., Gunnarsen, K. S., Høydahl, L. S., Andersen, J. T., Berntzen, G., Pharo, A.,... & Nielsen, E. W. (2013). Chimeric anti-CD14 IGG2/4 hybrid antibodies for therapeutic intervention in pig and human models of inflammation. The Journal of Immunology, 191(9), 4769-4777.

Figure 6 In vitro binding of recombinant IgG2/4 hybrid Abs to complement and Fc-receptors.

In creasing concentrations of rMil2 (s), r18D11 (O) or raNIP (N) were incubated with (A) immobilized human C1q, (B) the human Fcg receptors FcgRI, (C) FcgRIIa (allotype His131), (D) FcgRIIb, (E) FcgRIIIa (allotype Val158), (F) FcgRIIIb, and (G, H) human (hFcRn) or (I, J) porcine FcRn (pFcRn) at acidic (pH 6.0) and neutral pH (pH 7.4). Human IgG1 ()) (A-H) or porcine IgG (pIgG; ♦) (I, J) served as positive controls. Binding was measured as absorbance at 450 nm. Data are given as mean and SD (n = 3 independent experiments).

Lau, C., Gunnarsen, K. S., Høydahl, L. S., Andersen, J. T., Berntzen, G., Pharo, A.,... & Nielsen, E. W. (2013). Chimeric anti-CD14 IGG2/4 hybrid antibodies for therapeutic intervention in pig and human models of inflammation. The Journal of Immunology, 191(9), 4769-4777.