Method for the diagnosis of c3nef-mediated membranoproliferative glomerulonephritis
A glomerulonephritis, proliferative technology, applied in the direction of disease diagnosis, biological testing, biomaterial analysis, etc., can solve the problem of not describing the evaluation of C3 convertase decay
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no. 1 approach
[0024] In a first embodiment, the method according to the invention comprises:
[0025] a) immobilizing C3b in plate wells, preferably 96 microwell plates;
[0026] b) adding IgG purified from biological samples isolated from patients and / or healthy subjects to the wells to form C3bBb complexes in the presence of FB, FD, BSA and magnesium ions;
[0027] c) detachment of the complex formed;
[0028] d) the complex is subjected to electrophoresis, and due to denaturing conditions (presence of SDS), the two components of C3 convertase, C3b and Bb, dissociate and are transferred to the membrane;
[0029] e) Detection of the protein of interest Bb to obtain a defined baseline value
[0030] It is characterized in that, after said step b) of adding purified IgG, in one or more wells, after a washing step, further incubation in the presence or absence of FH, wherein in the sample subjected to said further incubation , the ratio of the measured intensity of the Bb band to the basel...
no. 2 approach
[0039] The second embodiment includes the following steps:
[0040] a) immobilization of C3b in the wells of the plate;
[0041] b) adding FB, FD, BSA and magnesium ions in the pores;
[0042] c) in at least a first of said wells, a complex formation and decay step comprising washing and incubating in the absence and / or presence of FH with IgG purified from a biological sample of a subject, and in said In at least a second of the wells, the complex formation and decay step comprises washing and incubation with IgG purified from a control sample in the absence and / or presence of FH;
[0043] d) detachment of the complex formed;
[0044] e) electrophoresis of the complex under denaturing conditions to dissociate the two components C3b and Bb and transfer to the membrane;
[0045] f) detection of the protein of interest Bb to obtain values for samples obtained in wells in which the decay step was performed in the presence of IgG isolated from the patient, and for said decay ...
Embodiment 1
[0051] Example 1: Analysis of C3NeF activity in samples from healthy or pathological subjects
[0052] In the presence of C3NeF-IgG purified from MPGN patients (IgG patients) or from healthy controls (IgG controls), in FB, FD and MgCl 2 In this case, the C3 convertase complex C3bBb (Mg 2+ ). Spontaneous or FH-mediated decay was monitored after further incubation at 25°C for 32 min in the absence or presence of FH, respectively. The results of WB analysis are shown in figure 1 middle. The amount of C3 convertase formed was calculated as the ratio of the density measurement of the Bb band after decay to the density measurement of the band corresponding to the baseline (*100).
[0053] Analysis Mean ± 2Dev Std of the results obtained by testing 35 samples from healthy donors in which a residual Bb higher than 36% relative to baseline was observed after decay in the absence of FH in which said decay occurred Samples were considered positive for bands, or in the case of said...
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