Polyethylene glycol (PEG) is an uncharged linear macromolecular polysaccharide that can nonspecifically cause protein precipitation. The precipitation is reversible, and the biological activity of the precipitated protein is not affected. Different concentrations of PEG can precipitate proteins with different molecular weights. When the pH value, ion concentration and other conditions are fixed, the larger the protein molecular weight, the smaller the PEG concentration used for precipitation. Because PEG6000 has good selectivity for protein precipitation, PEG6000 is mainly used in IC assay.
The mechanism of PEG precipitation of IC may lie in the precipitation by steric exclusion from the liquid phase. In addition, PEG can also inhibit the dissociation of CIC and promote the further polymerization of CIC into larger aggregates to be precipitated. Adding low-concentration PEG to the tested serum can precipitate the IC in the serum, and the existence and content of CIC can be detected by turbidimetric or nephelometry. This method is simple and easy to implement and has been widely used in China.
1. Reagents
(1) 0.1 mol/L pH8.4 borate buffer solution (BBS): 3.40 g of boric acid (H3B03), 4.29 g of borax (Na2B407 10H20), dissolved in distilled water, and added to 1 000 mL. Filter with G3 or G4 glass filter.
(2) PEG-NaF dilution solution: 40.9 g of PEG6000, 10.0 g of NaF, dissolved in BBS, added to 1000 mL, and filtered with a G3 or G4 glass filter.
(3) Thermally polymerized human IgG Human IgC (10mg/mL) was heated in a 63°C water bath for 15min, immediately placed in an ice bath, cooled and passed through a Sepharose4B column or a SephacrylS-300 column to collect the first protein peak. The obtained thermally polymerized human IgG can be used to measure the protein content by Coomassie brilliant blue method. It is also possible to take 10ml of human IgG (10mg/ml), add 100uL of 1.8% glutaraldehyde dropwise under stirring, add 0.4mol/L pH5.4 after 24h, 100uL of Tris-HCl buffer to terminate the reaction, and pass through Sepharose 4B or Sephacryl S-300 column , eluted with 0.01mol/L pH7.4 phosphate buffer to collect the first protein peak. Add bovine serum albumin to 0.5%, and store at -40°C after aliquoting. It can be used as a positive control and to prepare a standard curve in experiments.
2. Operation steps
(1) Take 0.15ml of serum to be tested, add 0.3ml of BBS (1:3 dilution);
(2) Add each solution according to Table 10-1 (the final dilution ratio of the serum to be tested is 1:33, and the final concentration of PEG is 3.64%);
(3) Put the test tube and the control tube in a 37°C water bath for 60 rrfin;
(4) Measure the absorbance of the spectrophotometer at the wavelength of 495nm, and use the control tube to zero.
3. Result judgment
Serum turbidity value to be measured: (measurement tube absorbance – control tube absorbance) × 100. CIC positive was defined as the mean plus 2 standard deviations of turbidity values greater than normal.
Reference value: 4.3 ± 2.0, CIC positive if greater than or equal to 8.3. Or prepare a standard curve with different concentrations of thermally polymerized human IgG according to the above method, check the standard curve according to the serum absorbance value to be tested, and then obtain the IC content (equivalent to ug/mL of thermally polymerized human IgG).
4. Matters needing attention
(1) Low-density lipoprotein can cause increased turbidity, so fasting blood should be collected;
(2) Hypergammaglobulinemia or high blood lipid content and repeated freezing and thawing of specimens can lead to false positives in IC detection;
(3) This method is fast and simple, but the specificity is poor, and it is more suitable for the screening of serum samples.
