Kit for detecting NGAL content and preparation method thereof
A kit and reagent technology are applied in the field of kits for detecting NGAL content and the field of preparation thereof, which can solve the problem that immune enhancement turbidimetry and latex immune turbidimetry have poor specificity, results are easily affected by subjective factors, and are easily affected by lipidemia. It can achieve the effect of no radioactive contamination, high sensitivity and strong specificity.
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Embodiment 1
[0038] The preparation method of embodiment 1NGAL antibody-coated magnetic microspheres
[0039] 1. Antibody pretreatment:
[0040] (1) Add 0.1mg of antibody (NGAL-coated antibody) into an ultrafiltration centrifuge tube (50kDa), and centrifuge at 8000r / min for 5-6min. Add 200uL Binding Buffer to the antibody concentrated by centrifugation, centrifuge at 8000-9000r / min for 5-6min, discard the filtrate, then add 200uL Binding Buffer, centrifuge as above, repeat this step 5-6 times.
[0041] (2) Take out the centrifuge tube after several times of centrifugation, discard the filtrate, invert the spin column, centrifuge at 2000-3000r / min for 1min, collect the filtrate, and collect about 200 μL of antibody during the whole process.
[0042] 2. Processing of magnetic microspheres:
[0043] (1) Use a vortex mixer to fully mix and suspend the magnetic microspheres, and take 4.0 mg of the suspension into a micro-volume thick-walled reaction bottle;
[0044] (2) Place the micro thick...
Embodiment 2
[0061] Example 2 Preparation method of acridinium ester-labeled NGAL antibody
[0062] 1. Antibody pretreatment:
[0063] (1) Add 0.13mg of the antibody into an ultrafiltration centrifuge tube (50kDa), centrifuge at 8000r / min for 5-6min to concentrate. Add the concentrated antibody to 200uL PB Buffer, centrifuge at 8000-9000r / min for 5-6min, discard the filtrate, then add 200uL PB Buffer, centrifuge as above, repeat this step 5-6 times.
[0064] (2) Take out the centrifuge tube after several times of centrifugation, discard the filtrate, invert the spin column, centrifuge at 2000-3000r / min for 1min, collect the filtrate, and collect about 200 μL of antibody during the whole process.
[0065] Antibody labeling: Mix the pretreated antibody and acridinium ester salt at a mass ratio of 50:1, and shake for 16-20 hours. Then the obtained product is purified by a purifier. Dilute the antibody labeled with acridinium ester salt to the final concentration with R2 diluent, and store ...
Embodiment 3
[0066] The usage method of embodiment 3 kit
[0067] 1. Loading Reagents
[0068] The 1.1M reagent is yellow-brown magnetic microsphere suspension, and precipitation is a normal phenomenon. Before loading the NGAL quantitative determination reagent on the Caris200 system for the first time, the M reagent bottle needs to be inverted and mixed.
[0069] 1.2 If the magnetic microspheres are still attached to the bottle wall by visual inspection, it is necessary to continue to flip until the magnetic microspheres are resuspended. The reagent cannot be used if it has not been suspended for a long time.
[0070] 1.3 Load the NGAL reagents on the Caris200 according to the requirements of the instrument to ensure that all the reagents required for the detection are loaded on the machine. Make sure all reagent bottle caps are removed.
[0071] 2. Calculate the required sample size
[0072] 2.1 When the NGAL test is performed for the first time, the sample needs to be diluted 1:10 ...
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