[박상규] Antigen-KLH coupling method

Antigen-KLH coupling method

1.     Dissolve 1 mg antigen in 100ul H2O to make 10 mg/ml solution

2.     In 15ml polypropylene tube,

80 ul of KLH  (125mg/ml in 50% glycerol): 10mg

520 ul H2O

100 ul of 1M Na phosphate, pH7.0

300 ul of antigen: 3mg

Vortex.

3.     Dilute 10 ul of 25% glutaraldehyde into 1ml of 0.1 M Na phosphate, pH7.0 for final 21 mM glutaraldehyde.

4.     Add 0.5 ml of diluted glutaraldehyde solution dropwise to KLH/antigen mixture while vortexing…..(very important)

5.     Let incubate overnight at RT

6.     Use 1ml of KLH-coupled antigen for primary injection (2mg). Freeze remainder at -20oC

7.     Mix 1ml (2mg antigen-KLH conjugate) with 1ml complete Freund’s adjuvant. Emulsify until stiff. Inject 1ml emulsion into each of two rabbits in multiple intradermal sites.