CN116068200A

CN116068200A - Bovine serum albumin detection kit

Info

Publication number: CN116068200A
Application number: CN202211705978.9A
Authority: CN
Inventors: 张娅玲; 尹国维
Original assignee: Suzhou Haiao Siker Biotechnology Co ltd
Current assignee: Suzhou Haiao Siker Biotechnology Co ltd
Priority date: 2022-12-29
Filing date: 2022-12-29
Publication date: 2023-05-05

Abstract

The invention discloses a bovine serum albumin detection kit, which comprises: the detection kit provided by the invention is convenient and simple to operate, can be directly used for analyzing the residual quantity of BSA in a sample, has good reproducibility and high stability, and compared with the existing commercial products, the capture-detection antibodies of the kit prepared by the invention are monoclonal antibodies, have the characteristics of good specificity, sensitivity and stability, and have a wider linear quantitative range of 0-60ng/mL.

Description

Bovine serum albumin detection kit

Technical Field

The invention relates to the field of biotechnology and immunoassay, in particular to a bovine serum albumin detection kit.

Background

Bovine Serum Albumin (BSA) is an albumin in serum consisting of 583 amino acid residues and having a molecular weight of 66.43KDa. BSA has wide application in numerous fields, particularly in the production of biologicals: in the production of vaccines by cultured cells, the serum medium contains a large amount of BSA, and most commercial serum-free media also contain significant BSA, although the majority of BSA in the preparation can be removed after purification, trace amounts of BSA remain in the finished biological preparation. Especially BSA exceeding a certain concentration is liable to cause immunopathogenic reactions in humans, which presents challenges for the safety of biologics. Therefore, detection of residual BSA in vaccines is very important. The 2015 edition Chinese pharmacopoeia clearly prescribes the residual quantity of BSA in biological products, which should not be higher than 50ng/mL.

At present, 2 BSA content detection kits are widely used by vaccine manufacturers, namely a BSA detection kit (product number F030) of Cygnus corporation in the United states, and a quantitative detection bovine serum albumin no-face immune kit (product number RB 0001) of the biological technology development limited company of Xionbo biomedical technology in China. The former linear detection range is 0-32ng/mL, and the latter linear detection range is 0-40ng/mL. Both of these kits have a small linear detection range. In the biological product detection process, the sample may need to be diluted appropriately, which further causes the complex detection process and the inaccuracy of the detection result. BSA detection kits are also put forward by other companies in China, but the BSA detection kits are not widely used by vaccine manufacturers, and the linear detection range is also mostly 0-32ng/mL.

Disclosure of Invention

In order to overcome the defect of smaller linear detection range, the invention provides a BSA detection kit with a linear detection range of 0-60ng/mL.

A BSA detection kit with a linear detection range of 0-60ng/mL according to an embodiment of the present invention comprises: the anti-BSA monoclonal antibody is coated on a microplate, an enzyme-labeled antibody, a BSA standard solution, a sample diluent, a concentrated washing solution, an HRP labeling reagent diluent, a chromogenic solution and a stop solution.

Preferably, the anti-BSA monoclonal antibody coating microplate comprises the following steps:

A. coating: 100. Mu.L/well of primary antibody (anti-BSA monoclonal antibody) diluted to 1-10. Mu.g/mL with coating buffer was added to ELISA plates and coated overnight at 4 ℃; or coated for 2h at 37 ℃.

B. Closing: after washing with washing liquid for 2 times, adding blocking liquid, 200. Mu.L/well, and blocking at 37deg.C for 2-4 hr.

C. Protection: the blocking solution was discarded, a protective solution was added thereto, 250. Mu.L/well, and incubated at 37℃for 2 hours.

D. And (3) baking: discarding the protective solution, drying the ELISA plate in a drying room after beating, and vacuum packaging with aluminum foil bags.

Preferably, the coating buffer is a 0.5M carbonate buffer at ph 9.6.

Preferably, the blocking solution is a 10mM PBS solution containing 1% gelatin at a pH of 7.2-7.4.

Preferably, the protectant is a commercial enzyme label plate protectant.

Preferably, the enzyme-labeled antibody is an HRP conjugated anti-BSA monoclonal antibody.

Preferably, the BSA standard solution is a series of standard solution with the concentration of 60ng/mL, 50ng/mL, 30ng/mL, 40ng/mL, 20ng/mL, 10ng/mL and 5ng/mL in sequence. Wherein 30ng/mL is the internal reference solution.

Preferably, the sample diluent is a 10mM PBS solution having pH7.2-7.4 containing 0.05% (v/v) surfactant, 0.05% (v/v) preservative, 2% (m/v) trehalose.

Preferably, the concentrated wash solution is a 150mM PBS solution having a pH of 7.2-7.4 containing 0.75% surfactant.

Preferably, the HRP-labeled reagent diluent is a 10mM PBS solution having a pH of 7.2-7.4 containing 0.05% surfactant, 0.1% gelatin, and 10% glycerol.

Preferably, the color developing solution is TMB color developing solution.

Preferably, the stop solution is 2M H ₂ SO ₄ 。

Preferably, the surfactant is tween-20.

Preferably, the preservative is one of thimerosal or Proclin 300.

A preparation method of a Bovine Serum Albumin (BSA) detection kit with a linear detection range of 0-60ng/mL specifically comprises the following steps:

(1) Preparing a BSA-resistant monoclonal antibody coated microplate: diluting the primary antibody (anti-BSA monoclonal antibody) to 1-10 mu g/mL by using a coating buffer solution, uniformly mixing, adding the mixture into an empty ELISA plate according to 100 mu L/hole, sealing a plate membrane, and coating at 4 ℃ overnight; or coating for 2h at 37 ℃; after washing the washing liquid, adding a blocking solution at a concentration of 200 mu L/hole, and incubating at 37 ℃ for 2 hours; after the sealing is finished, washing the washing liquid, adding a protective solution according to 250 mu L/hole, and incubating for 90min at room temperature; and (5) spin-drying the liquid in the ELISA plate, drying for 2-4 hours in a drying box, and vacuumizing and plastic packaging.

Wherein, preparation of coating buffer: weigh 1.59g NaCO ₃ 、2.93g NaHCO ₃ Deionized water is added to fix the volume to 1L.

Preparation of the lotion: 8g of NaCl, 0.2g of KCl and 1.42g of Na are weighed ₂ HPO ₄ 、0.27g KH ₂ PO4, adding deionized water about 800mL, fully stirring and dissolving, adjusting the pH to 7.2-7.4, adding 0.5mL of surfactant, uniformly mixing, and then using deionized water to fix the volume to 1L.

Preparation of a sealing liquid: weighing 8g of NaCl, 0.2g of KCl and 1.42g of Na ₂ HPO ₄ 、0.27g KH ₂ PO4, deionized water of about 800mL is added, the mixture is fully stirred and dissolved, the pH is adjusted to 7.2-7.4, 1g of gelatin is added, and after the mixture is fully dissolved, deionized water is used for fixing the volume to 1L.

(2) Configuration of ELISA kit components: preparing an enzyme-labeled antibody, a BSA standard solution, a sample diluent, a concentrated washing solution, an HRP labeling reagent diluent, a color development solution and a termination solution in the kit:

wherein, the enzyme-labeled antibody is HRP conjugated anti-BSA monoclonal antibody which is diluted according to a certain proportion.

The BSA standard solutions were standard solutions with concentrations of 60ng/mL, 50ng/mL, 40ng/mL, 20ng/mL, 10ng/mL, 5ng/mL and 0ng/mL, in that order, and an internal reference solution with a concentration of 30 ng/mL. The BSA stock solution was diluted sequentially with sample dilutions to standard solution concentrations of 60ng/mL, 50ng/mL, 30ng/mL, 40ng/mL, 20ng/mL, 10ng/mL, 5ng/mL.

Preparation of sample dilutions: 8g NaCl, 3g KCl and 1.42g Na are weighed ₂ HPO ₄ 、0.27g KH ₂ PO4, deionized water of about 800mL is added, the mixture is fully stirred and dissolved, the pH is adjusted to 7.2-7.4, 20g of trehalose is added, and 0.5mL of surfactant is fully dissolved, and deionized water is used for fixing the volume to 1L.

Preparation of concentrated washing liquid: 120g NaCl, 0.2g KCl and 21.3g Na are weighed ₂ HPO ₄ 、4.05g KH ₂ PO4, deionized water of about 800mL is added, the mixture is fully stirred and dissolved, the pH is adjusted to 7.2-7.4, 7.5mL of surfactant is added for fully dissolving, and deionized water is used for fixing the volume to 1L.

HRP-labeled reagent diluentIs prepared from the following steps: 8g NaCl, 3g KCl and 1.42g Na are weighed ₂ HPO ₄ 、0.27g KH ₂ PO4, deionized water about 800mL, stirring thoroughly to dissolve, adjusting pH to 7.2-7.4, adding 1g gelatin, 100mL glycerol, and 0.5mL surfactant, dissolving thoroughly, and then fixing volume to 1L with deionized water.

The color development liquid is TMB color development liquid.

Termination liquid of 2M H ₂ SO ₄ 。

Preferably, the coated antibody concentration is 2 μg/mL.

Preferably, the stock solution of the BSA standard solution has a concentration of 100. Mu.g/mL.

Preferably, the enzyme-labeled antibody is an HRP conjugated anti-BSA monoclonal antibody to 1:2000 dilution.

The detection method for detecting BSA by the kit comprises the following steps:

(1) All reagents were equilibrated for 30min at room temperature.

(2) Washing liquid preparation: the concentrated washing solution was diluted 15-fold with ultrapure water or water for injection.

(3) The coated microplate was removed and 350 μl of wash solution was added to each well, tapped dry and repeated 2 times.

(4) 0, 5, 10, 20, 30, 40, 50, 60ng/mL standard solution and sample to be tested are added into each well, 50 mu L/well, and 2-3 compound wells are made for each concentration standard solution and sample.

(5) Sealing the sealing plate film, and standing at 25+/-3 ℃ in dark place for incubation for 60min.

(6) The wells of the microplate were discarded, tapped dry, 350 μl of wash was added to each well, tapped dry, and repeated 2 times.

(7) mu.L of pre-diluted HRP-labeled antibody was added to each well.

(8) Sealing the sealing plate film, and standing at 25+/-3 ℃ in a dark place for 30min.

(9) After the incubation, the plate was washed according to step 6 and repeated 4 times.

(10) After washing the plate, 50 mu LTMB substrate solution was added to each well, and the mixture was allowed to stand at 25.+ -. 3 ℃ for 15min in a dark place.

(11) 50. Mu.L of reaction termination solution was added to each well.

(12) Absorbance was read using a microplate reader at either dual wavelengths (450 nm and 630 nm) or single wavelength (450 nm). Detection was completed within 30 minutes after termination of the reaction.

The incubation time of the sample is 60min, the incubation time of the enzyme-labeled antibody is 30min, and the incubation time of the color development solution is 15min; the upper limit of quantitative detection of the kit is 60ng/mL, the lower limit of detection is 0.3ng/mL, and the linear quantitative range is 0-60ng/mL.

The invention has the beneficial effects that:

the invention uses double antibody sandwich enzyme-linked immunosorbent assay technology to measure the trace residue of BSA in the sample. The transfer kit is used for quantitatively detecting the residual quantity of BSA in intermediate products, semi-finished products and finished products of various biological products. The kit is convenient and simple to operate, can be directly used for sample analysis, and has good reproducibility and high stability.

Compared with the method which uses the monoclonal antibody-polyclonal antibody as the coating-detecting antibody pair (widely used detection kit of Cygnus and bosin), the method uses the mouse monoclonal antibody pair as the coating-detecting antibody, has the advantages of more detection effect and wider linear quantitative range, and can reach 0-60ng/mL.

Therefore, the kit prepared by the invention has the characteristics of good specificity, sensitivity and stability, and the linear quantitative range is 0-60ng/mL, and can be used for quantitative detection of BSA in biological products.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings may be obtained according to the drawings without inventive effort to those skilled in the art.

FIG. 1 is a graph showing the standard curve of BSA detection by the double-antibody sandwich ELISA detection kit;

FIG. 2 is a graph of the analysis of the linear correlation of the theoretical concentration and the back calculated concentration of the present invention;

FIG. 3 is a graph showing the Hook effect test results of the kit of the present invention.

Detailed Description

In order that the above-recited objects, features and advantages of the present invention will be more clearly understood, a more particular description of the invention will be rendered by reference to the appended drawings and appended detailed description. It should be noted that, in the case of no conflict, the embodiments of the present application and the features in the embodiments may be combined with each other.

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which are intended to be encompassed by the present invention by those of ordinary skill in the art, are intended to be within the scope of the present invention as they are not to be inventive, and numerous specific details are set forth in the following description to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those described herein, and the scope of the invention is therefore not limited to the specific embodiments disclosed below.

Example 1 optimization of kit-related reagents and incubation conditions

(1) Determination of first antibody coating concentration and dilution ratio of detection antibody

Preparation of anti-BSA monoclonal antibody coated microplate: diluting the primary antibody (anti-BSA monoclonal antibody) to 1-10 mu g/mL by using a coating buffer solution, uniformly mixing, adding the mixture into an empty ELISA plate according to 100 mu L/hole, sealing a plate, and coating at 4 ℃ overnight; after washing the washing liquid, adding a blocking solution at a concentration of 200 mu L/hole, and incubating at 37 ℃ for 2 hours; after the sealing is finished, washing the washing liquid, adding a protective solution according to 250 mu L/hole, and incubating for 90min at room temperature; and (5) spin-drying the liquid in the ELISA plate, drying for 2 hours in a drying oven, and vacuumizing and plastic packaging.

Balance: all reagents were equilibrated for 30min at room temperature.

Washing liquid preparation: the concentrated washing solution was diluted 15-fold with ultrapure water or water for injection.

Washing the plate: the coated microplate was removed and 350 μl of wash solution was added to each well, tapped dry and repeated 2 times.

Incubating a standard: 0 and 60ng/mL of standard solution were added well by well, 50. Mu.L/well, and 2 multiplex wells were made. Sealing the sealing plate film, and standing at 25+/-3 ℃ in dark place for incubation for 60min.

Washing the plate: the wells of the microplate were discarded, tapped dry, 350 μl of wash was added to each well, tapped dry, and repeated 2 times.

Incubating the detection antibody: after 1000-fold, 2000-fold, 4000-fold, 6000-fold dilutions of the HRP-labeled antibody stock were made with HRP-labeled reagent dilutions, 100 μl was added per well. Sealing the sealing plate film, and standing at 25+/-3 ℃ in a dark place for 30min.

Washing the plate: after the incubation, the plate was washed according to step 6 and repeated 4 times.

Color development: after washing the plate, 50 mu LTMB substrate solution was added to each well, and the mixture was allowed to stand at 25.+ -. 3 ℃ for 15min in a dark place.

And (3) terminating: 50. Mu.L of reaction termination solution was added to each well.

Reading OD value: absorbance was read using a microplate reader at two wavelengths (450 nm and 630 nm).

Based on the results shown in Table 1, subsequent experiments were performed with a concentration of 2. Mu.g/mL of coated antibody and a dilution ratio of 1:2000 of detection antibody in combination with background values, signal values, etc.

TABLE 1 OD values at various dilution factors of antibodies

(2) Determination of first antibody coating temperature and coating time

Diluting the primary antibody (anti-BSA monoclonal antibody) to 2 mug/mL by using a coating buffer solution, adding the primary antibody (anti-BSA monoclonal antibody) into an empty ELISA plate according to 100 mug/hole after uniformly mixing, and coating overnight at 4 ℃ after sealing a plate membrane; or coating for 2h at 37 ℃; after washing the washing liquid, adding a blocking solution at a concentration of 200 mu L/hole, and incubating at 37 ℃ for 2 hours; after the sealing is finished, washing the washing liquid, adding a protective solution according to 200 mu L/hole, and incubating for 90min at room temperature; and (5) spin-drying the liquid in the ELISA plate, drying for 2 hours in a drying oven, and vacuumizing and plastic packaging.

Balance: all reagents were equilibrated for 30min at room temperature.

Incubating the detection antibody: after 2000-fold dilution of the HRP-labeled antibody stock with HRP-labeled reagent dilution, 100 μl was added per well. Sealing the sealing plate film, and standing at 25+/-3 ℃ in a dark place for 30min.

The results are shown in Table 2, and the overall background value, signal value, and signal to noise ratio were selected at a coating temperature and a coating time of 4℃overnight.

TABLE 2 OD, mean and SNR at different coating temperatures and times

(3) Selection of coating buffer

Respectively taking 50mM carbonate buffer solution (CBS, pH 9.6) and 10mM phosphate buffer solution (PBS, pH 7.2-7.4) as coating buffer solutions, diluting the primary antibody (anti-BSA monoclonal antibody) to 2 mug/mL, adding 100 mug/hole into an empty ELISA plate after uniformly mixing, and coating at 4 ℃ overnight after sealing the plate; after washing the washing liquid, adding a blocking solution at a concentration of 200 mu L/hole, and incubating at 37 ℃ for 2 hours; after the sealing is finished, washing the washing liquid, adding a protective solution according to 200 mu L/hole, and incubating for 90min at room temperature; and (5) spin-drying the liquid in the ELISA plate, drying for 2 hours in a drying oven, and vacuumizing and plastic packaging.

Balance: all reagents were equilibrated for 30min at room temperature.

The results are shown in Table 3, and 50mM carbonate buffer was selected as the coating buffer, combining the background value, signal value, and signal to noise ratio.

TABLE 3 OD, mean and SNR at different coating buffers

(4) Determination of optimal sealing liquid

Diluting the primary antibody (anti-BSA monoclonal antibody) to 2 mug/mL by using a coating buffer solution, adding the primary antibody (anti-BSA monoclonal antibody) into an empty ELISA plate according to 100 mug/hole after uniformly mixing, and coating overnight at 4 ℃ after sealing a plate membrane; after washing the washing liquid, adding a blocking solution at a concentration of 200 mu L/hole, and incubating at 37 ℃ for 2 hours; after the sealing is finished, washing the washing liquid, adding a protective solution according to 200 mu L/hole, and incubating for 90min at room temperature; and (5) spin-drying the liquid in the ELISA plate, drying for 2 hours in a drying oven, and vacuumizing and plastic packaging.

Wherein 5%, 2%, 1% and 0.5% gelatin were selected as blocking solutions.

Balance: all reagents were equilibrated for 30min at room temperature.

As shown in Table 4, when 1% gelatin was used as the blocking solution, the signal value was high, the background value was constant, and the signal-to-noise ratio was highest. Thus, 1% gelatin was selected as the blocking solution, combining the background value, signal value, and signal to noise ratio.

TABLE 4 OD, mean and SNR at different confining liquids

(5) Determination of the protection liquid of the ELISA plate

Diluting the primary antibody (anti-BSA monoclonal antibody) to 2 mug/mL by using a coating buffer solution, adding the primary antibody (anti-BSA monoclonal antibody) into an empty ELISA plate according to 100 mug/hole after uniformly mixing, and coating overnight at 4 ℃ after sealing a plate membrane; after washing the washing liquid, adding a blocking solution at a concentration of 200 mu L/hole, and incubating at 37 ℃ for 2 hours; after the sealing is finished, washing the washing liquid, adding an ELISA plate protecting liquid according to 200 mu L/hole, and incubating for 90min at room temperature; and (5) spin-drying the liquid in the ELISA plate, drying for 2 hours in a drying oven, and vacuumizing and plastic packaging. The enzyme label plate after plastic package is placed at 37 ℃ for 3 days, 5 days, 7 days and 10 days.

Wherein, 3% sucrose solution, 5% trehalose solution and commercial ELISA plate protecting solution are selected as ELISA plate protecting solution.

Balance: all reagents were equilibrated for 30min at room temperature.

As a result, the decrease rate of the signal value was calculated from the OD value of the ELISA plate treated with the different protectants after 3 days, 5 days, 7 days, and 10 days at 37℃as compared with the ELISA plate placed at 4 ℃. It can be seen that the protection effect of the commercial protection liquid is optimal.

TABLE 5 ELISA plate Heat stability degradation rate measurement Table for different ELISA plate protectants

(6) Determination of drying plate time

Diluting the primary antibody (anti-BSA monoclonal antibody) to 2 mug/mL by using a coating buffer solution, adding the primary antibody (anti-BSA monoclonal antibody) into an empty ELISA plate according to 100 mug/hole after uniformly mixing, and coating overnight at 4 ℃ after sealing a plate membrane; after washing the washing liquid, adding a blocking solution at a concentration of 200 mu L/hole, and incubating at 37 ℃ for 2 hours; after the sealing is finished, washing the washing liquid, adding an ELISA plate protecting liquid according to 200 mu L/hole, and incubating for 90min at room temperature; and (5) spin-drying the liquid in the ELISA plate, drying for 2-4 hours in a drying box, and vacuumizing and plastic packaging.

Balance: all reagents were equilibrated for 30min at room temperature.

As shown in Table 6, after the ELISA plate is dried for 2 hours or 4 hours at 37 ℃, the background value and the signal value of 4 hours are better, and the signal to noise ratio is better, so that the drying plate time is 4 hours.

TABLE 6 OD values, means and SNR for different baking plate durations

Example 2 preparation of double antibody sandwich ELISA kit

(1) Preparation of anti-BSA monoclonal antibody coated microplate

ELISA plate: costar

Coating buffer (50 mM carbonate buffer pH 9.6): 1.59g NaCO was weighed out ₃ 、2.93g NaHCO ₃ Deionized water is added to fix the volume to 1L.

Wash solution (10 mM PBS solution pH 7.2-7.4): weigh 8g NaCl, 0.2g KCl, 1.42g Na ₂ HPO ₄ 、0.27g KH ₂ PO ₄ Adding deionized water about 800mL, stirring thoroughly to dissolve, adjusting pH to 7.2-7.4, adding Tween-20 0.5mL, mixing well, and then fixing volume to 1L with deionized water.

Preparation of a sealing liquid: weigh 8g NaCl, 0.2g KCl, 1.42g Na ₂ HPO ₄ 、0.27g KH ₂ PO ₄ Adding deionized water about 800mL, stirring thoroughly to dissolve, adjusting pH to 7.2-7.4, adding 1g gelatin, dissolving thoroughly, and then fixing volume to 1L with deionized water.

Coating of capture antibodies: diluting the primary antibody (anti-BSA monoclonal antibody) to 2 mug/mL by using a coating buffer solution, adding the primary antibody (anti-BSA monoclonal antibody) into an empty ELISA plate according to 100 mug/hole after uniformly mixing, and coating overnight at 4 ℃ after sealing a plate membrane;

closing: after washing the washing liquid, adding a blocking solution at a concentration of 200 mu L/hole, and incubating at 37 ℃ for 2 hours;

protection: after the sealing is finished, washing the washing liquid, adding a protective solution according to 250 mu L/hole, and incubating for 90min at room temperature;

and (3) baking: spin-drying the liquid in the ELISA plate, and drying in a drying oven for 4 hours;

and (5) plastic packaging and preservation: and (5) vacuumizing, plastic packaging and preserving at 4 ℃.

(2) The configuration of each component: preparing an enzyme-labeled antibody, a BSA standard solution, a sample diluent, a concentrated washing solution, an HRP labeling reagent diluent, a color development solution and a termination solution in the kit:

wherein, the ELISA monoclonal antibody (HRP labeling reagent) is prepared by diluting HRP conjugated anti-BSA monoclonal antibody with labeling reagent stock solution at 1:20, and diluting with HRP labeling reagent diluent at 1:100.

The BSA standard solutions were standard solutions with concentrations of 60ng/mL, 50ng/mL, 40ng/mL, 20ng/mL, 10ng/mL, 5ng/mL and 0ng/mL, in that order, and an internal reference solution with a concentration of 30 ng/mL. The BSA stock solution (100. Mu.g/mL) was diluted sequentially with the sample dilution to standard solution concentration.

Preparation of sample dilutions: 8g NaCl, 3g KCl and 1.42g Na are weighed ₂ HPO ₄ 、0.27g KH ₂ PO ₄ Adding deionized water about 800mL, stirring thoroughly to dissolve, adjusting pH to 7.2-7.4, adding 20g trehalose, dissolving 0.5mL Tween-20 thoroughly, and metering volume to 1L with deionized water.

Preparation of concentrated washing liquid: 120g of NaCl, 0.2g of KCl and 21.3g of Na are weighed ₂ HPO ₄ 、4.05g KH ₂ PO ₄ Adding deionized water about 800mL, stirring thoroughly to dissolve, adjusting pH to 7.2-7.4, adding Tween-20 7.5mL, dissolving thoroughly, and then fixing volume to 1L with deionized water.

Preparation of a labeling reagent dilution: 8g NaCl, 3g KCl and 1.42g Na are weighed ₂ HPO ₄ 、0.27g KH ₂ PO ₄ Adding deionized water about 800mL, stirring thoroughly to dissolve, adjusting pH to 7.2-7.4, adding gelatin 1g, glycerol 100mL, and Tween-20 0.5mL, and diluting with deionized water to 1L.

The color development liquid is TMB color development liquid.

Termination liquid of 2M H ₂ SO ₄ 。

The specific components and storage conditions are shown in Table 7.

TABLE 7 list of kit Components

(3) The specific experimental method for detecting BSA by using the kit comprises the following steps:

A. all reagents were equilibrated for 30min at room temperature.

B. Washing liquid preparation: the concentrated washing solution was diluted 15-fold with ultrapure water or water for injection.

C. The coated microplate was removed and 350 μl of wash solution was added to each well, tapped dry and repeated 2 times.

D. 0, 5, 10, 20, 30, 40, 50, 60ng/mL standard solution, 50. Mu.L/well, 2-3 multiplex wells were added per concentration of standard solution and sample.

E. Sealing the sealing plate film, and standing at 25+/-3 ℃ in dark place for incubation for 60min.

F. The wells of the microplate were discarded, tapped dry, 350 μl of wash was added to each well, tapped dry, and repeated 2 times.

G. mu.L of HRP-labeled reagent working fluid diluted 100-fold with HRP-labeled reagent diluent was added to each well.

H. Sealing the sealing plate film, and standing at 25+/-3 ℃ in a dark place for 30min.

I. After the incubation, the plate was washed according to step 6 and repeated 4 times.

J. After washing the plate, 50 mu LTMB substrate solution was added to each well, and the mixture was allowed to stand at 25.+ -. 3 ℃ for 15min in a dark place.

K. 50. Mu.L of reaction termination solution was added to each well.

L. absorbance was read using a microplate reader at either dual wavelengths (450 nm and 630 nm) or single wavelength (450 nm). Detection was completed within 30 minutes after termination of the reaction.

(4) Linear range

Diluting BSA standard with sample diluent to 60-5ng/mL, detecting with the above kit, repeating for 3 times, and OD with BSA concentration (ng/mL) as abscissa _450-630 Values are on the ordinate and a standard curve is drawn.The results are shown in Table 8 and FIG. 1, the BSA standard solution has a good linear correlation in the measurement range, and the regression equation of the standard curve is: y=0.0177x+0.0649, r ² ＝0.9966。

TABLE 8 standard curve OD and Coefficient of Variation (CV), mean and standard deviation

(5) Analysis of linear correlation between theoretical concentration and back-calculated concentration

The concentration of the standard solution was calculated according to the test data in Table 8 and the standard curve shown in FIG. 1, and the calculated value of 30ng/mL of the reference solution in BSA is 29.33ng/mL, the coefficient of variation between the theoretical concentration and the calculated concentration is 1.16%, and the calculation accuracy is 97.75%. The linear correlation analysis result is shown in fig. 2, the theoretical concentration and the calculated concentration have good linear correlation, the pearson coefficient r=0.9997, and r ² ＝0.9995。

TABLE 9 theoretical concentration, calculated concentration, coefficient of variation and back calculation accuracy of the two

(6) Kit Hook effect detection

BSA stock (100. Mu.g/mL) was diluted with sample dilution to 100. Mu.g/mL, 10. Mu.g/mL, 1. Mu.g/mL, 100ng/mL, 60ng/mL, 50ng/mL, 40ng/mL, 30ng/mL, 20ng/mL, 10ng/mL, 5ng/mL and 0ng/mL. The detection is carried out by using the kit, the test results are shown in Table 10 and FIG. 3, when the BSA concentration reaches 100 mug/mL, the measured OD values are higher than the OD value of 60ng/mL of the highest concentration in the standard curve, and therefore, the kit has no Hook effect when the BSA concentration reaches 100 mug/mL.

TABLE 10 hook Effect test OD value, coefficient of variation, mean and standard deviation

(7) Cross-reaction test with Human Serum Albumin (HSA)

Human serum albumin HSA was diluted to 50mg/mL, 20mg/mL, 10mg/mL, 5mg/mL, 2mg/mL, 1mg/mL, 500. Mu.g/mL, 200. Mu.g/mL, 100. Mu.g/mL, 50. Mu.g/mL, 20. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL, 1. Mu.g/mL, 100ng/mL, 50ng/mL with the sample dilution. The detection is carried out by using the kit, the test results are shown in Table 11, and the OD values measured when the concentration of HSA is diluted as described above are all close to the background value, so that the kit has no cross effect with HSA when the concentration of HSA reaches 50 mg/mL.

TABLE 11 OD value for HSA Cross-reaction test

(8) Detection limit

The sample dilution was used as a sample for detection, and the measurement was repeated 20 times to obtain absorbance (OD _450-630 ) And calculating the Mean value (Mean) and Standard Deviation (SD) of the sample solution, obtaining a light absorption value corresponding to the mean+2SD, taking the light absorption value corresponding to the mean+2SD into a standard curve equation according to a standard Qu Quxian equation of a standard solution used by the kit, and obtaining a corresponding concentration value, namely the detection limit.

TABLE 12 calculation of OD value and detection limit for sample Diluent detection

(9) Standard deviation of

The average value of the results obtained after 3 times repeated measurement of high (50 ng/mL), medium (30 ng/mL) and low (10 ng/mL) concentration BSA standard solutions was designated as M according to the formula: measurement deviation = (M-theoretical)/theoretical x 100%,

the detection results of the standard deviation are shown as the OD value measurement deviation of three different concentrations is far less than 15%, and the results show that the kit is good in accuracy.

TABLE 12 measurement deviation results for high, medium and low concentration standard solutions

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A bovine serum albumin assay kit, comprising: the anti-BSA monoclonal antibody is coated on a micro-porous plate, an ELISA monoclonal antibody (HRP labeling reagent), a BSA standard solution, a sample diluent, a concentrated washing solution, an HRP labeling reagent diluent, a chromogenic solution and a stop solution.

2. The bovine serum albumin detection kit according to claim 1, wherein the anti-BSA monoclonal antibody is coated on a microplate, and the preparation method thereof is as follows:

A. coating: 100. Mu.L/well of primary antibody diluted to 1-10. Mu.g/mL with coating buffer was added to ELISA plates and coated overnight at 4 ℃; or coated for 2h at 37 ℃.

3. The bovine serum albumin detection kit according to claim 1, wherein the elisa monoclonal antibody is an HRP-conjugated anti-BSA monoclonal antibody.

4. The bovine serum albumin assay kit according to claim 1, wherein the BSA standard solution is a series of standard solutions having a concentration of 60ng/mL, 50ng/mL, 30ng/mL, 40ng/mL, 20ng/mL, 10ng/mL, 5ng/mL in that order, wherein 30ng/mL is an internal reference solution.

5. The bovine serum albumin assay kit according to claim 1, wherein the sample diluent is a 10mm pbs solution having a pH of 7.2-7.4 containing 0.05% (v/v) surfactant, 0.05% (v/v) preservative, 2% (m/v) trehalose.

6. The bovine serum albumin assay kit according to claim 1, wherein the concentrated wash solution is a 150mm pbs solution having a pH of 7.2-7.4 containing 0.75% surfactant.

7. The bovine serum albumin assay kit according to claim 1, wherein the HRP labeling reagent diluent is a 10 mmsbs solution having a pH of 7.2-7.4 containing 0.05% surfactant, 0.1% gelatin, 10% glycerol.

8. The bovine serum albumin assay kit according to claim 1, wherein the color development solution is TMB color development solution.

9. The bovine serum albumin assay kit according to claim 2, wherein the coating buffer is a 0.5M carbonate buffer at ph 9.6.

10. The bovine serum albumin assay kit according to claim 2, wherein the blocking solution is a 10mm pbs solution containing 1% gelatin and having a pH of 7.2-7.4.