Cryoglobulin kits

DETECTION OF CRYOGLOBULINS

 Cryoglobulins can be detected by Immunofixation or by Immunodiffusion

  IMMUNODIFFUSION (OUCHTERLONY  METHOD) 

1. Take blood in a centrifuge tube without anticoagulant.
2. Immediately incubate the tube with the blood  30’/37 ⁰C.
3. Centrifuge the blood 5 minutes /3-4000rpm
4. Collect the serum
5. Incubate the serum for 7 days at 4-8^oC
6. On 8^th day check if there are sediments on the bottom of the tube. If not it is negative. If yes than,
7. Centrifuge 5 minutes /3-4000 rpm, carefully discard the serum, mix the sediment with 8ml PBS, incubate it 30 minutes/37^oC and than incubate it at 4-8^oC O/N.
8. Centrifuge 5 minutes /3-4000 rpm, discard the fluid, mix the sediment with 5ml PBS, incubate it  30 minutes/37^oC and than incubate it at 4-8^oC O/N.
9. Centrifuge 5 minutes/3-4000 rpm, carefully discard the serum, mix the sediment with 1ml PBS, incubate it 30’/37^oC and run  the immunodiffusion  test:
10. Cut the gel envelop.
11. Take the gel out of the envelop carefully. Uncover the gel.
12. Apply the sample and the corresponding antisera ( 10-15μl).
13. Put the gel into a humid incubator in a horizontal position overnight.
14. Put on the gel  2 drying blotter sheets and press it ( 2Kg)  for 5 min.
15. Put the gel in the washing Solution bath (0.1% Tween 20* in 0.9% NaCl)  for 10 min.
16. Repeat  steps 14 and 15  ones again.
17. Dry the gel in hot air ( less than 60 ⁰C) .
18. Put the  gel in the staining solution for 60 seconds.
19. Distain the gel in three sub sequential washing baths (5% Acetic Acid) 

a)10μl sample, b) anti IgG, c) anti IgA, d) anti IgM, e) anti kappa, f) anti lambda, anti C’ or anti Albumin.

 Incubate it O/N at room temperature in humid incubator, press it with filter papers, put it in saline for 10 min. repeat the pressing and washing procedure 2 times, dry it, stain  it by amido black and distain with 5% acetic acid.