CN113777320A - Secretion-Based Human Immunity Test Method - Google Patents

Abstract

The invention provides a secretion-based human immunity test method, which belongs to the technical field of biomedical detection. The double-antibody sandwich ELISA method is adopted, an enzyme-labeled antibody is used, and the reaction is terminated after adding a substrate TMB for color development with the help of the catalytic action of the enzyme. Detect the OD value of each well of the secretion sample to be tested at a wavelength of 450 nm, and the size of the OD value is proportional to the content of the antibody to be detected; use the detection results of the secretion standard to perform curve fitting to obtain the IgE standard curve; Combine the drawn IgE The standard curve is interpolated and calculated to obtain the IgE content of the secretion sample to be detected. The invention adopts the double-antibody sandwich ELISA method, which can quantitatively detect the IgE content in the saliva. When sampling, it is not necessary to collect the blood of the subject, and only the saliva can be obtained to detect the IgE content. Inside, the strength of human immunity can be characterized by quantitative detection results, and changes in IgE content due to changes in ambient temperature can be measured.

Description

Secretion-Based Human Immunity Test Method

technical field

The invention relates to the technical field of biomedical testing, in particular to a secretion-based human immunity testing method.

Background technique

Immunity is the body's own defense mechanism, and it is the body's ability to recognize and eliminate any foreign intrusion (viruses, bacteria, etc.) .

Modern immunology believes that immunity is the physiological response of the human body to identify and exclude "others". Low immunity is the root cause of most disease invasions. When the body's immune function is dysfunctional, or the immune system is not sound, diseases such as colds, tonsillitis, asthma, bronchitis, pneumonia, diarrhea, allergies will recur repeatedly, and may even lead to a series of tumors and cancers.

Detection of the content of immune globulin in the body can understand the state of the body's humoral immune function and help diagnose various diseases such as immune hyperplasia, immune deficiency, infection and autoimmunity, which has important clinical significance. Enzyme-linked immunosorbent assay (ELISA), as a labeling immune technology, has the characteristics of high sensitivity, strong specificity, stable reagents, low price, easy operation, and no environmental pollution. It is widely used in antigens and antibodies of infectious pathogens, tumors Determination of markers, hormones, special proteins, cytokines, therapeutic drugs, etc. Serum (plasma), saliva, fecal cerebrospinal fluid and other body fluids can be used as test samples.

The concentration of immunoglobulin E (IgE) in serum is extremely low, accounting for about 0.002% of immunoglobulin, mainly in blood vessels, skin, and mucous membranes. It is produced by plasma cells and can bind to mast cells and basophilic polymorphonuclear granulocytes in the blood. Within the normal range of IgE content, the higher the IgE content, the stronger the body's resistance to some external bacteria, viruses, parasites, etc., and the stronger the body's immunity. At present, the detection of the content of IgE in the serum mainly judges the immunity of the human body by detecting the content of IgE in the serum. This method for detecting the content of IgE in the serum has low detection efficiency, complicated process and poor accuracy.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method to solve at least one technical problem existing in the above background technology.

In order to achieve the above object, the present invention has adopted the following technical solutions:

The present invention provides a secretion-based human immunity test method, comprising:

The double-antibody sandwich ELISA method is used, using enzyme-labeled antibody, and with the help of enzyme catalysis, the reaction is terminated after adding the substrate TMB for color development, and the OD value of each well of the secretion sample to be tested is detected at a wavelength of 450 nm; among them, the size of the OD value It is proportional to the content of the antibody to be detected;

Utilize the detection result of secretion standard substance, carry out curve fitting to obtain IgE standard curve;

Combined with the drawn IgE standard curve interpolation calculation, the IgE content of the secretion sample to be detected is obtained.

Preferably, detecting the OD value of each well of the secretion standard comprises the following steps:

Take the standard stock solution dilution series, secretion standard and the secretion sample to be detected with different dilution concentrations and add them to different well plate strips, and add PBS to the well as a control;

Add Antibody Cocktail to each well, seal the plate and incubate on a plate shaker;

After the incubation, add 1X Wash Buffer PT to each well to wash the wells, then turn the well plate belt over and spin dry, and use a clean paper towel to dry up the excess liquid, and the above operations are repeated 3 times;

Add TMB Substrate (substrate, item name in English) to each well and incubate in the dark on a plate shaker;

After incubation, add Stop Solution to each well, shake the well plate on a plate shaker, and place the well plate on a microplate reader to measure the OD value at a wavelength of 450 nm.

Preferably, prepare 1X Wash Buffer PT (1X washing solution); prepare Antibody Cocktail (antibody mixture); add Sample Diluent NS (sample diluent) to Human IgE Lyophilized Protein (human IgE purified protein), mix well, and obtain standard stock Solution; using Sample Diluent NS, the standard stock solution was diluted to obtain multiple dilution series of standard stock solution of different concentrations.

Preferably, 10X Wash Buffer PT is prepared by diluting 10X Wash Buffer PT with distilled water; Antibody Cocktail is prepared by diluting capture and detection antibodies in Antibody Diluent 5BI; 1 mL of SampleDiluent NS is added to a test tube containing a certain amount of Human IgE Lyophilized Protein, and placed in the test tube Gently mix on a rotator for 10 minutes to obtain standard stock solutions.

Preferably, 50 mL of 1X Wash Buffer PT is prepared, and 5 mL of 10X Wash Buffer PT is uniformly mixed with 45 mL of distilled or deionized water to obtain 1X Wash Buffer PT.

Preferably, 3 mL of Antibody Cocktail is prepared, and 300 uL of 10X Capture Antibody and 300 uL of 10X Detector Antibody are uniformly mixed with 2.4 mL of Antibody Diluent 5BI to obtain Antibody Cocktail.

Preferably, the collection of the sample to be tested is to collect the saliva of the tested person by spitting, transfer the collected saliva into a disposable sterile test tube, and seal it with a Profile plastic wrap; when the sample to be tested is immediately subjected to OD testing During value detection, the sealed test tube needs to be stored in a -80°C environment for preservation.

Preferably, before the stored samples to be tested are tested, the samples to be tested are thawed, a certain amount of thawed saliva is taken into a centrifuge tube and centrifuged by 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 has been added to the disposable sterile test tube in advance, and the configuration of the mixed solution is PBS+PMSF+distilled water.

Preferably, the mixed solution is 1 mL, the content of PBS is 100 times that of PMSF, and the rest is distilled water.

Beneficial effects of the present invention: by collecting the saliva of the subject, the double-antibody sandwich ELISA method can be used to quantitatively detect the IgE content in the saliva, which does not depend on the medical detection of the IgE content in the serum, and does not need to be collected during sampling. The blood of the subject can only be obtained from saliva. Within the normal range of IgE content in the human body, the strength of human immunity can be characterized by quantitative test results, and this test method can measure the IgE content caused by changes in ambient temperature. The change.

Additional aspects and advantages of the present invention will be set forth in part in the following description, which will be apparent from the following description, or may be learned by practice of the present invention.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.

FIG. 1 is a flow chart of the secretion-based human immunity testing method according to the embodiment of the present invention.

FIG. 2 is a schematic diagram of the dilution method of the standard stock solution dilution series according to the embodiment of the present invention.

FIG. 3 is a schematic diagram of the measurement results of IgE at different temperatures according to the embodiment of the present invention.

Detailed ways

Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals refer to the same or similar elements or elements having the same or similar functions throughout. The embodiments described below through the accompanying drawings are exemplary and are only used to explain the present invention, but not to be construed as a limitation of the present 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 should also be understood that terms such as those defined in general dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art and, unless defined as herein, are not to be taken in an idealized or overly formal sense. explain.

It will be understood by those skilled in the art that the singular forms "a", "an", "the" and "the" as used herein can include the plural forms as well, unless expressly stated otherwise. It should be further understood that the word "comprising" used in the description of the present invention refers to the presence of stated features, integers, steps, operations, elements and/or components, but does not exclude the presence or addition of one or more other features, Integers, steps, operations, elements and/or groups thereof.

In the description of this specification, description with reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples", etc., mean specific features described in connection with the embodiment or example , structure, material or feature is included in at least one embodiment or example of the present 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, those skilled in the art may combine and combine the different embodiments or examples described in this specification, as well as the features of the different embodiments or examples, without conflicting each other.

In the description of this specification, the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with "first", "second" may expressly or implicitly include at least one of that feature. In the description of the present invention, "plurality" means two or more, unless otherwise expressly and specifically defined.

In order to facilitate the understanding of the present invention, the present invention will be further explained and described below with reference to the accompanying drawings with specific embodiments, and the specific embodiments do not constitute limitations to the embodiments of the present invention.

Those skilled in the art should understand that the accompanying drawings are only schematic diagrams of the embodiments, and the components in the accompanying drawings are not necessarily necessary to implement the present invention.

Example 1

In the present embodiment 1, a secretion-based human immunity test method is provided, comprising:

The double-antibody sandwich ELISA method is used, using enzyme-labeled antibody, and with the help of enzyme catalysis, the reaction is terminated after adding the substrate TMB for color development, and the OD value of each well of the secretion sample to be tested is detected at a wavelength of 450 nm; among them, the size of the OD value It is proportional to the content of the antibody to be detected;

Utilize the detection result of secretion standard substance, carry out curve fitting to obtain IgE standard curve;

Combined with the drawn IgE standard curve interpolation calculation, the IgE content of the secretion sample to be detected is obtained.

Wherein, detecting the OD value of each well of the secretion standard includes the following steps:

Take the standard stock solution dilution series, secretion standard and the secretion sample to be detected with different dilution concentrations and add them to different well plate strips, and add PBS to the well as a control;

Add Antibody Cocktail to each well, seal the plate and incubate on a plate shaker;

After the incubation, add 1X Wash Buffer PT to each well to wash the wells, then turn the well plate belt over and spin dry, and use a clean paper towel to dry up the excess liquid, and the above operations are repeated 3 times;

Add TMB Substrate to each well and incubate in the dark on a plate shaker;

After incubation, add Stop Solution to each well, shake the well plate on a well plate shaker, and place the well plate 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; Sample Diluent NS was added to Human IgE Lyophilized Protein, and mixed well to obtain a standard stock solution; Sample Diluent NS was used to dilute the standard stock solution to obtain a number of different concentrations of Standard stock solution dilution series.

Among them, dilute 10X Wash Buffer PT with distilled water to prepare 1X Wash Buffer PT; prepare Antibody Cocktail by diluting capture and detection antibodies in Antibody Diluent 5BI; add 1 mL of SampleDiluent NS to a test tube containing a certain amount of Human IgE Lyophilized Protein, and rotate the test tube Gently mix for 10 minutes on a sterilizer to obtain standard stock solutions.

Among them, prepare 50mL of 1X Wash Buffer PT, and evenly mix 5mL of 10X Wash Buffer PT with 45mL of distilled or deionized water to obtain 1X Wash Buffer PT. To prepare 3mL Antibody Cocktail, mix 300uL 10X Capture Antibody and 300uL 10X Detector Antibody with 2.4mL Antibody Diluent 5BI to obtain Antibody Cocktail.

In the present embodiment 1, the collection of the sample to be tested is to use the spit method to collect the saliva of the tested person, transfer the collected saliva into a disposable sterile test tube, and seal it with Profile plastic wrap; When the sample is tested for OD value, the sealed test tube needs to be stored in a -80°C environment for preservation. Before testing the stored samples to be tested, perform a thawing operation on the samples to be tested, take a certain amount of thawed saliva into a centrifuge tube and perform a centrifugation operation with a centrifuge, and take the centrifuged supernatant as a sample to be tested. tested samples. Before the sample to be tested is transferred into the disposable sterile test tube, a mixed solution has been added to the disposable sterile test tube in advance, and the configuration of the mixed solution is PBS+PMSF+distilled water.

In Example 1, the mixed solution added into the disposable sterile test tube was 1 mL, the content of PBS was 100 times that of PMSF, and the rest was distilled water.

Example 2

As shown in FIG. 1 , in this Example 2, a rapid test method for immunity based on human secretions is provided, which can be used for clinical diagnosis and non-diagnostic purposes, does not involve ethical issues, and is convenient for sample collection .

In the present embodiment 2, the described rapid test method for immunity based on human secretions includes the following steps:

Step 1, sample collection and preservation;

Step 2, thawing and processing of samples;

Step 3, the detection of IgE;

Step 4, IgE quantitative calculation and analysis,

Among them, in step 1, the collection and preservation of the sample is as follows: first, the sample is obtained, and the collection of the sample is to use the spitting method to allow the subject to spit saliva into a disposable urine cup, and then transfer it to a disposable urine cup. In a sterile test tube, and sealed with Profile plastic wrap; secondly, when the sample cannot be processed in step 2 and detected in step 3 immediately, the sample needs to be stored for a short time, and the sealed test tube Stored in -80℃ environment,

In step 2, the method for thawing and processing the sample is as follows: when there is a preservation operation for the sample described in step 1, the sample must be thawed before the sample is detected, and the sample is thawed. thawing refers to the natural thawing of the frozen test tube at room temperature, or thawing in warm water with a temperature equal to room temperature; the sample processing operation is as follows: use a pipette to take a certain amount of saliva from the test tube Put it into the centrifuge tube ① and seal it, then put the centrifuge tube ① on the centrifuge for 5 minutes. After the centrifugation operation, use a pipette to take the supernatant in the centrifuge tube ① and put it into the centrifuge tube ② inside.

In step 3, the detection method of IgE is as follows: using double antibody sandwich ELISA method, using enzyme-labeled antibody, with the help of enzyme catalysis, adding substrate TMB for color development to terminate the reaction, and measuring the OD value of each hole at a wavelength of 450nm, OD The magnitude of the value is proportional to the amount of the antibody to be detected.

In step 4, IgE quantitative calculation and analysis, the method is as follows: use the detection result of standard substance to carry out curve fitting to obtain standard curve or relational expression, the IgE detection result of described sample can adopt to calculate from the standard curve of drawing to obtain or utilize relation formula to obtain.

In this Example 2, in the collection and storage of the sample in step 1, the sample collection volume is generally not less than 1 mL. Before the sample is transferred into the disposable sterile test tube, the disposable sterile test tube 1mL of mixed solution has been added in advance, and the configuration of the mixed solution is PBS+PMSF+distilled water, wherein the content of PBS is 100 times that of PMSF, and the rest is distilled water, for example, 3mL mixed solution is configured, 3mL mixed solution=300uL PBS+3uL PMSF +2697uL distilled water;

In the present embodiment 2, the detection of IgE in step 3 comprises the following steps:

The first step, preparation of 1X Wash Buffer PT: Dilute 10X Wash Buffer PT with distilled water to prepare 1X Wash Buffer PT;

Step 2, Preparation of Antibody Cocktail: Antibody Cocktail is prepared by diluting capture and detection antibodies in Antibody Diluent 5BI;

The third step is to reconstitute Human IgE Lyophilized Protein: Add 1mL of Sample DiluentNS to a 0# test tube containing a certain amount of Human IgE Lyophilized Protein with a pipette, and mix gently on a test tube rotator for 10 minutes to obtain 25ng/ mL of standard stock solution;

The fourth step, mark 8 test tubes, number 1#, 2#... Diluent NS, then follow the procedure shown in Figure 2 to prepare the following dilution series.

Step 5: Preparation of well strips: The kit comes with 96 well strips (8*12) available, unused strips should be immediately put back in the foil bag with desiccant pack, resealed, and placed in the desiccant bag. Store at 4°C. For each experiment performed, in order to see the parallelism of the samples, at least two wells must be used as null controls, and at least two replicate experiments are performed for each sample;

The sixth step, the detection program:

1) At room temperature, use a pipette to take 50uL of 1#-8# test tube standards and 50uL of samples and add them to different orifice strips, and add 50uL of PBS to the well as a control. It should be noted that each time it is used The pipette needs to be replaced with a new tip;

2) Add 50uL Antibody Cocktail to each well, seal the well plate and incubate for 1 hour on a plate shaker set at 400 rpm;

3) After the incubation, add 350uL of 1X Wash Buffer PT to each well with a pipette to wash the wells, then turn the well plate over and spin dry, and use a clean paper towel to dry up the excess liquid, and the above operations are repeated 3 times. ;

4) Then add 100uL of TMB Substrate to each well and incubate in the dark for 10 minutes on a plate shaker set to 400 rpm;

5) After incubation, add 100uL of Stop Solution to each well, shake the well plate on a plate shaker for 1 minute, and then place the well plate on a microplate reader to measure the OD value at a wavelength of 450nm.

in,

1) Preparation of 1X Wash Buffer PT: Prepare 50 mL of 1X Wash Buffer PT, and mix 5 mL of 10X Wash Buffer PT with 45 mL of distilled or deionized water evenly;

2) Preparation of Antibody Cocktail: To prepare 3mL Antibody Cocktail, mix 300uL 10XCapture Antibody and 300uL 10X Detector Antibody with 2.4mL Antibody Diluent 5BI evenly;

3) The 8# test tube does not contain protein, which is a blank control;

4) Detection procedure: Before starting the IgE detection, equilibrate all materials and prepared reagents to room temperature, and prepare all standards and samples in duplicate.

In this embodiment 2, the test method uses human saliva to measure the IgE content in the human body, which is more convenient than collecting human blood for measurement, and the sample is easier to obtain. The test method can measure changes in human body IgE content due to changes in environmental parameters (mainly changes in ambient temperature).

Example 3

In this Example 3, the method for rapid immunity test based on human secretions, taking the detection of IgE in 10 subjects in September 2020 as an example, the specific steps will be described.

Step 100, prepare 30 sterile urine cups and 30 disposable sterile test tubes. The test tubes are numbered with the subject's "name + temperature" for easy identification. Add 1 mL of mixed solution to each disposable sterile test tube. The configuration of the mixed solution is PBS+PMSF+distilled water, wherein, the PBS content is 100 times that of PMSF, and the rest is water, for example, configure 3mL mixed solution, 3mL mixed solution=300uL PBS+3uL PMSF+2697uL distilled water;

Step 101, the subject sits for 40 minutes at an ambient temperature of 18°C to fully adapt to the environment;

Step 102, each subject spit into no less than 1mL of saliva in the disposable sterile urine cup;

Step 103, move the saliva into a disposable sterile test tube corresponding to the subject's "name + temperature", seal it with Profile plastic wrap, and then store it in a -80°C freezer for short-term storage;

Step 104, the subject sits quietly for 40 minutes at 24°C to fully adapt to the environment, and other environmental parameters are the same as those at 18°C;

Step 105, implement step 102;

Step 106, implement step 103;

Step 107, the subject sits for 40 minutes at 30°C to fully adapt to the environment, and other environmental parameters are the same as those at 18°C and 24°C;

Step 108, implement step 102;

Step 109, implement step 103;

Step 110, performing a thawing operation on all the above frozen samples, placing the frozen test tubes at room temperature to thaw naturally, or placing them in warm water with a temperature equal to room temperature for accelerated thawing;

Step 111, preprocessing the thawed sample, taking the sample numbered "name+18°C" as an example, the operation is as follows: (1) Take 20 empty centrifuge tubes, 10 of which are in accordance with [name+1+18 ℃] format, and the other 10 are numbered according to the format of [name+2+18℃]; (2) Take 10 tips of 300uL pipettes and 10 tips of 100uL pipettes; (3) ) Use a pipette to take 300uL of samples from 10 thawed test tubes and put them into 10 centrifuge tubes corresponding to the number [name+1+18℃]. Note that each pipette tip can only be used once; (4 ) Put 10 centrifuge tubes numbered [name+1+18°C] on the centrifuge, set the rotation speed to 40,000 rpm, and perform centrifugation for 5 minutes; (5) After the centrifugation operation, use a pipette Take 100uL of the supernatant from the centrifuge tubes numbered [name+1+18℃] and put them into 10 centrifuge tubes corresponding to the number [name+2+18℃]. Note that each pipette tip can only be used once; the number is The sample processing operations of "Name+24℃" and "Name+30℃" are the same as those under "Name+18℃";

Step 112, uniformly mix 5mL 10X Wash Buffer PT with 45mL distilled water or deionized water to prepare 50mL 1X Wash Buffer PT;

Step 113, uniformly mix 400uL 10X Capture Antibody and 400uL 10X Detector Antibody with 3.2mL Antibody Diluent 5BI to prepare 4mL Antibody Cocktail;

Step 114, prepare a 25ng/mL standard stock solution, add 1mL of Sample Diluent NS to a test tube No. 0 containing a certain amount of Human IgE Lyophilized Protein with a pipette, and mix gently for 1 minute on a test tube shaker;

Step 115, label 8 test tubes, number 1#, 2#...7#, 8# in sequence, add 427uL Sample Diluent NS to the 1# test tube, and add 150uL Sample Diluent NS to the 2#-8# test tubes respectively Diluent NS, then transfer 300uL liquid from 1# test tube to 2# test tube, and so on, and finally transfer 6# to 7# test tube with 300uL, 8# test tube is used as a blank control, then the solution concentrations in 1#-8# test tubes are respectively 1.33ng/mL, 0.89ng/mL, 0.59ng/mL, 0.40ng/mL, 0.26ng/mL, 0.18ng/mL, 0.11ng/mL, 0ng/mL;

Step 116, 78 orifice strips are left, and the remaining unused strips are immediately put back into the foil bag containing the desiccant pack, resealed, and stored at 4°C;

Step 117, take 39 50uL pipette tips, (1) use the pipette to take twice the 50uL solution from the 1#-8# test tube into the two rows (16) of the orifice strips. The operation needs to use 8 pipette tips; (2) Use a pipette to take 50uL of supernatant twice from the centrifuge tube numbered [name + temperature + 2] into 60 orifice plates. Among these 60, 10 is a group, 3 groups are needed at 3 temperatures, plus the control, a total of 60, this operation requires 30 pipette tips; (3) Add 50uL of PBS to the remaining two wells respectively, need to use a gun head;

Step 118, change a new 50uL pipette tip, add 50uL AntibodyCocktail to each well with a pipette, seal the well plate and incubate for 1 hour on a plate shaker set at 400 rpm;

Step 119: After the incubation is completed, change to a new 350uL pipette tip, add 350uL of 1X Wash Buffer PT to each well with a pipette, wash the wells, and then spin the well plate to dry and place it upside down on a clean paper towel , suck up the excess liquid, and perform the above operations 3 times in a row;

Step 120, change a new 100uL pipette tip, add 100uL TMBSubstrate to each well with a pipette, and incubate in the dark on a plate shaker set to 400 rpm for 10 minutes;

Step 121, after the incubation is completed, change to a new 100uL pipette tip, add 100uLStop Solution to each well with a pipette, and shake the well plate on a plate shaker for 1 minute;

Step 122, then place the well plate on a microplate reader to measure the OD value of each well at a wavelength of 450 nm;

Step 123, using the OD value detection result of the standard product and the corresponding concentration to perform curve fitting to obtain a relational formula between the concentration and the OD value, and the IgE detection result of the sample is obtained by adding the OD value into the relational formula.

As shown in Figure 3, it is a summary of the results of the test in Example 3, in which the gray bars from left to right in each test tube corresponding to the abscissa correspond to 18°C, 24°C, and 30°C in turn. As can be seen from the figure, the method described in this Example 3 can detect IgE through the saliva of the experimenter; can detect the change of IgE content due to the change of the ambient temperature of the experimenter .

To sum up, the secretion-based human immunity test method described in the embodiment of the present invention can quantitatively detect the IgE content in the saliva by collecting the saliva of the subject and adopting the double-antibody sandwich ELISA method, independent of In medicine, it is mainly for the detection of IgE content in serum. It is not necessary to collect the blood of the subject during sampling, but only to obtain saliva. Within the normal range of IgE content in the human body, quantitative detection results can characterize the strength of human immunity. , and this test method can measure changes in IgE content due to changes in ambient temperature.

Although the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, they do not limit the scope of protection of the present invention. Those skilled in the art should understand that on the basis of the technical solutions disclosed in the present invention, those skilled in the art do not need to pay Various modifications or deformations that can be made by creative work shall be covered within the protection scope of 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.

Images