The precipitation by perchloric acid is usually the first step in the isolation of β2GPI used in the ELISA for anti-β2GPI antibodies. Perchloric acid inhibits plasmin and denaturates most proteins except for those being very basic.
The aim of our study was to evaluate the common procedure for the isolation of β2GPI with special emphasis on the precipitation step with perchloric acid.
The precipitation by perchloric acid, performed with different timing (3, 18 or 50 minutes) was followed by affinity chromatography on heparin, concluded by cationic exchange chromatography. Elution with the Na+ gradient (linear 0.05-0.65 M) led to three distinct protein peaks. Each peak was isolated and analysed separately by 1./denaturated polyacrylamide electrophoresis, 2./rocket electrophoresis with rabbit polyclonal anti-β2GPI and 3./ELISA with 6 SLE and/or APS patients' sera, previously determined by the standard anticardiolipin and anti-β2GPI ELISA. In the ELISA all 9 proteins were used at the same concentration, determined by colorimetric reaction.
The protein from the 2nd peak exhibited a molecular weight of 50 kDa, corresponding to both molecular weight markers and reference β2GPI (Tincani, Brescia Italy). Both the protein from the 1st and 3rd peak exhibited a molecular weight of about 55 kDa. The same result was observed after all three different precipitations. In rocket electrophoresis, the proteins from the 2nd and 3rd but not from the 1st peak reacted with polyclonal rabbit anti-β2GPI antibody. There were no diferences in the activities among the isolates obtained by the different precipitation timing. In the ELISA, the proteins from the 2nd and 3rd but not from the 1st peak reacted with human anti-β2GPI antibodies. Differences among the isolates obtained by different precipitation timing were observed. The protein from the 2ndpeak obtained after 3-minute precipitation reacted 2 to 10 times stronger with different patients' sera than the protein from the 3rd peak (from the same isolation). Proteins obtained after 18-minute precipitation reacted more weakly than did the proteins from the isolation after 3-minute precipitation, but the protein from the 2nd peak gave still 2 to 6 time higher results than the protein from the 3rd peak. The proteins from the 2nd and 3rd peaks after 50-minute precipitation reacted almost equally; the protein from the 3rd peak after 50-minute precipitation reacted stronger than the same protein either after 18-minute or 3-minute precipitation.
In conclusion, the precipitation with perchloric acid followed by affinity purification on heparin and cation exchange chromatography with linear Na+ gradient (0.05 to 0.65 M) yielded three different proteins, out of which one was antigenically nonactive and two were antigenically active with anti-β2GPI antibodies. The time of precipitation influenced the antigenic properties of the two proteins with the same antigenic specificity but different molecular weight.