Fig 1.
Diagrammatic representation of animal immunization protocol.
Fig 2.
FTIR spectra of (a) native HSA (b) HSA modified with 400 mg/dL of glucose.
Fig 3.
Mass spectroscopic profile of (a) native HSA (b) standard carboxymethyl lysine (c) HSA+100 mg/dL glucose (d) HSA+200 mg/dL glucose (e) HSA+300 mg/dL glucose (f) HSA+400 mg/dL glucose.
Table 1.
Heamtological and rectal temperature analysis of preimmune and immune rabbits demonstrating the inflammatory response after immunization with glycated HSA (antigen).
Table 2.
Biochemical analysis of preimmune and immune rabbits after immunization with glycated HSA (antigen).
Fig 4.
(a) Level of induced antibodies against native HSA. Direct binding ELISA of native HSA with pre-immune and immune sera. The microtitre plates were coated with native HSA (10 μg/mL). (b) Level of induced antibodies against glucose modified HSA. Direct binding ELISA of glucose modified HSA with pre-immune and immune sera. The microtitre plates were coated with native HSA (10 μg/mL).
Fig 5.
(a) Inhibition ELISA of anti-HSA immune and pre-immune sera with native HSA. Microtitre plates were coated with native HSA (10 μg/mL). (b) Inhibition ELISA of anti-glucose-HSA immune and pre-immune sera with glucose modified HSA. Microtitre plates were coated with native HSA (10 μg/mL).
Fig 6.
(a) Binding of affinity purified anti-HSA IgG and pre-immune IgG to native HSA. Microtitre plates were coated with native HSA (10 μg/mL). (b) Binding of affinity purified anti-glucose modified-HSA IgG and pre-immune IgGto glucose modified- HSA. Microtitre plates were coated with glucose modified- HSA(10 μg/mL).
Fig 7.
(a) Inhibition of binding of affinity purified anti-HSA IgG and pre-imune IgG to native HSA. Microtitre plates were coated with native HSA (10 μg/mL). (b) Inhibition of binding of affinity purified anti-glucose-HSA IgG and pre-imune IgG to glucose modified HSA. Microtitre plates were coated with native HSA (10 μg/mL).
Fig 8.
Direct binding ELISA of serum auto-antibodies in (a) T2DM (n = 50) (c) T1DM (n = 50) (e) GDM (n = 50) (g) T2DM+CKD (n = 50) with native and 400 mg/dL glucose modified HSA and normal human sera (NHS) served as control in (b) (n = 50), (d) (n = 50), (f) (n = 50) and (h) (n = 50).
The plate was coated with the respective antigens (10μg/mL). A p value <0.05 considered to be significant. ns = non-significant
Fig 9.
(a) Ultraviolet absorption spectra of native HSA+IgG immune complex and AGEs HSA+IgG immune complex with 400 mg/dL D-glucose.
Fluorescence emission profile of immune complex (native HSA+IgG) shown by black colour and immune complex (AGE-HSA+IgG) shown by red colour.
Fig 10.
X-ray diffraction pattern under scan range of 10–500 (a) affinity purified IgG alone (b) immune complex of native HSA with IgG (c) immune complex of AGE-HSA (modified with 400 mg/dL D-glucose) with IgG.