Method for Detecting Human Soluble Asialoglycoprotein Receptor
20200319174 ยท 2020-10-08
Inventors
Cpc classification
C07K14/705
CHEMISTRY; METALLURGY
G01N2400/00
PHYSICS
G01N33/5308
PHYSICS
International classification
Abstract
The disclosure discloses a method for detecting a human soluble asialoglycoprotein receptor, and belongs to the field of immunological detection. The disclosure provides an economical, rapid, accurate and highly practical ELISA method for detecting a human sASGPR. Based on the specific recognition between a specific ligand of ASGPR and ASGPR, the method selects galactosylated human serum albumin (GSA) as the specific ligand of ASGPR. GSA is prepared from human serum albumin, and has the advantages of cheap price, easy operation, easy storage and the like, and the limit of detection of the method is suitable for the detection of sASGPR in human serum samples. The method can be carried out in ordinary laboratories without using special, large-scale instruments and equipment. The method has the advantages of high specificity, good stability, simple and convenient operation, low cost and the like, and provides a certain reference value for clinical liver function evaluation.
Claims
1. A method for detecting a human soluble asialoglycoprotein receptor (ASGPR), comprising using an (enzyme-linked immunosorbent assay (ELISA method) to detect galactosylated human serum albumin (GSA) as a specific ligand of ASGPR.
2. The method according to claim 1, wherein the using the ELISA method to detect GSA comprises: coating an ELISA plate with GSA diluted with a CBS buffered solution at a temperature of 0 to 4 C. for 12 to 24 h, and then washing with a washing buffer; adding a blocking buffer to the coated ELISA plate to block at a temperature of 35 to 37 C. for 1.5 to 2.5 h; discarding the blocking buffer, and washing with a washing buffer; adding a sample to the ELISA plate, incubating at a temperature of 35 to 37 C. for 2 to 2.5 h, then washing with a washing buffer, and patting dry; adding an ASGPR1 primary antibody and incubating at a temperature of 35 to 37 C. for 2 to 2.5 h, and then washing with a washing buffer; adding an HRP-labeled goat anti-mouse enzyme-labeled secondary antibody, incubating at a temperature of 35 to 37 C. for 1 to 2 h, and washing with a washing buffer; developing with a TMB development solution, incubating for 10 to 20 min in the dark, and then immediately stopping the development with H.sub.2SO.sub.4; and determining absorbance at 445 to 455 nm.
3. The method according to claim 2, wherein a concentration of the CBS buffered solution is 0.03 to 0.08 M.
4. The method according to claim 2, wherein a coating concentration of the GSA is 15 to 25 g/m L.
5. The method according to claim 2, wherein the enzyme-labeled secondary antibody is added after being diluted 2000 times.
6. The method according to claim 2, wherein a concentration of the CBS buffered solution is 0.03 to 0.08 M; a coating concentration of the GSA is 15 to 25 g/mL; and the enzyme-labeled secondary antibody is added after being diluted 2000 times.
7. A kit for detecting a human soluble asialoglycoprotein receptor, comprising a GSA-coated ELISA plate, a blocking buffer, an ASGPR standard product, an ASGPR primary antibody, a goat anti-mouse enzyme-labeled secondary antibody, a stop buffer and a washing buffer.
8. The kit according to claim 7, wherein the washing buffer comprises a first washing buffer and a second washing buffer; the first washing buffer is a PBS buffered solution; and the second washing buffer is a PBST buffered solution.
9. The kit according to claim 7, wherein the blocking buffer is a mixture of skim milk and a PBST buffered solution; and the stop buffer is an H.sub.2SO.sub.4 solution.
10. The kit according to claim 7, wherein the washing buffer comprises a first washing buffer and a second washing buffer; the first washing buffer is a PBS buffered solution; the second washing buffer is a PBST buffered solution; the blocking buffer is a mixture of skim milk and the PBST buffered solution; and the stop buffer is an H.sub.2SO.sub.4 solution.
Description
BRIEF DESCRIPTION OF FIGURES
[0035]
[0036]
[0037]
[0038]
[0039]
DETAILED DESCRIPTION
Example 1 Preparation of Ligand GSA
[0040] 20 mg of human serum albumin was added to a clean reaction flask, then 20 mL of 0.5 M MES biological buffer (pH 5.25) was added to fully dissolve the human serum albumin, then 108.6 mg of galactosamine and 62 mg of EDC were sequentially added to fully dissolve, and after the sample addition was fully completed, the reaction flask was put in a 37 C. oil bath pan to react for 16 h; after the reaction was finished, 3.4 mL of 1 M CH.sub.3COOH solution (pH 4.5) was added to the reaction flask to stop the reaction; after the reaction was stopped, a reaction solution was transferred to an Amicon MLtra centrifugal filter for centrifugation at 5000g for 20 min, a filtrate in the lower centrifuge tube was discarded, a 10 mM CH.sub.3COOH solution (pH 7) was added to the upper filter to restore to the original sample volume, and centrifuging was performed again at 5000g, and ultrafiltration was repeated many times to remove unreacted galactosamine and reduce the concentration of sodium acetate; a GSA solution was finally obtained after performing ultrafiltration many times, subpackaged and put in a refrigerator at 80 C. for storing for later use. The protein concentration of GSA was detected by using a BCA kit (Takara, T9300A). Before determining, a solution A and a solution B of a BCA reagent were mixed according to the ratio of 100:1 and prepared into a working solution. The BSA standard solution was respectively diluted to 2000 g/mL, 1500 g/mL, 1000 g/mL, 750 g/mL, 500 g/mL, 250 g/mL, and 125 g/mL, and then the diluted BSA standard solution and the GSA sample solution to be measured were sequentially added to a 96-well plate with 10 L per well, two parallel samples were taken for each concentration, 200 L of working solution was added to each well, the solutions were immediately mixed well, and then put in a 37 C. water bath to react for 30 min, and cooled to room temperature, and the absorbance at 562 nm was detected by an ELISA. The standard curve of a BSA standard solution was drawn, and the protein concentration of GSA protein to be measured was calculated according to the standard curve.
Example 2 Purification of ASGPR Standard Product
[0041] 150 mL of HepG2 cell supernatant collected in advance was taken and centrifuged for 5 min (3500g) by a centrifuge, and the centrifuged supernatant was taken and added to a 50 mL Amicon MLtra centrifugal filter (3500g) for concentration, and finally concentrated to a final volume of 8 mL. 8 mL of lysis buffer was added to the concentrated HepG2 cell supernatant and mixed well, and was lysed for 2 to 3 h at a temperature of 4 C. After lysis was completed, centrifuging was performed at 20,000g for 30 min, the supernatant was taken into a new centrifuge tube, and 800 L of 1 M CaCl.sub.2 was added to incubate on ice for 30 min. Centrifuging was performed again at 20000g for 30 min, and a precipitate was discarded. A lactose agarose bead column was equilibrated with 50 mL of washing buffer I in advance, and lactose agarose beads were fully transferred to the centrifuge tube and combined with the centrifuged cell supernatant at a temperature of 4 C. overnight. The combined lactose agarose beads were fully transferred to the column, and the column was sequentially washed with 10 mL of washing buffer I and 5 mL of washing buffer II. The lactose agarose bead column was eluted with 14.4 mL of elution buffer, and 1.6 mL of 1 M Tris-HCl (pH 7.8) buffered solution was added to the collected eluent. The concentration of purified sASGPR was detected by a BCA method, and specificity was verified by Western blot.
Example 3 Selection of Primary Antibodies
[0042] Two ASGPR1 mouse-derived primary antibodies, namely, ASGPR1/2 (E-1) (Santa Cruz Biotechnology, sc-166633) and ASGPR1 (A-5) (Santa Cruz Biotechnology, sc-393849), were purchased, hereinafter referred to as a mouse anti-human ASGPR-1 monoclonal antibody and a mouse anti-human ASGPR-2 monoclonal antibody. Two 96-well ELISA plates were coated with 10 g/mL GSA, 100 L of GSA was added to each well, coating was performed at a temperature of 4 C. for 24 h, after coating was finished, washing was performed with PBST 5 times, 3 min each time and patting dry was performed. 300 L of 1% BSA was added to each well to block the whole well, blocking was performed overnight at a temperature of 4 C., after blocking was finished, washing was performed with PBST 3 times, 3 min each time and patting dry was performed. An ASGPR diluent was set as a negative control well and a quality control product well respectively, and 0.15 g/mL, 0.50 g/mL, 2.00 g/mL, and 8.00 g/mL ASGPR standard products were respectively set as sample wells, the negative control and samples were sequentially added to the ELISA plate, 4 replicate wells were made in the sample, and the sample adding volume for each well is 100 L. Incubation was performed for 1 h in a 37 C. incubator after sample addition. After the sample incubation was finished, washing was performed with PBST 3 times, 3 min each time and patting dry was performed. The mouse anti-human ASGPR-1 monoclonal antibody and the mouse anti-human ASGPR-2 monoclonal antibody diluted according to a ratio of 1:500 were respectively added to the two ELISA plates, the sample addition volume was 100 L in each well, and incubation was performed in a 37 C. incubator for 1 h. After the primary antibody incubation was finished, washing was performed with PBST 4 times, 3 min each time and patting dry was performed. The goat anti-mouse IgG-HRP diluted according to a ratio of 1:2000 was added to the two plates, the sample adding volume for each well was 100 L, and incubation was performed in a 37 C. incubator for 1 h. After the secondary antibody incubation was finished, washing was performed with PBST 4 times, 3 min each time and patting dry was performed. 200 L of TMB development solution was added to each well to develop in a 37 C. incubator for 15 min in the dark. After the development was finished, 50 L of 2M H.sub.2SO.sub.4 was quickly added to each well to stop the development and mixed well. The absorbance of each well at 450 nm was immediately measured with the ELISA and the results were analyzed.
TABLE-US-00001 TABLE 1 Detection results of different ASGPR primary antibodies Primary Negative ASGPR concentration (g/mL) antibodies control 0.15 0.50 2.00 8.00 ASGPR-1 0.263 0.301 0.380 1.112 2.204 ASGPR-2 0.218 0.202 0.218 0.238 0.569
[0043] It can be seen from the data in the table that when using the mouse anti-human ASGPR-1 monoclonal antibody, the value of OD.sub.450 will have a significant gradient increase with the increase of the concentration of ASGPR, so the mouse anti-human ASGPR-1 monoclonal antibody was selected as the detection primary antibody for the indirect ELISA method.
Example 4 Selection of Coating Buffers
[0044] A phosphate buffered solution (0.01 M PBS), a Tris hydrochloride buffered solution (0.01 M TBS), a carbonate buffered solution (0.05 M CBS), and 0.9% NaCl were respectively used as coating buffers to be selected. 10 g/mL GSA was coated with 0.01 M PBS, 0.01 M TBS, 0.05 M CBS, and 0.9% NaCl into a 96-well ELISA plate, the volume of GSA was 100 L in each well, and coating was performed at a temperature of 4 C. for 24 h. After coating was finished, washing was performed with PBST 5 times, 3 min each time and patting dry was performed. 300 L of 1% BSA was added to each well to block the whole well, blocking was performed overnight at a temperature of 4 C., after blocking was finished, washing was performed with PBST 3 times, 3 min each time and patting dry was performed. A negative control well and standard ASGPR1 (80 g/mL) and standard ASGPR2 (40 g/mL) sample wells were respectively set, the negative control and the samples were sequentially added to the ELISA plate, 4 replicate wells were made in the sample, and the sample adding volume for each well is 100 L. Incubation was performed for 1 h in a 37 C. incubator after sample addition. After the sample incubation was finished, washing was performed with PBST 3 times, 3 min each time and patting dry was performed. The mouse anti-human ASGPR-1 monoclonal antibody diluted according to a ratio of 1:500 was respectively added to the two plates, the sample adding volume for each well was 100 L, and incubation was performed in a 37 C. incubator for 1 h. After the primary antibody incubation was finished, washing was performed with PBST 4 times, 3 min each time and patting dry was performed. The goat anti-mouse IgG-HRP diluted according to a ratio of 1:2000 was added to the two plates, the sample adding volume for each well was 100 L, and incubation was performed in a 37 C. incubator for 1 h. After the secondary antibody incubation was finished, washing was performed with PBST 4 times, 3 min each time and patting dry was performed. 200 L of TMB development solution was added to each well to develop in a 37 C. incubator for 15 min in the dark. After the development was finished, 50 L of 2 M H.sub.2SO.sub.4 was quickly added to each well to stop the development and mixed well. The absorbance of each well at 450 nm was immediately measured with the ELISA.
TABLE-US-00002 TABLE 2 Detection effects of different coating buffers Samples Negative P/N Coating buffers ASGPR1 ASGPR2 control P1/N P2/N 0.01M PBS 1.682 0.958 0.234 7.19 4.09 0.01M TBS 1.637 0.933 0.229 7.15 4.07 0.05M CBS 1.882 1.042 0.202 9.32 5.16 0.9% NaCl 1.598 0.932 0.212 7.54 4.40
[0045] It can be seen from the data in the table that when using 0.05 M CBS as the coating buffer, the value of positive sample OD.sub.450/negative control OD.sub.450 (P/N) is the highest.
Example 5 Selection of Different Coating Concentrations
[0046] GSA was diluted with 0.05 M CBS to 5 g/mL, 10 g/mL, 15 g/mL, 20 g/mL, and 25 g/mL, respectively, and coated on different ELISA plates, with 100 L volume per well. The rest of the steps are the same as above, and the OD.sub.450 values of the negative control well and the standard product ASGPR1 (80 g/mL) and standard product ASGPR2 (40 g/mL) sample wells were detected respectively. 4 replicate wells were set in each sample for detecting.
TABLE-US-00003 TABLE 3 Test results of different coating concentrations GSA coating Samples concentrations Negative P/N (g/mL) ASGPR1 ASGPR2 control P1/N P2/N 5 g/mL 1.207 0.427 0.146 8.26 2.92 10 g/mL 1.256 0.464 0.147 8.57 3.17 15 g/mL 1.404 0.577 0.159 8.83 3.63 20 g/mL 1.432 0.711 0.151 9.48 4.71 25 g/mL 1.435 0.720 0.164 8.86 4.45
[0047] The results show that when coating 20 g/mL GSA, the P/N value is the highest.
Example 6 Selection of Coating Time
[0048] 20 g/mL GSA was coated onto the ELISA plate with 0.05 M CBS. The coating conditions were respectively set as follows: coating at a temperature of 37 C. for 1 h, coating at a temperature of 37 C. for 2 h, coating at a temperature of 4 C. for 12 h, and coating at a temperature of 4 C. for 24 h. The rest of the steps are the same as above. The OD.sub.450 values of the negative control well and standard product ASGPR1 (80 g/L) and standard product ASGPR2 (40 g/L) sample wells were respectively detected, 4 replicate wells were set in each sample for detecting, and the optimal GSA coating time was determined according to the P/N value.
TABLE-US-00004 TABLE 4 Detection results of different coating time Samples Negative P/N Coating time ASGPR1 ASGPR2 control P1/N P2/N 1 h at 37 C. 1.225 0.722 0.255 4.80 2.83 3 h at 37 C. 1.375 0.776 0.209 6.57 3.71 12 h at 4 C. 1.007 0.608 0.151 6.68 4.04 24 h at 4 C. 1.106 0.679 0.149 7.43 4.56
[0049] The results show that when coating at a temperature of 4 C. for 24 h, the P/N value is the highest. Therefore, the coating time of 24 h at a temperature of 4 C. is selected as the coating time for the indirect ELISA method.
Example 7 Selection of Blocking Buffers
[0050] 20 g/mL GSA was coated with 0.05 M CBS at a temperature of 4 C. for 24 h. Six different blocking buffers, i.e., 1% BSA, 5% BSA, 1% skim milk powder, 5% skim milk powder, 1% FBS, and 5% FBS were respectively used for blocking. 300 L of blocking buffer was added to each well to block at a temperature of 37 C. for 1 h. The rest of the steps are the same as above, the OD.sub.450 values of the negative control well and standard product ASGPR1 (80 g/L) and standard product ASGPR2 (40 g/L) sample wells were respectively detected, 4 replicate wells were set in each sample for detecting, and finally the optimal blocking buffer was determined according to the P/N value.
TABLE-US-00005 TABLE 5 Detection results of different blocking buffers Samples Negative P/N Blocking buffers ASGPR1 ASGPR2 control P1/N P2/N 1% BSA 1.323 0.567 0.149 8.91 3.82 5% BSA 1.403 0.613 0.155 9.08 3.97 1% skim milk 1.512 0.726 0.134 11.32 5.43 powder 5% skim milk 1.429 0.577 0.134 10.70 4.32 powder 1% FBS 1.26 0.496 0.150 8.43 3.32 5% FBS 1.217 0.492 0.151 8.06 3.26
[0051] When using 1% skim milk powder as the blocking buffer, the P/N value is the highest.
Example 8 Selection of Blocking Time
[0052] 20 g/mL GSA was coated with 0.05 M CBS at a temperature of 4 C. for 24 h, and blocked with 1% skim milk powder. The blocking time was set as 1 h at a temperature of 37 C., 1.5 h at a temperature of 37 C., 2 h at a temperature of 37 C., 2.5 h at a temperature of 37 C., and 3 h at a temperature of 37 C. The rest of the steps are the same as above. The OD.sub.450 values of the negative control well and standard product ASGPR1 (80 g/L) and standard product ASGPR2 (40 g/L) sample wells were respectively detected, 4 replicate wells were set in each sample for detecting, and finally the optimal blocking time was determined according to the P/N value.
TABLE-US-00006 TABLE 6 Detection effects of different blocking time Samples Negative P/N Blocking time ASGPR1 ASGPR2 control P1/N P2/N 1 h 1.412 0.823 0.186 7.61 4.43 1.5 h 1.552 0.934 0.163 9.52 5.73 2 h 1.519 0.885 0.123 12.35 7.20 2.5 h 1.626 0.935 0.163 10.01 5.75 3 h 1.392 0.857 0.197 7.08 4.36
[0053] The results show that when the blocking time is 2 h, the P/N value is the highest.
Example 9 Selection of Sample Incubation Time
[0054] Coating and blocking were performed by selecting the optimum conditions determined by the above experiments. The negative control and standard product ASGPR1 (80 g/L) and standard product ASGPR2 (40 g/L) samples were respectively detected, 4 replicate wells were set in each sample for detecting, the sample incubation time was respectively set as 1 h, 1.5 h, 2 h, 2.5 h, 3 h, and finally the optimal sample incubation time was determined according to the OD value and P/N value.
TABLE-US-00007 TABLE 7 Detection results of different sample incubation time Samples Incubation Negative P/N time ASGPR1 ASGPR2 control P1/N P2/N 1 h 1.155 0.798 0.166 6.96 4.81 1.5 h 1.208 0.945 0.167 7.23 5.66 2 h 1.407 1.151 0.184 7.82 6.39 2.5 h 1.498 1.221 0.191 7.84 6.39
[0055] When the incubation time reaches 2 h, the P/N value reaches the highest, and when the incubation time increases again, although the OD value of the sample well will increase, the corresponding negative control value will also increase, and the P/N value remains stable.
Example 10 Optimization of Extent of Dilution of Primary and Secondary Antibodies
[0056] Coating and blocking were performed by selecting the optimum conditions determined by the above experiments. The negative control well and ASGPR sample wells were respectively detected. 4 replicate wells were set in each sample for detecting. The primary antibodies were respectively diluted according to 1:30, 1:50, 1:100, 1:200, and 1:400, and the secondary antibodies were respectively diluted according to 1:1000, 1:2000, 1:4000, and 1:6000. Finally, the optimal extend of dilution of the secondary antibody was determined according to the P/N value.
TABLE-US-00008 TABLE 8 Detection effects of different extents of dilution of primary and secondary antibodies Extent of dilution of primary antibody Extent of dilution of ASGPR positive samples ASGPR negative samples secondary antibody 1:30 1:50 1:100 1:200 1:400 1:30 1:50 1:100 1:200 1:400 1:1000 1.587 1.437 1.134 0.878 0.456 0.297 0.263 0.197 0.185 0.155 1:2000 1.347 1.104 0.996 0.746 0.376 0.261 0.186 0.172 0.153 0.135 1:4000 0.896 0.702 0.615 0.518 0.315 0.243 0.174 0.159 0.147 0.117 1:6000 0.587 0.474 0.397 0.328 0.267 0.185 0.167 0.146 0.135 0.105
TABLE-US-00009 TABLE 9 P/N value of checkerboard titration P/N Extent of dilution Extent of dilution of primary antibody of secondary antibody 1:30 1:50 1:100 1:200 1:400 1:1000 5.34 5.46 5.76 4.75 2.94 1:2000 5.16 5.94 5.79 4.88 2.79 1:4000 3.69 4.03 3.87 3.52 2.69 1:6000 3.17 2.84 2.72 2.43 2.54
[0057] It can be seen from the results that when the extent of dilution of the primary antibody is 1:50 and the extent of dilution of the secondary antibody is 1:2000, the P/N value reaches the maximum value.
Example 11 Selection of TMB Development Time
[0058] The indirect ELISA test was performed under the above optimized optimal conditions. The final TMB development time was respectively set as 5 min, 10 min, 15 min, and 20 min, and then the development was stopped with 2 M H.sub.2SO.sub.4, the OD.sub.450 value was determined with an ELISA, and finally the TMB development time was determined according to the P/N value.
TABLE-US-00010 TABLE 10 Detection results of different development time Samples Development Negative P/N time ASGPR1 ASGPR2 control P1/N P2/N 5 min 1.125 0.658 0.136 8.27 4.83 10 min 1.478 0.945 0.174 8.52 5.45 15 min 1.787 1.220 0.202 8.87 6.05 20 min 2.185 1.471 0.256 8.54 5.74 25 min 2.339 1.603 0.289 8.09 5.55
[0059] When the development time is 15 min, the P/N value reaches the highest point. As the development time increases, the P/N value will decrease slightly.
Example 12 Drawing of Standard Curve by ELISA Method
[0060] (1) Coating: GSA was diluted with a 0.05 M CBS buffered solution to 20 g/mL to coat a 96-well ELISA plate with 100 L per well at a temperature of 4 C. for 24 h, then washing was performed with a washing buffer solution (PBS containing 0.1% tween-20) three times, 3 min each time, and patting dry was performed.
[0061] (2) Blocking: 1% skim milk was added to the coated ELISA well plate with 300 L per well, and the plate as sealed with a microplate sealer, and blocked at a temperature of 37 C. for 2 h; a blocking buffer was blocked, washing was performed with a washing buffer solution three times, 3 min each time, and patting dry was performed;
[0062] (3) Sample adding: an ASGPR negative control well and ASGPR standard product wells were respectively set, and the standard products were respectively diluted with a standard diluent to seven concentrations, i.e., 5 g/L, 10 g/L, 20 g/L, 40 g/L, 60 g/L, 80 g/L, and 100 g/L. The negative control and ASGPR standard products were sequentially added to the 96-well ELISA plate, replicate wells were made, and the sample adding volume for each well was 100 L. After adding the sample, the plate was sealed with a microplate sealer, and incubation was performed at a temperature of 37 C. for 2 h.
[0063] (4) Primary antibody incubation: a mouse-derived primary antibody of ASGPR1/2 was incubated by diluting with a blocking buffer according to a ratio of 1:50, with 100 L per well, at a temperature of 37 C. for 2 h, then washing was performed with a washing buffer solution four times, 3 min each time, and patting dry was performed;
[0064] (5) Secondary antibody incubation: an HRP-labeled goat anti-mouse enzyme-labeled secondary antibody (Kangwei Century Biotechnology Co., Ltd., CW0102) was incubated by diluting with a blocking buffer according to a ratio of 1:2000, with 100 L per well, at a temperature of 37 C. for 1 h, then washing was performed with a washing buffer solution four times, 3 min each time, and patting dry was performed;
[0065] (6) TMB development: the development was performed with a TMB development solution (Biyuntian Biotechnology Co., Ltd., P0209), with 200 L per well, incubation was performed for 15 min in the dark, and then immediately the development was stopped with 2 M H.sub.2SO.sub.4, with 50 L per well; after the development was stopped, the absorbance of each well at 450 nm was measured with an ELISA as soon as possible.
[0066] (7) Standard curve drawing: the OD.sub.450 values of different concentrations of ASGPR standard products were read and a standard curve was drawn.
Example 13 Determination of Minimum Limit of Detection
[0067] According to the method of Example 12, the ASGPR negative control sample was repeatedly determined on the same coated ELISA plate for 20 wells, and the determination was repeated for five batches. The average value M and standard deviation SD of the OD.sub.450 values of the negative control sample were calculated, the value of M3SD was calculated, and substituted into the equation of the standard curve to obtain the minimum limit of detection of the ELISA method. The result shows that the minimum limit of detection is 4 g/mL.
TABLE-US-00011 TABLE 11 Detection results of repeated 5 batches First Second Third Fourth Fifth time time time time time Average value 0.151 0.162 0.169 0.155 0.153 Standard deviation 0.005 0.005 0.004 0.004 0.005 Minimum limit of 3.41 3.75 3.56 2.95 3.36 detection (g/L)
Example 14 Inter-Batch and Intra-Batch Repeat Tests
[0068] By using GSA coated ELISA plates prepared in the same batch and different batches, the repeat tests were performed on three different concentrations of ASGPR positive samples, negative samples, and serum samples according to the method of Example 12, three times for each; after the OD value was determined, the average values of coefficients of variation of the inter-batch and intra-batch repeat tests were both less than 7% by calculating, so the method has good repeatability.
TABLE-US-00012 TABLE 12 Inter-batch repeated tests Inter-batch coefficient Repeat times of First Second Third Average Standard variation Samples time time time value deviation (%) Negative 0.184 0.169 0.164 0.173 0.009 5.28% control ASGPR1 0.229 0.251 0.225 0.240 0.014 5.64% ASGPR2 0.833 0.906 0.801 0.853 0.041 4.76% ASGPR3 1.444 1.459 1.369 1.410 0.076 5.40% Serum 0.766 0.722 0.752 0.755 0.019 2.56% sample 1
TABLE-US-00013 TABLE 13 Intra-batch repeated tests Intra-batch coefficient Repeat times of First Second Third Average Standard variation Sample time time time value deviation (%) Negative 0.154 0.145 0.164 0.158 0.009 5.68% control ASGPR1 0.211 0.191 0.223 0.211 0.013 5.91% ASGPR2 0.713 0.716 0.731 0.730 0.016 2.14% ASGPR3 1.245 1.25 1.269 1.226 0.042 3.44% Serum 0.689 0.653 0.623 0.644 0.029 4.56% sample 1
Example 15 Linear Range
[0069] Three batches of coated ELISA plates were used to detect 5 different concentrations (5 g/L, 10 g/L, 20 g/L, 40 g/L, 70 g/L, and 100 g/L) of ASGPR standard products, the determined concentration and the theoretical concentration were linearly fitted according to the method of Example 12, and the detection was repeated three times. The linearly dependent coefficients r of fitted straight lines obtained by determining three times are all greater than 0.990, so the method satisfied the expected set linear range of 4 to 100 g/mL.
Example 16 Accuracy (Recovery Test)
[0070] Three serum samples were taken, and ASGPR standard products having high (20 g/mL), medium (10 g/mL) and low (5 g/mL) concentrations were respectively added to the three serum samples, the sASGPR concentrations of the original serum sample and the samples added with ASGPR were detected according to the method of Example 12, each sample was detected three times repeatedly, and the recovery volume and recovery rate of ASGPR were calculated.
TABLE-US-00014 TABLE 14 Recovery test ASGPR (g/L) Addition Detection Recovery Recovery Samples volume volume volume percentage (%) Serum sample 1 0 78.86 Serum sample 1 5 83.43 4.57 91.40% Serum sample 1 10 87.89 9.03 90.30% Serum sample 1 20 97.30 18.44 92.20% Serum sample 2 0 85.22 Serum sample 2 5 90.10 4.88 97.60% Serum sample 2 10 94.46 9.24 92.40% Serum sample 2 20 104.20 18.98 94.90% Serum sample 3 0 108.87 Serum sample 3 5 113.20 4.33 86.60% Serum sample 3 10 117.66 8.79 87.90% Serum sample 3 20 128.89 20.02 100.10% Average recovery 92.60% rate
Example 17 Detection of Clinical Serum Samples by ELISA Method
[0071] According to the method of Example 12, the ASGPR negative control sample, the ASGPR standard products and the samples to be detected (3 serum samples) were respectively detected, the standard products were respectively diluted with a standard diluent to seven concentrations, i.e., 5 g/L, 10 g/L, 20 g/L, 40 g/L, 60 g/L, 80 g/L, and 100 g/L, and the serum samples were diluted two times with 1PBS for later use. A standard curve was drawn and the ASGPR content of the corresponding sample was calculated according to the OD.sub.450 value of the sample well.
TABLE-US-00015 TABLE 15 Detection of serum samples by ELISA Standard (g/L) OD.sub.450 Serum samples (g/L) OD.sub.450 0 0.164 80 1.563 5 0.239 100 1.866 10 0.364 Serum sample 1 0.793 20 0.494 Serum sample 2 0.857 40 0.806 Serum sample 3 0.684 60 1.188 Serum sample 3 0.924
[0072] It can be seen from the above table that the OD values of the serum samples are within the test range of the standard curve, so the method can achieve the expected detection effect.
Example 18 Detection of Clinical Serum Samples by ELISA Method
[0073] A total of 533 serum samples were detected by the indirect ELISA method of Example 12, including 489 serum samples of healthy people and 44 serum samples of liver injury patients. The detection results show that the content of sASGPR in the serum samples of healthy people is 85.9459.69 g/mL, and the content of sASGPR in the serum samples of liver injury patients is 20.4110.59 g/mL. By statistical analysis, there was a statistically significant difference in content of serum sASGPR between the healthy samples and liver injury samples (P<0.001).
[0074] Although the disclosure has been disclosed in the above as preferred embodiments, it is not intended to limit the disclosure. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure. The protection scope of the disclosure shall be defined by the claims.