WO2020211317A1 - 一种检测人体可溶性去唾液酸糖蛋白受体的方法 - Google Patents

一种检测人体可溶性去唾液酸糖蛋白受体的方法 Download PDF

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WO2020211317A1
WO2020211317A1 PCT/CN2019/112461 CN2019112461W WO2020211317A1 WO 2020211317 A1 WO2020211317 A1 WO 2020211317A1 CN 2019112461 W CN2019112461 W CN 2019112461W WO 2020211317 A1 WO2020211317 A1 WO 2020211317A1
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buffer
wash
asgpr
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胡静
尹健
施奇敏
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江南大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the invention relates to a method for detecting human soluble asialoglycoprotein receptors, and belongs to the field of immunological detection.
  • Liver disease has been an important and high-incidence disease worldwide for decades. Liver damage such as cirrhosis, hepatitis, and fatty liver is the only way for liver cancer. Liver damage is a serious threat to the health of people in my country and the world. And life.
  • the main detection methods for liver damage include histopathological diagnosis, serological diagnosis and imaging diagnosis.
  • the main clinical serum markers for serological diagnosis are alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and serum bilirubin (BIL), but these indicators have low specificity
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • ALP alkaline phosphatase
  • BIL serum bilirubin
  • Soluble asialoglycoprotein receptor is a soluble protein specifically expressed in the liver. Soluble galactose/N-acetylgalactosamine will appear in the blood due to normal tissue metabolism, injury, diet, disease and other factors. Glycoligands competitively bind to lectins between cells and tissues. The sASGPR in the blood is mainly involved in the elimination of these harmful glycoligands, thereby maintaining the stability of galactose levels in the body. Studies have shown that the content of sASGPR in human blood maintains a certain constant level in healthy people, and when liver damage occurs, it will cause changes in the content of sASGPR. Therefore, as a new potential liver function marker, sASGPR is attracting the attention of researchers. If the link between sASGPR and liver injury can be established, it will provide great help for clinical testing of liver function.
  • kits for detecting human sASGPR there are very few kits for detecting human sASGPR, and the existing ELISA kits for detecting human ASGPR are all double antibody sandwich method kits.
  • the double-antibody sandwich method kit requires two different antibodies directed against different sites of ASGPR.
  • the antibody preparation process is time-consuming and laborious, and the preparation cost is relatively high, which leads to more expensive kits and higher user testing costs; in addition,
  • the detection limits of some existing ELISA kits are also not suitable for the detection of sASGPR in human serum, which makes the detection of serum samples inconvenient and the results are not accurate enough.
  • this study established an ELISA method to detect human sASGPR, which uses the specific binding of ligand and receptor for detection It greatly reduces the cost and preparation difficulty of the detection method, and the detection limit of this method can be well applied to the detection of serum samples. It is an economical, rapid, accurate and highly practical ELISA method.
  • the invention provides an economical, rapid, accurate and highly practical ELISA method for detecting human sASGPR.
  • the ELISA method is an indirect ELISA method based on the specific recognition between the specific ligand of ASGPR and ASGPR. Compared with the common double antibody sandwich ELISA method, this method selects galactosylated human serum albumin (GSA) as the specific ligand of ASGPR. GSA is prepared from human serum albumin, which is cheap and easy to prepare. , Easy to store, etc., and the detection limit of this method is suitable for the detection of sASGPR in human serum samples, and has high practicability. This method does not require the use of special and large-scale equipment and can be carried out in ordinary laboratories.
  • the first object of the present invention is to provide a method for detecting human soluble asialoglycoprotein receptors.
  • the method uses galactosylated human serum albumin (GSA) as the specific ligand for ASGPR and uses ELISA method Perform testing.
  • GSA galactosylated human serum albumin
  • the preparation method of the galactosylated human serum albumin is as follows;
  • the method specifically includes: coating the ELISA plate with GSA diluted with CBS buffer, coating at 4°C for 12-24 hours, and then washing with washing buffer; Add blocking solution to the standard plate and block at 35 ⁇ 37°C for 1.5 ⁇ 2.5h; discard the blocking solution and wash with washing buffer; add sample to the plate, incubate at 35 ⁇ 37°C for 2 ⁇ 2.5h, then use washing buffer Wash and pat dry; add ASGPR1 primary antibody and incubate at 35 ⁇ 37°C for 2 ⁇ 2.5h, then wash with washing buffer; add HRP-labeled goat anti-mouse enzyme-labeled secondary antibody, incubate at 35 ⁇ 37°C for 1 ⁇ 2h, wash with washing buffer Develop the color with TMB color developing solution, and incubate for 10-20 min in the dark, then immediately stop the color development with H 2 SO 4 ; measure the absorbance at 450 nm.
  • the concentration of the CBS buffer is 0.05M.
  • the coating concentration of the GSA is 15-25 ⁇ g/mL.
  • the blocking solution is phosphate Tween buffered saline (PBST) containing 1% skimmed milk.
  • PBST phosphate Tween buffered saline
  • the ASGPR primary antibody is diluted 50 times and then added; the ASGPR primary antibody is purchased from Santa Cruz Biotechnology, the catalog number is sc-166633.
  • the enzyme-labeled secondary antibody is diluted 2000 times and then added; the enzyme-labeled secondary antibody is purchased from Kangwei Century Biotechnology Co., Ltd., and the product number is CW0102.
  • the color development is terminated with H 2 SO 4 at a concentration of 2M.
  • Blocking Add 1% skimmed milk to the coated ELISA well plate according to 300 ⁇ L/well, seal the plate with sealing film, and block at 37°C for 2h; discard the blocking solution and wash with PBS containing 0.1% tween-20 for three times , 3min/time, pat dry;
  • TMB color development use TMB color development solution (Biyuntian Biotechnology Company, P0209) to develop color, 200 ⁇ L/well, incubate in the dark for 15 minutes, and then immediately stop color development with 2M H 2 SO 4 , 50 ⁇ L/well. After stopping the color development, measure the absorbance of each well at 450nm with a microplate reader as soon as possible.
  • TMB color development solution Biyuntian Biotechnology Company, P0209
  • the ELISA plate is prepared by using a 96-well plate.
  • the second object of the present invention is to provide a detection kit, including GSA-coated enzyme-labeled plate, blocking solution, ASGPR standard, ASGPR primary antibody, goat anti-mouse enzyme-labeled secondary antibody, stop solution and washing buffer .
  • the GSA-coated ELISA plate is prepared as follows: GSA is diluted to 20 ⁇ g/mL with a coating solution to coat a 96-well ELISA plate, 100 ⁇ L/well, 4°C After coating for 24 hours, it was washed three times with washing buffer (PBS containing 0.1% tween-20), 3 min/time, and patted dry.
  • washing buffer PBS containing 0.1% tween-20
  • the coating solution is a CBS buffer, and its preparation method is as follows: weigh 1.59g Na 2 CO 3 and 2.93g NaHCO 3 and dissolve them in 800 mL ddH 2 O, and adjust the solution with NaOH The pH reaches 9.6, and finally the volume is accurately adjusted to 1L with ddH 2 O and stored at 4°C.
  • the washing buffer includes a first washing buffer and a second washing buffer;
  • the first washing buffer is 0.01M PBS buffer, and the preparation method is: weighing 2.9 g Na 2 HPO 4 ⁇ 12H 2 O, 0.2g KH 2 PO 4 , 8g NaCl, 0.2g KCl dissolved in 800mL ddH 2 O, adjust the pH of the solution to 7.4 with HCl, and finally use ddH 2 O to accurately dilute to 1L, Store at 4°C;
  • the second washing buffer is PBST buffer, and its preparation method is: add 1 mL of Tween 20 to 1 L of a prepared 0.01M PBS buffer, mix well, and store at 4°C.
  • the method for preparing the blocking solution is as follows: Weigh 1g of skimmed milk and dissolve it in 100mL PBST buffer. After it is fully dissolved, filter it with a 0.22 ⁇ m sterile filter membrane at -20°C. save.
  • the preparation method of the ASGPR standard diluent is: take 1 mL of 1M NaCl solution prepared in advance, 100 ⁇ L 1M Tris-HCl solution (pH 7.4), 100 ⁇ L 1M CaCl 2 solution in a beaker Then accurately weigh 0.1g BSA and add it to a beaker. Use ddH 2 O to accurately dilute to 10 mL. After being fully dissolved, filter and sterilize with a 0.22 ⁇ m sterile filter membrane and store at -20°C.
  • the ASGPR primary antibody was purchased from Santa Cruz Biotechnology, the article number is sc-166633; the enzyme-labeled secondary antibody was purchased from Kangwei Century Biotechnology Co., Ltd., the article number is CW0102; the TMB The color developing solution was purchased from Biyuntian Biotechnology, the article number is P0209.
  • the stop solution is 2M H 2 SO 4
  • the preparation method is: add 89.15 mL ddH 2 O in a beaker, and then add 10.85 mL of concentrated sulfuric acid (98%) dropwise, while adding While stirring slowly.
  • the present invention also claims to protect the application of the kit in detecting liver function indexes.
  • This method selects galactosylated human serum albumin (GSA) as the specific ligand for ASGPR, instead of expensive antibody-coated enzyme-labeled plates, and optimizes the conditions of ELISA detection, which is suitable for human serum samples.
  • GSA galactosylated human serum albumin
  • the detection method of sASGPR in China can reach the detection limit of 4 ⁇ g/L; the average coefficient of variation of the inter-batch repeat test and the intra-batch repeat test are both less than 7%, which has good repeatability; and the linear linear correlation coefficient obtained by the repeated test r is greater than 0.990, the method meets the expected linear range of 4-100 ⁇ g/L; the ELISA plate coated with the method of the present invention can still achieve good repeatability and linear range after stable storage at 37°C for 10 days , The detection limit meets the standard and has important application prospects.
  • Figure 1 is a schematic diagram of the ELISA method for detecting human sASGPR.
  • Figure 2 shows the SDS-PAGE gel electrophoresis (A) and Western blot (B) of the purified ASGPR standard.
  • Figure 3 is a schematic diagram of the ASGPR standard curve.
  • Figure 4 is a linear fitting diagram between the measured concentration and the theoretical concentration.
  • Figure 5 shows the detection of sASGPR in clinical serum samples.
  • reaction solution is transferred to Centrifuge at 5000 ⁇ g for 20 minutes in the MLtra centrifugal filter, discard the filtrate in the lower centrifuge tube, add 10mM CH 3 COOH solution (pH 7) to the upper filter to restore the original volume of the sample, centrifuge again at 5000 ⁇ g, repeat several times Ultrafiltration to remove unreacted galactosamine and reduce the concentration of sodium acetate.
  • the final GSA solution obtained after multiple ultrafiltrations is divided into aliquots and stored in a refrigerator at -80°C for later use.
  • the BCA kit (Takara, T9300A) was used to detect the protein concentration of GSA.
  • BCA reagent B solution 100:1 to prepare a working solution.
  • the solution was sequentially added to a 96-well plate, 10 ⁇ L/well, two parallel samples were taken for each concentration, 200 ⁇ L working solution was added to each well, mixed immediately, and then placed in a 37°C water bath for 30 minutes, cooled to room temperature, and labeled with an enzyme
  • the meter detects the absorbance at 562nm. Draw the standard curve of the BSA standard solution, and calculate the protein concentration of the GSA protein to be tested according to the standard curve.
  • the lactose sepharose bead column was pre-equilibrated with 50 mL washing buffer I, and the lactose sepharose beads were fully transferred to a centrifuge tube and combined with the centrifuged cell supernatant at 4°C overnight. The combined lactose sepharose beads were fully transferred to the column, and the column was washed with 10 mL washing buffer I and 5 mL washing buffer II in sequence.
  • the lactose sepharose bead column was eluted with 14.4 mL of elution buffer, and 1.6 mL of 1M Tris-HCl (pH 7.8) buffer was added to the collected eluate.
  • the concentration of purified sASGPR was detected by the BCA method, and the specificity was verified by Western blot.
  • ASGPR1 mouse-derived primary antibodies namely: ASGPR1/2(E-1) (Santa Cruz Biotechnology, sc-166633) and ASGPR1(A-5) (Santa Cruz Biotechnology, sc-393849), referred to as It is mouse anti-human ASGPR-1 monoclonal antibody, mouse anti-human ASGPR-2 monoclonal antibody.
  • Coat two 96-well microtiter plates with 10 ⁇ g/mL GSA add 100 ⁇ L GSA to each well, and coat at 4°C for 24h. After coating, wash with PBST 5 times, 3min each time, and pat dry. Add 300 ⁇ L of 1% BSA to each well to seal the whole well, and seal it overnight at 4°C.
  • mice anti-human ASGPR-1 monoclonal antibody and mouse anti-human ASGPR-2 monoclonal antibody at a dilution of 1:500 to the two ELISA plates, respectively, with 100 ⁇ L per well, and incubate in a 37°C incubator for 1h. After the primary antibody incubation, wash 4 times with PBST for 3 minutes each time and pat dry.
  • Add 1:2000 diluted goat anti-mouse IgG-HRP to the two plates, with 100 ⁇ L per well, and incubate in a 37°C incubator for 1 hour. After the secondary antibody incubation, wash 4 times with PBST for 3 minutes each time and pat dry.
  • mouse anti-human ASGPR-1 monoclonal antibody is selected as the indirect ELISA method.
  • the detection primary antibody is selected as the indirect ELISA method.
  • Phosphate buffer (0.01M PBS), Tris hydrochloride buffer (0.01M TBS), carbonate buffer (0.05M CBS), 0.9% NaCl were selected as the coating solution to be selected.
  • GSA was diluted to 5 ⁇ g/mL, 10 ⁇ g/mL, 15 ⁇ g/mL, 20 ⁇ g/mL, 25 ⁇ g/mL with 0.05M CBS, and coated on different microtiter plates, each well was coated with 100 ⁇ L.
  • the rest of the steps are the same as above, and the OD 450 values of the negative control wells and the standard ASGPR1 (80 ⁇ g/L) and standard ASGPR2 (40 ⁇ g/L) sample wells are tested respectively, and each sample is set to 4 replicate wells for testing.
  • Embodiment 6 Selection of coating time
  • Different blocking liquids were used for sealing. 300 ⁇ L of blocking liquid was added to each well and blocked at 37°C for 1 hour. The rest of the steps are the same as above, and the OD 450 values of the negative control wells and the standard ASGPR1 (80 ⁇ g/L) and standard ASGPR2 (40 ⁇ g/L) sample wells are tested respectively. Each sample is set with 4 replicate wells for testing, and finally according to P/N Value to determine the best blocking solution.
  • the P/N value is the highest.
  • TMB color development time is set to 5min, 10min, 15min, 20min, then 2M H 2 SO 4 is used to stop the color development, and the OD 450 value is measured with a microplate reader , And finally determine the TMB color time according to the P/N value.
  • the P/N value reaches the highest point. As the color development time increases, the P/N value will decrease.
  • Blocking Add 1% skimmed milk to the coated ELISA well plate according to 300 ⁇ L/well, seal the plate with membrane, and block at 37°C for 2h; discard the blocking solution, wash with washing buffer three times, 3min/time, pat dry.
  • TMB color development use TMB color development solution (Biyuntian Biotechnology Company, P0209) to develop color, 200 ⁇ L/well, incubate in the dark for 15 minutes, and then immediately stop color development with 2M H 2 SO 4 , 50 ⁇ L/well. After stopping the color development, measure the absorbance of each well at 450nm with a microplate reader as soon as possible.
  • TMB color development solution Biyuntian Biotechnology Company, P0209
  • Standard curve drawing Read the OD 450 values of ASGPR standard products of different concentrations and draw the standard curve.
  • the ASGPR negative control sample was repeatedly tested for 20 wells on the same coated ELISA plate, and the test was repeated for five batches. Calculate the average value M and standard deviation SD of the OD 450 value of the negative control sample, calculate the value of M ⁇ 3SD, and put it into the equation of the standard curve to get the lowest detection limit of the ELISA method, and the result shows the lowest detection limit It is 4 ⁇ g/L.
  • Embodiment 15 Linear range
  • Example 17 ELISA method to detect clinical serum samples
  • the ASGPR negative control sample, the ASGPR standard and the sample to be tested (3 serum samples) were respectively detected, and the standard was diluted to 5 ⁇ g/L, 10 ⁇ g/L, 20 ⁇ g/L, With 7 concentrations of 40 ⁇ g/L, 60 ⁇ g/L, 80 ⁇ g/L, and 100 ⁇ g/L, the serum samples were diluted twice with 1 ⁇ PBS for use. Draw a standard curve and calculate the ASGPR content of the corresponding sample according to the OD 450 value of the sample hole.
  • Example 18 ELISA method to detect clinical serum samples
  • a total of 533 serum samples were detected by the indirect ELISA method of Example 12, including 489 healthy people's serum samples and 44 liver injury patients' serum samples.
  • the test results showed that the content of sASGPR in serum samples of healthy people was 85.94 ⁇ 59.69 ⁇ g/L, and the content of sASGPR in serum samples of liver injury patients was 20.41 ⁇ 10.59 ⁇ g/L.
  • serum sASGPR between healthy samples and liver injury samples There are significant statistical differences in content (P ⁇ 0.001).

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Abstract

一种检测人体可溶性去唾液酸糖蛋白受体(sASGPR)的ELISA方法。该方法基于ASGPR的特异性配体与ASGPR之间的特异性识别，选取半乳糖基化人血清白蛋白(GSA)作为ASGPR的特异性配体，GSA由人血清白蛋白制备得来，具有价格便宜、容易制备、易于保存等优点，且该方法的检测限度适用于人血清样品中sASGPR的检测，不需要使用特殊、大型仪器设备，在普通实验室即可开展，具有特异性高、稳定性好、操作简便、费用低廉等优势，为临床肝功能评估提供一定的参考价值。

Description

一种检测人体可溶性去唾液酸糖蛋白受体的方法 技术领域
本发明涉及一种检测人体可溶性去唾液酸糖蛋白受体的方法，属于免疫学检测领域。
背景技术
数十年来肝脏疾病一直是全球范围内一项重要且发病率高的疾病，肝硬化、肝炎、脂肪肝等肝损伤都是肝癌的必经之路，肝脏损伤严重威胁着我国乃至全球人民的健康和生命。目前，肝脏损伤的主要检测手段有组织病理学诊断、血清学诊断及影像学诊断。其中血清学诊断的主要临床血清标志物有谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)及血清胆红素(BIL)等，但这些指标存在着特异性不高的缺点，除了肝损伤会对这些指标存在影响，其他一些疾病也会引起这些指标的异常变化。因此需要发掘一种新的肝损伤血清标志物，从而可以特异性的指示肝功能的异常。
可溶性去唾液酸糖蛋白受体(sASGPR)是肝脏特异性表达的一种可溶性蛋白，血液中因机体正常组织代谢、损伤、饮食、疾病等因素会出现可溶性的半乳糖/N-乙酰半乳糖胺糖配体来竞争性的结合细胞组织间的凝集素，血液中的sASGPR主要参与这些有害的糖配体的清除，从而维持体内半乳糖水平的稳定。有研究表明人体血液中的sASGPR的含量在健康人群中保持一定的恒定水平，并且当肝损伤发生时，会引起sASGPR含量的变化。因此，sASGPR作为一个新型的潜在的肝功能标志物正在受到研究者的关注，如果能建立sASGPR与肝损伤之间的联系，将为肝功能临床检测提供极大的帮助。
目前，可以买到的检测人体sASGPR的试剂盒产品很少，且已有的检测人体ASGPR的ELISA试剂盒均为双抗夹心法试剂盒。双抗夹心法试剂盒需要用到两种针对ASGPR不同位点的不同抗体，抗体制备过程比较费时费力，制备成本也相对较高，从而导致试剂盒价格比较昂贵，使用者检测成本增高；另外，现有的一些ELISA试剂盒的检测限度也不适用于对人体血清中sASGPR的检测，使得对血清样本的检测操作不便，同时结果不够准确。为了更好的检测人体血清中的sASGPR，为肝功能检测提供新的可能的检测方法，本研究建立了一种检测人体sASGPR的ELISA方法，本方法利用配体与受体的特异性结合进行检测，大大降低了检测方法的成本和制备难度，并且本方法的检测限度可以很好的适用于血清样品的检测，是一种经济、快速、准确、实用性高的ELISA法。
发明内容
本发明提供了一种经济、快速、准确、实用性高用于检测人体sASGPR的ELISA法。 该ELISA方法是基于ASGPR的特异性配体与ASGPR之间的特异性识别所建立的间接ELISA法。相对于常见的双抗夹心ELISA法而言，本方法选取半乳糖基化人血清白蛋白(GSA)作为ASGPR的特异性配体，GSA由人血清白蛋白制备得来，具有价格便宜、容易制备、易于保存等优点，且该方法的检测限度适用于人血清样品中sASGPR的检测，实用性较高。该方法不需要使用特殊、大型仪器设备，在普通实验室即可开展。
本发明的第一个目的是提供一种检测人体可溶性去唾液酸糖蛋白受体的方法，所述方法以半乳糖基化人血清白蛋白(GSA)作为ASGPR的特异性配体，应用ELISA法进行检测。
在本发明的一种实施方式中，所述半乳糖基化人血清白蛋白(GSA)的制备方法如下；
在干净的反应瓶中加入20mg人血清白蛋白，再加入20mL 0.5M MES生物缓冲液(pH 5.25)将人血清白蛋白充分溶解，再依次加入108.6mg氨基半乳糖和62mg EDC充分溶解，加样全部完成后将反应瓶放入37℃油浴锅中，反应16h；反应结束后，向反应瓶中加入3.4mL 1M CH 3COOH溶液(pH 4.5)来终止反应；反应终止后将反应液转移至
Figure PCTCN2019112461-appb-000001
ΜLtra离心过滤器中，5000×g离心20min，倒掉下层离心管中滤液，在上层过滤器中加入10mM CH 3COOH溶液(pH 7)复原至样本原体积，再次5000×g离心，重复多次超滤来除去未反应的氨基半乳糖及降低醋酸钠的浓度；多次超滤后最终得到的GSA溶液。
在本发明的一种实施方式中，所述方法具体为：用CBS缓冲液稀释的GSA包被酶标板,4℃包被12～24h，随后用洗涤缓冲液洗涤；在包被好的酶标板中加入封闭液，35～37℃封闭1.5～2.5h；弃封闭液，用洗涤缓冲液洗涤；向酶标板中加入样品，35～37℃孵育2～2.5h，随后用洗涤缓冲液洗涤，拍干；加入ASGPR1一抗35～37℃孵育2～2.5h，随后洗涤缓冲液洗涤；加入HRP标记的羊抗鼠酶标二抗，35～37℃孵育1～2h，洗涤缓冲液洗涤；用TMB显色液显色，避光孵育10～20min，随后立即用H 2SO 4终止显色；测定450nm处吸光度。
在本发明的一种实施方式中，所述CBS缓冲液的浓度为0.05M。
在本发明的一种实施方式中，所述GSA的包被浓度为15～25μg/mL。
在本发明的一种实施方式中，所述封闭液是含1％脱脂牛奶的磷酸盐吐温缓冲液(PBST)。
在本发明的一种实施方式中，所述ASGPR一抗稀释50倍后添加；所述ASGPR一抗购 自Santa Cruz Biotechnology，货号为sc-166633。
在本发明的一种实施方式中，所述酶标二抗稀释2000倍后添加；所述酶标二抗购自康为世纪生物科技有限公司，货号为CW0102。
在本发明的一种实施方式中，用浓度为2M的H 2SO 4终止显色。
在本发明的一种实施方式中，所述方法的具体步骤如下：
(1)包被：用0.05M CBS缓冲液将GSA稀释到20μg/mL包被96孔酶标板，100μL/孔，4℃包被24h，随后用含0.1％tween-20的PBS洗涤三次，3min/次，拍干；
(2)封闭：在包被好的ELISA孔板中按照300μL/孔加入1％脱脂牛奶，封板膜封板，37℃封闭2h；弃封闭液，用含0.1％tween-20的PBS洗涤三次，3min/次，拍干；
(3)加样：待测样品加入96孔酶标板中，每孔加样量为100μL；加样后封板膜封板，37℃孵育2h。
(4)孵育一抗：用封闭液以1:50稀释ASGPR1/2的鼠来源一抗进行孵育，100μL/孔，37℃孵育2h，随后用含0.1％tween-20的PBS洗涤四次，3min/次，拍干；
(5)孵育二抗：用封闭液以1:2000稀释HRP标记的羊抗鼠酶标二抗进行孵育，100μL/孔，37℃孵育1h，用含0.1％tween-20的PBS洗涤四次，3min/次，拍干；
(6)TMB显色：用TMB显色液(碧云天生物技术公司，P0209)显色，200μL/孔，避光孵育15min，随后立即用2M H 2SO 4终止显色，50μL/孔。终止显色后用酶标仪尽快测定450nm处各孔吸光度。
在本发明的一种实施方式中，所述酶标板应用96孔板制备。
本发明的第二个目的是提供一种检测试剂盒，包括经GSA包被的酶标板、封闭液、ASGPR标准品、ASGPR一抗、羊抗鼠酶标二抗、终止液和洗涤缓冲液。
在本发明的一种实施方式中，所述GSA包被的酶标板是按如下方法制备：用包被液将GSA稀释到20μg/mL包被96孔酶标板,100μL/孔，4℃包被24h，随后用洗涤缓冲液(含0.1％tween-20的PBS)洗涤三次，3min/次，拍干。
在本发明的一种实施方式中，所述包被液为CBS缓冲液，其配制方法为：称取1.59g Na 2CO 3、2.93g NaHCO 3溶于800mL ddH 2O中，用NaOH调节溶液pH至9.6，最后用ddH 2O准确定容至1L，4℃保存。
在本发明的一种实施方式中，所述洗涤缓冲液包括第一洗涤缓冲液和第二洗涤缓冲液；所述第一洗涤缓冲液为0.01M PBS缓冲液，其配制方法为：称取2.9g Na 2HPO 4·12H 2O、0.2g KH 2PO 4、8g NaCl、0.2g KCl溶解于800mL ddH 2O中，用HCl调节溶液pH至7.4，最 后用ddH 2O准确定容至1L，4℃保存；所述第二洗涤缓冲液为PBST缓冲液，其配制方法为：将1mL吐温20加入1L配置好的0.01M PBS缓冲液中，混合均匀，4℃保存。
在本发明的一种实施方式中，所述封闭液的配制方法为：称取1g脱脂牛奶溶解于100mL PBST缓冲液中，充分溶解后，用0.22μm的无菌滤膜过滤后于-20℃保存。
在本发明的一种实施方式中，所述ASGPR标准品稀释液的配制方法为：取1mL提前配制好的1M NaCl溶液、100μL 1M Tris-HCl溶液(pH 7.4)、100μL 1M CaCl 2溶液于烧杯中，并准确称取0.1g BSA加入烧杯中，用ddH 2O准确定容至10mL，充分溶解后，用0.22μm的无菌滤膜过滤除菌，-20℃保存。
在本发明的一种实施方式中，所述ASGPR一抗购自Santa Cruz Biotechnology，货号为sc-166633；所述酶标二抗购自康为世纪生物科技有限公司，货号为CW0102；所述TMB显色液购自碧云天生物技术，货号为P0209。
在本发明的一种实施方式中，所述终止液为2M H 2SO 4，配制方法为：在烧杯中加入89.15mL ddH 2O，之后逐滴加入浓硫酸(98％)10.85mL，边加边缓慢搅拌。
本发明还要求保护所述试剂盒在检测肝功能指标中的应用。
有益效果：本方法选取半乳糖基化人血清白蛋白(GSA)作为ASGPR的特异性配体，代替价格高昂的抗体包被酶标板，并优化ELISA检测的条件，建立了适用于人血清样品中sASGPR的检测方法，可达到检测限4μg/L；批间重复试验和批内重复试验的变异系数均值均小于7％，有较好的重复性；且重复检测获得的拟合直线线性相关系数r均大于0.990，该方法满足预期设定的4-100μg/L线性范围；应用本发明的方法包被好的酶标板在37℃下稳定保存10天后仍能够达到良好的重复性、线性范围，检测限符合标准，具有重要的应用前景。
附图说明
图1为检测人体sASGPR的ELISA法的原理图。
图2为ASGPR标准品纯化后的SDS-PAGE凝胶电泳(A)及Western Blot(B)。
图3为ASGPR标准曲线示意图。
图4为测定浓度与理论浓度的线性拟合图。
图5为对临床血清样本中sASGPR的检测。
具体实施方式
实施例1 配体GSA的制备
在干净的反应瓶中加入20mg人血清白蛋白，再加入20mL 0.5M MES生物缓冲液 (pH 5.25)将人血清白蛋白充分溶解，再依次加入108.6mg氨基半乳糖和62mg EDC充分溶解，加样全部完成后将反应瓶放入37℃油浴锅中，反应16h。反应结束后，向反应瓶中加入3.4mL 1M CH 3COOH溶液(pH 4.5)来终止反应。反应终止后将反应液转移至
Figure PCTCN2019112461-appb-000002
ΜLtra离心过滤器中，5000×g离心20min，倒掉下层离心管中滤液，在上层过滤器中加入10mM CH 3COOH溶液(pH 7)复原至样本原体积，再次5000×g离心，重复多次超滤来除去未反应的氨基半乳糖及降低醋酸钠的浓度。多次超滤后最终得到的GSA溶液，将其分装后放入-80℃冰箱中储存待用。用BCA试剂盒(Takara,T9300A)检测GSA的蛋白浓度。测定前，按照BCA试剂A液：BCA试剂B液＝100：1的比例混合后配制成工作液。将BSA标准品溶液分别稀释至2000μg/mL、1500μg/mL、1000μg/mL、750μg/mL、500μg/mL、250μg/mL、125μg/mL，分别将稀释后的BSA标准品溶液以及待测GSA样品溶液依次加入96孔板中，10μL/孔，每个浓度取两个平行样，每孔加入200μL工作液，立即混匀，随后放入37℃水浴槽中反应30min，冷却至室温，用酶标仪检测562nm处吸光值。绘制BSA标准品溶液的标准曲线，并根据标准曲线计算出待测GSA蛋白的蛋白浓度。
实施例2 ASGPR标准品的提纯
取150mL提前收集好的HepG2细胞上清液用离心机离心5min(3500×g)，取离心后的上清液加入到50mL的
Figure PCTCN2019112461-appb-000003
ΜLtra离心过滤器中(3500×g)进行浓缩，最终浓缩至终体积为8mL。向浓缩后的HepG2细胞上清液中加入8mL裂解缓冲液，混合均匀，4℃裂解2-3h。裂解完成后，20000×g离心30min，取上清至新的离心管中，加入800μL 1M CaCl 2，冰上孵育30min。再次20000×g离心30min，弃沉淀。将乳糖琼脂糖珠柱预先用50mL洗涤缓冲液Ⅰ平衡，将乳糖琼脂糖珠充分转移至离心管中，与离心后的细胞上清4℃过夜结合。将结合后的乳糖琼脂糖珠充分转移至柱子中，依次用10mL洗涤缓冲液Ⅰ和5mL洗涤缓冲液Ⅱ洗柱。用14.4mL洗脱缓冲液洗脱乳糖琼脂糖珠柱，收集的洗脱液中加入1.6mL 1M Tris-HCl(pH 7.8)缓冲液。用BCA法检测提纯的sASGPR的浓度，并用Western blot进行特异性验证。
实施例3 一抗的选择
购买两个ASGPR1鼠来源的一抗，分别为：ASGPR1/2(E-1)(Santa Cruz Biotechnology,sc-166633)和ASGPR1(A-5)(Santa Cruz Biotechnology,sc-393849)，以下分别称为鼠抗人ASGPR-1单抗、鼠抗人ASGPR-2单抗。用10μg/mL的GSA包被2块96孔酶标板，每孔加入100μL GSA，4℃条件下包被24h，包被结束后，用PBST洗涤5次，每次3min，拍干。每孔加入300μL 1％BSA进行封闭全孔，4℃条件下封闭过夜，封闭结束后，用PBST洗涤3 次，每次3min，拍干。分别设置ASGPR稀释液为阴性对照孔、质控品孔以及0.15μg/mL、0.50μg/mL、2.00μg/mL、8.00μg/mL的ASGPR标准品为样品孔，将阴性对照及样品依次加入酶标板中，样品做4个重复孔，每孔加样量为100μL。加样后37℃恒温箱中孵育1h。样品孵育结束后，用PBST洗涤3次，每次3min，拍干。两块酶标板中分别加入1:500稀释的鼠抗人ASGPR-1单抗，鼠抗人ASGPR-2单抗，每孔加样量为100μL，37℃恒温箱中孵育1h。一抗孵育结束后，用PBST洗涤4次，每次3min，拍干。两块板中加入1:2000稀释的山羊抗鼠IgG-HRP，每孔加样量为100μL，37℃恒温箱中孵育1h。二抗孵育结束后，用PBST洗涤4次，每次3min，拍干。每孔加入200μL TMB显色液，37℃恒温箱中避光显色15min。显色结束后，每孔迅速加入50μL 2M H 2SO 4终止显色并混匀。立即用酶标仪测各孔450nm处吸光度值，分析结果。
表1 不同ASGPR一抗的检测效果
Figure PCTCN2019112461-appb-000004
从表中数据可以看出使用鼠抗人ASGPR-1单抗时，OD 450的值会随着ASGPR浓度的增加有明显的梯度增加，因此选择鼠抗人ASGPR-1单抗作为本间接ELISA法的检测一抗。
实施例4 包被缓冲液的选择
分别选择磷酸盐缓冲液(0.01M PBS)、Tris盐酸盐缓冲液(0.01M TBS)、碳酸盐缓冲液(0.05M CBS)、0.9％NaCl作为待选包被液。分别用0.01M PBS、0.01M TBS、0.05M CBS和0.9％NaCl将10μg/mL的GSA包被到96孔酶标板中，每孔100μL的GSA，4℃条件下包被24h，包被结束后，用PBST洗涤5次，每次3min，拍干。每孔加入300μL 1％BSA进行封闭全孔，4℃条件下封闭过夜，封闭结束后，用PBST洗涤3次，每次3min，拍干。分别设置阴性对照孔及标准品ASGPR1(80μg/L)、标准品ASGPR2(40μg/L)样品孔，将阴性对照及样品依次加入酶标板中，样品做4个重复孔，每孔加样量为100μL。加样后37℃恒温箱中孵育1h。样品孵育结束后，用PBST洗涤3次，每次3min，拍干。两块酶标板中分别加入1:500稀释的鼠抗人ASGPR-1单抗，每孔加样量为100μL，37℃恒温箱中孵育1h。一抗孵育结束后，用PBST洗涤4次，每次3min，拍干。两块板中加入1:2000稀释的山羊抗鼠IgG-HRP，每孔加样量为100μL，37℃恒温箱中孵育1h。二抗孵育结束后，用PBST洗涤4次，每次3min，拍干。每孔加入200μL TMB显色液，37℃恒温箱中避光显色15min。 显色结束后，每孔迅速加入50μL 2M H 2SO 4终止显色并混匀。立即用酶标仪测各孔450nm处吸光度值。
表2 不同包被缓冲液的检测效果
Figure PCTCN2019112461-appb-000005
从表中数据可以看出，使用0.05M CBS作为包被缓冲液，阳性样品OD 450/阴性对照OD 450(P/N)值最高。
实施例5 不同包被浓度的选择
用0.05M CBS分别将GSA稀释为5μg/mL、10μg/mL、15μg/mL、20μg/mL、25μg/mL，并分别包被在不同的酶标板上，每孔包被100μL。其余步骤同上，分别检测阴性对照孔及标准品ASGPR1(80μg/L)、标准品ASGPR2(40μg/L)样品孔的OD 450值，每个样品设置4个重复孔进行检测。
表3 不同包被浓度的检测结果
Figure PCTCN2019112461-appb-000006
结果显示。包被20μg/mL GSA时，P/N值最高。
实施例6 包被时间的选择
用0.05M CBS将20μg/mL GSA包被到酶标板上，包被条件分别设定为：37℃条件下包被1h、37℃条件下包被2h、4℃条件下包被12h、4℃条件下包被24h。其余步骤同上，分别检测阴性对照孔及标准品ASGPR1(80μg/L)、标准品ASGPR2(40μg/L)样品孔的OD 450值，每个样品设置4个重复孔进行检测，最终根据P/N值确定最佳GSA包被时间。
表4 不同包被时间的检测结果
Figure PCTCN2019112461-appb-000007
结果显示，4℃条件下包被24h，P/N值最高。因此，选择4℃条件下包被24h作为间接ELISA法的包被时间。
实施例7 封闭液的选择
用0.05M CBS将20μg/mL GSA包被在4℃条件下包被24h，分别用1％BSA、5％BSA、1％脱脂奶粉、5％脱脂奶粉、1％FBS、5％FBS这6种不同的封闭液进行封闭，每孔加入300μL封闭液，37℃封闭1h。其余步骤同上，分别检测阴性对照孔及标准品ASGPR1(80μg/L)、标准品ASGPR2(40μg/L)样品孔的OD 450值，每个样品设置4个重复孔进行检测，最终根据P/N值确定最佳封闭液。
表5 不同封闭液的检测效果
Figure PCTCN2019112461-appb-000008
使用1％脱脂奶粉作为封闭液时，P/N值最高。
实施例8 封闭时间的选择
用0.05M CBS将20μg/mL GSA包被在4℃条件下包被24h，用1％脱脂奶粉封闭，封闭时间分别设置为37℃条件下封闭1h、37℃条件下封闭1.5h、37℃条件下封闭2h、37℃条件下封闭2.5h、37℃条件下封闭3h。其余步骤同上，分别检测阴性对照孔及标准品ASGPR1(80μg/L)、标准品ASGPR2(40μg/L)样品孔的OD 450值，每个样品设置4个重复孔进行 检测，最终根据P/N值确定最佳封闭时间。
表6 不同封闭时间的检测效果
Figure PCTCN2019112461-appb-000009
结果显示，封闭时间为2h时，P/N值最高。
实施例9 样品孵育时间的选择
选择上述实验确定的最佳条件进行包被、封闭。分别检测阴性对照以及标准品ASGPR1(80μg/L)、标准品ASGPR2(40μg/L)样品，每个样品设置4个重复孔进行检测，样品孵育时间分别设置为1h、1.5h、2h、2.5h、3h，最终根据OD值及P/N值确定最佳样品孵育时间。
表7 不同样品孵育时间的检测效果
Figure PCTCN2019112461-appb-000010
当孵育时间达到2h时，P/N值达到最高，孵育时间再增加，虽然样品孔OD值会增大，但相应的阴性对照值也会增大，P/N值维持稳定。
实施例10 一抗二抗稀释度的优化
选择上述实验确定的最佳条件进行包被、封闭。分别检测阴性对照孔及ASGPR样品孔，每个样品设置4个重复孔进行检测。一抗分别按照为1:30、1:50、1:100、1:200、1:400稀释，二抗分别按照1:1000、1:2000、1:4000、1:6000稀释。最终根据P/N值确定最佳二抗稀释度。
表8 不同一抗二抗稀释度的检测效果
Figure PCTCN2019112461-appb-000011
表9 棋盘滴定法的P/N值
Figure PCTCN2019112461-appb-000012
从结果中看到，当一抗稀释度为1:50，二抗稀释度为1:2000时，P/N值达到最大。
实施例11 TMB显色时间的选择
用上述优化好的各个最适条件进行间接ELISA检验，最后一步TMB显色时间分别设置为5min、10min、15min、20min，之后用2M H 2SO 4终止显色，并用酶标仪测定OD 450值，最终根据P/N值确定TMB显色时间。
表10 不同显色时间的检测效果
Figure PCTCN2019112461-appb-000013
当显色时间为15min的时候，P/N值达到最高点，随着显色时间的增加，P/N值会有所下降。
实施例12 ELISA法标准曲线的绘制
(1)包被：用0.05M CBS缓冲液将GSA稀释到20μg/mL包被96孔酶标板,100μL/孔，4℃包被24h，随后用洗涤缓冲液(含0.1％tween-20的PBS)洗涤三次，3min/次，拍干。
(2)封闭：在包被好的ELISA孔板中按照300μL/孔加入1％脱脂牛奶，封板膜封板，37℃封闭2h；弃封闭液，洗涤缓冲液洗涤三次，3min/次，拍干。
(3)加样：分别设置ASGPR阴性对照孔以及ASGPR标准品孔，标准品分别用标准品稀释液稀释5μg/L、10μg/L、20μg/L、40μg/L、60μg/L、80μg/L、100μg/L这7个浓度，将阴性对照及ASGPR标准品依次加入96孔酶标板中，做复孔，每孔加样量为100μL。加样后封板膜封板，37℃孵育2h。
(4)孵育一抗：用封闭液以1:50稀释ASGPR1/2的鼠来源一抗(Santa Cruz Biotechnology)进行孵育，100μL/孔，37℃孵育2h，随后洗涤缓冲液洗涤四次，3min/次，拍干。
(5)孵育二抗：用封闭液以1:2000稀释HRP标记的羊抗鼠酶标二抗(康为世纪生物科技有限公司，CW0102)进行孵育，100μL/孔，37℃孵育1h，洗涤缓冲液洗涤四次，3min/次，拍干。
(6)TMB显色：用TMB显色液(碧云天生物技术公司，P0209)显色，200μL/孔，避光孵育15min，随后立即用2M H 2SO 4终止显色，50μL/孔。终止显色后用酶标仪尽快测定450nm处各孔吸光度。
(7)标准曲线绘制：读取不同浓度ASGPR标准品的OD 450值，绘制标准曲线。
实施例13 最低检测限的确定
按照实施例12的方法，将ASGPR阴性对照样品在包被好的同一个酶标板上重复测定20孔，且重复测定五批。计算阴性对照样品OD 450值的平均值M以及标准差SD，计算出M±3SD的值，并将其带入标准曲线的方程中即可得到该ELISA法的最低检测限，结果显示最低检测限为4μg/L。
表11 重复5个批次的检测结果
Figure PCTCN2019112461-appb-000014
实施例14 批间及批内重复性试验
用同一批次和不同批次制备的GSA包被酶标板，按照实施例12的方法，对3个不同浓度的ASGPR阳性样品、阴性样品及血清样品进行重复性试验，各进行三次重复，测定OD值之后，计算得到批间重复试验和批内重复试验的变异系数均值均小于7％，因此该方法有较好的重复性。
表12 批间重复性试验
Figure PCTCN2019112461-appb-000015
表13 批内重复性试验
Figure PCTCN2019112461-appb-000016
实施例15 线性范围
用三个批次包被的酶标板分别检测5个不同浓度(5μg/L、10μg/L、20μg/L、40μg/L、70μg/L、100μg/L)的ASGPR标准品，按照实施例12的方法，对测定浓度与理论浓度做线性拟合，重复检测三次。三次测定获得的拟合直线线性相关系数r均大于0.990，因此该方法满足预期设定的4-100μg/L线性范围。
实施例16 准确性(回收试验)
取三个血清样品，并向三个血清样品中分别添加高(20μg/L)、中(10μg/L)、低(5μg/L)三个浓度的ASGPR标准品，按照实施例12的方法检测原始血清样品及添加ASGPR后的样品中的sASGPR浓度，每个样品重复检测三次，计算ASGPR的回收量及回收率。
表14 回收试验
Figure PCTCN2019112461-appb-000017
实施例17 ELISA法检测临床血清样品
按照实施例12的方法，分别检测ASGPR阴性对照样品、ASGPR标准品和待测样品(3个血清样品)，标准品分别用标准品稀释液稀释至5μg/L、10μg/L、20μg/L、40μg/L、60μg/L、80μg/L、100μg/L这7个浓度，血清样品用1×PBS稀释两倍待用。绘制标准曲线，根据样品孔的OD 450值计算出对应样品的ASGPR含量。
表15 ELISA检测血清样本
Figure PCTCN2019112461-appb-000018
从上述表中可以看到，血清样品的OD值处于标准曲线的测试范围内，该方法可以 达到预期的检测效果。
实施例18 ELISA法检测临床血清样品
用实施例12的间接ELISA法检测共533个血清样本，其中含489例健康人的血清样本及44例肝损伤病人的血清样本。检测结果显示健康人血清样本中sASGPR的含量为85.94±59.69μg/L，肝损伤病人血清样本中sASGPR的含量为20.41±10.59μg/L，经统计学分析，健康样本与肝损伤样本间血清sASGPR含量有显著统计学差异(P＜0.001)。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (13)

  1. 一种检测人体可溶性去唾液酸糖蛋白受体的方法，其特征在于，以半乳糖基化人血清白蛋白作为ASGPR的特异性配体，应用ELISA法进行检测；所述方法具体包括如下步骤：用CBS缓冲液稀释的GSA包被酶标板，4℃包被12～24h，随后用洗涤缓冲液洗涤；在包被好的酶标板中加入封闭液，35～37℃封闭1.5～2.5h；弃封闭液，用洗涤缓冲液洗涤；向酶标板中加入样品，35～37℃孵育2～2.5h，随后用洗涤缓冲液洗涤，拍干；加入ASGPR1一抗35～37℃孵育2～2.5h，随后洗涤缓冲液洗涤；加入HRP标记的羊抗鼠酶标二抗，35～37℃孵育1～2h，洗涤缓冲液洗涤；用TMB显色液显色，避光孵育10～20min，随后立即用H 2SO 4终止显色；测定450nm处吸光度；所述ASGPR1一抗在NCBI上的登录号为NP_001662.1。
  2. 一种检测人体可溶性去唾液酸糖蛋白受体的方法，其特征在于，以半乳糖基化人血清白蛋白作为ASGPR的特异性配体，应用ELISA法进行检测。
  3. 根据权利要求2所述的方法，其特征在于，所述方法具体为：用CBS缓冲液稀释的GSA包被酶标板，4℃包被12～24h，随后用洗涤缓冲液洗涤；在包被好的酶标板中加入封闭液，35～37℃封闭1.5～2.5h；弃封闭液，用洗涤缓冲液洗涤；向酶标板中加入样品，35～37℃孵育2～2.5h，随后用洗涤缓冲液洗涤，拍干；加入ASGPR1一抗35～37℃孵育2～2.5h，随后洗涤缓冲液洗涤；加入HRP标记的羊抗鼠酶标二抗，35～37℃孵育1～2h，洗涤缓冲液洗涤；用TMB显色液显色，避光孵育10～20min，随后立即用H 2SO 4终止显色；测定450nm处吸光度。
  4. 根据权利要求3所述的方法，其特征在于，所述CBS缓冲液的浓度为0.03～0.08M。
  5. 根据权利要求3所述的方法，其特征在于，所述GSA的包被浓度为15～25μg/mL。
  6. 根据权利要求3所述的方法，其特征在于，所述ASGPR一抗购自Santa Cruz Biotechnology，货号为sc-166633；所述酶标二抗稀释2000倍后添加；所述酶标二抗购自康为世纪生物科技有限公司，货号为CW0102。
  7. 一种检测人体可溶性去唾液酸糖蛋白受体的试剂盒，其特征在于，包括经GSA包被的酶标板、封闭液、ASGPR标准品、ASGPR一抗、羊抗鼠酶标二抗、终止液和洗涤缓冲液。
  8. 根据权利要求7所述的试剂盒，其特征在于，所述洗涤缓冲液包括第一洗涤缓冲液和第二洗涤缓冲液；所述第一洗涤缓冲液为PBS缓冲液；所述第二洗涤缓冲液为PBST缓冲液。
  9. 根据权利要求7所述的试剂盒，其特征在于，所述封闭液是脱脂牛奶和PBST缓冲液的混合物；所述终止液为H 2SO 4溶液。
  10. 权利要求1～6任一所述方法在检测肝功能指标中的应用。
  11. 权利要求7～9任一所述的试剂盒在生物、医学检测领域中的应用。
  12. 根据权利要求11所述的应用，其特征在于，用于检测肝功能指标。
  13. 根据权利要求10或12所述的应用，所述肝功能指标包括血清中sASGPR含量。
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