Study On The Detection Method Of(1,3)-beta-D Glucan

In human serum, the concentration of(1,3)-beta-D glucan(?G) is a clinical testing index of invasive fungal infection. In this paper, a beta-glucan binding protein of horseshoe crab factor G-subunit ? fragment-a(G?a) was cloned. Instead of using hemocyte extract of horsehoe crad. There is not concern about depletion of resources and stability of the raw material. The DNA fragment of the binding protein gene was cloned into an expressional plasmid pET-15 b. G?a protein was purified with Ni-column. The purified protein was 43 kDa on SDS-PAGE and concentration was 2mg/mL with the purity more than 95 %.Using the characteristics of G?a specific binding ?G, we explored 3 methods to detect the content of ?G: ELISA, Turbidimetry and CDs fluorescent biological method.The experimental results of ELISA showed that the limit of detection was 20ng/mL, indicating that we have successfully established a ELISA method for detection of the ?G.Using the ultraviolet spectrophotometer to detect the absorbance at 340 nm, the results showed that the absorbance value of positive linear correlation with the concentration of ?G. The range was 3.125-200 ?g/mL, R2 was 0.997, and limit of detection was 3.125 ?g/mL. We established a Turbidimetry method for detection of the ?G.Coupling of amination retouching carbon dots with G?a protein and establishing a“carbon dots/ G?a protein” fluorescent sensor for ?G detection. The range was9.375~150 pg/mL, the limit of detection was 9.375 pg/mL, the result showed a good linear relationship. The CV values within batch and between batches were all less than 5 %, accuracy(recovery) were between 97 % ~ 102 %. These results showed that we established a CDs fluorescent biological method for detection of the ?G.The three methods will pave a new way for the determination of ?G. These were low cost, rapid, high sensitivity and specificity.