CN117169517A - Method and kit for detecting CD28 antibody residues in T lymphocyte preparation - Google Patents
Method and kit for detecting CD28 antibody residues in T lymphocyte preparation Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a method and a kit for detecting CD28 antibody residues in a T lymphocyte preparation. The invention provides a method for detecting CD28 antibody residues in a T lymphocyte preparation and a kit for detecting the CD28 antibody residues in the T lymphocyte preparation. The CD28 antibody residue measured by the method disclosed by the invention has high accuracy, and can accurately reflect and measure trace residues of the sample CD28 antibody. The detection range of the invention can reach 12.5-800 pg/mL, the relative recovery rate (RE%) is 80-120%, and more accurate data reference is provided for objectively evaluating the quality of the T lymphocyte preparation.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method and a kit for detecting CD28 antibody residues in a T lymphocyte preparation.
Background
T cell immunotherapy is one of the most interesting and fastest growing tumor treatments in clinical trials today. The basic operation is that the tumor specific T cells of the patient or the tumor specific T cells after genetic modification are purified, activated, amplified and cultured in vitro to prepare a T lymphocyte preparation, and then the T lymphocyte preparation is returned to the patient, and the purpose of targeted tumor cell elimination is realized by utilizing the specific killing effect of the T lymphocytes.
CD28 antibodies are the most commonly used antibodies for T cell activation in vitro. Studies have shown that the T cells, after activation by the first signal, are non-specific co-stimulatory signals, which are generated by interaction of pairs of co-stimulatory molecules on the surface of APCs with corresponding receptors on the T cells (e.g., CD28, CTLA-4 and CD80, CD86,4-1BB and 4-1BBL, CD40 and CD40L, PD-1 and PD-L1, etc.), wherein CD28 is the most important co-stimulatory molecule, and the second signal allows complete activation of the T cells, secretion of cytokines and expression of cytokine receptors; the combined use of CD28 antibodies in vitro to stimulate T cells mimics the signaling effects of T cell activation is the most widely used method for T cell activation and expansion. The content of CD28 antibody residues in T lymphocyte preparations is one of the important parameters for the quality evaluation of T cell preparations, and is very important for the safety and quality controllability level of cell preparation products. The currently existing methods are difficult to detect with less CD28 antibody residues, especially CD28 antibody residues on the ng/mL scale are less accurate. Therefore, it is very important to establish a method for detecting CD28 antibody residues in a T lymphocyte preparation with high efficiency, simple operation and high accuracy.
Disclosure of Invention
In view of the above, the present invention aims to establish a method for detecting CD28 antibody residues in T lymphocyte preparations with high efficiency, simple operation and high accuracy. The invention provides a method and a kit for detecting CD28 antibody residues in a T lymphocyte preparation. The CD28 antibody residue measured by the method disclosed by the invention has high accuracy, and can accurately reflect and measure trace residues of the sample CD28 antibody. The detection range of the invention can reach 12.5-800 pg/mL, the relative recovery rate (RE%) is 80-120%, and more accurate data reference is provided for objectively evaluating the quality of the T lymphocyte preparation.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for detecting CD28 antibody residues in a T lymphocyte preparation, which comprises the following steps:
step 1, coating an antigen on a carrier, cleaning and sealing;
step 2, cleaning;
respectively adding standard substance solutions with different concentrations into the standard substance solutions;
adding a test solution into the test group;
incubating and cleaning;
step 3, respectively adding a labeled antibody into a blank control group, a standard substance group and a test group, incubating, cleaning, developing, and stopping, and respectively obtaining absorbance data of the blank control group, the standard substance group and the test group at the wavelength of 450 nm;
step 4, subtracting the absorbance data of the blank control group from the absorbance data of the standard substance group to obtain a standard curve by using Logistic curve fitting, and obtaining a detection result of the CD28 antibody residues through the standard curve according to the absorbance data of the test group;
the antigen is a CD28E & CD28D heterodimeric protein;
the standard substance is Anti-Human CD28 mAB;
the test sample is a T lymphocyte preparation taking CD28 as a raw material.
In some embodiments of the invention, the T lymphocyte preparation comprises a central memory T lymphocyte preparation.
In some embodiments of the invention, the coating amount of the CD28E & CD28D heterodimeric protein is 0.1 μg/unit carrier;
the antigen coating condition is that the antigen is coated overnight at 2-8 ℃;
the washing liquid for washing is sodium phosphate;
the number of times of the washing was 3.
In some embodiments of the invention, the carrier is an elisa plate;
in some embodiments of the invention, the coating amount of the CD28E & CD28D heterodimeric protein is 0.1 μg/well.
In some embodiments of the invention, the sodium phosphate salt is 1 XPBST.
In some embodiments of the invention, the method of preparing 1 XPBST comprises: taking a PBS powder 1 bag, adding 2L sterilization injection water, and fully dissolving; then, 1 mL of Tween20 was added and mixed well to obtain the 1 XPBST.
In some embodiments of the invention, the conditions for the washing are 3 times, each at 300. Mu.L/unit of carrier.
In some embodiments of the invention, the period of closure is 1 hour;
the blocking solution used for the blocking was either 2% (w: v) BSA or 5% (w: v) skimmed milk powder in PBST.
In some embodiments of the invention, the blocking fluid is used in an amount of 300 μl/unit of carrier.
In some embodiments of the invention, the blocking solution used for the blocking is a 2% (w: v) BSA solution in PBST.
In some embodiments of the invention, the incubation described in step 2 is for 2 hours at 25 ℃ ± 5 ℃.
In some embodiments of the invention, the incubation in step 3 is at 25 ℃ ± 5 ℃ for 1 hour in the absence of light.
In some embodiments of the invention, the concentration of the labeled antibody solution is 0.05 μg per unit carrier.
In some embodiments of the invention, the label of the labeled antibody is horseradish peroxidase (HRP), biotin (Biotin), fluorescein Isothiocyanate (FITC), or Alkaline Phosphatase (AP).
In some embodiments of the invention, the source of the labeled antibody is rabbit, mouse, sheep or recombinant IgG antibodies.
In some embodiments of the invention, the labeled antibody is HRP-labeled goat anti-mouse IgG2a.
In some embodiments of the invention, the labeled antibody solution is used in an amount of 100 μl.
In some embodiments of the invention, the color development time is 10-15 minutes;
the color development liquid adopted by the color development is color development liquid TMB.
In some embodiments of the invention, the blocking solution is used in an amount of 100 μl/unit of carrier.
In some embodiments of the invention, the termination liquid is used in an amount of 50. Mu.L/unit of carrier.
In some embodiments of the invention, the detection method specifically comprises the steps of:
step 1, taking CD28E & CD28D heterodimer protein, adjusting the concentration of the protein to 600 mug/mL by using sterilized water for injection, and diluting the 1X coating liquid 600 times to prepare a coating protein solution with the final concentration of 1 mug/mL; adding 1 mug/mL of coating protein solution into an empty ELISA plate, and coating 100 mug/mL of coating protein solution in each hole at 2-8 ℃ overnight to obtain a coated ELISA plate; taking out the coated ELISA plate, recovering to 25+/-5 ℃, washing the washing liquid for 3 times, 300 mu L/hole, and if residual liquid exists in the hole after 1-time liquid discarding, beating to dry on absorbent paper; adding 300 mu L of blocking solution into each hole, and blocking for 1 hour at 25+/-5 ℃;
step 2, washing the washing liquid for 3 times, 300 mu L/hole, and if residual liquid exists in the hole after the liquid is discarded for the last 1 time, beating the washing liquid on absorbent paper; adding a standard substance solution into a standard substance group, adding a test substance solution into a test group, setting 2 compound holes in each group at 100 mu L/hole, sealing a plate membrane sealing plate, and incubating at 25+/-5 ℃ for 2 hours;
step 3, washing the washing liquid for 3 times, 300 mu L/hole, and if residual liquid exists in the hole after the liquid is discarded for the last 1 time, beating the washing liquid on absorbent paper; adding a labeled antibody solution into each hole, and sealing a plate membrane sealing plate at 25+/-5 ℃ for light-shielding incubation for 1 hour at 100 mu L/hole; washing the washing liquid for 3 times, 300 mu L/hole, and if residual liquid exists in the hole after the last 1 times of liquid discarding, beating the washing liquid on absorbent paper; adding a developing solution TMB,100 mu L/hole and a sealing plate membrane sealing plate into each hole of the blank control group, the standard substance group and the test group respectively, and incubating for 10-15 minutes at 25+/-5 ℃ in a dark place; adding a stop solution into each hole, and measuring the absorbance value at the wavelength of 450 nm by using an enzyme-labeling instrument after uniformly mixing 50 mu L/hole;
step 4, subtracting the mean value of the blank control group from the mean value of the absorbance value of the standard substance group to be the ordinate Y, taking the theoretical concentration of the standard substance to be the abscissa X, and fitting and drawing by using a Logistic curve (four parameters) to obtain a standard curve regression equation and a correlation coefficient R 2 The method comprises the steps of carrying out a first treatment on the surface of the Calculating X from Y on a standard curve to obtain an actual measurement concentration value of the sample; if the sample is pre-diluted, multiplying the concentration of the sample obtained according to the standard curve by the dilution multiple of the concentration;
the standard substance is Anti-Human CD28 mAB;
the test sample is a T lymphocyte preparation taking CD28 as a raw material.
In some embodiments of the invention, the detection range of the detection method is 12.5-800 pg/mL.
The invention also provides a kit for detecting trace amounts of CD28 antibody residues in T lymphocyte preparations, comprising CD28E & CD28D heterodimer proteins, a blocking solution and a labeled antibody;
the mass ratio of the CD28E & CD28D heterodimeric protein and the labeled antibody is 2:1, a step of;
the blocking solution was either 2% (w: v) BSA or 5% (w: v) PBST solution of skimmed milk powder.
In some embodiments of the invention, color-developing solution TMB is also included.
The invention includes, but is not limited to, achieving the following beneficial effects:
the detection range of the invention can reach 12.5-800 pg/mL, the relative recovery rate (RE%) is 80-120%, and more accurate data reference is provided for objectively evaluating the quality of the T lymphocyte preparation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a standard curve of an embodiment;
FIG. 2 shows a fitted curve for comparative example 1;
FIG. 3 shows a fitted curve for comparative example 2;
fig. 4 shows a fitted curve of comparative example 3.
Detailed Description
The invention discloses a method and a kit for detecting CD28 antibody residues in a T lymphocyte preparation, and a person skilled in the art can refer to the content of the invention to properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
Interpretation of related terms:
elisa: in the quantitative determination, a series of reference standard substances with different concentrations are needed to be used for preparing a standard curve under the same condition for each batch of test.
Relative recovery (RE%): the relative recovery rate was mainly examined for accuracy. Accuracy refers to the degree of closeness between the result of the measurement with the method and a true or approved reference value. Sometimes also referred to as realism. Certain accuracy is a necessary condition for quantitative determination, so that detection items related to quantitative determination all need to verify accuracy, such as content determination, impurity quantitative test and the like.
Relative recovery RE (%) = (measured concentration/true concentration) ×100%
Four parameter fitting: refers to the law that the dependent variable (y) varies with the independent variable (x) by an equation containing four parameters.
The four parameter pattern is Y= (a-d)/[ 1+ (x/c) b ] +d
a asymptote estimation on a
d, estimating the asymptote under the curve
Slope of curve b
c corresponding dose at maximum half-combined
The result output requirement obtains four parameters of a, b, c and d to optimize the solutions of the groups and ensure R 2 0.95 and the curve between the maximum point and the minimum point is a monotonic curve.
The invention aims to provide a method for detecting trace CD28 antibody residues of T lymphocyte preparations, in particular to central memory type T lymphocyte preparations.
CD28 is a transmembrane protein of relative molecular mass 44000 belonging to the immunoglobulin family, and its mediated co-stimulatory signals can induce T cell differentiation, proliferation and secretion of IL-2. The anti-CD 28 monoclonal antibody can provide effective co-stimulation signals, excite T cell proliferation, and has good application value in tumor immunity, treatment of autoimmune diseases and immune intervention for resisting transplant rejection. At present, the research at home and abroad mostly uses the anti-CD 3/CD28 antibody in combination for the in vitro activation of T lymphocytes, and the stimulation activity of the anti-CD 3 antibody is obviously improved under the synergistic effect of the anti-CD 28 antibody, so that the T lymphocytes are activated in vitro by clinically adopting the anti-CD 28 antibody in combination with the anti-CD 3 antibody. Human CD28 monoclonal antibody is usually added in the production process of T lymphocyte preparation for activation and proliferation of cells in early stage. Through the technological operations of cell activation and proliferation, target cell collection, cleaning and the like, most of CD28 monoclonal antibodies can be removed, and only a trace amount of CD28 monoclonal antibodies remain. The content of CD28 antibody residues in T lymphocyte preparations is one of the important parameters for the quality evaluation of T cell preparations, and is very important for the safety and quality controllability level of cell preparation products.
The currently existing methods are difficult to detect with less CD28 antibody residues, especially CD28 antibody residues on the ng/mL scale are less accurate. Therefore, it is very important to establish a method for detecting CD28 antibody residues in a T lymphocyte preparation with high efficiency, simple operation and high accuracy.
Detection of CD28 residues is often performed by indirect ELISA. Coating an ELISA plate with CD28E & CD28D heterodimer protein, wherein the CD28 antibodies in the test sample and the standard sample can be combined with the protein to form antigen-antibody complexes, and free components are removed; adding goat anti-mouse IgG2a detection antibody (secondary antibody) marked by horseradish peroxidase (HRP), combining the antibody with the antibody in the complex to form an antigen-antibody complex, removing free components, adding chromogenic substrate (TMB), enabling the colorless chromogenic agent to turn blue by horseradish peroxidase combined on the complex, enabling the color to be in direct proportion to the concentration of the CD28 antibody, turning yellow after adding a stopping solution, enabling the solution to have an absorption peak at 450 nm, detecting the OD value of the solution at 450 nm, and drawing a standard curve to calculate the concentration of the CD28 antibody in the test sample.
ELISA assays involve multiple links. The inventor continuously optimizes ELISA reaction systems and parameters, and the inventor continuously optimizes ELISA reaction systems and parameters to summarize and obtain the detection method based on a large number of tests. The detection range of the invention can reach 12.5-800 pg/mL, the relative recovery rate (RE%) is 80-120%, and more accurate data reference is provided for objectively evaluating the quality of the T lymphocyte preparation.
According to the invention, an ELISA method is adopted to carry out gradient dilution on CD28 antibody standard products with known concentration according to 800pg/mL, 400 pg/mL, 200 pg/mL, 100 pg/mL, 50 pg/mL, 25 pg/mL and 12.5pg/mL, links such as coating protein concentration, closing time, plate washing times, incubation time, secondary antibody concentration, reaction time, color development time and the like are optimized, a standard curve is obtained through logic fitting according to OD450 reading, the detection range of the invention can reach 12.5-800 pg/mL, and the relative recovery rate (RE%) can reach 80% -120%.
In the present invention, the comparison with the standard substance with the known concentration is performed with the curve of the standard substance with the known concentration, preferably by performing a parallel test with the biological sample to be tested by using the anti-CD 28 antibody standard substance, so as to obtain the curve of the standard substance with the known concentration. Specifically, by using a blank sample containing PBST to gradiently dilute the CD28 antibody standard, and loading,the enzyme label plate after sample addition was subjected to the reaction operation according to the same procedure as above, and OD450 was read, and a standard curve was obtained by logical fitting according to all the readings. The standard curve requires R 2 >0.95. Among them, dilution of CD28 antibody standards is generally graded as follows: 800pg/mL, 400 pg/mL, 200 pg/mL, 100 pg/mL, 50 pg/mL, 25 pg/mL, 12.5 pg/mL.
The biological sample is: t lymphocyte preparation using CD28 as raw material.
The sample buffer solution to be tested is a common buffer solution, such as phosphate buffer solution and the like.
Preferably, 0.1% BSA may be added to the buffer.
Preferably, tween20 may be added to the buffer.
The conditions for coating the enzyme-labeled plates were 1. Mu.g/mL of CD28 protein solution and allowed to stand overnight at 4 ℃.
The concentration of the coating protein is 0.1 mug/hole, the sealing concentration is 300 mug/hole, and the sealing time is 1 hour;
the washing liquid for washing the plate is sodium phosphate, the concentration is 300 mu L/hole, and the number of times of washing the plate is 3.
The sealing liquid is as follows: 2% BSA-PBST or 5% skim milk powder or BSA or other common blocking solution.
The labeled antibody is a luminescent substance labeled human IgG antibody, and the labels include, but are not limited to, horseradish peroxidase (HRP), biotin (Biotin), fluorescein Isothiocyanate (FITC), alkaline Phosphatase (AP) and the like, and the detection forms can be chemiluminescence, fluorescence, conventional visible light and the like. The anti-human IgG antibody can be prepared from common mammal, such as rabbit, mouse, sheep, etc., or recombinant IgG antibody. One non-limiting example is for example: the labeled antibody is an enzyme-linked antibody, such as, but not limited to, specifically an enzyme-linked labeled rabbit anti-human IgG. In addition, the enzyme-linked antibodies may also be used with other labeled anti-human IgG.
The standing reaction of the sample-added ELISA plate is 2 hours.
The color development liquid is TMB (3, 3', 5' -tetramethyl benzidine), the concentration and time of the color development reaction are 100 mu L/hole respectively, the reaction temperature is 25+/-5 ℃ for 10-15 minutes; the stop solution is a conventional stop solution.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to be all but practical. Reagents and equipment not specifically identified in the examples are generally those conventionally commercially available in the art; the experimental procedure, without specific conditions noted in the examples, is generally carried out under conventional conditions or under conditions recommended by the manufacturer.
Specifically, the following table (table 1) relates to the raw material manufacturers in the following examples:
table 1 raw materials table
Unless otherwise specified, the method for detecting CD28 antibody residues in the T lymphocyte preparation and the raw materials and reagents used in the kit can be purchased from the market.
The invention is further illustrated by the following examples:
example reaction System and method
1.1 Preparation of coated plates
Preparing a 1X coating liquid: taking coating liquid (10X), diluting with sterilized water for injection to obtain 1X coating liquid, and fully and uniformly mixing for later use;
preparing coating protein: taking one piece of Human CD28E & CD28D Heterdimei Protein protein, adding sterilized water for injection to make the concentration of the water 600 mug/mL, fully and uniformly mixing, and separately storing at-70+/-10 ℃ for later use; taking the protein with the concentration, and diluting the protein with the concentration by 600 times by using a 1X coating liquid to prepare a coating protein solution with the final concentration of 1 mug/mL;
and (3) coating an ELISA plate: 1 mug/mL of coating protein is added into an empty ELISA plate, 100 mug/hole is filled, and the coating is carried out at 2-8 ℃ overnight.
1.2 Reagent preparation
1.2.1 Wash (1×pbst): taking a PBS powder 1 bag, adding 2L sterilization injection water, and fully dissolving; then adding 1 mL of Tween20, and fully and uniformly mixing;
1.2.2 Blocking solution (2% BSA-PBST): prepared according to the proportion of 0.6 g BSA powder and 30 mL 1 XPBST, and fully dissolved;
1.2.3 Sample dilutions (0.1% BSA-PBST): adding 1 XPBST into 2% BSA-PBST for 20-time dilution, and dissolving completely;
1.2.4 Secondary antibody solution: goat anti-mouse IgG2a (HRP) was taken and diluted with sample dilution (0.1% BSA-PBST) to a final concentration of 0.5 μg/mL of secondary antibody solution.
1.3 Operational flow
1.3.1 Washing the plate: taking out the coated ELISA plate, recovering to 25+/-5 ℃, washing the plate with washing liquid for 3 times, 300 mu L/hole, discarding liquid for the last 1 time, and if residual liquid exists in the hole, beating to dry on absorbent paper;
1.3.2 Closing: adding 300 mu L of blocking solution into each hole, and blocking for 1 hour at 25+/-5 ℃;
1.3.3 Washing the plate: 1.3.1;
1.3.4 Sample adding: adding standard substances, test substances, quality control substances (Blank) and the like into corresponding holes according to plate arrangement, wherein no reagent is added into the Blank, 100 mu L/hole, arranging 2 compound holes in each group, sealing a plate membrane sealing plate, and incubating at 25+/-5 ℃ for 2 hours;
1.3.5 Washing the plate: 1.3.1;
1.3.6 Adding a secondary antibody (labeled antibody) solution: adding a secondary antibody solution into each hole, sealing a plate membrane sealing plate at 25+/-5 ℃ for 1 hour in a dark place at 100 mu L/hole;
1.3.7 Washing the plate: 1.3.1;
1.3.8 Color development: adding TMB color development liquid into each hole, 100 mu L/hole, sealing a plate membrane sealing plate, and carrying out light-shielding incubation at 25+/-5 ℃ for 10-15 minutes, wherein the specific incubation time is determined according to the actual color development result;
1.3.9 And (3) terminating: stop solution was added to each well, 50. Mu.L/well, OD values at a wavelength of 450. 450 nm were measured using an enzyme-labeled instrument after mixing, and the OD values were derived as Excel and analyzed as initial data.
1.4 And (3) data processing:
1.4.1 And calculating the average absorbance value of each sample compound hole, subtracting the average absorbance value of the Blank compound hole, and calculating the compound hole CV value.
Complex CV (%) =complex SD/complex Mean
1.4.2 Standard curve fitting: subtracting the Blank mean value from the absorbance mean value of the standard substance to obtain a standard curve regression equation and a correlation coefficient R, and fitting and drawing by using a Logistic curve (four parameters) with the standard substance theoretical concentration as an ordinate 2 。
Calculating X from Y on a standard curve to obtain measured concentration values of a standard substance, a quality control substance, a test substance and the like; if the sample is pre-diluted, the concentration of the sample obtained according to the standard curve is multiplied by the dilution ratio.
1.4.3 And calculating the recovery rate RE (%) of the standard curve, quality control and standard addition test sample.
Recovery rate RE (%) = (measured concentration/theoretical concentration) ×100%
TABLE 2 data for the examples of the invention
Wherein:
S1-S7 are standard substances with the concentration of 800pg/mL, 400 pg/mL, 200 pg/mL, 100 pg/mL, 50 pg/mL, 25 pg/mL and 12.5pg/mL respectively;
BL is blank control;
HQC, MQC, LQC is a quality control product with three different concentrations of high, medium and low, wherein the concentrations are 600 pg/mL, 250 pg/mL and 37.5 pg/mL respectively;
a1 and A2 are samples to be tested (test samples).
According to R 2 The standard curve is shown in fig. 1, which is a CD28 antibody standard curve, and the standard curve regression equation is: y= [ 47.99640- (-0.00130) ]/[ 1+ (x/31820.15752) ^ (-0.99833) + (-0.00130) where R 2 The detection limit is 12.5 pg/mL-800 pg/mL.
The X-axis represents CD28 antibody concentration values (pg/mL) and the Y-axis represents OD450 values.
The standard curve meets the recovery standard at multiple points, and the standard curve is qualified. According to the standard curve, the relative recovery rate of the measured quality control product concentration is 89.76-100.30%, and the difference between the measured quality control product concentration and the actual concentration of the quality control product is small, so that the data measured by the method can reflect the actual concentration of the quality control product.
Comparative example reaction System and method
The basic procedure and flow of this comparative example was carried out as in the examples, with the relevant differences as shown in the following table:
TABLE 3 comparative examples of parameters of examples and comparative examples of the invention
According to the four-parameter fitting method, standard curves of comparative example 1, comparative example 2 and comparative example 3 are obtained, and related data and standard curves are shown in tables 4-6 and fig. 2-4:
table 4 comparative example 1 test data
Wherein:
S1-S7 are standard substances with the concentration of 800pg/mL, 400 pg/mL, 200 pg/mL, 100 pg/mL, 50 pg/mL, 25 pg/mL and 12.5pg/mL respectively;
BL is blank control;
HQC, MQC, LQC is a quality control product with three different concentrations of high, medium and low, and the concentrations are 600 pg/mL, 250 pg/mL and 37.5 pg/mL respectively.
The standard curve recovery rate refers to the ratio of the fitted concentration to the standard point concentration after each point of the standard curve is returned to the standard curve, is reflected as the fitting degree of each standard point and the standard curve, and can be used as the basis for judging whether the standard curve is qualified or not. Comparative example 1 the concentration of the secondary antibody was adjusted relative to the example, and the standard curve regression equation fitted to comparative example 1 was: y= [ 1.72527- (-0.00752) ]/[ 1+ (x/583.09213) ^ (-0.93627) + (-0.00752) wherein R 2 Is 0 to996, the multi-point standard curve does not meet the recovery standard, the standard curve is unqualified, the recovery rate of quality control points is lower, the absorbance of the whole plate hole is higher, and the experiment is not established.
Table 5 comparative example 2 test data
Wherein:
S1-S7 are standard substances with the concentration of 800pg/mL, 400 pg/mL, 200 pg/mL, 100 pg/mL, 50 pg/mL, 25 pg/mL and 12.5pg/mL respectively;
BL is blank control;
HQC, MQC, LQC is a quality control product with three different concentrations of high, medium and low, and the concentrations are 600 pg/mL, 250 pg/mL and 37.5 pg/mL respectively.
Comparative example 2 was adjusted with respect to the examples in terms of coating protein concentration, blocking time, plate washing times, incubation time, secondary antibody concentration and reaction time, color development time, etc., the standard curve regression equation fitted in comparative example 2 was: y= [ 0.43915-0.00189 ]/[ 1+ (x/136.52649) ^ (-0.88860) 0.00189, wherein R 2 The standard curve is 0.991, the multiple points of the standard curve do not meet the recovery standard, the standard curve is not qualified, the recovery rate is low, and the experiment is not established.
Table 6 comparative example 3 test data
Wherein:
S1-S7 are standard substances with the concentration of 800pg/mL, 400 pg/mL, 200 pg/mL, 100 pg/mL, 50 pg/mL, 25 pg/mL and 12.5pg/mL respectively;
BL is blank control;
HQC, MQC, LQC is a quality control product with three different concentrations of high, medium and low, and the concentrations are 600 pg/mL, 250 pg/mL and 37.5 pg/mL respectively.
Comparative example 3 relative to the examples in coating protein concentration, blocking time, plate wash times, incubation time, secondary antibody concentrationAnd links such as reaction time, color development time and the like are adjusted, and a standard curve regression equation fitted in the comparative example 3 is as follows: y= [ 1.50367-0.07660 ]/[ 1+ (x/89.46388) ^ (-1.90089) A+ 0.07660 wherein R 2 For 0.994, the fitting standard curve has multiple points which do not meet the recovery standard, the standard curve is unqualified, the recovery rate of quality control points is lower, the absorbance of the whole plate hole is too high, and the experiment is not established.
From the comparison results, the embodiment of the invention can form a standard curve with higher accuracy, and the standard curve with higher accuracy can ensure that the value measured by the test sample is true and reliable. From the condition of the relative recovery rate (RE%), the relative recovery rate (RE%) of the quality control product is 89.76-100.30%, which is obviously better than that of comparative examples 1-3, and the RE% of the quality control product of comparative examples 1-3 is 75-112%, 68-73%, 49-82% respectively, and the detection values are obviously deviated from the actual values, which shows that the quality control product has outstanding test effects.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
- A method for detecting CD28 antibody residues in a t lymphocyte preparation, comprising the steps of:step 1, coating an antigen on a carrier, cleaning and sealing;step 2, cleaning;respectively adding standard substance solutions with different concentrations into the standard substance solutions;adding a test solution into the test group;incubating and cleaning;step 3, respectively adding a labeled antibody into a blank control group, a standard substance group and a test group, incubating, cleaning, developing, and stopping, and respectively obtaining absorbance data of the blank control group, the standard substance group and the test group at the wavelength of 450 nm;step 4, subtracting the absorbance data of the blank control group from the absorbance data of the standard substance group to obtain a standard curve by using Logistic curve fitting, and obtaining a detection result of the CD28 antibody residues through the standard curve according to the absorbance data of the test group;the antigen is a CD28E & CD28D heterodimeric protein;the standard substance is Anti-Human CD28 mAB;the test sample is a T lymphocyte preparation taking CD28 as a raw material.
- 2. The assay of claim 1, wherein said T lymphocyte preparation comprises a central memory T lymphocyte preparation.
- 3. The method of claim 1, wherein the CD28E & CD28D heterodimeric protein is coated in an amount of 0.1 μg/unit carrier;the antigen coating condition is that the antigen is coated overnight at 2-8 ℃;the washing liquid for washing is sodium phosphate;the number of times of the washing was 3.
- 4. A method of detection according to claim 3, wherein the period of closure is 1 hour;the blocking solution used for the blocking was either 2% (w: v) BSA or 5% (w: v) skimmed milk powder in PBST.
- 5. The method of claim 4, wherein the incubation in step 2 is at 25 ℃ ± 5 ℃ for 2 hours.
- 6. The method according to claim 5, wherein the incubation in step 3 is carried out at 25 ℃ + -5 ℃ for 1 hour under light-shielding conditions.
- 7. The method according to claim 6, wherein the concentration of the labeled antibody solution is 0.05. Mu.g/unit carrier.
- 8. The detection method according to claim 7, wherein the color development time is 10 to 15 minutes;the color development liquid adopted by the color development is color development liquid TMB.
- 9. A kit for the detection of CD28 antibody residues in a T lymphocyte preparation, comprising a CD28E & CD28D heterodimer protein, a blocking solution and a labeled antibody;the mass ratio of the CD28E & CD28D heterodimeric protein and the labeled antibody is 2:1, a step of;the blocking solution was either 2% (w: v) BSA or 5% (w: v) PBST solution of skimmed milk powder.
- 10. The kit of claim 9, further comprising a chromogenic solution TMB.
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