ELISA CASPR1 IN CIDPRIT COHORT (04/04/2024)

AIM: to test for anti-CASPR1 antibodies in patients with CIDP (Italian cohort) at baseline.

MATERIALS REQUIRED:

• Microtiter plate (96-well plate) with coating antigen (Caspr1)
• Blocking buffer (milk 5% with PBS-tween 0.1%)
• Enzyme-conjugated secondary antibody (RAH IgG HRP)
• Substrate solution (TMB)
• Stop solution (sulfuric acid 25%)
• Positive control serum for CASPR1
• Negative controls serum
• Washing buffer (PBS-Tween 0.1%)
• Plate Reader

SAMPLES: 13 Samples from the Italian CIDP cohort at baseline.

BLOCKING:

• Decant the coating solution from the plate.
• Wash the plate 3 times with washing buffer to remove unbound antigen.
• Add blocking buffer to each well (200 µl per well).
• Incubate the plate at room temperature for 1 hour to block nonspecific binding sites.

SAMPLES INCUBATION:

• Decant the blocking solution from the plate. Wash the plate 3 times with washing buffer.
• Prepare the serum and control dilutions in blocking buffer at 1/100.
• Add the serum and control solution to each well (100 µl per well).
• Incubate the plate at room temperature for 1 hour.

SECONDARY ANTIBODY INCUBATION:

• Decant the serum/control solution from the plate. Wash the plate 3 times with washing buffer.
• Dilute the enzyme-conjugated secondary antibody in blocking buffer at 1/3000.
• Add the secondary antibody solution to each well (100 µl per well).
• Incubate the plate at room temperature for 45 minutes.

SUBSTRATE INCUBATION:

• Decant the secondary antibody solution from the plate. Wash the plate 3 times with washing buffer.
• Prepare the substrate solution (TMB 1:1).
• Add the substrate solution to each well (100 µl per well).
• Incubate the plate at room temperature in the dark for 5 minutes.

STOPPING THE REACTION:

• Add stop solution to each well (50µl per well) to halt the enzymatic reaction.

READING THE PLATE:

Measure the absorbance of each well at a wavelength of 450 and 570 nm using a plate reader.

	1	2	3	4
A prot	C neg	24-2-0267	24-2-0315	24-2-0326
B empty	C neg	24-2-0267	24-2-0315	24-2-0326
C prot	C pos	24-2-0306	24-2-0318	24-2-0329
D empty	C pos	24-2-0306	24-2-0318	24-2-0329
E prot	24-2-0229	24-2-0309	24-2-0321
F empty	24-2-0229	24-2-0309	24-2-0321
G prot	24-2-0243	24-2-0312	24-2-0323	24-2-0330
H empty	24-2-0243	24-2-0312	24-2-0323	24-2-0330

	1	2	3	4
A prot	0,008	0,011	0,008	0,01
B empty	0,007	0,01	0,012	0,011
C prot	0,284	0,013	0,009	0,012
D empty	0,008	0,013	0,016	0,017
E prot	0,012	0,009	0,011	0
F empty	0,019	0,012	0,012	0
G prot	0,011	0,082	0,012	0,016
H empty	0,01	0,01	0,012	0,015

RESULTS:

24-2-0229: negative

24-2-0243: negative

24-2-0267: negative

24-2-0306: negative

24-2-0309: negative

24-2-0312: negative (<0.1)

24-2-0315: negative

24-2-0318: negative

24-2-0321: negative

24-2-0323: negative

24-2-0326: negative

24-2-0329: negative