Kit and method for quantitative detection of HBsAg
11543413 · 2023-01-03
Assignee
Inventors
- Shunhua Wen (Xiamen, CN)
- Liuwei Song (Xiamen, CN)
- Zuxing Weng (Xiamen, CN)
- Haijun Lin (Xiamen, CN)
- Feihai Xu (Xiamen, CN)
- Xudong Sun (Xiamen, CN)
- Shengxiang Ge (Xiamen, CN)
Cpc classification
International classification
Abstract
A kit for quantitatively detecting HBsAg and a method for quantitatively detecting an HBsAg content in a sample containing HBsAg. The kit comprises a first antibody specifically binding to HBsAg and a reagent composition. The reagent composition comprises tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and urea.
Claims
1. A kit, comprising a first antibody capable of specifically binding to HBsAg, and a reagent composition, the reagent composition comprising tris(2-carboxyethyl)phosphine hydrochloride (TCEP), urea, a non-ionic surfactant, an inorganic salt and a buffer, wherein: the TCEP is present in an amount of 1 to 100 mM; the urea is present in an amount of 0.5 to 8 M; the non-ionic surfactant is present in an amount of 0 to 10% (v/v), the inorganic salt is present in an amount of 0.5 to 8 M, and the buffer is present in an amount of 0 to 200 mM.
2. The kit of claim 1, wherein the reagent composition has one or more of the following characteristics: (i) the non-ionic surfactant is selected from the group consisting of Chaps, sulfobetaine type surfactants, Triton type detergents, Tween type detergents, and any combination thereof; (ii) the inorganic salt is selected from NH.sub.4SO.sub.4 and NaCl; (iii) the buffer is a carbonate buffer.
3. The kit of claim 1, wherein the reagent composition comprises: TCEP, urea, an inorganic salt, a non-ionic surfactant, a buffer, and a balance of water.
4. The kit of claim 1, wherein the kit further comprises a second antibody capable of specifically binding to HBsAg; and optionally the second antibody bears a detectable label.
5. The kit of claim 4, wherein, the detectable label is selected from an enzyme, a chemiluminescent reagent, or a fluorescent dye.
6. The kit of claim 1, wherein the kit further comprises a solid support, optionally surface of which is coated with the first antibody.
7. The kit of claim 6, wherein the solid support is a microtiter plate or a magnetic bead.
8. The kit of claim 1, wherein the kit further comprises one or more reagents or devices selected from the group consisting of: a standard; a positive control sample; a negative control sample; a stop solution for stopping a color reaction of substrate catalyzed by enzyme; a blocking solution for inhibiting a non-specific binding; and, a blood collection device.
9. The kit of claim 1, wherein the non-ionic surfactant is selected from SB14, SB 16, Tween-20, Tween-40, Triton X-100, and any combination thereof.
10. The kit of claim 1, wherein TCEP is present in an amount of 10 to 50 mM, and/or, urea is present in an amount of 1 to 8 M.
11. A method for quantitatively detecting the amount of HBsAg in a sample containing HBsAg, comprising the following steps: (1) contacting the sample with a first antibody capable of specifically binding to HBsAg in a reagent composition to obtain an immune complex, wherein the reagent composition comprises tris(2-carboxyethyl)phosphine hydrochloride (TCEP), urea, a non-ionic surfactant, an inorganic salt and a buffer, wherein: the TCEP is present in an amount of 1 to 100 mM; the urea is present in an amount of 0.5 to 8 M; the non-ionic surfactant is present in an amount of 0 to 10% (v/v), the inorganic salt is present in an amount of 0.5 to 8 M, and the buffer is present in an amount of 0 to 200 mM; and (2) determining the amount of the immune complex obtained in step (1); wherein the sample is a blood sample.
12. The method of claim 11, wherein the method has one or more of the following characteristics: (i) the blood sample is selected from whole blood, plasma or serum; (ii) the blood sample is undiluted; (iii) the first antibody is coated on the surface of a solid support; (iv) a step of washing the immune complex to remove unreacted substance is further comprised before step (2).
13. The method of claim 12, wherein, the first antibody is coated on the surface of a microtiter plate or a magnetic bead.
14. The method of claim 11, wherein in step (2), the amount of the immune complex is determined by an immunological detection.
15. The method of claim 14, wherein the immunological detection is an enzyme immunoassay or a chemiluminescence immunoassay.
16. The method of claim 14, wherein the immunological detection is selected from a CLEIA method and a CLIA method.
17. The method of claim 14, in step (2), the amount of the immune complex is detected using a second antibody capable of specifically binding to HBsAg, the second antibody bears a detectable label.
18. The method of claim 17, wherein the detectable label is selected from an enzyme, a chemiluminescent reagent or a fluorescent dye.
Description
DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
SPECIFIC MODELS FOR CARRYING OUT THE INVENTION
(5) The invention will now be described with reference to the following examples which are intended to illustrate the invention without limiting it.
(6) Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F M Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes was in accordance with conditions recommended by the product manufacturers. Those skilled in the art know that the embodiments describe the present invention by way of example and are not intended to limit the scope of the invention as claimed.
PREPARATION EXAMPLE 1
Preparation of Dissociation Solution
(7) Formulation: 40 mM TCEP+2M urea+600 mM NaCl+100 mM CB9.6+0.1% Tween-20.
(8) Reagents: TCEP (Cat. No. 68957), Tween-20 (Cat. No. P1379), NaCl (Cat. No. V900058), sodium bicarbonate (Cat. No. S5761-500 g), urea (V800441), and sodium carbonate (V800371) were purchased from Sigma-Aldrich.
(9) Preparation Steps:
(10) 1. 31.8 g of sodium carbonate and 58.6 g of sodium bicarbonate were weighed, dissolved in deionized water to a volume of 1000 mL, so as to prepare a 1M carbonate buffer (CB) with a pH value of 9.6.
(11) 2. 87.66 g of NaCl was weighed, dissolved in deionized water to a volume of 500 mL, so as to prepare a 3M NaCl solution.
(12) 3. 240.24 g of urea was weighed, dissolved in deionized water to a volume of 500 mL, so as to prepare an 8M urea solution.
(13) 4. 5.733 g of TCEP was weighed, dissolved in 100 mL of deionized water, and then added with 50 mL of 1M carbonate buffer (pH 9.6), 125 mL of 8M urea, 100 mL of 3M NaCl solution, and 0.5 mL of Tween-20, mixed well and added with deionized water to a volume of 500 mL.
PREPARATION EXAMPLE 2
Preparation of HBsAg Detection Kit for Chemiluminescence Enzyme-Linked Immunoassay (CLEIA)
(14) 1. Preparation of Immobilized Antibody
(15) (1-1) Mouse anti-HBsAg monoclonal antibody (purchased from Xiamen Wantai Canghai Biotechnology Co., Ltd.) was diluted with 50 mM CB buffer (NaHCO.sub.3/Na.sub.2CO.sub.3 buffer, final concentration 50 mM, pH 9.6) at pH 9.6, to a final concentration of 4 μg/ml to prepare a coating solution.
(16) (1-2) 100 μL of the coating solution prepared in the previous step (1) was added to each well of a 96-well plate for chemiluminescence reaction to perform coating at 2-8° C. for 16-24 hours.
(17) (1-3) Washing was performed once with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) to remove uncoated mouse anti-HBsAg monoclonal antibody. Then, 200 μl of blocking solution (20 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer solution that had a pH of 7.4 and contained 20% infant bovine serum, 1% BSA and 1% casein) was added to each well to perform blocking at 37° C. for 2 hours; and then the blocking solution was discarded. After drying, the plate was put into an aluminum foil bag and stored at 2-8° C. for later use.
(18) 2. Preparation of Labeled Antibody
(19) HRP labeling of anti-HBsAg polyclonal antibody was performed using a modified sodium periodate method. The following example was performed to label 10 mg of anti-HBsAg polyclonal antibody.
(20) (2-1) Goat anti-HBsAg polyclonal antibody (5 mL) (purchased from Xiamen Wantai Canghai Biotechnology Co., Ltd., Cat. No. G1051) at a concentration of 2 mg/mL was placed in a dialysis bag, and dialyzed against 20 mM CB buffer at 4° C. for 4 hours, in which the dialysis buffer was changed every 2 hours.
(21) (2-2) 40 mg of Horseradish Peroxidase (HRP) (Sigma-Aldrich/77332) was weighed accurately and dissolved in 2 mL of ddH.sub.2O, followed by addition of 2 ml of 20 mg/mL NaIO.sub.4 and reaction at room temperature for 30 minutes. 40 μL of ethylene glycol was added and reacted at 4° C. for 30 minutes to prepare an HRP activation solution (10 mg/mL, 4 mL).
(22) (2-3) The HRP activation solution prepared in step (2-2) was added to a dialysis bag containing goat anti-HBsAg polyclonal antibody, mixed well and dialysis against 20 mM CB buffer continued at 4° C. in the dark for 6-8 hours, and the dialysis buffer was changed every 2 hours during the process.
(23) (2-4) 0.5 mL of NaBH.sub.4 solution (20 mg/mL) was prepared, added with the label reaction solution prepared in step (2-3), and mixed well. The mixture was reacted at 4° C. in the dark for 2 hours, and mixed well every 30 minutes.
(24) (2-5) After step (2-4) was completed, the label reaction solution was loaded again into a new dialysis bag, and dialyzed against PBS buffer at 4° C. for 4 hours.
(25) (2-6) After step (2-5) was completed, 50% saturated ammonium sulfate (50% referred to the concentration of ammonium sulfate in the dialysis bag, and the main purpose was to precipitate the goat polyclonal antibody-HRP labeled product). After centrifugation at 12000 rpm for 10 min, the supernatant was discarded, the precipitate was dissolved in 50% glycerol+10% NBS (final concentration) (NBS was the abbreviation of newborn bovine serum) by pipetting up and down, mixed well and stored at −20° C. for later use.
(26) (2-7) The goat polyclonal antibody-HRP labeled product obtained in step (2-6) was diluted into enzyme label dilution buffer (20 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer solution having a pH of 7.4 and containing 20% fetal bovine serum, 1% casein, 10% sucrose and 0.05% aminopyrine) by a dilution rate of 1/4000 by volume, to prepare an enzyme label reaction solution, and after mixing, it was stored at 2-8° C. for later use.
(27) 3. Quantitation Standards
(28) The quantitation standards for the quantitative detection of middle- and high-concentration HBsAg samples were a series of samples containing different concentrations of hepatitis B virus surface antigen. A total of 8 standards were included, the concentrations of which were 100,000 IU/mL, 50,000 IU/mL, 10,000 IU/mL, 5,000 IU/mL, 1,000 IU/mL, 500 IU/mL, 100 IU/mL, 20 IU/mL, respectively, with a volume of 500 μl per tube. The surface antigen was provided by Xiamen Wantai Kairui Biotechnology Co., Ltd., and could be traced back to the WHO standard (Code: 00/588) issued by NIBSC. The solution used to dilute the standards could be HBsAg- and HBsAb-negative healthy blood donor's plasma or serum, or a PBS solution containing 20% newborn bovine serum.
(29) 4. Dissociation Solution
(30) It was prepared in Preparation Example 1.
PREPARATION EXAMPLE 3
Preparation of HBsAg Detection Kit for Chemiluminescent Immunoassay (CLIA) Based on Microparticles in Tube
(31) 1. Preparation of Antibody-Labeled Magnetic Particles
(32) (1-1) 4 mg of magnetic beads (Magnosphere MS300/Carboxyl of JSR Company, Japan, particle size was 3 μm) were washed 2 times with 1 mL of activation buffer system (50 mM MES 5.0), and the supernatant was discarded. 4 mg of EDC and 4 mg of NHS reagents (both were formulated to 10 mg/mL with 50 mM MES 5.0) were added, and mixed well, followed by activation with shaking at room temperature for 20 minutes;
(33) (1-2) The activated magnetic beads were washed 3 times with 1 mL of activation buffer system (50 mM MES 5.0) to remove excess EDC and NHS, and the supernatant was discarded. 1 mL of 50 mM phosphate buffer with a pH of 6.0 and 160 μg of a mouse anti-HBsAg monoclonal antibody were added, and mixed well, followed by reaction with shaking at room temperature for 3 hours;
(34) (1-3) The magnetic beads obtained in (1-2) were washed 3 times with 1 ml of PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20), and then added with 1 ml of blocking buffer (20 mM PB7.4, 1% glycine, 0.1% BSA, 0.05% Tween-20), and mixed well, followed by reaction with shaking at room temperature for 3 hours;
(35) (1-4) The blocked magnetic particles obtained in step (1-3) were washed 3 times with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20), and then added with 1 ml of storage buffer (20 mM Tris-HCl buffer pH 8.0, 0.5% BSA, 150 mM NaCl, 0.5% casein, 0.1% preservative), stored at 2-8° C. for later use;
(36) (1-5) The coated magnetic particles were diluted by 10 times with storage buffer (20 mM Tris-HCl buffer pH 8.0, 0.5% BSA, 150 mM NaCl, 0.5% casein, 0.1% preservative), stored at 2-8° C. for later use.
(37) 2. Acridinium Ester Labeling of Goat Anti-HBsAg Polyclonal Antibody
(38) (2-1) 100 μg of goat anti-HBsAg polyclonal antibody was added to 300 μl of labeling buffer system (50 mM phosphate buffer, pH 8.0), added with 12 μL of acridinium ester (5 mM NHS-SAE), and reacted at room temperature for 30 minutes in the dark.
(39) (2-2) 200 μL of stopping buffer (phosphate buffer containing 100 mM glycine, pH 8.0) was added with the labeled product prepared in step (2-1), and reacted at room temperature for 30 minutes in the dark.
(40) (2-3) The labeled product from step (2-2) was loaded into a dialysis bag, and dialyzed against dialysis buffer (20 mM phosphate buffer, pH 7.4) at 4° C. for 6-8 hours in the dark, in which the dialysis buffer was changed every 2 hours.
(41) (2-4) The labeled product obtained in step (2-3) was transferred into a storage tube, added with 2% BSA and 50% glycerol, and stored at −20° C. for later use.
(42) (2-5) The acridinium ester labeled product as prepared in step (2-4) was subjected to dilution by volume ratio of 1/3000 into an acridinium ester label dilution buffer (20 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer solution having a pH value of 7.4 and containing 0.5% BSA, 0.5% casein, 0.05% Tween-20 and 0.1% preservative) to prepare a luminescent label reaction solution, then it was stored at 2-8° C. for later use after mixing.
(43) 3. Quantitation Standards
(44) The quantitation standards of the kit of this Preparation Example were the same as in step 3 of Preparation Example 2.
(45) 4. Dissociation Solution
(46) It was prepared as Preparation Example 1.
EXAMPLE 1
Quantitative Detection of HBsAg in Clinical Samples
(47) 1. Experimental Reagent/Kit
(48) Kit of Preparation Example 2.
(49) 2. Experimental Method
(50) 38 serum samples from patients with chronic hepatitis B (numbered P1 to P38) were subjected to HBsAg quantitative detection according to the following steps.
(51) (1) Sample reaction: to each well of a coated chemiluminescent reaction plate, was added 90 μL of dissociation solution, then 10 μL of sample or standard, followed by mixing with shaking and reaction in a 37° C. incubator for 30 minutes.
(52) (2) Enzyme label reaction: After step (1) was completed, the chemiluminescent reaction plate was washed 5 times with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) to remove the reaction dissociation solution and unreacted sample. 100 μL of the enzyme label reaction solution prepared in step (2-7) in Preparation Example 2 was added to each well, and reacted in a 37° C. incubator for 30 minutes.
(53) (3) Luminescence reaction and measurement: After step (2) was completed, the chemiluminescent reaction plate was washed 5 times with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) to remove the excess enzyme label reaction solution. 100 μL of PICO Chemiluminescent Substrate produced by Pierce Company was added to each well, and the luminescence value (RLU) of each reaction well was immediately read with Orin II chemiluminescence detector.
(54) (4) Generation of standard curves for quantitation: After step (3) was completed, the linear regression was performed on the measured values of the 8 quantitation standards and their corresponding concentrations to obtain a standard curve. The results were shown in
(55) (5) Obtaining the HBsAg concentration of the samples to be tested: After the samples P1 to P38 were measured through steps (1) to (4), the corresponding RLU values of the samples were obtained. The measured values were substituted into the formula for calculating HBsAg concentration obtained in step (4) to calculate the concentrations of hepatitis B virus surface antigen in the samples.
(56) At the same time, the quantitative detection kit for hepatitis B virus surface antigen of Roche (100 tests/box, Cat. No.: 07143737190) and the quantitative detection kit for hepatitis B virus surface antigen of Abbott (100 tests/box, Cat. No.: 6C36) were used for detecting the samples from the same batch, in which the operations were performed according to the instructions of the kits, and the Roche and Abbott kits were used with the full-automatic chemiluminescence instruments of their respective companies, and the dilution and detection of the samples were automatically performed by the instruments.
(57) 3. Experimental Results
(58) The detection results of the samples from the same batch using the kit of Preparation Example 2, the Roche kit (100 tests/box, Cat. No.: 07143737190) and the Abbott kit were shown in Table 1.
(59) TABLE-US-00001 TABLE 1 Determination of concentrations of hepatitis B virus surface antigen in samples P1 to P38 Abbott Roche The present invention Sample No. (IU/mL) (IU/mL) (IU/mL) P1 444 1240 439.99 P2 794 1173 1584.89 P3 501 1034 994.78 P4 3162 11407 11416.55 P5 631 3074 1376.94 P6 7943 14211 18805.41 P7 32 100 125.89 P8 6310 19686 15738.38 P9 63 100 125.89 P10 8188 7930 9004.31 P11 288 445 198.27 P12 1348 1541 2110.13 P13 12696 11977 18575.77 P14 1872 9794 3391.13 P15 208 1170 677.70 P16 2868 8123 4306.91 P17 9740 15792 12752.08 P18 12656 14364 11261.68 P19 4528 3287 7654.29 P20 12360 18142 15281.08 P21 424 738 807.52 P22 6284 10602 15878.31 P23 964 1778 1232.46 P24 80 137 158.49 P25 10300 28063 14426.89 P26 16 25 31.62 P27 17856 20136 21193.46 P28 500 1130 591.92 P29 176 177 139.06 P30 8116 15300 12414.66 P31 8192 10217 9340.80 P32 1224 2920 2492.14 P33 6284 9276 8587.02 P34 292 688 829.49 P35 7928 12090 11518.21 P36 4704 3489 4590.60 P37 14672 16810 21535.62 P38 612 429 458.12
(60) Quantitation correlation analysis was performed on the detection results in Table 1, and the results were shown in
(61) In addition, the inventors detected 747 clinical HBsAg-positive samples by using the kit of Preparation Example 2, and the concentration distribution range of the samples was calculated. The results were shown in Table 2, in which there were 92.1% of the clinical samples having a concentration within the detection range of 20 to 100,000 IU/mL of the kit of the present invention; in contrast, there were only 25.97% of the samples having a concentration within the detection ranges (0.05 to 250 IU/mL) of conventional HBsAg quantitative detection reagents.
(62) TABLE-US-00002 TABLE 2 Concentration distribution ranges of clinical HBsAg-positive samples number of IU/mL samples(n = 747) % of the total <20 55 7.4 20 < 250 139 8.4 250 < 1000 160 31.6 1000 < 10000 248 33.2 10000 < 100000 141 18.9 100000< 4 0.5
COMPARATIVE EXAMPLE 1
Effects of Dissociation Solutions of Different Formulas on the Upper Limit of Detection
(63) 1. Experimental reagents: hepatitis B virus surface antigen assay kit (chemiluminescence microparticle immunoassay method) (purchased from Xiamen Wantai Kairui Biotechnology Co., Ltd.); dissociation solution of Preparation Example 1, 40 mM TCEP solution, 600 mM NaCl solution, 2M urea solution and 6M urea.
(64) 2. Experimental samples: A sample with a HBsAg concentration of 100,000 IU/mL was 3-fold serially diluted until 0.03 IU/mL with a sample that was negative for both HBsAg and HBsAb.
(65) 3. Experimental Steps:
(66) (1) Sample reaction: 20 μL of the sample was added into a reaction tube, followed by addition of 100 μL of the dissociation solution prepared in Preparation Example 1, 2M urea, 6M urea, 600 mM NaCl, 40 mM TCEP solution, respectively. 50 μl of the magnetic particle reagent of the hepatitis B virus surface antigen determination kit (chemiluminescence microparticle immunoassay method) produced by Xiamen Wantai Kairui Biotechnology Co., Ltd. was added, mixed well by shaking, and then reacted in a 37° C. incubator for 15 minutes.
(67) (2) Luminescent label reaction: After step (1) was completed, the chemiluminescence reaction tube was washed twice with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween-20), and 50 μL of the acridinium ester label reagent of the hepatitis B virus surface antigen assay kit (chemiluminescence microparticle immunoassay method) produced by Xiamen Wantai Kairui Biotechnology Co., Ltd. was added to each well, and reacted in a 37° C. incubator for 10 minutes.
(68) (3) Measurement of luminescence reaction: After step (2) was completed, the chemiluminescence reaction tube was washed 4 times with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween-20). A Sirius-L single-tube chemiluminescence detector was used, wherein an excitation solution was added by in-situ injection, and light intensity detection was carried out at the same time.
(69) (4) Generation of standard curves for quantitation: After step (3) was completed, linear regression was performed on the measured values and corresponding concentrations of the series of samples obtained by 3-fold gradient dilution of the 100,000 IU/mL HBsAg sample to obtain standard curves for quantitation.
(70) The results were shown in
(71) Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that according to all the teachings that have been disclosed, various modifications and changes can be made to the details, and these changes are all within the protection scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.