CN111781346B - Enzyme-linked immune diluent for whole blood and preparation method and using method thereof - Google Patents
Enzyme-linked immune diluent for whole blood and preparation method and using method thereof Download PDFInfo
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Abstract
The invention discloses an enzyme-linked immune diluent for whole blood, a preparation method and a use method thereof, wherein the diluent consists of disodium hydrogen phosphate, monopotassium phosphate, a surfactant S9, PEG-6000, calcium chloride, glycerol, an anti-rheumatoid factor antibody and a preservative. According to the sample diluent for diluting whole blood, the whole blood is treated by the diluent, so that the influence of the whole blood sample on a detection result can be effectively removed, the background can be effectively reduced, false positives can be removed, the detection sensitivity can be improved, the positive and negative contrast can be increased, and the performance of the sample diluent is obviously superior to that of the serum or plasma diluted by the traditional sample diluent. The diluent can be used for directly diluting whole blood, reduces the process of extracting serum or plasma, is convenient for simplifying operation, can also be used for diluting serum, and has good effect.
Description
Technical Field
The invention relates to the technical field of biological detection reagents, in particular to an enzyme-linked immunosorbent assay diluent for whole blood and a preparation method and a using method thereof.
Background
An enzyme-linked immunosorbent assay kit (ELISA kit) is the most widely applied detection technology in enzyme immunoassay technology. The basic method is to adsorb known antigen or antibody onto the surface of solid phase carrier (polystyrene micro reaction plate), add the substance to be measured, then use enzyme-labeled antigen antibody to react on the surface of solid phase, and wash away the free components in liquid phase by washing method. The enzyme-linked immunoassay kit generally comprises reagents used in the detection process, such as a coating plate, a sample diluent, a washing solution, an enzyme-labeled antibody diluent, a stop solution and the like.
Most of current enzyme-linked immunosorbent assay product detection samples are serum or plasma, and a whole blood sample is rarely used directly, the serum can be separated better after blood coagulation, the blood coagulation is generally placed for several hours or even longer at 4 ℃, and the serum can be obtained after the blood coagulation through centrifugation, so that the process is complicated and time-consuming. And certain components of the whole blood sample can affect the detection result, so that the background is increased, false positive is caused, and the detection sensitivity is reduced. Therefore, there is an urgent need for an enzyme-linked immunodeficient solution for whole blood that reduces the process of extracting serum or plasma, facilitates simplified operation, reduces background, and improves detection sensitivity.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an enzyme-linked immunosorbent assay diluent for whole blood;
a second object of the present invention is to provide a process for the preparation of such a dilution;
a third object of the present invention is to provide a method for using such a diluent.
The purpose of the invention is realized by the following technical scheme: an enzyme-linked immune diluent for whole blood, which comprises the following raw materials:
disodium hydrogen phosphate: 0.4 to 0.8 percent; potassium dihydrogen phosphate: 0.04 to 0.08 percent; surfactant S9:0.05 to 0.1 percent; PEG-6000:0.05 to 0.1 percent; calcium chloride: 0.01 to 0.03 percent; glycerol: 5-10 mL/L; anti-rheumatoid factor antibody: 0.1-0.3 mg/L; preservative: 100-400 mu L/L.
Further, the feed comprises the following raw materials:
disodium hydrogen phosphate: 0.5 to 0.6 percent; potassium dihydrogen phosphate: 0.05 to 0.06 percent; surfactant S9:0.05 to 0.08 percent; PEG-6000:0.05 to 0.08 percent; calcium chloride: 0.01 to 0.02 percent; glycerol (b): 5-8 mL/L; anti-rheumatoid factor antibody: 0.1-0.2 mg/L; preservative: 200-400 mu L/L.
Preferably, the method comprises the following raw materials:
disodium hydrogen phosphate: 0.58 percent; potassium dihydrogen phosphate: 0.052 percent; surfactant S9:0.05 percent; PEG-6000:0.05 percent; calcium chloride: 0.01 percent; glycerol: 5mL/L; anti-rheumatoid factor antibody: 0.2mg/L; preservative: 300 μ L/L.
Further, the anti-rheumatoid factor antibody is a mouse anti-human rheumatoid factor polyclonal antibody.
Further, the preservative is Proclin-300.
The preparation method of the enzyme-linked immune diluent for whole blood comprises the following steps:
s1, weighing: weighing the raw materials according to the formula proportion for later use;
s2, dissolving: adding disodium hydrogen phosphate, potassium dihydrogen phosphate, surfactant S9, PEG-6000 and calcium chloride into pure water for dissolving, and then adding glycerol and an anti-rheumatoid factor antibody;
s3, volume fixing: after the substances are dissolved, adding purified water to a constant volume to a corresponding volume, finally adding the preservative, and storing at 2-8 ℃.
The use method of the enzyme-linked immune diluent for whole blood comprises the following steps:
s1, subpackaging: subpackaging the diluent into EP tubes with 0.5mL per tube;
s2, dilution: collecting whole blood according to a conventional method, and adding 10-50 mu L of whole blood into the subpackaged diluent in the step S1;
s3, precipitation: placing the diluent EP tube added with the whole blood at the temperature of 2-8 ℃ for more than or equal to 1h for natural precipitation;
s4, storage: and (3) if the precipitated sample is placed in an environment of 2-8 ℃ for detection within 5 days, and if the sample is stored for a long time, the supernatant needs to be sucked and loaded into an EP tube, and the sample is placed at-20 ℃ for freezing storage.
In the invention, disodium hydrogen phosphate and potassium dihydrogen phosphate form a phosphate buffer system, so that blood cells stably exist and cannot autolyze; the anti-rheumatoid factor antibody can effectively remove the influence of the rheumatoid factor, the surfactant S9, calcium chloride, glycerol and the like can effectively remove false reaction to reduce background, and the PEG-6000 can effectively promote antigen-antibody reaction and improve the positive detection rate.
The invention has the following advantages: according to the sample diluent for diluting the whole blood, the whole blood (including venous blood and peripheral whole blood) is treated by the diluent, so that the influence of the whole blood sample on a detection result can be effectively removed, meanwhile, the background can be effectively reduced, false positive can be removed, the detection sensitivity is improved, the positive and negative contrast ratio is increased, and the performance of the sample diluent is obviously superior to that of the serum or plasma diluted by the traditional sample diluent. The diluent can be used for directly diluting whole blood, reduces the process of extracting serum or plasma, is convenient for simplifying operation, can also be used for diluting serum, and has good effect.
Detailed Description
The invention is further described below with reference to examples, without limiting the scope of the invention to the following:
example 1: an enzyme-linked immune diluent for whole blood, which comprises the following raw materials:
disodium hydrogen phosphate: 0.4 percent; potassium dihydrogen phosphate: 0.04 percent; surfactant S9:0.05 percent; PEG-6000:0.05 percent; calcium chloride: 0.01 percent; glycerol: 5mL/L; anti-rheumatoid factor antibody: 0.1mg/L; preservative: 100 mu L/L;
wherein the anti-rheumatoid factor antibody is a mouse anti-human rheumatoid factor polyclonal antibody.
Example 2: an enzyme-linked immune diluent for whole blood, which comprises the following raw materials:
disodium hydrogen phosphate: 0.8 percent; potassium dihydrogen phosphate: 0.08 percent; surfactant S9:0.1 percent; PEG-6000:0.1 percent; calcium chloride: 0.03 percent; glycerol (b): 10mL/L; anti-rheumatoid factor antibody: 0.3mg/L; preservative: 400 mu L/L;
wherein the anti-rheumatoid factor antibody is a mouse anti-human rheumatoid factor polyclonal antibody.
Example 3: an enzyme-linked immune diluent for whole blood, which comprises the following raw materials:
disodium hydrogen phosphate: 0.5 percent; potassium dihydrogen phosphate: 0.05 percent; surfactant S9:0.05 percent; PEG-6000:0.05 percent; calcium chloride: 0.01 percent; glycerol (b): 5mL/L; anti-rheumatoid factor antibody: 0.1mg/L; preservative: 200 mu L/L;
wherein the anti-rheumatoid factor antibody is a mouse anti-human rheumatoid factor polyclonal antibody.
Example 4: an enzyme-linked immune diluent for whole blood, which comprises the following raw materials:
disodium hydrogen phosphate: 0.6 percent; potassium dihydrogen phosphate: 0.06 percent; surfactant S9:0.08 percent; PEG-6000:0.08 percent; calcium chloride: 0.02 percent; glycerol (b): 8mL/L; anti-rheumatoid factor antibody: 0.2mg/L; preservative: 400 mu L/L;
wherein the anti-rheumatoid factor antibody is a mouse anti-human rheumatoid factor polyclonal antibody.
Example 5: an enzyme-linked immune diluent for whole blood, which comprises the following raw materials:
disodium hydrogen phosphate: 0.58 percent; potassium dihydrogen phosphate: 0.052 percent; surfactant S9:0.05 percent; PEG-6000:0.05 percent; calcium chloride: 0.01 percent; glycerol (b): 5mL/L; anti-rheumatoid factor antibody: 0.2mg/L; preservative: 300 mu L/L;
wherein the anti-rheumatoid factor antibody is a mouse anti-human rheumatoid factor polyclonal antibody.
Example 6: a preparation method of enzyme-linked immune diluent for whole blood comprises the following steps:
s1, weighing: weighing the raw materials according to the formula proportion in the embodiment 1 for later use;
s2, dissolving: adding disodium hydrogen phosphate, potassium dihydrogen phosphate, surfactant S9, PEG-6000 and calcium chloride into pure water for dissolving, and then adding glycerol and an anti-rheumatoid factor antibody;
s3, volume fixing: after the substances are dissolved, adding purified water to a constant volume to a corresponding volume, finally adding the preservative, and storing at 2-8 ℃.
Example 7: a preparation method of enzyme-linked immune diluent for whole blood comprises the following steps:
s1, weighing: weighing the raw materials according to the formula proportion of the embodiment 3 for later use;
s2, dissolving: adding disodium hydrogen phosphate, potassium dihydrogen phosphate, surfactant S9, PEG-6000 and calcium chloride into pure water for dissolving, and then adding glycerol and an anti-rheumatoid factor antibody;
s3, volume fixing: after the substances are dissolved, adding purified water to a constant volume to a corresponding volume, finally adding the preservative, and storing at 2-8 ℃.
Example 8: a preparation method of enzyme-linked immune diluent for whole blood comprises the following steps:
s1, weighing: weighing the raw materials according to the formula proportion of the embodiment 4 for later use;
s2, dissolving: adding disodium hydrogen phosphate, potassium dihydrogen phosphate, surfactant S9, PEG-6000 and calcium chloride into pure water for dissolving, and then adding glycerol and an anti-rheumatoid factor antibody;
s3, constant volume: after the substances are dissolved, adding purified water to a constant volume, finally adding a preservative, and storing at 2-8 ℃.
Example 9: a method of using an enzyme-linked immunodiluent for whole blood comprising the steps of:
s1, subpackaging: the diluent prepared in example 6 was dispensed into EP tubes at 0.5 mL/tube;
s2, dilution: collecting whole blood according to a conventional method, and adding 10 mu L of whole blood into the diluent subpackaged in the step S1;
s3, precipitation: placing the dilution EP tube added with the whole blood at 2-8 ℃ for 1h for natural precipitation;
s4, storage: and (4) placing the precipitated sample in an environment of 2-8 ℃ for 5 days for detection.
Example 10: a method of using an enzyme-linked immunodiluent for whole blood comprising the steps of:
s1, subpackaging: the diluent prepared in example 7 was dispensed into EP tubes at 0.5 mL/tube;
s2, dilution: collecting whole blood according to a conventional method, and adding 50 mu L of whole blood into the diluent subpackaged in the step S1;
s3, precipitation: placing the dilution EP tube added with the whole blood at the temperature of between 2 and 8 ℃ for 1.5 hours for natural precipitation;
s4, storage: the supernatant fluid is required to be sucked and put into an EP tube, and the EP tube is frozen and stored at the temperature of minus 20 ℃.
Example 11: a method of using an enzyme-linked immunodiluent for whole blood comprising the steps of:
s1, subpackaging: the dilution prepared in example 8 was dispensed into EP tubes at 0.5 mL/tube;
s2, dilution: collecting whole blood according to a conventional method, and adding 30 mu L of whole blood into the diluent subpackaged in the step S1;
s3, precipitation: placing the diluent EP tube added with the whole blood at 2-8 ℃ for 2h for natural precipitation;
s4, storage: and (4) placing the precipitated sample in an environment of 2-8 ℃ for 5 days for detection.
The following experiments illustrate the beneficial effects of the present invention:
1. preparation of sample dilutions
Weighing 5.8g of disodium hydrogen phosphate, 0.52g of potassium dihydrogen phosphate, 0.5g of surfactant S9,0.5g of macromolecular polymer PEG-6000 and 0.1g of calcium chloride, dissolving the materials in purified water, adding 5mL of glycerol and 0.1mg of anti-internal-rheumatism factor, adding the purified water to the solution to reach a constant volume of 1L after the solution is dissolved, finally adding 200 mu L of preservative Proclin-300, and storing the solution at the temperature of between 2 and 8 ℃.
2. Use of the Diluent
1. The prepared sample diluent can be subpackaged into an EP tube with 0.5mL per tube and stored in a refrigerator at 2-8 ℃.
2. Collecting whole blood (such as venous blood or fingertip blood) by conventional method, adding 10-50 μ L of whole blood into the sample diluted solution, and diluting at a ratio substantially equal to that of serum.
3. The sample diluent EP tube with whole blood added was placed on an EP tube stand and placed in a refrigerator.
4. After being placed for 1 hour, the whole blood cells basically sink to the bottom of the tube, and the blood cells are basically separated by the natural sedimentation method, and the detected soluble antibodies (such as IgG, igM, igA and the like) and related proteins are uniformly dissolved in the buffer solution.
5. Due to the addition of the preservative, the detection of the sample has no obvious difference within 5 days at the temperature of 2-8 ℃. If long-term storage is required, the supernatant can be sucked and put into another EP tube for long-term cryopreservation at the temperature of minus 20 ℃.
3. Detecting the effect
The commonly used sample diluent is PBS buffer containing Tween-20. The method comprises the following steps of diluting serum or plasma by 1. The effects of dilution of serum or plasma with sample dilutions include: the target components in the sample can be more effectively diffused in the buffer solution to facilitate the reaction by thinning the sample; reducing background and false positive reaction; the detection effect of the effective substances is improved, and the negative and positive contrast is increased.
However, the traditional diluent often has the problems of high background, individual false positive reaction, undesirable positive detection result and the like after whole blood is diluted.
The detection effects of the two diluents are compared, the serum and the whole blood are respectively diluted by the two diluents, and then 10 positive samples and 10 negative samples are detected, and the results are compared. The substances detected were respectively: 1. human blood antibodies to mycobacterium tuberculosis IgG. 2. Human blood troponin (cTnI). 3. Alpha-fetoprotein (AFP) in human blood. The process and results are as follows:
1. comparison of IgG antibodies against Mycobacterium tuberculosis in human blood by dilution and detection
1) The detection principle of the IgG antibody of the mycobacterium tuberculosis in human blood by adopting an enzyme-linked immunosorbent assay is as follows: the method comprises the steps that tuberculosis proteins TB-SA with a certain concentration are coated on a microporous plate, a sample is diluted by a diluent and then added into the microporous plate, incubation is carried out at 37 ℃ for 10min, if anti-tuberculosis proteins TB-SA antibodies exist in the sample, corresponding antibodies IgG can be specifically combined with the TB-SA on the enzyme label plate and firmly attached to the enzyme label plate, an enzyme-labeled secondary antibody is added into the microporous plate after washing, incubation and washing are carried out, and finally a color developing agent is added, so that the existence and the content of the antibodies are judged through color development.
In the whole process, the components are the same except for the sample diluent, so that the effects of the sample diluent are compared.
2) Samples of 10 tuberculosis patients and 10 health examinees were collected, including serum and peripheral whole blood. The dilution was carried out with two dilutions as shown in table 1:
table 1: dilution of human blood for IgG antibody against Mycobacterium tuberculosis
And placing the diluted serum and peripheral whole blood samples in a refrigerator at the temperature of 2-8 ℃ for 1 hour for detection.
After the peripheral whole blood is diluted by the sample diluent prepared by the method, the red blood cells basically sink to the bottom of the tube, and the upper part of the tube is clear and transparent. After peripheral whole blood was diluted with PBS buffer containing 0.05% Tween-20, red blood cells were also dispersed in the solution, and the solution was slightly red.
Adding 100 mu L of supernatant into the coated microporous plate, and detecting according to a detection process, wherein the detection results are shown in Table 2:
table 2: results of detection of Mycobacterium tuberculosis IgG antibody in human blood
Note: the test results showed that the test was positive when the OD value was 0.3 or more and negative when the OD value was less than 0.3. The statistics are shown in table 3:
table 3: statistical results for detecting human blood mycobacterium tuberculosis IgG antibodies
From the detection results, it can be seen that:
1) Compared with the traditional diluent, the diluent has improved detection rate in terms of dilution of serum or whole blood.
2) On the effect of whole blood dilution detection, the traditional diluent has larger interference, which is reflected in that the detection rate of negative and positive is not high, and the discrimination of negative and positive is greatly reduced. The diluent can remove the influences, the detection rate of negative and positive is higher than the result of diluting serum by the traditional diluent, and the discrimination of negative and positive is improved.
3) After the diluent is used for diluting the serum and the whole blood, the detection effect has no obvious difference, the background seems to be slightly low, and the discrimination is better.
2. Dilution and detection of human blood troponin (cTnI) comparison
1) The method for detecting human blood central troponin (cTnI) by using a double monoclonal antibody sandwich method has the following detection principle: the microplate is coated with mouse anti-human cardiac troponin (cTnI) monoclonal antibody IgG with a certain concentration, a blood sample is diluted by a diluent and then added into the microplate, the incubation is carried out for 30min at 37 ℃, if the sample contains the human cardiac troponin (cTnI), the corresponding cardiac troponin (cTnI) can be specifically combined with the mouse anti-human cardiac troponin (cTnI) monoclonal antibody IgG on the microplate and firmly attached to the microplate, after washing, the other monoclonal antibody IgG of the enzyme-labeled mouse anti-human cardiac troponin (cTnI) is added into the microplate, after the incubation and the washing, finally, a color developing agent is added, and the existence and the content of the antibody are judged by color development.
In the whole process, the components are the same except for the sample diluent, so that the effects of the sample diluent are compared.
2) Samples of 10 patients with myocardial infarction and 10 healthy subjects, including serum and peripheral whole blood, were collected. The dilution was carried out with two dilutions as shown in table 4:
table 4: dilution of human blood-central troponin
The diluted serum and peripheral whole blood samples were placed in a refrigerator at 2-8 ℃ for 1 hour for testing.
After the peripheral whole blood is diluted by the sample diluent prepared by the method, the red blood cells basically sink to the bottom of the tube, and the upper part of the tube is clear and transparent. After peripheral whole blood was diluted with PBS buffer containing 0.05% Tween-20, red blood cells were also dispersed in the solution, and the solution was slightly red.
100 mul of supernatant was added to the coated plate and the assay was performed according to the assay protocol, with the results shown in table 5:
table 5: results of detection of cardiac troponin in human blood
Note: positive values were determined by measuring the OD value at 0.25 or more, and negative values were determined by measuring the OD value at 0.25 or less.
Statistics are shown in table 6:
table 6: statistical results of troponin detection in human blood
From the detection results, it can be seen that:
1) Compared with the traditional diluent, the total detection rate of the diluent, regardless of the diluted serum or whole blood, is not lower than the traditional serum dilution effect.
2) In the effect of whole blood dilution detection, the traditional diluent has larger interference, the high value of the positive is not enough, the low value of the positive is not enough, a sample is negative in other dilutions and positive in the dilution, a non-specific reaction is required to cause background rise and positive, and the negative detection result shows that the whole blood diluted by the traditional diluent causes false positive caused by two non-specific reactions.
3) From the contrast (P/N) of the detection, the diluent has no obvious difference in detection effect after diluting the serum and the whole blood, but has better discrimination.
4) When the conventional diluent is used for diluting serum, the number of positive and negative detections is not different, but the contrast ratio (P/N) of the detection is slightly poor, and when whole blood is diluted, more serious interference occurs.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and the inventive concept within the technical scope of the present invention.
Claims (5)
1. An enzyme-linked immune diluent for whole blood, which is characterized by comprising the following raw materials:
disodium hydrogen phosphate: 0.58 percent; potassium dihydrogen phosphate: 0.052 percent; surfactant S9:0.05 percent; PEG-6000:0.05 percent; calcium chloride: 0.01 percent; glycerol: 5mL/L; anti-rheumatoid factor antibody: 0.2mg/L; preservative: 300 μ L/L.
2. The ELISA diluent of claim 1 wherein the anti-rheumatoid factor antibody is a mouse anti-human rheumatoid factor polyclonal antibody.
3. The ELISA diluent of claim 1 wherein the preservative is Proclin-300.
4. A method of preparing an enzyme-linked immunodiluent for whole blood according to any of claims 1-3, comprising the steps of:
s1, weighing: weighing the raw materials according to the formula proportion for later use;
s2, dissolving: adding disodium hydrogen phosphate, potassium dihydrogen phosphate, surfactant S9, PEG-6000 and calcium chloride into pure water for dissolving, and then adding glycerol and an anti-rheumatoid factor antibody;
s3, constant volume: after the substances are dissolved, adding purified water to a constant volume, finally adding a preservative, and storing at 2-8 ℃.
5. Method of use of an enzyme-linked immunodeficient diluent for whole blood for non-diagnostic purposes according to any of claims 1-3, characterized in that it comprises the following steps:
s1, subpackaging: subpackaging the diluent into EP tubes, wherein each tube is 0.5 mL;
s2, dilution: collecting whole blood according to a conventional method, and adding 10-50 mu L of whole blood into the diluent subpackaged in the step S1;
s3, precipitation: placing the diluent EP tube added with the whole blood at the temperature of 2-8 ℃ for more than or equal to 1h for natural precipitation;
s4, storage: and (3) if the precipitated sample is placed in an environment of 2-8 ℃ for detection within 5 days, and if the sample is stored for a long time, the supernatant needs to be sucked and filled into an EP tube, and the sample is frozen and stored at the temperature of-20 ℃.
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