CN112858687B - Serum amyloid A detection reagent and preparation method thereof - Google Patents
Serum amyloid A detection reagent and preparation method thereof Download PDFInfo
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- CN112858687B CN112858687B CN202011620572.1A CN202011620572A CN112858687B CN 112858687 B CN112858687 B CN 112858687B CN 202011620572 A CN202011620572 A CN 202011620572A CN 112858687 B CN112858687 B CN 112858687B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a serum amyloid A detection reagent and a preparation method thereof, comprising a reagent 1 and a reagent 2; the composition and concentration of reagent 1 are as follows: acetic acid buffer: 100-300mM; sodium chloride: 10-20g/L; magnesium sulfate: 10-20g/L; polyethylene glycol disuccinimide acid: 2-4g/L; lauroyl sarcosine isopropyl ester: 1-5g/L; niu Tuoyang sodium cholate: 0.1-0.5g/L; sodium deoxycholate: 1-5g/L; sodium azide: 0.1g/L; the solvent is purified water, and the PH of the reagent 1 is 5.5-6.0; the composition and concentration of reagent 2 are as follows: piperazine-1, 4-diethyl sulfonic acid buffer: 50-200mM; coating latex particles with rabbit anti-human serum amyloid A antibody: 1-3%; sodium starch octenyl succinate: 1-5g/L; polyvinylpyrrolidone: 2-5g/L; sodium alginate: 2-5g/L; sodium azide: 0.1g/L; the solvent is purified water, and the PH of the reagent 2 is 6.5-7.5. The reagent provided by the invention has the advantages of wide reporting range and good stability, and can effectively inhibit interference of rheumatoid factors.
Description
Technical Field
The invention relates to the field of analysis and detection, in particular to a serum amyloid A detection reagent and a preparation method thereof.
Background
Serum Amyloid A (SAA), an acute phase response protein, is markedly elevated 4 hours at the very fast time when the body is infected, and rapidly decreases to normal levels after pathogen clearance. SAA has very high sensitivity to virus infection, and is commonly used for early diagnosis, differential diagnosis, disease condition monitoring, prognosis evaluation and other aspects of infectious diseases in clinic at present.
The detection methods of SAA in the current market include enzyme-linked immunosorbent assay, radioimmunoassay, immunochromatography, turbidimetry and the like. The enzyme-linked immunosorbent assay is complex in operation, time-consuming and low in automation level. Radioimmunoassay is characterized by high specificity and high sensitivity, but is also subject to potential radioactive contamination and is therefore increasingly unacceptable. Because immunochromatography is limited by the methodology of the immunochromatography, qualitative diagnosis can be achieved generally, quantitative requirements of doctors cannot be met, and in addition, because the test paper is difficult to achieve uniform and consistent structure, the material, thickness and density degree are difficult to be completely the same, so that the repeatability of a sample detection result on different test cards is poor. Compared with an immunochromatography method, the latex-enhanced immunoturbidimetry method has higher sensitivity and accuracy, belongs to a derivative technology of a common immunoturbidimetry technology, overcomes the defect that the signal quantity of the common immunoturbidimetry technology is very limited, increases the volume of an antibody by orders of magnitude, and greatly improves the detectability, but the method has the defects that the method is easy to be interfered by a foreign antibody existing in a test sample, particularly by rheumatoid factors, so that false positive results are caused; in addition, the particle size of the latex microsphere also has a great influence on the testing performance of the reagent, and the particle size is too large, so that the small ratio of the surface area of the microsphere to the volume of the latex microsphere is easy to cause small antibody coating amount in unit volume, so that the antigen is excessive, the analyte with high concentration cannot be detected, the ratio of the surface area of the latex to the volume of the latex is large, the aggregation phenomenon is not easy to generate, the absorbance change is slow, and the detection sensitivity of the reagent is reduced.
Disclosure of Invention
The invention aims to provide a latex immunoturbidimetry SAA detection reagent which has high detection sensitivity, wide reporting range and good stability and can effectively inhibit interference of rheumatoid factors and a preparation method thereof.
The invention provides the following technical scheme:
an SAA detection reagent and a preparation method thereof, comprising a reagent 1 and a reagent 2;
the composition and concentration of reagent 1 are as follows:
acetic acid buffer: 100-300mM
Sodium chloride: 10-20g/L
Magnesium sulfate: 10-20g/L
Polyethylene glycol disuccinimide acid: 2-4g/L
Lauroyl sarcosine isopropyl ester: 1-5g/L
Niu Tuoyang sodium cholate: 0.1-0.5g/L
Sodium deoxycholate: 1-5g/L
Sodium azide: 0.1g/L
The solvent is purified water, and the PH of the reagent 1 is 5.5-6.0;
the composition and concentration of reagent 2 are as follows:
piperazine-1, 4-diethyl sulfonic acid buffer: 50-200mM
Rabbit anti-human SAA antibodies coat latex particles: 1-3%
Sodium starch octenyl succinate: 1-5g/L
Polyvinylpyrrolidone: 2-5g/L
Sodium alginate: 2-5g/L
Sodium azide: 0.1g/L
The solvent is purified water, and the PH of the reagent 2 is 6.5-7.5.
Preferably, the rabbit anti-human SAA antibody coating latex particles in the reagent 2 are a mixture of two latex particles, and the larger diameter particle size is 180-200 nm; the particle diameter of the latex particles with smaller diameter is 30 nm-40 nm;
preferably, the number of small diameter latex particles to the number of large diameter latex particles = 1:7.
The preparation method of the SAA detection reagent specifically comprises the following steps:
s1, preparation of a reagent 1:
adding sodium chloride, magnesium sulfate, polyethylene glycol disuccinimide acid, isopropyl lauroyl sarcosinate, niu Tuoyang sodium cholate, sodium deoxycholate and sodium azide into an acetic acid buffer solution in sequence according to the component amount of the reagent 1, fully stirring and uniformly mixing, adding solvent water, continuously stirring and uniformly mixing, regulating the pH value of the solution to 5.5-6.0, and filtering the solution by using a microporous filter membrane to obtain filtrate, namely the reagent 1;
s2, preparation of a reagent 2:
s21, sequentially adding sodium starch octenyl succinate, polyvinylpyrrolidone, sodium alginate and sodium azide into a piperazine-1, 4-diethyl sulfonic acid buffer solution, uniformly stirring, adding small-diameter SAA antibody latex particles, ultrasonically vibrating for 30-60 minutes, adding solvent water, continuously stirring for 30-60 minutes, filtering by using a microporous filter membrane, and preserving filtrate.
S22, dripping the SAA antibody latex particles with the same volume and large diameter into the S21 liquid, and uniformly mixing after ultrasonic vibration for 2-4 hours to obtain a mixed liquid 1; then dripping SAA antibody latex particles with large diameter, which are equal to the volume of the mixed solution 1, and carrying out ultrasonic vibration for 2-4 hours until all the SAA antibody latex particles are uniformly mixed to obtain a mixed solution 2; and then dripping SAA antibody latex particle liquid with the same volume as the mixed liquid 2 and large diameter, and carrying out ultrasonic vibration for 2-4 hours until all the SAA antibody latex particle liquid and the mixed liquid are uniformly mixed, thus obtaining a reagent 2 solution.
Preferably, the test conditions for the detection of SAA in a sample with the detection reagent are as follows: temperature: 37 ℃; the cuvette optical path was 1.0cm. The main wavelength was 546nm and the sub-wavelength was 700nm.
Preferably, the method for determining SAA in a sample with the reagent is as follows: after the sample is uniformly mixed with the reagent 1, placing at 37 ℃ for incubation for 5 minutes, then adding the reagent 2, uniformly mixing, incubating at 37 ℃ for 30 seconds, reading absorbance A1, continuously incubating for 4 minutes and 30 seconds, and reading absorbance A2; Δa=a2-A1. Wherein the amount of the sample is 2. Mu.l, the amount of the reagent 1 is 200. Mu.l, and the amount of the reagent 2 is 40. Mu.l.
The invention has the beneficial effects that:
(1) The combined application of Niu Tuoyang sodium cholate, deoxysodium cholate and lauroyl sarcosine isopropyl ester is beneficial to dissociating the nonspecific combination between the rheumatoid factor and the protein and weakening the interference of the rheumatoid factor on the detection result.
(2) The combined application of sodium chloride and magnesium sulfate is helpful for promoting and maintaining the stability of a sample and a reaction system and preventing the turbidity of the system.
(3) The rabbit anti-human SAA antibody coating latex particles in the reagent 2 are a mixture of two types of latex particles, so that the reagent has low blank absorbance, high sensitivity and large reporting range; and the equal incremental method is adopted for mixing operation, so that two kinds of latex particles can be uniformly distributed, and the detection stability of the reagent is good.
(4) The combined application of the sodium starch octenyl succinate, the polyvinylpyrrolidone and the sodium alginate in the reagent 2 is beneficial to promoting and maintaining the stability of the reagent 2 and improving the stability of the reagent.
Detailed Description
Example 1:
an SAA detection reagent and a preparation method thereof, comprising a reagent 1 and a reagent 2; the composition and concentration of reagent 1 are as follows:
acetic acid buffer: 100mM; sodium chloride: 10g/L; magnesium sulfate: 10g/L; polyethylene glycol disuccinimide acid: 2g/L; lauroyl sarcosine isopropyl ester: 1g/L; niu Tuoyang sodium cholate: 0.1g/L; sodium deoxycholate: 1g/L; sodium azide: 0.1g/L; the solvent is purified water, and the PH of the reagent 1 is 5.5; the composition and concentration of reagent 2 are as follows: piperazine-1, 4-diethyl sulfonic acid buffer: 50mM; rabbit anti-human SAA antibodies coat latex particles: 1%; sodium starch octenyl succinate: 1g/L; polyvinylpyrrolidone: 2g/L; sodium alginate: 2g/L; sodium azide: 0.1g/L; the solvent was purified water and reagent 2 had a pH of 6.5. The rabbit anti-human SAA antibody coating latex particles in the reagent 2 are a mixture of two latex particles, and the particle diameter of the larger diameter is 180nm; the particle size of the smaller diameter latex particles is 30nm; small diameter latex particle number: large diameter latex particle number = 1:7.
The preparation method of the SAA detection reagent specifically comprises the following steps:
s1, preparation of a reagent 1:
adding sodium chloride, magnesium sulfate, polyethylene glycol disuccinimide acid, isopropyl lauroyl sarcosinate, niu Tuoyang sodium cholate, sodium deoxycholate and sodium azide into an acetic acid buffer solution in sequence according to the component amount of the reagent 1, fully stirring and uniformly mixing, adding solvent water, continuously stirring and uniformly mixing, regulating the pH value of the solution to 5.5, and filtering the solution by using a microporous filter membrane to obtain filtrate, namely the reagent 1;
s2, preparation of a reagent 2:
s21, sequentially adding sodium starch octenyl succinate, polyvinylpyrrolidone, sodium alginate and sodium azide into a piperazine-1, 4-diethyl sulfonic acid buffer solution, uniformly stirring, adding small-diameter SAA antibody latex particles, ultrasonically vibrating for 30 minutes, adding solvent water, continuously stirring for 30 minutes, filtering by using a microporous filter membrane, and preserving filtrate.
S22, dripping SAA antibody latex particles with the same volume and large diameter into the S21 liquid, and uniformly mixing after ultrasonic vibration for 2 hours to obtain a mixed liquid 1; then dripping SAA antibody latex particles with large diameter, which are equal to the volume of the mixed solution 1, and carrying out ultrasonic vibration for 2 hours until all the SAA antibody latex particles are uniformly mixed to obtain a mixed solution 2; and then dripping SAA antibody latex particle liquid with large diameter, which is equal to the volume of the mixed liquid 2, and carrying out ultrasonic vibration for 2 hours until all the SAA antibody latex particle liquid and the mixed liquid are uniformly mixed, thus obtaining the reagent 2 solution.
The test conditions for the detection of SAA in a sample by the detection reagent are as follows: temperature: 37 ℃; the cuvette optical path was 1.0cm. The main wavelength was 546nm and the sub-wavelength was 700nm. The method for determining SAA in a sample by using the reagent comprises the following steps: after the sample is uniformly mixed with the reagent 1, placing at 37 ℃ for incubation for 5 minutes, then adding the reagent 2, uniformly mixing, incubating at 37 ℃ for 30 seconds, reading absorbance A1, continuously incubating for 4 minutes and 30 seconds, and reading absorbance A2; Δa=a2-A1. Wherein the amount of the sample is 2. Mu.l, the amount of the reagent 1 is 200. Mu.l, and the amount of the reagent 2 is 40. Mu.l.
Example 2:
an SAA detection reagent and a preparation method thereof, comprising a reagent 1 and a reagent 2; the composition and concentration of reagent 1 are as follows:
acetic acid buffer: 300mM; sodium chloride: 20g/L; magnesium sulfate: 20g/L; polyethylene glycol disuccinimide acid: 4g/L; lauroyl sarcosine isopropyl ester: 5g/L; niu Tuoyang sodium cholate: 0.5g/L; sodium deoxycholate: 5g/L; sodium azide: 0.1g/L; the solvent is purified water, and the PH of the reagent 1 is 6.0; the composition and concentration of reagent 2 are as follows: piperazine-1, 4-diethyl sulfonic acid buffer: 200mM; rabbit anti-human SAA antibodies coat latex particles: 3%; sodium starch octenyl succinate: 5g/L; polyvinylpyrrolidone: 5g/L; sodium alginate: 5g/L; sodium azide: 0.1g/L; the solvent was purified water and reagent 2 had a pH of 7.5. The rabbit anti-human SAA antibody coating latex particles in the reagent 2 are a mixture of two latex particles, and the particle diameter of the larger diameter is 200nm; the smaller diameter latex particles have a particle size of 40nm; small diameter latex particle number: large diameter latex particle number = 1:7.
The preparation method of the SAA detection reagent specifically comprises the following steps:
s1, preparation of a reagent 1:
adding sodium chloride, magnesium sulfate, polyethylene glycol disuccinimide acid, isopropyl lauroyl sarcosinate, niu Tuoyang sodium cholate, sodium deoxycholate and sodium azide into an acetic acid buffer solution in sequence according to the component amount of the reagent 1, fully stirring and uniformly mixing, adding solvent water, continuously stirring and uniformly mixing, regulating the pH value of the solution to 6.0, and filtering the solution by using a microporous filter membrane to obtain filtrate, namely the reagent 1;
s2, preparation of a reagent 2:
s21, sequentially adding sodium starch octenyl succinate, polyvinylpyrrolidone, sodium alginate and sodium azide into a piperazine-1, 4-diethyl sulfonic acid buffer solution, uniformly stirring, adding small-diameter SAA antibody latex particles, ultrasonically vibrating for 60 minutes, adding solvent water, continuously stirring for 60 minutes, filtering by using a microporous filter membrane, and preserving filtrate.
S22, dripping SAA antibody latex particles with the same volume and large diameter into the S21 liquid, and uniformly mixing after ultrasonic vibration for 4 hours to obtain a mixed liquid 1; then dripping SAA antibody latex particles with large diameter, which are equal to the volume of the mixed solution 1, and carrying out ultrasonic vibration for 4 hours until all the SAA antibody latex particles are uniformly mixed to obtain a mixed solution 2; and then dripping SAA antibody latex particle liquid with large diameter, which is equal to the volume of the mixed liquid 2, and carrying out ultrasonic vibration for 4 hours until all the SAA antibody latex particle liquid and the mixed liquid are uniformly mixed, thus obtaining the reagent 2 solution.
The test conditions for the detection of SAA in a sample by the detection reagent are as follows: temperature: 37 ℃; the cuvette optical path was 1.0cm. The main wavelength was 546nm and the sub-wavelength was 700nm. The method for determining SAA in a sample by using the reagent comprises the following steps: after the sample is uniformly mixed with the reagent 1, placing at 37 ℃ for incubation for 5 minutes, then adding the reagent 2, uniformly mixing, incubating at 37 ℃ for 30 seconds, reading absorbance A1, continuously incubating for 4 minutes and 30 seconds, and reading absorbance A2; Δa=a2-A1. Wherein the amount of the sample is 2. Mu.l, the amount of the reagent 1 is 200. Mu.l, and the amount of the reagent 2 is 40. Mu.l.
Example 3:
an SAA detection reagent and a preparation method thereof, comprising a reagent 1 and a reagent 2; the composition and concentration of reagent 1 are as follows: acetic acid buffer: 200mM; sodium chloride: 15g/L; magnesium sulfate: 15g/L; polyethylene glycol disuccinimide acid: 3g/L; lauroyl sarcosine isopropyl ester: 3g/L; niu Tuoyang sodium cholate: 0.3g/L; sodium deoxycholate: 3g/L; sodium azide: 0.1g/L; the solvent is purified water, and the PH of the reagent 1 is 5.7; the composition and concentration of reagent 2 are as follows: piperazine-1, 4-diethyl sulfonic acid buffer: 125mM; rabbit anti-human SAA antibodies coat latex particles: 2%; sodium starch octenyl succinate: 3g/L; polyvinylpyrrolidone: 3.5g/L; sodium alginate: 3.5g/L; sodium azide: 0.1g/L; the solvent was purified water and reagent 2 had a pH of 7. The rabbit anti-human SAA antibody coating latex particles in the reagent 2 are a mixture of two latex particles, and the particle diameter of the larger diameter is 190nm; the particle size of the smaller diameter latex particles is 35nm; small diameter latex particle number: large diameter latex particle number = 1:7.
The preparation method of the SAA detection reagent specifically comprises the following steps:
s1, preparation of a reagent 1:
adding sodium chloride, magnesium sulfate, polyethylene glycol disuccinimide acid, isopropyl lauroyl sarcosinate, niu Tuoyang sodium cholate, sodium deoxycholate and sodium azide into an acetic acid buffer solution in sequence according to the component amount of the reagent 1, fully stirring and uniformly mixing, adding solvent water, continuously stirring and uniformly mixing, regulating the pH value of the solution to 5.7, and filtering the solution by using a microporous filter membrane to obtain filtrate, namely the reagent 1;
s2, preparation of a reagent 2:
s21, sequentially adding sodium starch octenyl succinate, polyvinylpyrrolidone, sodium alginate and sodium azide into a piperazine-1, 4-diethyl sulfonic acid buffer solution, uniformly stirring, adding small-diameter SAA antibody latex particles, ultrasonically vibrating for 45 minutes, adding solvent water, continuously stirring for 45 minutes, filtering by using a microporous filter membrane, and preserving filtrate.
S22, dripping SAA antibody latex particles with the same volume and large diameter into the S21 liquid, and uniformly mixing after ultrasonic vibration for 3 hours to obtain a mixed liquid 1; then dripping SAA antibody latex particles with large diameter, which are equal to the volume of the mixed solution 1, and carrying out ultrasonic vibration for 3 hours until all the SAA antibody latex particles are uniformly mixed to obtain a mixed solution 2; and then dripping SAA antibody latex particle liquid with large diameter, which is equal to the volume of the mixed liquid 2, and carrying out ultrasonic vibration for 3 hours until all the SAA antibody latex particle liquid and the mixed liquid are uniformly mixed, thus obtaining the reagent 2 solution.
The test conditions for the detection of SAA in a sample by the detection reagent are as follows: temperature: 37 ℃; the cuvette optical path was 1.0cm. The main wavelength was 546nm and the sub-wavelength was 700nm. The method for determining SAA in a sample by using the reagent comprises the following steps: after the sample is uniformly mixed with the reagent 1, placing at 37 ℃ for incubation for 5 minutes, then adding the reagent 2, uniformly mixing, incubating at 37 ℃ for 30 seconds, reading absorbance A1, continuously incubating for 4 minutes and 30 seconds, and reading absorbance A2; Δa=a2-A1. Wherein the amount of the sample is 2. Mu.l, the amount of the reagent 1 is 200. Mu.l, and the amount of the reagent 2 is 40. Mu.l.
The performance of the kits obtained in examples 1 to 3 of the present invention will be described below with reference to the tables.
1. Blank absorbance
The test of the reagent with distilled water shows that the absorbance of the reagent blank of the technology is obviously smaller than that of the reagent of the conventional technology on the market, which indicates that the reagent blank of the technology is lower.
2. Analytical sensitivity test
The reagent was tested with a sample at a concentration of 20mg/L and repeated 2 times, and the change in absorbance at the prescribed parameters of the reagent was recorded, and the average value of the difference was calculated. The results show that the absorbance difference of the test of the technical reagent of the invention is obviously larger than that of the conventional technical reagent sold in the market, thus indicating that the analysis sensitivity of the technical reagent of the invention is better.
HOOK Effect evaluation
And dissolving standard substances by using normal saline, adding the standard substances into high-value samples, respectively preparing high-value samples with 4 different concentrations, such as high value in a linear range, high value in the linear range of 1.25 times, high value in the linear range of 1.5 times, high value in the linear range of 2 times, and the like, repeatedly measuring the high-value samples, wherein the concentration with reduced absorbance is the inflection point value of the hook effect, and determining the concentration of the highest point of the hook effect of the reagent according to the high-value samples. The results show that the commercial conventional technical reagent showed a lower concentration of absorbance than the technical reagent of the present invention, indicating a broader reportable range of the technical reagent of the present invention.
4. Precision test
Under the condition of repeatability, the technical reagent of the invention is tested by using a low-value quality control (30.7 mg/L) and a high-value quality control (80.6 mg/L), the test is repeated for 10 times, and the average value of the measured values is calculated respectivelyAnd standard deviation(s). The Coefficient of Variation (CV) was calculated as follows.
The result shows that the variation coefficients of the quality control products with low and high values are respectively 1.63% and 1.00% by the detection reagent test of the technology, which shows that the detection stability of the reagent of the invention is good.
From the above results, it is clear that the SAA detection reagent of the present invention has the advantages of low blank absorbance, high sensitivity, large reportable range and good test stability.
The above is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that the present invention is described in detail with reference to the foregoing embodiments, and modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. A serum amyloid a detection reagent, characterized in that: comprising reagent 1 and reagent 2;
the composition and concentration of reagent 1 are as follows:
acetic acid buffer: 100-300mM
Sodium chloride: 10-20g/L
Magnesium sulfate: 10-20g/L
Polyethylene glycol disuccinimide acid: 2-4g/L
Lauroyl sarcosine isopropyl ester: 1-5g/L
Niu Tuoyang sodium cholate: 0.1-0.5g/L
Sodium deoxycholate: 1-5g/L
Sodium azide: 0.1g/L
The solvent is purified water, and the PH of the reagent 1 is 5.5-6.0;
the composition and concentration of reagent 2 are as follows:
piperazine-1, 4-diethyl sulfonic acid buffer: 50-200mM
Coating latex particles with rabbit anti-human serum amyloid A antibody: 1-3%
Sodium starch octenyl succinate: 1-5g/L
Polyvinylpyrrolidone: 2-5g/L
Sodium alginate: 2-5g/L
Sodium azide: 0.1g/L
The solvent is purified water, and the PH of the reagent 2 is 6.5-7.5;
the rabbit anti-human serum amyloid A antibody coating latex particles in the reagent 2 are a mixture of two latex particles, and the larger diameter particle size is 180 nm-200 nm; the particle size of the latex particles with smaller diameter is 30 nm-40 nm.
2. A serum amyloid a detection reagent according to claim 1 wherein: small diameter latex particle number: large diameter latex particle number = 1:7.
3. The method for preparing the serum amyloid a detection reagent according to claim 1, which is characterized by comprising the following specific steps: s1, preparation of a reagent 1:
adding sodium chloride, magnesium sulfate, polyethylene glycol disuccinimide acid, isopropyl lauroyl sarcosinate, niu Tuoyang sodium cholate, sodium deoxycholate and sodium azide into an acetic acid buffer solution in sequence according to the component amount of the reagent 1, fully stirring and uniformly mixing, adding solvent water, continuously stirring and uniformly mixing, adjusting the pH of the solution to 5.5-6.0, and filtering the solution by using a microporous filter membrane to obtain filtrate, namely the reagent 1;
s2, preparation of a reagent 2:
s21, sequentially adding sodium starch octenyl succinate, polyvinylpyrrolidone, sodium alginate and sodium azide into piperazine-1, 4-diethyl sulfonic acid buffer solution, uniformly stirring, adding small-diameter serum amyloid A antibody latex particles, ultrasonically vibrating for 30-60 minutes, adding solvent water, continuously stirring for 30-60 minutes, filtering by using a microporous filter membrane, and preserving filtrate;
s22, dripping equal-volume large-diameter serum amyloid A antibody latex particles into the solution S21, and uniformly mixing after ultrasonic vibration for 2-4 hours to obtain a mixed solution 1; dripping large-diameter serum amyloid A antibody latex particles which are equal to the volume of the mixed solution 1, and performing ultrasonic vibration for 2-4 hours until all the particles are uniformly mixed to obtain a mixed solution 2; and then dripping SAA antibody latex particle liquid with the same volume as the mixed liquid 2 and large diameter, and carrying out ultrasonic vibration for 2-4 hours until all the SAA antibody latex particle liquid and the mixed liquid are uniformly mixed, thus obtaining a reagent 2 solution.
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| CN202011620572.1A Active CN112858687B (en) | 2020-12-30 | 2020-12-30 | Serum amyloid A detection reagent and preparation method thereof |
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