The Development Of New Technology For 1,3-β-D-Glucan Detecting

In recent years,due to the comprehensive application of immunosuppressor and broad-spectrum antibiotic,organ transplantation,etc.,the number of invasive fungal infection increased year by year.The invasive fungal disease had already attracted extensive attention of hospitals and patients for its atypical clinical symptoms,low cure rate and high mortality.The traditional diagnosis method had many disadvantages,for example,it always need more time and also had a lot of false positives which would cause that the disease can not be timely diagnose and cure.Thus,hospitals need a method that could be used in the diagnosis of invasive fungal disease fast and effectively.In this paper,the 0.05% NEM and 0.015% EDTA were used as the anticoagulant.Highly active lysate was collected and extracted from horseshoe crabs hemocytes in millions of clean rooms which contain G factor,proclotting enzyme,etc.The1,3-β-D-glucan was the characteristic components in cell wall of the invasive fungus which is special to activate G factor.The biochemical basis of the horseshoe crabs amoebocyte lysate test is enzymatic with CMPS,initiating a series of cascading events which result in the activation of a proclotting enzyme present in the lyzate.The activated clotting enzyme hydrolyses Boc-Leu-Gly-Ary-PNA to release the PNA which make the solution shown the yellow color.This principle is used to establish chromogenic kinetic method to detect1,3-β-D-glucan and diagnose invasive fungal disease.The reagent was dried by the vacuum freeze drying technology because of the protein ingredients of the reaction system.In order to avoid the damage to protein in the freeze drying process,3.6% mannitol was added into the freeze-dried products as protein protectant and excipients.Because the effect of freeze-dried is good,the reagent can be kept for long time under 2-8℃.Processing fluid is mixed with the same volume of 0.02 mol/L KOH and 0.1 mol/L KCl,the cutoff value of CMPS was kept at 82.15 pg/mL,the method showed the better diagnostic value when the sensitivity and specificity are 94.3% and 81.9%,respectively.Endotoxin reaction pathways may be cut off by gel filtration to remove B factor.We can obtain the relationship between the B factor 280 nm absorption peaks and the liquid volume flowing through the column Superdex200 by a tracer protein.The two main agents were found that no B factor detection performance is slightly better after performance comparison,but it is difficult to remove the B factor.The agent containing B factor is easily applied to produce.