20140616 ELISA – NFASC155 Test 2 LAC sera

Objective

We want to see positive marks for LAC and test a new patients for IgG subclasses.

Materials

•  Patients sera
  • LAC
  • 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 without protein for negative controls o/n 4ºC in agitation
2. Wash with PBS-Tween 0.05% x3
3. Blocking with milk 5% in PBS-T 1h
4. Wash with PBS-Tween 0.05% x3
5. Incubate sera 1/100 1h
6. Wash with PBS-Tween 0.05% x3
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

New patient (Dal) is negative. New LAC sera (12-12) is more positive than old one and both have IgG4 subclasses.