CN113777320A - Human immunity test method based on secretion - Google Patents
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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Abstract
The invention provides a human immunity test method based on secretion, which belongs to the technical field of biomedical detection, adopts a double-antibody sandwich ELISA method, utilizes an enzyme-labeled antibody, adds a substrate TMB for color development by virtue of the catalytic action of enzyme, stops the reaction, and detects the OD value of each hole of a secretion sample to be detected under the wavelength of 450nm, wherein the OD value is in direct proportion to the content of the antibody to be detected; carrying out curve fitting by using the detection result of the secretion standard substance to obtain an IgE standard curve; and (4) combining the drawn IgE standard curve to carry out interpolation calculation to obtain the IgE content of the secretion sample to be detected. The invention adopts a double-antibody sandwich ELISA method, can carry out quantitative detection on the IgE content in the saliva, can carry out the detection on the IgE content only by obtaining the saliva without collecting the blood of a subject during sampling, can represent the strength of the immunity of a human body through a quantitative detection result in the normal IgE content range of the human body, and can measure the change of the IgE content caused by the change of the environmental temperature.
Description
Technical Field
The invention relates to the technical field of biomedical detection, in particular to a human immunity testing method based on secretion.
Background
The immunity is a defense mechanism of a human body, and the human body recognizes and eliminates any foreign matters (viruses, bacteria and the like) invaded from the outside; the ability to treat senescent, damaged, dead, degenerating self-cells, and to recognize and treat mutant and virally infected cells in vivo.
Modern immunology considers that immunity is the physiological response of the human body to recognize and eliminate "isohexia". Low immunity is the root cause of most diseases. When the human body has immune dysfunction or the immune system is not healthy, diseases such as cold, tonsillitis, asthma, bronchitis, pneumonia, diarrhea, allergy and the like can repeatedly attack, and even a series of tumor cancers can be caused.
The detection of the content of the immune globulin in the body can understand the humoral immune function state of the body, help diagnose various diseases such as immune hyperplasia, immune deficiency, infection, autoimmunity and the like, and has important clinical significance. Enzyme-linked immunosorbent assay (ELISA) is used as a labeling immunity technology, has the characteristics of high sensitivity, strong specificity, stable reagent, low price, simple and convenient operation, no environmental pollution and the like, and is widely used for the detection of antigens and antibodies of infectious pathogens, tumor markers, hormones, special proteins, cytokines, therapeutic drugs and the like. Serum (plasma), saliva, fecal cerebrospinal fluid and other body fluids can be used as the test sample.
Immunoglobulin E (IgE) is present in very low concentrations in serum, accounting for about 0.002% of immunoglobulin, and is mainly present in blood vessels, skin and mucous membrane, and is thought to be produced by plasma cells in the mucosa lining the nasopharynx, tonsils, bronchi and gastrointestinal tract, and to bind to mast cells and basophilic polymorphonuclear granulocytes in the blood. Within the normal content range of IgE, the higher the content of IgE, the stronger the resistance of a human body to partial bacteria, viruses, parasites and the like outside, and the stronger the immunity of the human body. At present, for detecting the content of middle IgE, the immunity of a human body is judged mainly by detecting the content of the IgE in serum, and the method for detecting the content of the IgE in the serum has the advantages of low detection efficiency, complex process and poor accuracy.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a human immunity testing method based on secretion, which comprises the following steps:
adopting a double-antibody sandwich ELISA method, using an enzyme-labeled antibody, adding a substrate TMB for color development by virtue of the catalytic action of enzyme, stopping the reaction, and detecting the OD value of each hole of a secretion sample to be detected at the wavelength of 450 nm; wherein the OD value is in direct proportion to the content of the antibody to be detected;
carrying out curve fitting by using the detection result of the secretion standard substance to obtain an IgE standard curve;
and (4) combining the drawn IgE standard curve to carry out interpolation calculation to obtain the IgE content of the secretion sample to be detected.
Preferably, the detection of the OD value of each well of the secretion standard comprises the following steps:
respectively taking standard stock solution dilution series with different dilution concentrations, adding the secretion standard substance and the secretion sample to be detected into different well plate strips, and adding PBS into a well serving as a control;
adding an Antibody Cocktail to each well, sealing the well plate and incubating on a plate shaker;
after incubation is finished, adding 1X Wash Buffer PT into each hole to clean the holes, then turning over the hole plate belt for spin-drying, sucking off redundant liquid by using a clean paper towel, and continuing the operation for 3 times;
TMB Substrate (Substrate, english is article name) was added to each well and incubated in the dark on a well plate shaker;
after incubation, Stop Solution was added to each well, the well plate was shaken on a well plate shaker, and the well plate was placed on a microplate reader to measure the OD value at a wavelength of 450 nm.
Preferably, 1X Wash Buffer PT (1X Wash) is prepared; preparing Antibody Cocktail (Antibody mixture); adding Sample Diluent NS into Human IgE lysolized Protein (Human IgE purified Protein), and mixing uniformly to obtain standard stock solution; the standard stock was diluted using Sample dilution NS to obtain multiple dilution series of standard stock at different concentrations.
Preferably, diluting 10XWash Buffer PT by using distilled water to prepare 1XWash Buffer PT; preparing an Antibody Cocktail by diluting the capture and detection Antibody in the Antibody Diluent 5 BI; 1mL of Sample Diluent NS was added to a tube containing an amount of Human IgE lysolized Protein and gently mixed on a tube rotator for 10 minutes to obtain a standard stock solution.
Preferably, 50mL of 1X Wash Buffer PT is prepared, and 5mL of 10X Wash Buffer PT is uniformly mixed with 45mL of distilled water or deionized water to obtain the 1X Wash Buffer PT.
Preferably, 3mL of the Antibody Cocktail was prepared and 300uL10X Capture Antibody and 300uL10X Detector Antibody were mixed uniformly with 2.4mL of Antibody dilution 5BI to obtain the Antibody Cocktail.
Preferably, the sample to be detected is collected by adopting a spitting method to collect saliva of a detected person, the collected saliva is transferred into a disposable sterile test tube, and a Profile preservative film is used for sealing; when the OD value of the sample to be detected is detected immediately, the sealed test tube needs to be stored in an environment at-80 ℃ for storage.
Preferably, before the stored sample to be detected is detected, the sample to be detected is unfrozen, a certain amount of unfrozen saliva is put into a centrifuge tube to be centrifuged by using a centrifuge, and the centrifuged supernatant is taken as the sample to be detected.
Preferably, before the sample to be detected is transferred into the disposable sterile test tube, a mixed solution is added into the disposable sterile test tube in advance, and the mixed solution is prepared from PBS + PMSF + distilled water.
Preferably, the mixed solution is 1mL, the PBS content is 100 times of that of PMSF, and the balance is distilled water.
The invention has the beneficial effects that: through collecting saliva of a subject, a double-antibody sandwich ELISA method is adopted, the content of IgE in the saliva can be quantitatively detected, the detection of the content of the IgE in serum is not depended on, the blood of the subject does not need to be collected during sampling, only the saliva is obtained, the strength of the immunity of the human body can be represented through a quantitative detection result in a normal IgE content range of the human body, and the change of the content of the IgE caused by the change of the environmental temperature can be measured by the testing method.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
Fig. 1 is a flowchart of a secretion-based human immunity test method according to an embodiment of the present invention.
FIG. 2 is a schematic diagram of a dilution series of a standard stock solution according to an embodiment of the present invention.
Fig. 3 is a schematic diagram of IgE measurement results at different temperatures according to the embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below by way of the drawings are illustrative only and are not to be construed as limiting the invention.
It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
As used herein, the singular forms "a", "an", "the" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
In the description of the present specification, the terms "first", "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
For the purpose of facilitating an understanding of the present invention, the present invention will be further explained by way of specific embodiments with reference to the accompanying drawings, which are not intended to limit the present invention.
It should be understood by those skilled in the art that the drawings are merely schematic representations of embodiments and that the elements shown in the drawings are not necessarily required to practice the invention.
Example 1
In this embodiment 1, there is provided a method for testing human immunity based on secretions, comprising:
adopting a double-antibody sandwich ELISA method, using an enzyme-labeled antibody, adding a substrate TMB for color development by virtue of the catalytic action of enzyme, stopping the reaction, and detecting the OD value of each hole of a secretion sample to be detected at the wavelength of 450 nm; wherein the OD value is in direct proportion to the content of the antibody to be detected;
carrying out curve fitting by using the detection result of the secretion standard substance to obtain an IgE standard curve;
and (4) combining the drawn IgE standard curve to carry out interpolation calculation to obtain the IgE content of the secretion sample to be detected.
Wherein, the OD value of each hole of the secretion standard product is detected, which comprises the following steps:
respectively taking standard stock solution dilution series with different dilution concentrations, adding the secretion standard substance and the secretion sample to be detected into different well plate strips, and adding PBS into a well serving as a control;
adding an Antibody Cocktail to each well, sealing the well plate and incubating on a plate shaker;
after incubation is finished, adding 1X Wash Buffer PT into each hole to clean the holes, then turning over the hole plate belt for spin-drying, sucking off redundant liquid by using a clean paper towel, and continuing the operation for 3 times;
TMB Substrate was added to each well and incubated in the dark on a well plate shaker;
after incubation, Stop Solution was added to each well, the well plate was shaken on a well plate shaker, and the well plate was placed on a microplate reader to measure the OD value at a wavelength of 450 nm.
In example 1, 1X Wash Buffer PT and Antibody Cocktail were prepared; adding Sample Diluent NS into Human IgE lysolized Protein, and uniformly mixing to obtain a standard stock solution; the standard stock was diluted using Sample dilution NS to obtain multiple dilution series of standard stock at different concentrations.
Wherein, diluting 10XWash Buffer PT by distilled water to prepare 1XWash Buffer PT; preparing an Antibody Cocktail by diluting the capture and detection Antibody in the Antibody Diluent 5 BI; 1mL of Sample Diluent NS was added to a tube containing an amount of Human IgE lysolized Protein and gently mixed on a tube rotator for 10 minutes to obtain a standard stock solution.
Wherein, 50mL of 1X Wash Buffer PT is prepared, and 5mL of 10X Wash Buffer PT is uniformly mixed with 45mL of distilled water or deionized water to obtain the 1X Wash Buffer PT. 3mL of an Antibody Cocktail was prepared by mixing 300uL10X Capture Antibody and 300uL10X Detector Antibody with 2.4mL of an Antibody dilution 5BI to obtain an Antibody Cocktail.
In this embodiment 1, the sample to be tested is collected by collecting saliva of a person to be tested by a spitting method, transferring the collected saliva into a disposable sterile test tube, and sealing the test tube with a Profile preservative film; when the OD value of the sample to be detected is detected immediately, the sealed test tube needs to be stored in an environment at-80 ℃ for storage. Before detecting a stored sample to be detected, unfreezing the sample to be detected, putting a certain amount of unfrozen saliva into a centrifuge tube, centrifuging by using a centrifuge, and taking the centrifuged supernatant as the sample to be detected. Before the sample to be detected is transferred into the disposable sterile test tube, mixed liquid is added into the disposable sterile test tube in advance, and the mixed liquid is prepared from PBS + PMSF + distilled water.
In this example 1, the amount of the mixed solution added to the disposable sterile test tube was 1mL, the PBS content was 100 times that of PMSF, and the balance was distilled water.
Example 2
As shown in fig. 1, in this example 2, a method for rapidly testing immunity based on human secretions is provided, which can be used for clinical diagnosis and non-diagnostic purposes, does not involve ethical problems, and is convenient for sample collection.
In this embodiment 2, the method for rapidly testing immunity based on human secretions includes the following steps:
in the step 1, the sample is collected and stored, and the method comprises the following steps: firstly, obtaining a sample, wherein the sample is collected by spitting saliva into a disposable urine cup by a detected person by adopting a spitting method, then transferring the disposable urine cup into a disposable sterile test tube, and sealing the disposable urine cup by using a Profile preservative film; secondly, when the sample can not be immediately processed in the step 2 and detected in the step 3, the sample needs to be stored for a short time, the sealed test tube is stored in an environment at-80 ℃,
in step 2, the sample is unfrozen and processed by the following method: when the sample in the step 1 is stored, before the sample is detected, unfreezing the sample, wherein the unfreezing of the sample refers to naturally unfreezing the frozen test tube at room temperature or unfreezing the frozen test tube in warm water at the temperature equal to the room temperature; the sample treatment operation was as follows: and taking a certain amount of saliva from the test tube by using a pipette gun, putting the saliva into a centrifugal tube I, sealing, putting the centrifugal tube I on a centrifugal machine, centrifuging for 5 minutes, and taking supernatant in the centrifugal tube I by using the pipette gun after the centrifugation is finished, and putting the supernatant into a centrifugal tube II.
In step 3, the method for detecting IgE is as follows: a double-antibody sandwich ELISA method is adopted, an enzyme-labeled antibody is utilized, a substrate TMB is added to the enzyme-labeled antibody under the catalytic action of the enzyme for color development, the reaction is stopped, the OD value of each hole is measured at the wavelength of 450nm, and the OD value is in direct proportion to the content of the antibody to be detected.
In step 4, quantitative calculation and analysis of IgE are carried out by the following method: and performing curve fitting by using the detection result of the standard substance to obtain a standard curve or a relational expression, wherein the IgE detection result of the sample can be obtained by interpolation calculation from the drawn standard curve or by using the relational expression.
In this example 2, the sample is collected and stored in step 1, the collection amount of the sample is generally not less than 1mL, before the sample is transferred into the disposable sterile tube, 1mL of mixed solution is added into the disposable sterile tube in advance, the mixed solution is configured by PBS + PMSF + distilled water, wherein the content of PBS is 100 times that of PMSF, and the rest is distilled water, for example, 3mL of mixed solution is configured, and 3mL of mixed solution is 300uL of PBS +3uL of PMSF +2697uL of distilled water;
in this example 2, the IgE detection in step 3 includes the following steps:
step one, preparing 1X Wash Buffer PT: diluting 10XWash Buffer PT by using distilled water to prepare 1XWash Buffer PT;
second step, preparation of Antibody Cocktail: preparing an Antibody Cocktail by diluting the capture and detection Antibody in the Antibody Diluent 5 BI;
thirdly, reconstructing Human IgE lysophilized Protein: add 1mL of Sample Diluent NS to a # 0 tube containing an amount of Human IgE lysophilized Protein using a pipette and mix gently on a tube rotator for 10 minutes to give a 25ng/mL standard stock solution;
fourthly, marking 8 test tubes, numbering 1#, 2 #. 7#, 8#, adding 427uL Sample dilution NS into the test tube 1#, adding 150uL Sample dilution NS into the test tube 2# -8 #, and preparing the following dilution series according to the operation shown in FIG. 2.
Fifthly, preparing the perforated plate strip: the kit is ready for use with a 96-well strip (8 x 12), the unused strip should be immediately replaced in a foil bag containing a desiccant pack, resealed and stored at 4 ℃, for each experiment performed, at least two wells must be used as zero controls to see the parallelism of the samples, and at least two replicates of each sample should be performed;
sixthly, detecting a program:
1) at room temperature, respectively taking 50uL 1# -8 # test tube standard substances and 50uL samples by using a pipette gun, adding the standard substances and the 50uL samples into different well plate strips, and adding 50uL PBS into a well serving as a reference, wherein a new pipette head needs to be replaced when the pipette gun is used once;
2) add 50uL of Antibody Cocktail to each well, seal the well plate and incubate for 1 hour on a plate shaker set at 400 rpm;
3) after incubation is finished, adding 350uL 1X Wash Buffer PT into each hole by using a pipette gun to clean the holes, then turning over the hole plate belt for spin-drying, sucking off redundant liquid by using a clean paper towel, and continuing the operation for 3 times;
4) then 100uL of TMB Substrate was added to each well and incubated in the dark for 10 minutes on a well plate shaker set at 400 rpm;
5) after incubation was complete, 100uL of Stop Solution was added to each well, the plates were shaken on a plate shaker for 1 minute, and then placed on a microplate reader to measure the OD at 450nm wavelength.
Wherein the content of the first and second substances,
1) preparation of 1X Wash Buffer PT: preparing 50mL of 1X Wash Buffer PT, and uniformly mixing 5mL of 10X Wash Buffer PT with 45mL of distilled water or deionized water;
2) preparation of Antibody Cocktail: preparing 3mL of an Antibody Cocktail, and uniformly mixing 300uL of 10XCapture Antibody and 300uL of 10X Detector Antibody with 2.4mL of Antibody dilution 5 BI;
3) no. 8 test tubes contain no protein and are blank controls;
4) and (3) detection procedures: all materials and prepared reagents were equilibrated to room temperature and all standards and samples were prepared in duplicate prior to starting IgE detection.
In this embodiment 2, the testing method utilizes saliva of a human body to measure the content of IgE in the human body, which is more convenient than the measurement of blood collection of the human body, and the sample is easier to obtain. The test method can measure the change of human IgE content under the change of environmental parameters (mainly the change of environmental temperature).
Example 3
In this example 3, a method for rapidly testing immunity based on human secretions is described, taking the detection of IgE on 10 subjects in 9 months in 2020 as an example, and specific procedures are described.
100, preparing 30 sterile urine cups, numbering the test tubes by name and temperature of a subject, and conveniently distinguishing, wherein 1mL of mixed liquid is added into each disposable sterile test tube, the mixed liquid is prepared from PBS (phosphate buffer solution) + PMSF (permanent phosphate buffer solution) + distilled water, the content of the PBS is 100 times that of the PMSF, and the balance of the mixed liquid is water, for example, 3mL of mixed liquid is prepared, and the 3mL of mixed liquid is 300uL of PBS (phosphate buffer solution) +3uL of PMSF +2697uL of distilled water;
step 101, the subject sits still at an ambient temperature of 18 ℃ for 40 minutes to fully acclimate to the environment;
102, spitting not less than 1mL of saliva into a disposable sterile urine cup by each subject;
103, transferring the saliva into a disposable sterile test tube corresponding to the name and the temperature of the subject, sealing the test tube by using a Profile preservative film, and then storing the test tube in a freezer at the temperature of-80 ℃ for short-time storage;
step 104, the subject sits still for 40 minutes at 24 ℃ to fully adapt to the environment, and other environmental parameters are the same as those at 18 ℃;
step 105, implementing step 102;
step 106, implementing step 103;
step 107, the subject sits still at 40 ℃ in an environment of 30 ℃ to fully adapt to the environment, and other environmental parameters are the same as those in the environment of 18 ℃ and 24 ℃;
step 108, implementing step 102;
step 109, implement step 103;
110, unfreezing all the frozen samples, and naturally unfreezing the frozen test tubes at room temperature or quickly unfreezing the frozen test tubes in warm water at the temperature equal to the room temperature;
step 111, preprocessing the unfrozen sample, taking the sample with the serial number of' name +18 ℃ as an example, and operating as follows: (1) taking 20 empty centrifuge tubes, wherein 10 centrifuge tubes are numbered according to a format of [ name +1+18 ℃ C ], and the other 10 centrifuge tubes are numbered according to a format of [ name +2+18 ℃ C ]; (2) taking 10 gun heads of 300uL liquid-transfering guns and 10 gun heads of 100uL liquid-transfering guns; (3) respectively taking 300uL samples from 10 unfrozen test tubes by using a pipette and placing the samples into 10 corresponding centrifugal tubes with the numbers of [ name +1+18 ℃), wherein each gun head can only be used once; (4) putting 10 numbered centrifugal tubes (name +1+18 ℃) on a centrifugal machine, setting the rotation speed of 40000 rpm, and carrying out centrifugal operation for 5 minutes; (5) after the centrifugal operation is finished, 100uL of supernatant is respectively taken from the 10 centrifugal tubes with the serial numbers of [ name +1+18 ℃ C ] by using a pipette and is put into the 10 corresponding centrifugal tubes with the serial numbers of [ name +2+18 ℃ C ], and each pipette head can only be used once; the sample processing operations numbered "name +24 ℃ and" name +30 ℃ are the same as those under "name +18 ℃;
step 112, uniformly mixing 5mL of 10X Wash Buffer PT with 45mL of distilled water or deionized water to prepare 50mL of 1X Wash Buffer PT;
step 113, uniformly mixing 400uL 10X Capture Antibody and 400uL 10X Detector Antibody with 3.2mL Antibody Diluent 5BI to prepare 4mL Antibody Cocktail;
step 114, preparing 25ng/mL standard stock solution, adding 1mL Sample Diluent NS to # 0 test tube containing a certain amount of Human IgE lysolized Protein using a pipette gun, and gently mixing for 1 minute on a tube shaker;
step 115, marking 8 test tubes, numbering 1#, 2# · · 7#, 8# in sequence, adding 427uL Sample Diluent NS into the 1# test tube, adding 150uL Sample Diluent NS into the 2# -8 # test tube respectively, then transferring 300uL liquid into the 2# test tube from the 1# test tube, repeating the steps, finally transferring 6# into the 7# test tube for 300uL, and using the 8# test tube as blank contrast, wherein the concentrations of the solutions in the 1# to 8# test tubes are 1.33ng/mL, 0.89ng/mL, 0.59ng/mL, 0.40ng/mL, 0.26/mL, 0.18ng/mL, 0.11ng/mL and 0ng/mL respectively;
step 116, leaving 78 pore plate strips, immediately putting the rest unused plate strips back into the foil bags filled with the desiccant bags, resealing and storing at 4 ℃;
step 117, taking 39 pipette tips of 50uL liquid transfer guns, (1) respectively taking 50uL prepared solution from 1# -8 # test tubes twice into two rows (16) of pore plate belts by using the liquid transfer guns, wherein 8 pipette tips are required for the operation; (2) taking 50uL of supernatant twice from a centrifugal tube with the serial number of [ name + temperature +2] to 60 pore plate belts by using a pipette, wherein 10 of the 60 supernatant are one group, three groups are needed at 3 temperatures, and the total number is 60 by adding contrast, and 30 pipette heads are needed in the operation; (3) adding 50uL PBS into the rest two holes respectively, and using a gun head;
step 118, replacing a new pipette tip of 50uL, adding 50uL of Antibody Cocktail to each well with a pipette, sealing the well plate, and incubating for 1 hour on a plate shaker set at 400 rpm;
step 119, after incubation is completed, replacing a new 350uL pipette head, adding 350uL 1X Wash Buffer PT into each hole by using a pipette gun, cleaning the holes, then spin-drying the hole plate belt, reversely buckling the hole plate belt on a clean paper towel, sucking off redundant liquid, and continuously performing the operation for 3 times;
step 120, replacing a new 100uL pipette tip, using a pipette tip, then adding 100uL TMB Substrate to each well, and incubating in the dark for 10 minutes on a well plate shaker set at 400 rpm;
step 121, after incubation is finished, changing a new gun head of 100uL, adding 100uL Stop Solution into each hole by using a pipette gun, and shaking the hole plate on a hole plate shaker for 1 minute;
step 122, placing the pore plate on an enzyme labeling instrument to measure the OD value of each pore under the wavelength of 450 nm;
and step 123, performing curve fitting by using the OD value detection result of the standard substance and the corresponding concentration to obtain a relational expression of the concentration and the OD value, and substituting the OD value into the relational expression to obtain the IgE detection result of the sample.
As shown in fig. 3, the results of the test in this example 3 are summarized, wherein the gray bars from left to right in each test tube corresponding to the abscissa correspond to the test results at 18 ℃, 24 ℃ and 30 ℃ respectively, and as can be seen from the figure, the method described in this example 3 can detect IgE through saliva of the subject; changes in IgE levels due to changes in the temperature of the environment in which the subject is exposed can be detected.
In summary, according to the method for testing human immunity based on secretions in the embodiment of the present invention, saliva of a subject is collected, a double antibody sandwich ELISA method is adopted, so that the content of IgE in the saliva can be quantitatively detected, the method does not depend on the detection of the content of IgE in serum medically, the blood of the subject does not need to be collected during sampling, only the saliva is obtained, the strength of human immunity can be represented by the quantitative detection result within the normal content range of IgE of a human body, and the test method can measure the content change of IgE caused by the change of environmental temperature.
Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the present invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive efforts based on the technical solutions disclosed in the present invention.
Claims (10)
1. A secretion-based human immunity test method is characterized by comprising the following steps:
adopting a double-antibody sandwich ELISA method, using an enzyme-labeled antibody, adding a substrate TMB for color development by virtue of the catalytic action of enzyme, stopping the reaction, and detecting the OD value of each hole of a secretion sample to be detected at the wavelength of 450 nm; wherein the OD value is in direct proportion to the content of the antibody to be detected;
carrying out curve fitting by using the detection result of the secretion standard substance to obtain an IgE standard curve;
and (4) combining the drawn IgE standard curve to carry out interpolation calculation to obtain the IgE content of the secretion sample to be detected.
2. The secretion-based human immunity test method according to claim 1, wherein detecting the OD value of each well of the secretion standard comprises the following steps:
respectively taking standard stock solution dilution series with different dilution concentrations, adding the secretion standard substance and the secretion sample to be detected into different well plate strips, and adding PBS into a well serving as a control;
adding an Antibody Cocktail to each well, sealing the well plate and incubating on a plate shaker;
after incubation is finished, adding 1X Wash Buffer PT into each hole to clean the holes, then turning over the hole plate belt for spin-drying, sucking off redundant liquid by using a clean paper towel, and continuing the operation for 3 times;
TMB Substrate was added to each well and incubated in the dark on a well plate shaker;
after incubation, Stop Solution was added to each well, the well plate was shaken on a well plate shaker, and the well plate was placed on a microplate reader to measure the OD value at a wavelength of 450 nm.
3. The secretion-based human immunity test method according to claim 2, wherein 1X Wash Buffer PT is prepared; preparing Antibody Cocktail; adding Sample Diluent NS into Human IgE lysolized Protein, and uniformly mixing to obtain a standard stock solution; the standard stock was diluted using Sample dilution NS to obtain multiple dilution series of standard stock at different concentrations.
4. The secretion-based human immunity test method according to claim 3, wherein 10XWash Buffer PT is diluted with distilled water to prepare 1XWash Buffer PT; preparing an Antibody Cocktail by diluting the capture and detection Antibody in the Antibody Diluent 5 BI; 1mL of Sample Diluent NS was added to a tube containing an amount of Human IgE lysolized Protein and gently mixed on a tube rotator for 10 minutes to obtain a standard stock solution.
5. The secretion-based human immunity test method according to claim 4, wherein 50mL of 1X Wash Buffer PT is prepared, and 5mL of 10X Wash Buffer PT is uniformly mixed with 45mL of distilled or deionized water to obtain 1X Wash Buffer PT.
6. The secretion-based human immunity test method according to claim 4, wherein 3mL of the Antibody Cocktail is prepared, and 300uL10X Capture Antibody and 300uL10X Detector Antibody are uniformly mixed with 2.4mL of Antibody dilution 5BI to obtain the Antibody Cocktail.
7. The secretion-based human immunity test method according to claim 1, wherein the sample to be tested is collected by collecting saliva of a subject by a spitting method, transferring the collected saliva into a disposable sterile test tube, and sealing the tube with a Profile preservative film; when the OD value of the sample to be detected is detected immediately, the sealed test tube needs to be stored in an environment at-80 ℃ for storage.
8. The secretion-based human immunity test method according to claim 7, wherein before the stored sample to be tested is tested, the sample to be tested is thawed, a certain amount of thawed saliva is put into a centrifuge tube and centrifuged by a centrifuge, and the centrifuged supernatant is used as the sample to be tested.
9. The secretion-based human immunity test method according to claim 8, wherein a mixed solution is added into the disposable sterile test tube in advance before the sample to be tested is transferred into the disposable sterile test tube, and the mixed solution is PBS + PMSF + distilled water.
10. The secretion-based human immunity test method according to claim 9, wherein the mixed solution is 1mL, the content of PBS is 100 times that of PMSF, and the rest is distilled water.
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