Preparation of ISCOMs Hydrophobic Interactions

Kidney Function Restoration Program

Natural Remedies for Kidney Disease

Get Instant Access

3.2.1. Amphipathic Antigens (Native or Lipidated) in Detergent

1. Make a preparation of the antigen in detergent.

2. Mix antigen, lipids, and saponin according to Table 4 and adjust the volume with PBS. Incubate for 1-2 h at room temperature prior to extensive dialysis against PBS.

3. Dialyze against 3-5 changes of buffer (24-48 h), the first 24 h at 20-25°C then at +4°C.

3.2.2. Antigen in 3-8 M urea (Gua-HCl, DMSO, EG, and so on)

1. If the antigen is solubilized in, e.g., urea, mix antigen, lipids, and saponin according to Table 4 and adjust the volume with 3-8 M urea. Incubate for 1-2 h at room temperature prior to extensive dialysis against PBS. If the antigen is provided in another buffer and urea is used to increase the solubility, the incubation time may need extension to 18-24 h.

2. Dialyze against 3-5 changes of buffer (24-48 h), the first 24 h at 20-25°C, then at +4°C.

3.2.3. Low pH Procedure

1. Mix antigen, lipids, and saponin according to Table 4 and add 1/10 of the final volume (e.g., 0.1 mL to 1 mL) of 1 M citrate pH 2.5. Mix thoroughly and incubate for 1-2 h at room temperature prior to extensive dialysis against PBS. Because of the low pH, a white precipitate forms. Dissolve the precipitate by resuspending the precipitate several times a day during dialysis until it is dissolved or diminished to minimum.

2. Dialyze against 3-5 changes of buffer (24-48 h), the first 24 h at 20-25°C, then at +4°C.

Table 4

Guideline for the Mixing of Antigens, Saponins, and Lipids for ISCOM Formation

Detergent Antigen CHOL1 PL2 Quillaja saponin3

3,5 (Iscoprep 703) Final 1-2% 0.2-1 mg/mL >1 mg/mL

Concentration (or more)

The final concentration of detergent should preferably not exceed 2% (see Notes 1 and 2). A higher concentration may be used, but it is important to realize that the time of dialysis required to remove the detergent and complete ISCOM (or ISCOM-matrix) formation often becomes substantially longer. Under these circumstances, it may happen that other components in the mixture, particularly the phospholipid and Quillaja saponin component(s), may also be partly lost. The final concentration of CHOL and PL depends on the antigen concentration and must be balanced with Quillaja saponin, as indicated in this table. 1Cholesterol 2Phospholipid

3Other Quillaja saponin preparations may balance with cholesterol different ratios. If the supplier of the saponin is unable to give this information this has to be tested by mixing 1 mL volumes containing 1 mg of cholesterol (containing 3H-cholesterol) with 1 mg of phospholipid (in 2% MEGA-10) with different amounts of the saponin preparation (e.g., 1, 3, and 10 mg). Proceed as described under Subheading 3.1. (1-3). Submit samples of the preparations for negative staining EM analysis (Note 6) and sucrose density gradient centrifugation (Note 7).

Was this article helpful?

0 0

Post a comment