ELISA CASPR1 IN CIDPRIT COHORT (22/02/2024)

AIM: to test for anti-CASPR1 antibodies in patients with CIDP (CIDPRIT 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: 10 Samples from the CIDPRIT 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
A prot	C neg	24-2-0229	24-2-0240
B empty	C neg	24-2-0229	24-2-0240
C prot	C pos	24-2-0232	24-2-0243
D empty	C pos	24-2-0232	24-2-0243
E prot	24-2-0222	24-2-0235	24-2-0246
F empty	24-2-0222	24-2-0235	24-2-0246
G prot	24-2-0224	24-2-0237	24-2-0249
H empty	24-2-0224	24-2-0237	24-2-0249

	1	2	3
A prot	0,125	0,157	0,124
B empty	0,175	0,433	0,241
C prot	1,747	0,188	0,188
D empty	0,149	0,252	0,4
E prot	0,143	0,213	0,113
F empty	0,187	0,299	0,17
G prot	0,201	0,177	0,135
H empty	0,275	0,196	0,168

RESULTS:

24-2-0222: negative

24-2-0224: negative

24-2-0229: negative

24-2-0232: negative

24-2-0235: negative

24-2-0237: negative

24-2-0240: negative

24-2-0243: negative

24-2-0246: negative

24-2-0249: negative

NOTES:

1. To repeat the test in samples 24-2-0229 and 24-2-0243 due to abnormal results (empty well significantly higher than the one with protein).