Method for quickly detecting lead poisoning based on magnetic separation and quantum dots marks and kit thereof
A kit and rapid technology, applied in measurement devices, analytical materials, instruments, etc., to achieve the effects of high photochemical stability, easy operation, and not easy to photolysis
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[0046] Example 1
[0047] 1) Pb immune antigen synthesis: Pb (National Center for Analysis and Testing of Non-ferrous Metals and Electronic Materials, GSB04-1742-2004), bifunctional chelating agent [(R)-2-thiocyano-3-(4-aminophenyl) )Propyl]-(SS)cyclohexane-1,2 diethylenetriaminepentaacetic acid (p-SCN-Bn-CHX-A"-DTPA) (Macroyclics, USA) and hemocyanin (KLH) (Sigma, United States, H7017) and triethylamine (TEA) (domestic analytical grade) according to Pb:p-SCN-Bn-CHX-A"-DTPA:KLH:TEA=9:12:200:13(W / W / W / W), the reaction was shaken at 25°C for 22h; the reaction product was washed with PBS buffer, and unreacted small molecules were filtered out through an ultrafiltration tube (30KD); the obtained Pb immune antigen was diluted with PBS buffer.
[0048] 2) Pb detection antigen synthesis: Pb, p-SCN-Bn-CHX-A"-DTPA, bovine serum albumin (BSA) (Sigma, USA, A1933) and TEA are combined according to Pb: p-SCN-Bn-CHX- A"-DTPA:BSA:TEA=9:12:200:13 (W / W / W / W) ratio of synthetic detection antigen. ...
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[0049] Example 2
[0050] Preparation and purification of anti-Pb monoclonal antibody (PbMAb):
[0051] The 1.0 mg / ml Pb immune antigen and an equal volume of Freund's complete adjuvant (FCA) are thoroughly mixed by the syringe double push method to form a white water-in-oil emulsion, that is, an antigen emulsifier. Multipoint immunization was used to immunize 6-week-old female Balb / C mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., 211), immunized once every 2 weeks, and immunized 5 times in total. In the second, third, and fourth immunizations, the Pb immune antigen was mixed with an equal volume of Freund’s incomplete adjuvant (FIA), and the same dose was used to boost the immunization. In the fifth immunization, only Pb was used to immunize the antigen without Add adjuvant. The titer and specificity of mouse serum after mouse immunization are as follows Figure 3-4 Shown. Spleen cells were prepared from the immunized mice with the highest antiserum tite...
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[0052] Example 3
[0053] Anti-Pb immunomagnetic beads (PbMAb-Fe 3 O 4 ) Preparation: Because nano magnetic beads ( M-280Tosylactivated, lifetechnologies, USA, 14203) has a low background value, and the antibody is covalently attached to the surface of the beads, which is an excellent choice for immunoprecipitation of protein complexes. The magnetic aggregation of the beads is gentle and fast and the incubation time is very short. Therefore, the choice of nano magnetic beads M-280Tosylactivated, its particle size is 280nm. The synthesis steps of anti-Pb immunomagnetic beads are as follows:
[0054] (1) Take 165 μL of nano magnetic beads, magnetically separate for 1 min, and remove the supernatant;
[0055] (2) Add 100μg of anti-lead monoclonal antibody and 0.1M pH7.4 phosphate buffer to make the volume become 150μL, vortex and shake;
[0056] (3) Add 100μL 3M(NH 4 ) 2 SO 4 Vortex the 0.1M pH7.4 phosphate buffer.
[0057] (4) Incubate with shaking at 37°C for 12-18h;
[0058] (5) Mag...
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