20140605 nfasc-elisa IgG subclasses and JML

Objective

We want to know if [Ab-a-nfasc] from JML serum decrease after the treatment (1 month and 3 months).

We also want to know the IgG subclasses for a new positive patient (with HEK transfected was IgG2).

Materials

•  Patients sera
  • LAC
  • JML (0, 1, 2)
  • Ctrl –
  • new patient (13)
• NFASC protein
• Milk
• PBS
• PBS-tween 0.05%
• TMB (reagent A+B)
• H2SO4 25%
• Buffer carbonate/bicarbonate
• Antibodies
  • RAH-IgG-HRP
  • MAH-IgG1-HRP
  • MAH-IgG2-HRP
  • MAH-IgG3-HRP
  • MAH-IgG4-HRP

Methods

1. Coating: neurofascin protein 1/350 (diluted in buffer carbonate/bicarbonate) + buffer with milk 5% for negative controls o/n 4ºC in agitation
2. Wash with PBS-Tween 0.05% x4
3. Blocking with milk 5% in PBS-T 1h
4. Wash with PBS-Tween 0.05% x4
5. Incubate sera 1/100 1h
6. Wash with PBS-Tween 0.05% x4
7. Secondary antibody (1) RAHIgG-HRP 1/10000 (2) MAHIgG1-HRP 1/10000 (3) MAHIgG2-HRP 1/10000 (4) MAHIgG3-HRP 1/10000 (5)MAHIgG4-HRP 1/10000

Results and conclusions

We can see that antibodies against neurofascin in JML decrease after treatment. We’ll do it again with sera from 6months after treatment.

Positive control (LAC) is negative in this case, so we’re going to repeat it.