CN111398600B - ELISA detection kit for human soluble BCMA protein, and use method and application thereof - Google Patents
ELISA detection kit for human soluble BCMA protein, and use method and application thereof Download PDFInfo
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Abstract
The invention relates to an ELISA detection kit of human soluble BCMA protein, a use method and application thereof. The ELISA detection kit comprises: the kit comprises an ELISA plate pre-coated with an anti-human BCMA rabbit monoclonal antibody, a biotin-labeled anti-human BCMA rabbit monoclonal antibody, peroxidase-labeled streptavidin, a human BCMA protein standard, a diluent, a stop reaction solution, an ELISA plate cleaning solution and a color development solution. The invention uses anti-human BCMA rabbit monoclonal antibody to detect human soluble BCMA protein by double antibody sandwich method, can accurately detect the concentration of human soluble BCMA protein in the sample to be detected, has accurate and reliable detection result when CV value is less than 10% in repeated test, has recovery rate of more than 85%, higher sensitivity and can detect human soluble BCMA protein with concentration of 31.25 pg/mL.
Description
Technical Field
The invention belongs to the technical field of immunoassay, and particularly relates to an ELISA detection kit for human soluble BCMA protein, and a use method and application thereof.
Background
BCMA (B cell maturation antigen ) is a type III transmembrane protein, mainly expressed on mature B cells and malignant B cells, is a member of the tumor necrosis factor receptor family, and is a receptor for B lymphocyte stimulators and proliferation-inducing ligands, capable of promoting B cell survival, playing an important role in humoral immune regulation.
Clinical studies indicate that BCMA and its ligands activate NF- κb signaling pathway and that a positive feedback loop can be formed between activation of JNK signaling pathway and BCMA expression, together promoting survival and proliferation of plasma and tumor cells. After the membrane protein BCMA is expressed by B cells, the gamma-secretase cuts off the extracellular region of the BCMA to enable the BCMA to fall off from the membrane to form soluble BCMA (sBCMA) which enters the peripheral blood circulation, and the process does not need to be activated or added with kinase to participate.
Multiple Myeloma (MM) is a malignant plasma cell disease in which tumor cells originate from plasma cells in the bone marrow, which are cells that develop from B lymphocytes to the final functional stage. Multiple myeloma is often accompanied by multiple osteolytic lesions, hypercalcemia, anemia, kidney lesions.
Studies have shown that MM patients have significantly higher concentration of sBCMA in peripheral blood than normal due to the presence of B-cell malignant proliferation. And further studies showed that in MM patients, sBCMA concentration can effectively reflect MM patient condition; changes in sBCMA concentration are consistent with changes in M protein and SFLC levels in MM patients, and changes in sBCMA concentration are correlated with post-treatment responses in MM patients. The above studies indicate that sBCMA plays an important role in MM and may serve as a biomarker for the disease or a therapeutic judgment for the disease.
On one hand, the detection method for human soluble BCMA provides a new reference index and detection method for clinical auxiliary diagnosis, differential diagnosis, observation of curative effect, recurrence detection and prognosis evaluation; on the other hand, compared with the semi-quantitative detection methods such as immunohistochemistry and western-blot detection, the ELISA detection method can quantitatively detect the protein content and has the advantages of being more direct and accurate. However, the commercially available ELISA detection kit for human soluble BCMA has the problems of poor stability, low detection sensitivity, poor repeatability, low reliability of detection results and the like.
Therefore, providing an ELISA detection kit with higher sensitivity and more accurate and reliable detection results is a problem to be solved in the art.
Disclosure of Invention
In view of the problems existing in the prior art, the invention aims to provide an ELISA detection kit for human soluble BCMA protein, and a use method and application thereof, wherein the ELISA detection kit has higher sensitivity and better reliability.
To achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides an ELISA assay kit of human soluble BCMA protein comprising:
the kit comprises an ELISA plate pre-coated with an anti-human BCMA rabbit monoclonal antibody, a biotin-labeled anti-human BCMA rabbit monoclonal antibody, peroxidase-labeled streptavidin, a human BCMA protein standard, a diluent, a stop reaction solution, an ELISA plate cleaning solution and a color development solution.
The invention uses anti-human BCMA rabbit monoclonal antibody coated on ELISA plate as solid phase capture antibody and biotin marked anti-human BCMA rabbit monoclonal antibody as detection antibody to detect human soluble BCMA protein quantitatively. The anti-human BCMA rabbit monoclonal antibody provided by the invention has better capturing capability on human BCMA protein, and can be used as a detection antibody and a capturing antibody, so that the sensitivity and the accuracy of a detection result can be improved.
As a preferred technical scheme of the invention, the preparation method of the anti-human BCMA rabbit monoclonal antibody comprises the following steps:
immunizing a rabbit by using purified human BCMA protein, extracting B lymphocytes in peripheral blood of the immunized rabbit, performing gene regulation to obtain an antibody gene with anti-human BCMA specificity, then carrying out transfection and expression on the antibody gene, and purifying to obtain the anti-human BCMA rabbit monoclonal antibody.
The specific operation of the anti-human BCMA rabbit monoclonal antibody in the invention can be as follows: the method comprises the steps of immunizing rabbits by using purified human BCMA protein to obtain peripheral blood reaching a required titer, extracting B lymphocytes in the peripheral blood to carry out gene modulation, obtaining an antibody gene with anti-human BCMA specificity, using the gene for transient transfection and expression of HEK293 cells, and purifying to obtain the antibody gene.
As a preferred technical scheme of the invention, the preparation method of the human BCMA protein comprises the following steps:
searching human BCMA genes and performing codon optimization, connecting target genes with an expression vector by using a homologous recombination method, and transforming competent cells; and performing expansion culture on the competent cells, and purifying after induced expression to obtain the human BCMA protein.
Preferably, the sequence of the gene encoding the human BCMA protein comprises the nucleotide sequence shown as seq id No. 1.
According to the human BCMA gene sequence (Q02223) queried in GENBANK, and by utilizing host organization preferred by escherichia coli: E.coil strain K12, codon optimization is carried out, and finally, an optimized gene sequence is obtained, wherein the sequence is shown as SEQ.ID.NO. 1, and then a target gene is synthesized.
The specific sequence of SEQ ID NO. 1 is:
ATGAATCACAAAGTGCATCATCATCATCATCATATGCTGCAGATGGCAGGCCAGTGTAGCCAGAACGAGTACTTCGACAGCCTGCTGCACGCTTGTATCCCTTGCCAGCTCCGCTGCAGTAGCAATACCCCTCCTCTGACTTGCCAGCGGTATTGCAACG CCAGCGTGAC CAACAGCGTGAAGGGAACCAACGCTTGA。
connecting a target gene with an expression vector by utilizing a homologous recombination method, transforming competent cells, performing expansion culture on the competent cells, adding IPTG in the expansion culture process for induction expression, and finally collecting thalli.
According to the anti-human BCMA rabbit monoclonal antibody, the stability of the detection kit can be improved, the detection value of the kit after unsealing can not be reduced along with the prolongation of the unsealing time, the accuracy of the detection result is ensured, the storage is convenient, and the denaturation is not easy to occur.
The first aspect of the present invention provides a method for detecting human soluble BCMA protein, wherein an anti-human BCMA rabbit monoclonal antibody coated on an ELISA ELISA plate is used as a solid phase capture antibody and a labeled anti-human BCMA rabbit monoclonal antibody is used as a detection antibody, and the enzyme-labeled substrate and the rabbit monoclonal antibody are combined and developed by coupling a Streptavidin (strepavidin) part of the enzyme-labeled substrate with biotin on the rabbit monoclonal antibody, so as to realize quantitative detection of human soluble BCMA.
As a preferred embodiment of the present invention, the peroxidase comprises horseradish peroxidase.
Preferably, the dilutions include a sample dilution, an antibody dilution, and a streptavidin dilution. In the present invention, the three dilutions may be PBS buffer with a mass fraction of 1% bsa.
Preferably, the color-developing solution comprises a tetramethylbenzidine solution.
Preferably, the termination reaction solution includes a sulfuric acid solution of 1.8 to 2.2mol/L (for example, 1.8mol/L, 1.85mol/L, 1.9mol/L, 1.95mol/L, 2mol/L, 2.1mol/L, 2.2mol/L, or the like).
In the present invention, the ELISA plate washing solution may be a PBS solution containing 0.05% Tween20 by mass, and has a pH of 7.2-7.4, such as 7.2, 7.25, 7.3, 7.35 or 7.4.
In a second aspect, the present invention provides a method for using the ELISA detection kit as described in the first aspect, comprising the steps of:
(1) Coating an anti-human BCMA rabbit monoclonal antibody on an ELISA plate, adding an ELISA plate cleaning solution, washing and drying;
(2) Adding the sample to be measured diluted in proportion into an ELISA plate, and incubating;
(3) Diluting a biotin-marked anti-human BCMA rabbit monoclonal antibody, adding the diluted anti-human BCMA rabbit monoclonal antibody into an ELISA plate, incubating, drying, adding an ELISA plate cleaning solution, washing and drying;
(4) Diluting the peroxidase-marked streptavidin, adding the diluted streptavidin into an ELISA plate, incubating, drying, adding an ELISA plate cleaning solution, washing and drying;
(5) And adding the color developing solution into the ELISA plate, adding a stop reaction solution after light-shielding incubation, measuring an OD value, and obtaining the concentration of the human soluble BCMA protein in the sample to be detected through a standard curve.
In a second aspect of the present invention, a method for using the ELISA detection kit of human soluble BCMA protein is provided, which can detect BCMA protein concentration in peripheral blood samples of healthy people and patients with multiple myeloma.
The healthy person refers to a person without a definite diagnosis of serious chronic diseases of various systems, systemic immune related diseases, infectious diseases, and a history of trauma and acute infection within 3 months.
The multiple myeloma comprises a stage I, a stage II and a stage III which are diagnosed as smoky (asymptomatic) multiple myeloma and active (symptomatic) multiple myeloma.
As a preferable technical scheme of the invention, the method for diluting the sample to be measured in proportion in the step (1) comprises the following steps: the sample to be tested is peripheral blood of a healthy person, and the volume ratio of the sample to be tested to the sample diluent is 1 (40-60), for example, 1:40, 1:42, 1:44, 1:46, 1:48, 1:50, 1:52, 1:54, 1:56, 1:58 or 1:60 and the like; the sample to be tested is peripheral blood of a patient with multiple myeloma, and the volume ratio of the sample to be tested to the sample diluent is 1 (80-120), for example, 1:80, 1:82, 1:85, 1:90, 1:92, 1:95, 1:98, 1:100, 1:102, 1:105, 1:108, 1:110, 1:115 or 1:120 and the like.
Preferably, the incubation in step (1) is carried out at a temperature of 35-38deg.C, which may be, for example, 35deg.C, 35.5 deg.C, 36.5 deg.C, 37 deg.C, 37.5 deg.C or 38deg.C; the time is 0.5-1.5h, and can be, for example, 0.5h, 0.8h, 1h, 1.05h, 1.1h, 1.15h, 1.2h, 1.3h, 1.4h or 1.5h, etc.
Preferably, the volume ratio of the diluted sample to be tested to the ELISA plate cleaning solution is 1 (2.5-3.5), for example, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4 or 1:3.5.
As a preferred embodiment of the present invention, the dilution in the step (2) is to dilute the biotin-labeled anti-human BCMA rabbit monoclonal antibody to a concentration of 150-250ng/mL, and may be, for example, 150ng/mL, 160ng/mL, 170ng/mL, 180ng/mL, 190ng/mL, 200ng/mL, 210ng/mL, 220ng/mL, 230ng/mL, 240ng/mL, 250ng/mL, or the like.
Preferably, the incubation in step (2) is carried out at a temperature of 35-38deg.C, which may be, for example, 35deg.C, 35.5 deg.C, 36.5 deg.C, 37 deg.C, 37.5 deg.C or 38deg.C; the time is 30-45min, such as 30min, 32min, 33min, 35min, 38min, 40min, 41min, 42min, 44min or 45min.
Preferably, the volume ratio of the diluted biotin-labeled anti-human BCMA rabbit monoclonal antibody to the ELISA plate washing solution is 1 (2.5-3.5), and can be 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4 or 1:3.5, for example.
Preferably, the volume ratio of the peroxidase-labeled streptavidin to the streptavidin diluent in step (3) is 1 (150-250), and may be, for example, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:210, 1:220, 1:230, 1:240, or 1:250, etc.
Preferably, the incubation in step (3) is carried out at a temperature of 35-38deg.C, which may be, for example, 35deg.C, 35.5 deg.C, 36.5 deg.C, 37 deg.C, 37.5 deg.C or 38deg.C; the time is 15-25min, such as 15min, 16min, 18min, 19min, 20min, 21min, 22min, 24min or 25min.
Preferably, the incubation in step (4) is carried out at a temperature of 35-38deg.C, which may be, for example, 35deg.C, 35.5 deg.C, 36.5 deg.C, 37 deg.C, 37.5 deg.C or 38deg.C; the time is 10-20min, such as 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min or 20min.
As a preferable technical scheme of the invention, the using method comprises the following steps:
(1) Diluting peripheral blood of a healthy person or peripheral blood of a patient with multiple myeloma by using a sample diluent, adding the diluted peripheral blood into an ELISA plate pre-coated with an anti-human BCMA rabbit monoclonal antibody, and incubating at 35-38 ℃ for 0.5-1.5h;
(2) Diluting biotin-labeled anti-human BCMA rabbit monoclonal antibody to a concentration of 150-250ng/mL, adding the diluted anti-human BCMA rabbit monoclonal antibody into an ELISA plate, incubating for 30-45min at 35-38 ℃, drying, adding an ELISA plate cleaning solution, washing and drying;
(3) Diluting peroxidase-labeled streptavidin according to the ratio of 1 (150-250), adding the diluted streptavidin into an ELISA plate, incubating for 15-25min at 35-38 ℃, drying, adding an ELISA plate cleaning solution, washing and drying;
(4) And adding the color developing solution onto an ELISA plate, incubating for 10-20min at 35-38 ℃ in the dark, adding a stop reaction solution, measuring an OD value, and obtaining the concentration of human soluble BCMA protein in the sample to be detected through a standard curve.
In the present invention, the kit may employ specific detection as follows:
(1) Diluting the anti-human BCMA rabbit monoclonal antibody to a concentration of 400ng/mL, 100 mu L of each well, and coating ELISA enzyme label plates at 4 ℃ overnight; removing liquid in each hole, adding ELISA plate cleaning liquid, washing for 5 times in 300 mu L of each hole, and beating dry on clean paper;
(2) Healthy human plasma was diluted 1:50 (or MM patient plasma was diluted 1:100), 100 μl per well, incubated for 1 hour at 37 ℃; simultaneously, incubating the gradient diluted human soluble BCMA standard protein at 37 ℃ for 1 hour at 100 mu L of each well; removing liquid in each hole, adding ELISA plate cleaning liquid, washing for 5 times in 300 mu L of each hole, and beating dry on clean paper;
wherein, human soluble BCMA standard protein is diluted into different concentrations by a gradient dilution method, and in a preferred embodiment of the invention, the concentration gradient is as follows: 2000pg/mL,1000pg/mL,500pg/mL,250pg/mL,125pg/mL,62.5pg/mL,31.25pg/mL,0pg/mL, and a standard curve was prepared;
(3) Biotin-labeled anti-human BCMA rabbit monoclonal antibody was diluted to 200ng/mL, 100. Mu.L per well, and incubated at 37℃for 40 min; removing liquid in each hole, adding ELISA plate cleaning liquid, washing for 5 times in 300 mu L of each hole, and beating dry on clean paper;
(4) Diluting enzyme-labeled substrate, namely peroxidase-labeled streptavidin according to a ratio of 1:200, and incubating for 20 minutes at 37 ℃ with 100 mu L of enzyme-labeled substrate per well; removing liquid in each hole, adding ELISA plate cleaning liquid, washing for 5 times in 300 mu L of each hole, and beating dry on clean paper;
(5) Adding TMB color development solution, and incubating for 15 minutes at 37 ℃ in a dark place with 100 mu L of each hole; the reaction was terminated by adding 50. Mu.L of 2mol/L sulfuric acid solution per well; the OD value is measured by an enzyme-labeled instrument at the wavelength of 450nm, and the concentration of the soluble BCMA protein in the blood plasma of the healthy person is obtained through quantitative analysis.
The use method provided by the invention is matched with the kit, so that the repeatability of the kit can be further improved when the human soluble BCMA protein is detected, and the detection result is more accurate.
In a third aspect, the present invention provides a use of the human soluble BCMA protein assay kit according to the first aspect for detecting sBCMA.
The numerical ranges recited herein include not only the above-listed point values, but also any point values between the above-listed numerical ranges that are not listed, and are limited in space and for the sake of brevity, the present invention is not intended to be exhaustive of the specific point values that the stated ranges include.
Compared with the prior art, the invention has at least the following beneficial effects:
the ELISA detection kit provided by the invention utilizes the anti-human BCMA rabbit monoclonal antibody to detect human soluble BCMA protein by a double-antibody sandwich method, and simultaneously, the concentration of human soluble BCMA protein in a sample to be detected can be accurately detected by quantitative analysis of an enzyme-labeled instrument, the CV value in repeated test is less than 10%, the detection result is accurate and reliable, the recovery rate is greater than 85%, the sensitivity is higher, and the human soluble BCMA protein with the concentration of 31.25pg/mL can be detected; meanwhile, the influence of interference elements can be reduced, better stability can be kept within a period of time after unsealing, subjectivity of semi-quantitative methods such as immunohistochemistry, immunofluorescence and the like is eliminated, and quantitative detection of human soluble BCMA proteins can be realized.
Drawings
FIG. 1 is a diagram showing SDS-PAGE electrophoresis of human BCMA protein expression (wherein, lane 1 is a protein before induction; lane 2 is a protein after induction; and lane M represents a protein marker).
FIG. 2 is a western-blot verification of human BCMA protein (lane 1 is human BCMA recombinant protein; lane M represents protein markers).
FIG. 3 shows SDS-PAGE of purified human BCMA protein (BSA in lane 1; recombinant human BCMA protein in lane 2; protein marker in lane M).
FIG. 4 shows SDS-PAGE (lane 1 shows the purified rabbit monoclonal antibody for capture; lane 2 shows the purified rabbit monoclonal antibody for detection; lane M shows a protein marker).
FIG. 5 is a western-blot verification chart of a rabbit monoclonal antibody (lane 1 is a purified capture rabbit monoclonal antibody; lane 2 is a purified detection rabbit monoclonal antibody; lane M represents a protein marker).
FIG. 6 is a standard curve of ELISA detection method for human soluble BCMA protein.
FIG. 7 is a graph showing the concentration profile of BCMA in plasma obtained from MM patients before treatment, using the ELISA test kit described in example 1.
FIG. 8 (a) is a graph showing the results of detection of D0, W1, W2, W4 after unsealing in a commercially available ELISA kit for human-derived soluble BCMA protein; fig. 8 (b) is a graph showing the detection results of D0, W1, W2, and W4 after unsealing in the detection kit provided in example 1.
Detailed Description
The following embodiments are further described with reference to the accompanying drawings, but the following examples are merely simple examples of the present invention and do not represent or limit the scope of the invention, which is defined by the claims.
In the examples below, experimental procedures not described in detail can be performed using experimental methods commonly used in the art.
Example 1
The embodiment provides an ELISA detection kit for preparing human soluble BCMA protein and a preparation method thereof.
First, a human soluble BCMA protein was prepared:
according to the human BCMA gene sequence (Q02223) inquired in GENBANK and by utilizing host organization preferred by escherichia coli to carry out codon optimization on E.coil strain K12, finally obtaining an optimized gene sequence, wherein the sequence is shown as SEQ.ID.NO. 1, and then synthesizing the target gene.
The specific sequence of SEQ ID NO. 1 is:
ATGAATCACAAAGTGCATCATCATCATCATCATATGCTGCAGATGGCAGGCCAGTGTAGCCAGAACGAGTACTTCGACAGCCTGCTGCACGCTTGTATCCCTTGCCAGCTCCGCTGCAGTAGCAATACCCCTCCTCTGACTTGCCAGCGGTATTGCAACG CCAGCGTGAC CAACAGCGTGAAGGGAACCAACGCTTGA。
connecting a target gene with an expression vector by utilizing a homologous recombination method, transforming competent cells, performing expansion culture on the competent cells, adding IPTG in the expansion culture process for induction expression, and finally collecting thalli. SDS-PAGE gel electrophoresis is carried out on the expressed protein to check whether the protein is correctly expressed, the result is shown in figure 1, and the result is shown in figure 2 after verification by using western-blot.
As can be seen from FIGS. 1 and 2, the target protein was successfully obtained, which had a size of 7.3KD. Purifying with AKTA instrument, performing SDS-PAGE gel electrophoresis of the purified protein, and checking the size and purity of the protein, and the result is shown in figure 3.
Then, rabbit monoclonal antibodies against human soluble BCMA protein were prepared:
immunizing rabbits by adopting purified human BCMA protein, and obtaining peripheral blood reaching a required titer when the serum dilution concentration is 1:72900 and the P/N is more than 2.0;
extracting B lymphocyte to take gene to obtain antibody gene with anti-human BCMA specificity, using the gene for transient transfection and expression of HEK293 cells, purifying culture solution by using AKTA instrument to obtain the rabbit monoclonal antibody, and verifying the purified rabbit monoclonal antibody by SDS-PAGE gel electrophoresis, wherein the result is shown in figure 4; performing western-blot verification on the monoclonal antibody, wherein the result is shown in FIG. 5; as can be seen from fig. 4 and 5, a purified rabbit monoclonal antibody was obtained.
Preparing the ELISA detection kit:
(1) Anti-human BCMA rabbit monoclonal antibodies were diluted to 400ng/mL with coating solution (1 XPBS, pH 7.4), 100 μl was added per well, and coated overnight at 4deg.C; discarding the liquid in the holes, adding 300 mu L of PBST into each hole, cleaning for 5 times, throwing away the liquid in the holes after each cleaning, and beating the liquid on clean absorbent paper; adding 300 mu L of PBS blocking solution containing 1% BSA into each hole for blocking, incubating for 30 minutes at 37 ℃, and washing to obtain an ELISA plate pre-coated with anti-human BCMA rabbit monoclonal antibody;
(2) The biotin-labeled anti-human BCMA rabbit monoclonal antibody, peroxidase-labeled streptavidin, human BCMA protein standard, diluent, stop reaction solution, ELISA plate cleaning solution and color development solution can be prepared by conventional methods in the art.
Finally, the resulting ELISA detection kit comprises: ELISA plates (5 blocks) pre-coated with anti-human BCMA rabbit monoclonal antibodies, biotin-labeled anti-human BCMA rabbit monoclonal antibodies (20 μg), peroxidase-labeled streptavidin (350 μl), human BCMA protein standards (40 ng), dilutions (10X) (25 mL), stop reactions (40 mL), ELISA plate washes (25X) (150 mL), chromogenic solution A (40 mL) and chromogenic solution B (40 mL).
Example 2
This example utilizes the ELISA detection kit provided in example 1 to detect the concentration of soluble BCMA standard protein.
Human soluble BCMA standard protein was diluted according to the concentration gradient shown in table 1 and tested as follows:
(1) Human soluble BCMA standard protein was diluted to different concentration gradients with PBS containing 1% BSA, incubated at 37℃for 1 hour,
(2) Washing: removing liquid in the holes, adding 300 mu L of PBST into each hole, washing for 5 times, throwing the liquid in the holes after each washing, and beating on clean absorbent paper.
(3) Adding a detection antibody: biotin-labeled anti-human BCMA rabbit monoclonal antibody was diluted to 200ng/mL with 1% BSA in PBS, 100. Mu.L was added to each well, incubated at 37℃for 40 minutes, and washed.
(4) Adding a substrate: HRP-labeled streptavidin was diluted 1:200 in PBS containing 1% BSA, 100. Mu.L per well was added, incubated at 37℃for 20min, and washed.
(5) Color development: 100. Mu.L of TMB color developing solution was added to each well, and developed for 15 minutes at 37℃in the absence of light.
(6) Terminating the reaction: after the development was completed, 50. Mu.L of 2mol/L H was immediately added to each well 2 SO 4 The absorbance was measured with a microplate reader at a single wavelength of 450nm over 20 minutes. Finally, the measured absorption values are shown in table 1:
TABLE 1 OD of human soluble BCMA proteins at different concentrations 450 Reading value
The result shows that the detection kit and the matched detection method thereof provided by the invention can detectTo a concentration of 31.25pg/mL of human soluble BCMA protein. The detection result shows that the four-parameter fitting of the standard curve (shown in figure 6) of the absorption value and the protein concentration at the single wavelength of 450nm has good linear relation, and the fitting curve is y= (2.931-0.06075)/[ 1+ (x/949.8) -1.133 ]+0.06075，r 2 >0.999。
Example 3
This example was used to evaluate the reproducibility of the ELISA test kit provided in example 1.
The ELISA method established by the invention is used for detecting 3 samples with different concentration levels, the specific detection method is the same as that of example 2, each sample is repeatedly tested for 10 times, the average value M and the standard deviation SD of the concentration results measured for 10 times are calculated, and the variation coefficient CV is obtained according to a formula (the results are shown in the table 2):
wherein: standard deviation of SD-10 measurements; CV-coefficient of variation; average of M-10 measurements.
TABLE 2 ELISA detection method reproducibility test of human soluble BCMA
According to the test results, 3 samples with different concentrations pass 10 repeated tests, and CV values are smaller than 10%.
Example 4
This example tests the recovery rate of the ELISA test kit provided in example 1.
Adding a high-level object (A) with known concentration into a low-concentration blood matrix (B), wherein the ratio of the added object A to the added object B is 1:9, repeatedly detecting 3 times by using an established ELISA detection method, taking an average value, and calculating the recovery rate according to a formula, wherein the result is shown in a table 3:
wherein: r-recovery rate; v-volume of sample A; v (V) 0 -volume of sample B fluid; c-average value of detection concentration of the sample B after the solution A is added; c (C) 0 -concentration average of sample B fluid; c (C) S Mean value of the detected concentration of sample a.
TABLE 3 ELISA detection method of human soluble BCMA recovery test
According to the test result, the recovery rate of the ELISA detection kit provided by the invention is 86.83% and is more than 85%.
Application example 1
This example utilizes the ELISA detection kit provided in example 1 to detect BCMA concentration in plasma of healthy people and pre-treatment MM patients.
45 healthy human plasma samples were collected, 17 pre-treatment plasma samples from MM patients, and healthy human plasma was diluted 1:50 and MM patient plasma was diluted 1:100 according to the procedure of example 1, and BCMA protein concentrations were measured separately.
Wherein, BCMA protein concentration in 45 healthy human blood is 3.88-23.94ng/mL, and average mean=11.97 ng/mL.
BCMA protein concentration in plasma of 17 MM patients was 4.57-312.93ng/mL, mean= 138.29ng/mL.
As shown in FIG. 7, the blood plasma of 45 healthy persons and the blood plasma of 17 pre-treatment MM patients, wherein the horizontal line represents the average mean, and BCMA proteins with different concentrations are detected, which indicates that the ELISA detection kit provided by the invention can detect the BCMA concentrations of normal persons and MM patients, and has good detection sensitivity.
Comparative example 1 was used
The stability of the commercial ELISA detection kit and the ELISA detection kit provided in example 1 were examined in this comparative example, respectively, wherein the capture antibody and the biotin-labeled antibody used in the commercial ELISA detection kit were goat polyclonal antibodies.
Specifically, the detection results of the day (D0), week 1 (W1), week 2 (W2), and week 4 (W4) after the unsealing of the kit were examined, respectively. The commercial kit detects standard substances with different concentrations at each time point according to the kit instruction, and the kit detection method provided in the example 1 is the same as that in the example 2, and the standard substances are detected at the same time point.
As a result, as shown in FIG. 8 (a), the detection OD value of the commercially available ELISA detection kit was reduced with the opening time, 4 weeks after the opening; the ELISA test kit provided in example 1 was subjected to unsealing for 4 weeks, and the results were shown in FIG. 8 (b), in which the detection OD values were kept substantially uniform. Therefore, the detection kit has obvious advantages in stability compared with commercial products.
The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.
SEQUENCE LISTING
<110> Michael conversion medical research (Suzhou) Co., ltd
<120> ELISA detection kit of human soluble BCMA protein, and use method and application thereof
<130> 20200326
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 198
<212> DNA
<213> Synthesis
<400> 1
atgaatcaca aagtgcatca tcatcatcat catatgctgc agatggcagg ccagtgtagc 60
cagaacgagt acttcgacag cctgctgcac gcttgtatcc cttgccagct ccgctgcagt 120
agcaataccc ctcctctgac ttgccagcgg tattgcaacg ccagcgtgac caacagcgtg 180
aagggaacca acgcttga 198
Claims (15)
1. An ELISA assay kit for human BCMA protein comprising:
an ELISA plate pre-coated with an anti-human BCMA rabbit monoclonal antibody, a biotin-labeled anti-human BCMA rabbit monoclonal antibody, peroxidase-labeled streptavidin, a human BCMA protein standard, a diluent, a stop reaction solution, an ELISA plate cleaning solution and a color development solution;
the preparation method of the anti-human BCMA rabbit monoclonal antibody comprises the following steps:
immunizing a rabbit by using purified human BCMA protein, extracting B lymphocytes in peripheral blood of the immunized rabbit, performing gene regulation to obtain an antibody gene with anti-human BCMA specificity, then carrying out transfection and expression on the antibody gene, and purifying to obtain the anti-human BCMA rabbit monoclonal antibody;
the preparation method of the human BCMA protein comprises the following steps:
searching human BCMA genes and performing codon optimization, connecting target genes with an expression vector by using a homologous recombination method, and transforming competent cells; performing expansion culture on the competent cells, and purifying after induced expression to obtain the human BCMA protein;
the sequence of the gene encoding the human BCMA protein is a nucleotide sequence shown as SEQ ID No. 1.
2. The ELISA detection kit of claim 1, wherein the preparation method of the ELISA plate pre-coated with anti-human BCMA rabbit monoclonal antibody comprises the following steps:
diluting the anti-human BCMA rabbit monoclonal antibody to the concentration of 300-500ng/mL, adding the diluted anti-human BCMA rabbit monoclonal antibody into an ELISA plate at the volume of 80-120 mu L per hole, and standing at the temperature of 4 ℃ for 10-14h to obtain the ELISA plate pre-coated with the anti-human BCMA rabbit monoclonal antibody.
3. The ELISA detection kit of claim 1, wherein the peroxidase comprises horseradish peroxidase.
4. The ELISA test kit of claim 1 wherein the dilutions comprise a sample dilution, an antibody dilution, and a streptavidin dilution.
5. The ELISA test kit of claim 1 wherein the chromogenic solution comprises a tetramethylbenzidine solution.
6. The ELISA detection kit of claim 1, wherein the stop reaction solution comprises 1.8-2.2mol/L sulfuric acid solution.
7. The method of using an ELISA detection kit for non-diagnostic purposes according to any one of claims 1-6, comprising the steps of:
(1) Adding a sample to be measured which is diluted in proportion into an ELISA plate pre-coated with an anti-human BCMA rabbit monoclonal antibody, and incubating;
(2) Diluting a biotin-marked anti-human BCMA rabbit monoclonal antibody, adding the diluted anti-human BCMA rabbit monoclonal antibody into an ELISA plate, incubating, drying, adding an ELISA plate cleaning solution, washing and drying;
(3) Diluting the peroxidase-marked streptavidin, adding the diluted streptavidin into an ELISA plate, incubating, drying, adding an ELISA plate cleaning solution, washing and drying;
(4) And adding the color developing solution into the ELISA plate, adding the stop reaction solution after light-shielding incubation, measuring an OD value, and obtaining the concentration of the human BCMA protein in the sample to be detected through a standard curve.
8. The method of claim 7, wherein the method of diluting the sample to be measured in proportion in step (1) is:
the sample to be measured is peripheral blood of a healthy person, and the volume ratio of the sample to be measured to the sample diluent is 1 (40-60); the sample to be measured is peripheral blood of a patient with multiple myeloma, and the volume ratio of the sample to be measured to the sample diluent is 1 (80-120).
9. The method of claim 7, wherein the incubation in step (1) is performed at a temperature of 35-38deg.C for a period of 0.5-1.5h;
the volume ratio of the diluted sample to be measured to the ELISA plate cleaning liquid is 1 (2.5-3.5).
10. The method of use of claim 7, wherein the dilution of step (2) is to dilute the biotin-labeled anti-human BCMA rabbit monoclonal antibody to a concentration of 150-250ng/mL;
the incubation temperature in the step (2) is 35-38 ℃ and the incubation time is 30-45min.
11. The method of claim 10, wherein the volume ratio of the diluted biotin-labeled anti-human BCMA rabbit monoclonal antibody to the elisa plate wash is 1 (2.5-3.5).
12. The method of claim 7, wherein the volume ratio of peroxidase-labeled streptavidin to streptavidin diluent in step (3) is 1 (150-250);
the incubation temperature in the step (3) is 35-38 ℃ and the incubation time is 15-25min.
13. The method of claim 7, wherein the incubation in step (4) is performed at a temperature of 35-38deg.C for a period of 10-20min.
14. Use according to claim 7, characterized in that it comprises the following steps:
(1) Diluting peripheral blood of a healthy person or peripheral blood of a patient with multiple myeloma by using a sample diluent, adding the diluted peripheral blood into an ELISA plate pre-coated with an anti-human BCMA rabbit monoclonal antibody, and incubating at 35-38 ℃ for 0.5-1.5h;
(2) Diluting biotin-labeled anti-human BCMA rabbit monoclonal antibody to a concentration of 150-250ng/mL, adding the diluted anti-human BCMA rabbit monoclonal antibody into an ELISA plate, incubating for 30-45min at 35-38 ℃, drying, adding an ELISA plate cleaning solution, washing and drying;
(3) Diluting peroxidase-labeled streptavidin according to the ratio of 1 (150-250), adding the diluted streptavidin into an ELISA plate, incubating for 15-25min at 35-38 ℃, drying, adding an ELISA plate cleaning solution, washing and drying;
(4) And adding the color developing solution onto an ELISA plate, incubating for 10-20min at 35-38 ℃ in the dark, adding a stop reaction solution, measuring an OD value, and obtaining the concentration of human BCMA protein in the sample to be detected through a standard curve.
15. Use of a human BCMA protein detection kit according to any one of claims 1-6 for the detection of non-diagnostic purposes in human BCMA.
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