Background[1-7]
Human Glypican 3 (GPC-3) ELISA KIT is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). Standards with known concentrations of the substance to be tested and samples with unknown concentrations are added to the microwells Detection in enzyme plate. First, the substance to be tested and the biotin-labeled antibody are incubated simultaneously. After washing, avidin-labeled HRP was added. After further incubation and washing, unbound enzyme conjugates are removed, and then substrates A and B are added, and the enzyme conjugates act simultaneously. The type and concentration of unknown samples are detected by using the proportional relationship between the color and the depth of the color and the concentration of the substance to be measured in the sample.
Operation Precautions
1. Reagents should be stored according to label instructions and returned to room temperature before use. The diluted standards should be discarded and should not be stored.
2. The slats not used in the experiment should be put back into the packaging bag immediately and sealed to avoid deterioration.
3. Other reagents that are not in use should be packed or covered. Do not mix reagents from different batch numbers. Use before expiration date.
4. Use disposable pipette tips to avoid cross-contamination. When pipetting the stop solution and substrate A and B solutions, avoid using a sample injector with metal parts.
5. Use a clean plastic container to prepare the washing liquid. Mix the components and samples in the kit thoroughly before use.
6. When washing the enzyme label plate, pat it dry thoroughly. Do not put absorbent paper directly into the enzyme label reaction well to absorb water.
7. Substrate A should evaporate, avoid opening the lid for a long time. Substrate B is light sensitive, avoid prolonged exposure to light. Avoid contact with hands, toxic. The OD value should be read immediately after the experiment is completed.
8. The order of adding reagents should be consistent to ensure that all wells of the reaction plate are incubated with the imported polymer flocculant for the same time.
9. Carry out the incubation operation according to the time, amount and sequence of added liquids indicated in the instructions.
Kit operation steps:
1. Mix all reagents thoroughly before use. Do not make the liquid produce a lot of foam to avoid adding a large number of bubbles when adding the sample, causing errors in the sample addition.
2. Determine the number of strips required based on the number of samples to be tested plus the number of standards. It is recommended to make duplicate holes for each standard and blank hole. Each sample is determined according to its own quantity. If multiple holes can be used, try to make multiple holes. Dilute the specimen 1:1 with specimen diluent and add 50ul into the reaction well.
3. Add 50ul of the diluted standard into the reaction well, and add 50ul of the sample to be tested into the reaction well. Immediately add 50ul of biotin-labeled antibody. Cover the membrane plate, shake gently to mix, and incubate at 37°C for 1 hour. 4. Shake off the liquid in the wells, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, and pat dry with absorbent paper. Repeat this 3 times. If washing with a plate washer, increase the number of washes by one.
4. Add 80ul of streptavidin-HRP to each well, mix gently, and incubate at 37°C for 30 minutes.
5. Shake off the liquid in the wells, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, and pat dry with absorbent paper. Repeat this 3 times. If washing with a plate washer, increase the number of washes by one.
6. Add 50ul of each of substrates A and B to each well, mix gently, and incubate at 37°C for 10 minutes. Avoid light.
7. Take out the enzyme plate and quickly add 50ul of stop solution. After adding the stop solution, measure the result immediately.
9. Measure the OD value of each well at a wavelength of 450nm.
Kit performance characteristics:
1. Sensitivity: The minimum detection concentration is less than the No. 1 standard. Linearity of dilution. The R value of the correlation coefficient between the sample linear regression and the expected concentration is 0.990.
2. Specificity: Does not react with other cytokines.
3. Repeatability: The intra-plate and inter-plate variation coefficients are both less than 10%.
Judgment and analysis of kit results:
1. Instrument value: Read the OD value of each well on a microplate reader with a wavelength of 450nm
2. Take the absorbance OD value as the ordinate (Y) and the corresponding standard concentration of the substance to be tested as the abscissa (X). Draw a corresponding curve. The content of the substance to be tested in the sample can be calculated from the standard according to its OD value. The curve is converted to the corresponding concentration.
Apply[8]
Human Glypican 3 (GPC-3) ELISA KIT can be used to detect human phosphatidylinositol proteoglycan 3 (GPC-3) ELISA KITImmunological activity of glypican 3:
In the construction of the eukaryotic expression vector of glypican 3 and the expression experiment in cells, the GPC3 gene was amplified by PCR technology, and the sequence was inserted into the p3XFLAG-CMV-14 vector using the restriction site to construct p CMV-gpc3 expression vector. The vector was transfected into HEK293 cells via liposomes, and Western blot technology was used to detect the final results. Collect stably expressing cells, break them, and pass them through an affinity chromatography column to obtain GPC3 protein with higher purity. However, it is necessary to detect the immune activity and concentration of glypican 3 before subsequent experiments to ensure the success of subsequent immune experiments.
References
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[2]Glypican‐3: a marker and a therapeutic target in hepatocellular carcinoma[J].Jorge Filmus,Mariana Capurro.FEBS J.2013(10)
[3]Diagnostic role of serum glypican‐3 in differentiating hepatocellular carcinoma from non‐malignant chronic liver disease and other liver cancers[J]. Hepatology.2009(1)
[4]Glypican-3 is a useful diagnostic marker for a component of hepatocellularcarcinoma in human liver cancer[J].Hirofumi Shirakawa,Toshimitsu Kuronuma,Yoshiko Nishimura,Takahiro Hasebe,Masayuki Nakano,Naoto Gotohda,Shinichiro Takahashi,Toshio Nakagohri ,Masaru Konishi,Nobuaki Kobayashi,Taira Kinoshita,Tetsuya Nakatsura.International Journal of Oncology.2009(3)
[5]Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma[J].Mariana Capurro,Ian R Wanless,Morris Sherman,Gerrit Deboer,Wen Shi,Eiji Miyoshi,Jorge Filmus.Gastroenterology.2003(1 )
[6]Glypican-3, overexpressed specifically in human hepatocellular carcinoma, is a novel tumor marker[J]. Tetsuya Nakatsura, Yoshihiro Yoshitake, Satoru Senju, Mikio Monji, Hiroyuki Komori, Yutaka Motomura, Seiji Hosaka, Toru Beppu, Takatoshi Ishiko,Hidenobu Kamohara,Hiroshi Ashihara,Toyomasa Katagiri,Yoichi Furukawa,Shigetoshi Fujiyama,Michio Ogawa,Yusuke Nakamura,Yasuharu Nishimura.Biochemical and Biophysical Rese Industrial Antioxidantsarch Communications.2003(1)
[7]Glypican 3 and glypican 4 are juxtaposed in Xq26.1[J].Reid Huber,Richard Mazzarella,Chun-Nan Chen,Ellson Chen,Maggie Ireland,Susan Lindsay,Giuseppe Pilia,Laura Crisponi.Gene.1998 (1)
[8] Cheng Hua, Xiang Xiangdong, Cui Shuo, Wu Meng, Zhang Zhaojun, Li Zexia. Construction of eukaryotic expression vector for glypican 3 and its expression in cells [J]. Biotechnology Bulletin, 2016,32(05):146-150.
