Objectives
To test some of the most positive neuron-staining Zika-GBS patients IgM positivity towards human CNTN-1+CASPR-1 and to test whether it’s a double positivity or just an unespecificity.
Patients
- ZN004A (lab id # 5)
- ZN005A (lab id # 11)–> remaining IP serum 1/100 (not further diluted in the experiment)
CNTN-1+CASPR-1 ICC
- Human CNTN-1+CASPR1 transfected HEKs 293 grown in coverslips (plates). Fixed with PFA 4% and blocked with Rabbit 1/40 in PBS
- PBS
- Blocking: Rabbit 1/40 in PBS
- Patient’s sera diluted 1/40 (IgM) with Rabbit 1/40 in PBS. Note:ZN005A (lab id # 11)–> remaining IP serum 1/100 (not further diluted in the experiment)
- Primary Ab: Goat CNTN1 (1/1000)
- Secondary Abs: RAG 488 (1/1000)+ RAH 594 (1/1000) IgM
- Vectashield mounting medium
NON-transfected HEKS ICC
- Human non-transfected HEKs 293 grown in coverslips. Fixed with PFA 4% and blocked with GOAT 5% in PBS.
- PBS
- Blocking: 5% goat serum in PBS
- Patient’s sera diluted 1/40 (IgM) with5% goat serum in PBS.Note:ZN005A (lab id # 11)–> remaining IP serum 1/100 (not further diluted in the experiment)
- Primary Ab: Mouse CASPR1 (1/1000)
- Secondary Abs: GAM 488 (1/1000)+ GAH 594 (1/1000) IgG or IgM
- Vectashield mounting medium
Note: Ideally cntn1 primary antibody should have been used in this condition. However, as it is produced in goat and cells had already been blocked with goat 5% in PBS (and double blocking has proved to be not useful in other experiments), mouse CASPR-1 was used instead.
RESULTS
All samples are negative, and therefore the staining seen in experiment 2017/05/30 for sample ZN004A (lab id 5) is totally unespecific.
