CN113820496A - Kit and method for quantitatively detecting human lipopolysaccharide binding protein - Google Patents
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Abstract
The invention relates to the technical field of immunoassay, in particular to a chemiluminiscence kit and a detection method for quantitatively detecting human Lipopolysaccharide Binding Protein (LBP). The kit comprises a well plate coated with human lipopolysaccharide binding protein antibodies. The detection method comprises the following steps: (1) adding a sample to be detected into a pore plate coated with a human lipopolysaccharide binding protein antibody; (2) adding a biotin-labeled human lipopolysaccharide binding protein antibody; (3) incubating, washing and drying; (4) adding horseradish peroxidase labeled with streptomycin and avidin or alkaline phosphatase labeled with streptomycin and avidin for reaction, washing and drying; (5) adding enzyme luminescent substrate, and reading the luminescent value. The method has higher detection sensitivity and stronger specificity; blood sample treatment is not needed, and the operation is simple and convenient; the method has no high requirement on required equipment, can realize large-flux detection, has low cost, can realize semi-automatic or full-automatic batch operation, and has good effect and application prospect.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a chemiluminiscence kit for quantitatively detecting human Lipopolysaccharide Binding Protein (LBP).
Background
Lipopolysaccharide Binding Protein (LBP) is a glycoprotein with a molecular weight of about 58KDa in size, which is synthesized by hepatocytes and belongs to the lipid binding protein family. The family also includes BPI, PLTP, CETP. LBP binds to the lipid A moiety of Lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, promotes the monomerization of LPS, catalyzes the binding of LPS monomer to the cytokine CD14, and induces an immune response that promotes LPS, an index that reflects the toxin loading level in the body.
The existing immunological research solution for detecting LBP, such as the conventional enzyme-linked immunosorbent assay (ELISA), has short detection range, generally ranges from 0.8 ng/mL to 50ng/mL, and has poor linearity; the sensitivity is not high, generally about 2ng/mL, and a blood sample needs to be pretreated and is diluted by 50-200 times for detection; meanwhile, the standard deviation is about 15%, and the precision is not high.
There is a need to develop a new method for detecting LBP, which overcomes the above-mentioned drawbacks and improves the sensitivity and detection range and precision.
Disclosure of Invention
The invention aims to provide a kit for quantitatively detecting human Lipopolysaccharide Binding Protein (LBP), in particular a chemiluminescence kit. The invention also provides a detection method. The method and the kit have the advantages of high detection sensitivity, good specificity, simple and convenient operation, high detection flux, low detection cost, small detection sample amount and no need of too many complex auxiliary equipment and reagents.
In order to realize the purpose, the invention adopts the following technical scheme:
a kit for quantitatively detecting human lipopolysaccharide binding protein comprises a pore plate coated with human lipopolysaccharide binding protein antibody.
The human lipopolysaccharide binding protein antibody is a human lipopolysaccharide binding protein monoclonal antibody or a human lipopolysaccharide binding protein polyclonal antibody. The amount of the human lipopolysaccharide binding protein antibody coated on each detection well or detection point of the well plate coated with the human lipopolysaccharide binding protein antibody is 0.1-0.5 mu g, preferably 0.2-0.4 mu g.
The pore plate coated with the human lipopolysaccharide binding protein antibody is sealed by protective protein, and the protective protein is casein, bovine serum albumin, gelatin, animal serum or human serum protein.
The kit for quantitatively detecting the human lipopolysaccharide binding protein further comprises at least one of the following reagents: (1) a biotin-labeled human lipopolysaccharide binding protein antibody; (2) streptavidin-labeled horseradish peroxidase or streptavidin-labeled alkaline phosphatase; (3) a luminescent substrate of horseradish peroxidase or alkaline phosphatase.
The biotin-labeled human lipopolysaccharide binding protein antibody is a monoclonal antibody or a polyclonal antibody.
The luminescent substrate of horseradish peroxidase HRP includes luminol, luminol derivatives and p-hydroxyphenylacetic acid. The luminescent substrate of alkaline phosphatase AP includes 3- (2-helical adamantane-4-methoxy-4-methyl-4- (3-phosphoxy) -phenyl-1, 2-dioxyethane (AMPPD), 4-methylumbelliferone phosphate (4-MUP, fluorogenic substrate), and the like.
The kit for quantitatively detecting the human lipopolysaccharide binding protein further comprises a washing solution, wherein the washing solution contains sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and tween-20; preferably, the composition further comprises a preservative.
The kit for quantitatively detecting the human lipopolysaccharide binding protein further comprises a diluent which is used for diluting a sample to be detected, a biotin-labeled human lipopolysaccharide binding protein antibody, a streptavidin-labeled horseradish peroxidase or streptavidin-labeled alkaline phosphatase.
The diluent is phosphate buffer (PB buffer), HEPES buffer, MOPS buffer or Tris buffer with the pH of 7.4 and 0.01-0.02mol/L, and contains 0.5% -2% of protein protective agent; preferably, the protein protective agent is casein, Bovine Serum Albumin (BSA), gelatin, animal serum, human serum albumin; preferably, the diluent also contains a preservative.
Preferably, the kit for quantitatively detecting the human lipopolysaccharide binding protein further comprises a human Lipopolysaccharide Binding Protein (LBP) standard.
The preparation method of the kit for quantitatively detecting the human lipopolysaccharide binding protein comprises the steps of coating the human lipopolysaccharide binding protein antibody on a pore plate and sealing the pore plate by using protective protein to obtain the pore plate coated with the human lipopolysaccharide binding protein antibody.
The protective protein is casein, bovine serum albumin, gelatin, animal serum, human serum and human serum protein.
A method for quantitatively detecting human lipopolysaccharide binding protein comprises the following steps:
(1) adding a sample to be detected into a pore plate coated with a human lipopolysaccharide binding protein antibody;
(2) adding a biotin-labeled human lipopolysaccharide binding protein antibody;
(3) incubating, washing and drying;
(4) adding horseradish peroxidase labeled with streptomycin and avidin or alkaline phosphatase labeled with streptomycin and avidin for reaction, washing and drying;
(5) adding enzyme luminescent substrate, and reading the luminescent value.
Preferably, the method further comprises the step (6): and comparing with the standard curve to obtain the concentration of the sample to be detected.
In the step (1), the sample to be detected is a blood sample, including a serum sample or a plasma sample.
Preferably, in step (1), the sample to be tested is diluted to 20-100 times and applied to a plate coated with the antibody against human lipopolysaccharide binding protein. Preferably, the sample to be tested is diluted to 50 fold.
In the well plate of step (1), the amount of the human lipopolysaccharide binding protein antibody coated on each detection well or detection spot is 0.1-0.5. mu.g, preferably 0.2-0.4. mu.g.
The well plate coated with the human lipopolysaccharide binding protein antibody is subjected to blocking treatment with a protective protein.
Preferably, in the step (4), the biotin-labeled human lipopolysaccharide binding protein antibody is dissolved in the diluent to obtain a working solution containing the biotin-labeled human lipopolysaccharide binding protein antibody, wherein the content of the biotin-labeled human lipopolysaccharide binding protein antibody is 0.1-2 μ g/mL, preferably 0.2-1 μ g/mL; dissolving streptavidin-labeled horseradish peroxidase or streptavidin-labeled alkaline phosphatase in a diluent to obtain a streptavidin-and-avidin-labeled horseradish peroxidase-labeled working solution or a streptavidin-and-avidin-labeled alkaline phosphatase-labeled working solution, wherein the content of the streptavidin-labeled horseradish peroxidase or alkaline phosphatase-labeled horseradish peroxidase-labeled alkaline phosphatase is 10-100mIU/mL, preferably 20-60 mIU/mL; in a preferred embodiment of the present invention, the content is 50 mIU/mL.
The diluent is phosphate buffer (PB buffer), HEPES buffer, MOPS buffer or Tris buffer with the pH of 7.4 and 0.01-0.02mol/L, and contains 0.5% -2% of protein protective agent; preferably, the protein protective agent is casein, Bovine Serum Albumin (BSA), gelatin, animal serum, human serum albumin; preferably, the diluent also contains a preservative.
Preferably, in step (5), the enzyme luminescent substrate is an enzymatic luminescent substrate of horseradish peroxidase or alkaline phosphatase. The luminescent substrate of horseradish peroxidase HRP includes luminol, luminol derivatives and p-hydroxyphenylacetic acid. The luminescent substrate of alkaline phosphatase AP includes 3- (2-helical adamantane-4-methoxy-4-methyl-4- (3-phosphoxy) -phenyl-1, 2-dioxyethane (AMPPD), 4-methylumbelliferone phosphate (4-MUP, fluorogenic substrate), and the like.
According to the kit and the detection method, the minimum detection limit in a sample is 0.23-0.27ng/mL, the linearity is good in the concentration range of 0-50ng/mL, the precision is high, the standard deviation is about 8%, and the standard deviation is far lower than 15% in the prior art.
The invention has the advantages that the human Lipopolysaccharide Binding Protein (LBP) monoclonal antibody or the human Lipopolysaccharide Binding Protein (LBP) polyclonal antibody is coated in the chemiluminescence plate, an enzymatic chemiluminescence method is adopted, an enzymatic catalytic luminescence substrate is utilized, and a biotin amplification system is utilized, so that the sensitivity and the accuracy of the kit are improved; experiments prove that the chemiluminescence method is an effective method for quantitatively detecting human Lipopolysaccharide Binding Protein (LBP) by matching with a biotin amplification system detection system. The method and the kit have the advantages of high detection sensitivity, good specificity, simple and convenient operation, small detection sample amount and no need of too many complex auxiliary equipment and reagents.
The kit and the detection method have the beneficial effects that compared with a human Lipopolysaccharide Binding Protein (LBP) enzyme-linked immunosorbent assay (ELISA) detection kit on the market, the kit and the detection method have higher detection sensitivity and stronger specificity; the kit and the method do not need blood sample treatment, so the operation is simple and convenient; the kit has no high requirement on required equipment, can realize large-flux detection, has low cost, can realize semi-automatic or full-automatic batch operation in the immunodiagnosis part, and has good effect and application prospect.
Drawings
FIG. 1 is a standard curve for detecting LBP using a chemiluminescent plate
Detailed Description
The present invention is described in more detail below with reference to specific embodiments.
EXAMPLE 1 preparation of the kit
1. Preparation of human Lipopolysaccharide Binding Protein (LBP) monoclonal antibody or polyclonal antibody coated chemiluminescence plate
Diluting a human Lipopolysaccharide Binding Protein (LBP) monoclonal antibody or a polyclonal antibody to 2-4 mu g/mL by using a phosphate buffer solution or a carbonate buffer solution, adding the diluted antibody into a chemiluminescence plate according to 100 mu l/hole, standing at 4 ℃ for 16-20 hours, taking out the chemiluminescence plate, spin-drying residual liquid, carrying out sealing treatment by using bovine serum albumin, casein, gelatin, animal or human serum and the like, adding sample according to 250 mu l/hole (the concentration is 5-20 mu g/mL), standing at 37 ℃ for 2 hours or 4 ℃ for 16-20 hours, spin-drying the residual liquid, and drying and storing at 4 ℃ for later use.
2. Preparation of Biotin-labeled human Lipopolysaccharide Binding Protein (LBP) monoclonal antibody or human Lipopolysaccharide Binding Protein (LBP) polyclonal antibody
A10 mM NHS-DPEG4-Biotin working solution was prepared using 0.1M PBS at pH 7.2. Adding human Lipopolysaccharide Binding Protein (LBP) monoclonal antibody or polyclonal antibody into a proper amount of 10mM NHS-DPEG4-Biotin working solution according to a certain proportion, reacting for 1 hour at room temperature, separating and purifying by using sephadex, and removing free Biotin. 0.1% BSA was added and left at 4 ℃ until use, wherein the antibody concentration was 500. mu.g/mL.
A biotin-labeled human Lipopolysaccharide Binding Protein (LBP) monoclonal antibody or a human Lipopolysaccharide Binding Protein (LBP) polyclonal antibody can also be prepared by purchasing a commercial biotin labeling kit for operation.
3. Preparation of biotin-labeled human Lipopolysaccharide Binding Protein (LBP) monoclonal antibody or human Lipopolysaccharide Binding Protein (LBP) polyclonal antibody working solution
1) Preparation of a diluent:
0.01M PB buffer (pH7.4), 1% Casein or 1% BSA, 0.1% proclin300
3) Preparing a working solution:
the biotin-labeled human Lipopolysaccharide Binding Protein (LBP) monoclonal antibody working solution is obtained after the biotin-labeled human Lipopolysaccharide Binding Protein (LBP) monoclonal antibody is diluted by a diluent, the dilution ratio in the embodiment is 1:2000, and the content of the biotin-labeled human Lipopolysaccharide Binding Protein (LBP) monoclonal antibody in the working solution is 0.25 mu g/mL.
4. Preparation of streptavidin-labeled horseradish peroxidase or alkaline phosphatase working solution
1) Preparation of a diluent:
0.1M TB buffer (pH7.4), 1% Casein or 1% BSA, 0.1% proclin300
2) Streptavidin-labeled horseradish peroxidase or alkaline phosphatase
Adopting a glutaric diol method, preparing horseradish peroxidase or alkaline phosphatase into 50IU/mL, taking a proper amount, adding the proper amount into PBS (phosphate buffer solution) containing 1.25% glutaraldehyde and having the pH value of 6.8, uniformly mixing, and reacting at room temperature overnight. Collecting reaction solution, dialyzing with PBS (pH7.2) for 4 times, dissolving appropriate amount of SA in 1mol/L carbonate buffer solution (pH 9.5), mixing with the dialyzed reaction solution, reacting at 4 deg.C, and adding appropriate amount of 0.2mol/L lysine solution. After mixing, the mixture was reacted at room temperature for 2 hours, and dialyzed 4 times against 0.05mol/L PBS (pH 7.2). The supernatant was centrifuged to 4 ℃ until use, at a concentration of 50IU/mL of streptavidin-labeled horseradish peroxidase.
Streptavidin-labeled horseradish peroxidase or alkaline phosphatase can also be prepared by purchasing a commercial streptavidin labeling kit protocol.
2) Preparing a working solution:
and diluting the streptomycin and the avidin-labeled horseradish peroxidase by using a diluent to obtain a streptavidin-labeled horseradish peroxidase working solution, wherein the dilution ratio is 1: 500-1: 5000, the dilution ratio in the experimental embodiment is 1:1000, and the contents of the streptomycin and the avidin-labeled horseradish peroxidase in the working solution are 50 mIU/mL. Streptavidin-labeled alkaline phosphatase working solutions were prepared in the same manner.
5. Preparation of calibrator
The antigen of the human Lipopolysaccharide Binding Protein (LBP) standard is a complete-sequence human lipopolysaccharide binding protein (1 to 481 amino acid sequence) with the gene recombination concentration of more than or equal to 95 percent or a recombinant sequence protein (functional section: 196 to 295 amino acid sequence) with the purity of more than or equal to 97 percent, and the diluent of the human Lipopolysaccharide Binding Protein (LBP) standard in the kit is HEPES containing 1 percent BSA or PB buffer containing 1 percent BSA, and contains 0.1 percent proclin 300. The dilution concentration gradient was: 0.78ng/mL, 1.56ng/mL, 3.13ng/mL, 6.25ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL for preparing a standard curve for detection.
6. Preparation of the washing solution
The washing solution is 0.01M PBST solution, and 1L of washing solution is prepared: 9.75g of sodium dihydrogen phosphate dihydrate, 51g of disodium hydrogen phosphate dodecahydrate, 155g of sodium chloride and Tween 2010 mL, wherein 0.1% proclin300 is contained in the sodium dihydrogen phosphate dihydrate, and the sodium dihydrogen phosphate dihydrate, the disodium hydrogen phosphate dodecahydrate, the sodium chloride and the Tween 2010 mL are diluted by 20 times with ultrapure water when in use to prepare a washing working solution.
Example 2 method and methodological evaluation of the detection of human Lipopolysaccharide Binding Protein (LBP) Using the kit
The method for quantitatively detecting the human lipopolysaccharide binding protein by using the kit in the embodiment 1 comprises the following steps:
(1) diluting human blood sample by 50 times with standard diluent, taking the gradient concentration of standard substance and serum/plasma sample according to 50 mu L/hole, adding into corresponding plate hole, and marking;
(2) adding biotin-labeled human Lipopolysaccharide Binding Protein (LBP) antibody working solution into a corresponding plate hole according to 50 mu L/hole;
(3) incubating at 37 ℃ for 1h, taking out, washing 250 mu L of working solution in each hole for 4 times, and patting to dry;
(4) adding streptavidin-labeled horseradish peroxidase working solution (with a concentration of 50mIU/mL) into corresponding reaction holes according to a concentration of 50 mu L/hole, and standing at 37 ℃ for reaction for 30 min; then taking out the luminescent plate, washing for 4 times by 250 mu L of washing liquid in each hole, and drying;
(5) adding 100 mu L of enzyme luminescent substrate luminol (the concentration is 50mIU/mL) into each hole, and reading the luminescent value;
respectively taking the concentration value of the gradient of the standard substance as an x value and a luminous value as a y value, and making a unitary linear equation standard curve to obtain a curve equation; and substituting the luminous value of the sample to be detected into the standard curve, and calculating the concentration value of the sample to be detected.
(II) methodological evaluation of the kit
1. Standard curve
The concentration of the calibrator (0ng/mL excluded, the value is considered as the background of the reaction plate and is not included in the range of the standard curve) is used as the X value, and the luminescence value RLU corresponding to concentration detection is used as the Y value, so that a linear equation of a unary is made.
As shown in fig. 1, the regression equation of the standard curve is plotted as y-5781.8 x-1176.6, R2＝0.9994
2. Minimum detection limit
The lowest detection limit reference substance (0.78ng/mL) was detected 10 times while the standard curve was detected, and the average concentration value (x) and Standard Deviation (SD) of the lowest detection limit reference substance were calculated from the standard curve. According to the formula: the detection limit was X ± 2SD, and the lowest detection limit of the sample was calculated.
As a result, as shown in Table 1, the minimum detection limit of the kit of the present invention for detecting human Lipopolysaccharide Binding Protein (LBP) in a blood sample was 0.25 ng/mL.
TABLE 1 minimum detection limits of the human Lipopolysaccharide Binding Protein (LBP) chemiluminescence detection kit
| Sample(s) | Mean value (ng/mL) | Standard deviation (ng/mL) | Minimum detection limit (ng/mL) |
| Reference product with minimum detection limit | 0.25 | 0.02 | 0.23-0.27 |
3. Precision of
As for the detection results in table 1 above, the ratio obtained by the Standard Deviation (SD)/average concentration value (x) of the kit of the present invention is the precision value, that is: the standard deviation 0.02ng/mL divided by the mean concentration value 0.25ng/mL, multiplied by 100%, was 8%, less than 15%.
The alkaline phosphatase is used for replacing horseradish peroxidase, or a monoclonal antibody is replaced by a human lipopolysaccharide binding protein polyclonal antibody, and the effect is the same.
In conclusion, compared with the ELISA detection kit in the current market, the kit for detecting human Lipopolysaccharide Binding Protein (LBP) has the advantages of simple and convenient operation, high operation sensitivity, good specificity, low detection cost, less sample usage amount, low requirement on equipment and the like.
The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. The invention belongs to the technical field of the invention, and a plurality of equivalent substitutions or obvious modifications can be made without departing from the concept of the invention, and the invention has the same or similar performance or use and shall belong to the protection scope of the invention.
Claims (9)
1. A kit for quantitatively detecting human lipopolysaccharide binding protein is characterized by comprising a pore plate coated with human lipopolysaccharide binding protein antibodies.
2. The kit for quantitative determination of human lipopolysaccharide binding protein of claim 1, wherein the amount of human lipopolysaccharide binding protein antibody coated on each detection well or spot of the well plate is 0.1-0.5. mu.g.
3. The kit for quantitative determination of human lipopolysaccharide binding protein according to claim 1 or 2, wherein the human lipopolysaccharide binding protein antibody is a human lipopolysaccharide binding protein monoclonal antibody or a human lipopolysaccharide binding protein polyclonal antibody.
4. The kit for quantitative determination of human lipopolysaccharide binding protein according to claim 1 or 2, characterized by further comprising at least one of the following reagents: (1) a biotin-labeled human lipopolysaccharide binding protein antibody; (2) streptavidin-labeled horseradish peroxidase or streptavidin-labeled alkaline phosphatase; (3) a luminescent substrate of horseradish peroxidase or alkaline phosphatase.
5. The method for preparing a kit for quantitative determination of human lipopolysaccharide binding protein according to claim 1, comprising coating a well plate with the human lipopolysaccharide binding protein antibody and blocking with a protective protein to obtain a well plate coated with the human lipopolysaccharide binding protein antibody.
6. A method for quantitatively detecting human lipopolysaccharide binding protein is characterized by comprising the following steps:
(1) adding a sample to be detected into a pore plate coated with a human lipopolysaccharide binding protein antibody;
(2) adding a biotin-labeled human lipopolysaccharide binding protein antibody;
(3) incubating, washing and drying;
(4) adding horseradish peroxidase labeled with streptomycin and avidin or alkaline phosphatase labeled with streptomycin and avidin for reaction, washing and drying;
(5) adding enzyme luminescent substrate, and reading the luminescent value.
7. The method for quantitatively detecting human lipopolysaccharide binding protein according to claim 6, further comprising the step (6): and comparing with the standard curve to obtain the concentration of the sample to be detected.
8. The method for quantitatively detecting human lipopolysaccharide binding protein according to claim 6, wherein in the step (1), the sample to be detected is a blood sample, including a serum sample or a plasma sample.
9. The method for quantitatively detecting human lipopolysaccharide binding protein according to claim 6, wherein in the step (4), the biotin-labeled human lipopolysaccharide binding protein antibody is dissolved in a buffer to obtain a working solution containing the biotin-labeled human lipopolysaccharide binding protein antibody; dissolving streptavidin-labeled horseradish peroxidase or streptavidin-labeled alkaline phosphatase in buffer solution to obtain a working solution containing streptomycin and avidin-labeled horseradish peroxidase or a working solution containing streptomycin and avidin-labeled alkaline phosphatase.
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| US5753504A (en) * | 1993-11-16 | 1998-05-19 | The Scripps Research Institute | Monoclonal antibodies immunoreactive with lipopolysaccharide binding protein (LBP) and methods of their use |
| KR100586567B1 (en) * | 1994-01-24 | 2006-09-22 | 조마 코포레이션 | How to Determine LBP in Body Fluids |
| US20030054422A1 (en) * | 1999-04-07 | 2003-03-20 | Badley Robert Andrew | Lipopolysaccharide immunoassay and test device |
| CN111965361A (en) * | 2020-08-05 | 2020-11-20 | 上海长征医院 | Chemiluminescence kit for quantitatively detecting human chitinase 3-like protein 1 |
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