Figure 2.

Human serum contains full-length GPI-APs which are bound to and displaced from serum proteins, among them the GPLD1, by PIGs (for structure and design of PIGs, see [146]) (a complete description of the methods and results is given in the Supplementary Materials). (a) After covalent immobilization (0–300 s) and subsequent blockade of unreacted carboxyl groups (300–400 s), anti-GPLD1 antibody (blue, yellow, brown, turquoise, red curves) or IgG (green curve) were injected into the chip channels. Following washing of the chips (600–700 s), 200 μL of human serum containing Pha (green curve) or lacking it (turquoise curve), which had been pretreated with bacterial PI-PLC (brown curve) or remained untreated (blue, yellow, turquoise, green curves) or PBS (red curve) were injected (700–900 s) in the absence (turquoise curve) or presence of Pha (all other curves) or PIG41 (yellow curve). After washing of the channels (900–1000 s), α-toxin (1000–1200 s), and subsequently anti-CD55 (1200–1500 s), anti-CD59 (1500–1800 s), anti-TNAP (1800–2100 s), anti-AChE (2100–2400 s) and anti-CD73 (2400–2700 s) antibodies were injected successively. After injection of PIG41 (2700–2850 s) together with Ca²⁺ and then TX-100 (2850–3000 s), anti-GPLD1 antibody was finally injected (3000–3300 s). Phase shift is given upon correction for unspecific interaction of serum components and altered viscosity of the sample fluid. (b) The experiment was performed as described (see (a); chips with immobilized anti-GPLD1) with human serum in the presence of Pha and subsequent injection of PIGs or buffer together with Ca²⁺ (blue curve). The measured phase shift was corrected (see (a)) and is only shown from the start of the last antibody (2400 s) to the end of TX-100 injection (3000 s). (c) After covalent immobilization (0–300 s) of human GPLD1 in contrast to buffer-treated chips (green curve), unreacted carboxyl groups were blocked (300–400 s). Following washing of the chips (400–600 s), human serum (blue, yellow, brown, turquoise, green curves) containing Pha (blue curve) or lacking it (turquoise curve) which had been pretreated with bacterial PI-PLC (brown curve) or remained untreated (blue, yellow, turquoise, green curves) or buffer (red curve) were injected (600–900 s) in the absence (turquoise curve) or presence of Pha (all other curves) or PIG41 (yellow curve). After washing of the channels (900–1000 s), α-toxin (1000–1200 s), and subsequently anti-CD55, anti-CD59, anti-TNAP, anti-AChE and anti-CD73 antibodies were injected successively. After injection of PIG41 (2700–2850 s) together with Ca²⁺ and then TX-100 (2850–3000 s), anti-GPLD1 antibody was finally injected (3000–3300 s). The measured phase shift was corrected (see (a)). (d) After covalent immobilization of full-length AChE or soluble AChE (grey curve) onto chips (0–300 s) and blockade of unreacted carboxyl groups (300–400 s), human serum containing Pha (blue curve) or lacking it (turquoise curve) which had been pretreated with bacterial PI-PLC (brown curve) or remained untreated (blue, yellow, turquoise, green curves) or PBS (red curve) were injected (400–600 s) in the absence (turquoise curve) or presence of Pha (all other curves) or PIG41 (yellow curve). After washing of the channels (600–700 s), monoclonal anti-GPLD1 antibody or IgG (green curve) (700–900 s), human annexin-V (900–1000 s), α-toxin (1000–1200 s), and subsequently anti-CD55 (1200–1500 s), anti-CD59 (1500–1800 s), anti-TNAP (1800–2100 s), and anti-CD73 (2100–2400 s) antibodies were injected successively. After injection of TX-100 (2400–2600 s) and then PIG41 together with Ca²⁺ (2600–2800 s), anti-AChE antibody (2800–2900 s), then buffer (2900–3000 s) and finally GPLD1 (3000–3300 s) together with Pha were injected. The measured phase shift was corrected (see (a)).