Novel crytococcus neoformans GXM (glucuronoxylomannan) antigen immunodetection kit as well as preparation method and application thereof
A cryptococcus neoformans detection kit technology, applied in the field of immune detection, can solve the problems of large limitations, poor sensitivity, and low positive detection rate
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Embodiment 1
[0101] The preparation of embodiment 1 anti-GXM polyclonal antibody
[0102] 1. Immunization of animals
[0103] Mix equal volumes of GXM antigen and complete Freund's adjuvant to an appropriate volume. After full emulsification, New Zealand big-eared rabbits were injected subcutaneously at multiple points, and the immune dose of each rabbit was controlled at 0.01-1 mg. Ear blood was collected 3 days before immunization, and serum was separated as a negative control. After the initial immunization, immunize once every 2 weeks, and the method is the same as the first time.
[0104] 2. Obtaining polyclonal antibodies
[0105] 1) Titer determination: During the immunization process, blood was collected every few days to measure the titer once after immunization, and the number of immunizations was not less than 3 times.
[0106] 2) Separation of antiserum: When the serum titer reaches the highest level, a large amount of blood is collected by carotid artery bleeding. After the...
Embodiment 2
[0117] The detection of embodiment 2 anti-GXM polyclonal antibodies
[0118] 1. SDS-PAGE electrophoresis detection
[0119] The anti-GXM polyclonal antibody prepared in Example 1 was subjected to SDS-PAGE electrophoresis, and the resulting gel was stained with Coomassie brilliant blue. See the experimental results figure 1 (The pAb swimming lane is the anti-GXM polyclonal antibody prepared in Example 1, and the M swimming lane is the protein Marker). As can be seen from the figure, there are clear and obvious bands in the 25kD and 50kD molecular weight regions, which are respectively the hydrogen chain and the heavy chain of the antibody protein, and there are no bands of foreign proteins, which illustrate the anti-GXM polyclonal antibody prepared in Example 1. The purity is very high.
[0120] 2. Potency determination
[0121] Antibody titers were determined by indirect ELISA. The enzyme-labeled secondary antibody used was horseradish peroxidase-labeled goat anti-rabbit ...
Embodiment 3
[0122] Example 3 Preparation of Cryptococcus neoformans capsular polysaccharide antigen immunoassay kit
[0123] 1. Preparation of enzyme-labeled carrier
[0124] ① Dilute GXM to 25ng / 100μL with coating buffer to obtain GXM coating solution and add it to the wells of the microplate, add 200μL of coating solution to each well, and place the microplate at 2-8°C for 8 hours;
[0125] ②Add blocking solution to the wells of the microplate, add 200 μL of blocking solution to each well, and place at 37°C for 30 minutes to block;
[0126] ③ Discard the blocking solution and place it at a constant temperature of 37°C for 30 minutes to obtain the GXM-coated enzyme-labeled carrier;
[0127] The coating buffer is a 0.01mol / L PBS buffer solution with a pH of 7.0-7.4;
[0128] The preparation of the blocking solution: 0.01mol / L PBS buffer solution containing 3% skimmed milk powder with a pH of 7.0-7.4;
[0129] 2. Preparation of GXM standard
[0130] Dilute GXM antigen with sample dilue...
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