Human LZM ELISA Kit

1. Sample processing and requirements:
1、The detection range of the kit is not equivalent to the concentration range of the test substance in the sampleIt is recommended to estimate the concentration of the test substance in the sample through relevant literature before the experiment and determine the actual concentration of the sample through pre-experiment. If the concentration of the test substance in the sample is too high or too low, please dilute or concentrate the sample appropriately.
2. If the sample tested is not among the sample types listed in the instructions, it is recommended to conduct pre-experiments to verify its detection effectiveness.
3、Serum：Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or 2-8 ℃ overnight, then centrifuge at 1000 × g for 20 minutes, take the supernatant, or store the supernatant at-20 ℃ or-80 ℃, but avoid repeated freezing and thawing.
4、Plasma：Collect samples with EDTA or heparin as anticoagulants, and centrifuge the samples at 2-8 ℃ 1000 × g for 15 minutes within 30 minutes after collection. Take the supernatant for detection, or place the supernatant at-20 ℃ or-80 ℃ for storage, but avoid repeated freezing and thawing.
5、Tissue homogenate：The tissue was washed with pre-cooled PBS (0.01 M, pH = 7.4) to remove residual blood (lysed red blood cells in the homogenate would affect the test results), and the tissue was weighed and cut into pieces. Combine the chopped tissue with the corresponding volume of PBS (generally according to the weight-to-volume ratio of 1: 9, for example, 1g of tissue sample corresponds to 9mL of PBS. The specific volume can be appropriately adjusted according to the experimental needs and recorded. It is recommended to add protease inhibitor to PBS) add to a glass homogenizer and grind thoroughly on ice or grind in a homogenizer. For further lysis of tissue cells, the homogenate can be sonicated, or freeze-thawed repeatedly. Finally, the homogenate was centrifuged at 5000 × g for 5-10 minutes, and the supernatant was taken for detection.
6、Cell culture supernatant:Please centrifuge at 1000 × g for 20 minutes, take the supernatant for detection, or store the supernatant at-20 ℃ or-80 ℃, but avoid repeated freezing and thawing.
7、Other biological samples:Centrifuge at 1000 × g for 20 minutes, and take the supernatant for detection.
8、Sample Appearance：The sample should be clear and transparent, and the suspension should be removed by centrifugation.
9、Sample preservation：If the sample is tested within 1 week after collection, it can be stored at 4 ℃. If it cannot be tested in time, please pack it according to the amount used once and store it frozen at-20 ℃ (test within 1 month) or-80 ℃ (test within 6 months) to avoid repeated freezing and thawing. Hemolysis of the sample will affect the final test results, so hemolyzed samples are not suitable for this test.
2. Sample dilution plan:
Please estimate the concentration range of the sample in advance, if your test sample needs dilution, referring to the dilution protocol asBelow:
Dilution 100 times ：One step dilution. Take 5uL sample into 495uL universal diluent and dilute it 100 times;
Dilution 1000 times:Two-step dilution. Take 5uL sample into 95uL universal diluent and do a 20-fold dilution, then take 5uL 20-fold dilution sample into 245uL universal diluent and do a 50-fold dilution, diluting a total of 1000 times;
Dilution 100000 times ：Three-step dilution. Take 5uL sample into 195uL universal diluent and do 40-fold dilution, then take 5uL 40-fold dilution sample into 245uL universal diluent and do 50-fold dilution, and finally take 5uL 2000-fold dilution sample into 245uL universal diluent and do 50-fold dilution, diluting 100,000 times in total;
The amount of liquid taken during each dilution step is not less than3uL, the dilution factor is not more than100 times。 Each step of dilution should be mixed evenly to avoid foaming.
3. Self-prepared test equipment required for the experiment:
1. Microplate reader (450nm)
2. High-precision pipette and tip: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL
3. 37 ℃ incubator
4. Distilled water or deionized water
4. Preparations before testing:
1. Please take out the kit from the refrigerator 10 minutes in advance and balance it to room temperature.
2. Preparation of standard gradient working solution: Add 1mL of universal diluent to the freeze-dried standard, let it stand for 15 minutes until it is completely dissolved, and then gently mix (the concentration is 80ng/mL), and then dilute according to the following concentrations: 80ng/mL, 26.66 ng/mL, 8.88 ng/mL, 2.96 ng/mL, 0.98 ng/mL, 0.32 ng/mL, 0.11 ng/mL, 0ng/mL.
Double dilution method: Take 7 EP tubes, add 400μL of universal diluent to each tube, draw 200μL of 80ng/mL standard working solution into the first EP tube and mix well to prepare 26.66 ng/mL standard working solution, according to this step, draw and mix well in sequence. The last tube is directly used as a blank hole, so there is no need to suck liquid from the penultimate tube, as shown in the figure below.

attention : This picture is for reference only The sample content should be calculated from the standard curve drawn from each experimental data.

8. Problem analysis:
If the experimental results are not good, please take photos of the color development results in time, save the experimental data, keep the used slats and unused reagents, and then contact our company's technical support to solve the problem for you. At the same time, you can also refer to the following information: