ELISA CASPR1 IN THE CIDPRIT COHORT (03/04/2024)

AIM: to test for anti-CASPR1 antibodies in patients with CIDP (Italian cohort) at baseline or 6m (24-2-0227).

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: 15 Samples from the Italian CIDP cohort at baseline or at 6 months due to the absence of a baseline sample (24-2-0227).

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.

	1	2	3	4	5
A prot	C neg	24-2-0255	24-2-0267	24-2-0282	24-2-0304
B empty	C neg	24-2-0255	24-2-0267	24-2-0282	24-2-0304
C prot	C pos	24-2-0258	24-2-0270	24-2-0285
D empty	C pos	24-2-0258	24-2-0270	24-2-0285
E prot	24-2-0227	24-2-0261	24-2-0273	24-2-0288
F empty	24-2-0227	24-2-0261	24-2-0273	24-2-0288
G prot	24-2-0252	24-2-0264	24-2-0279	24-2-0291
H empty	24-2-0252	24-2-0264	24-2-0279	24-2-0291

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	5
A prot	0,15	0,116	0,141	0,16	0,143
B empty	0,107	0,152	0,229	0,169	0,167
C prot	1,152	0,119	0,133	0,167	0,02
D empty	0,089	0,13	0,188	0,178	0
E prot	0,095	0,143	0,171	0,116	0
F empty	0,106	0,139	0,14	0,095	0
G prot	0,129	0,116	0,159	0,119	0
H empty	0,127	0,14	0,146	0,138	0

RESULTS:

24-2-0227: negative

24-2-0252: negative

24-2-0255: negative

24-2-0258: negative

24-2-0261: negative

24-2-0264: negative

24-2-0267: negative

24-2-0270: negative

24-2-0273: negative

24-2-0279: negative

24-2-0282: negative

24-2-0285: negative

24-2-0288: negative

24-2-0291: negative

24-2-0304: negative

NOTES:

1. To repeat the test in samples 24-2-0267 due to abnormal results (empty well almost significantly higher than the one with protein).