Generally speaking, due to the complex composition of blood, many substances, especially the plasma or serum of higher animals, have a strong interference effect on the reaction between TAL (LAL) reagent and endotoxin. Therefore, when we test the endotoxin of such samples, we must remove the interfering substances before testing.

At present, the interfering substances in plasma mainly include various protease inhibitors, endotoxin-binding proteins, and plasminogen inhibitors. For colorimetry, factor Xa can decompose the Boc-LEU-Gly-Arg-PNA peptide chain. Therefore, it is necessary to eliminate these interfering substances before determination.

The pretreatment methods for removing interfering substances in the blood are as follows:

(1) Dilution heating method: plasma is diluted by 3~10 times with dissolved water, and then heated in 37~100 ℃ water. The holding time is generally 5~10 minutes;

(2) Chloroform extraction method: add the same amount of chloroform to the plasma and vibrate for 3 hours, then take the upper layer for detection;

(3) Ether treatment method: add ether to plasma for treatment and remove ether at 40 ℃;

(4) Trifluoroacetic acid method: add acetic acid into a plasma to adjust pH 4.0, and then use K2HPO4 to adjust pH to 7~7.5;

(5) Surfactant treatment method: like the dilution heating method, only the dilution water is changed to surfactant;

(6) Gel filtration;

(7) Alkaline treatment method: the plasma was kept at 0.2NNaOH 37 ℃ for 10 minutes and then neutralized with HCl;

(8) Perchloric acid method (PCA) or new perchloric acid method (New PCA);

(9) Beads method: put the special resin absorbing endotoxin into the plasma, take it out, and add LAL reagent for reaction.

Among these methods, the commonly used methods are the chloroform extraction method, dilution heating method, perchloric acid (PCA) method, and new perchloric acid (New PCA) method. The first two methods need to be discussed during the determination due to the existence of endotoxin recovery rate, false positives, and other problems. The PCA method (New PCA method), due to the good recovery rate of added endotoxin, is currently mainly used in preclinical treatment in Japan.