CN112858687B - Serum amyloid A detection reagent and preparation method thereof - Google Patents

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

A serum amyloid A detection reagent and its preparation method

Technical field

The invention relates to the field of analysis and detection, and in particular to a serum amyloid A detection reagent and a preparation method thereof.

Background technique

Serum amyloid A (SAA) is an acute-phase reaction protein that can rise significantly as soon as 4 hours after the body is infected. When the pathogen is cleared, SAA can quickly drop to normal levels. SAA has a very high sensitivity to viral infections and is currently commonly used clinically for early diagnosis, differential diagnosis, condition monitoring and prognosis assessment of infectious diseases.

Currently, SAA detection methods on the market include enzyme-linked immunosorbent assay, radioactive immunoassay, immunochromatography, immunoturbidimetric method, etc. The enzyme-linked immunosorbent assay is cumbersome, time-consuming, and has a low level of automation. Although radioimmunoassay has the characteristics of strong specificity and high sensitivity, it also has potential radioactive contamination, so it is gradually not accepted. Due to the limitations of its own methodology, immunochromatography can generally only achieve qualitative diagnosis and cannot meet the quantitative needs of doctors. In addition, it is difficult for the test paper to have a uniform structure, and the material, thickness, and density are also difficult to be exactly the same, resulting in The repeatability of sample test results on different test cards is poor. The latex-enhanced immunoturbidimetric method has higher sensitivity and accuracy than the immunochromatographic method. This method is a derivative technology of the ordinary immunoturbidimetric technology. It overcomes the shortcomings of the very limited signal volume of the ordinary immunoturbidimetric technology and makes the antibody volume more efficient. It has increased by orders of magnitude and greatly improved the detectability. However, this method also has many shortcomings. For example, it is susceptible to interference from heterophilic antibodies present in the test sample, especially the interference from rheumatoid factor, which leads to false positives. Positive result; in addition, the particle size of latex microspheres will also have a great impact on the test performance of the reagent. If the particle size is too large, due to the small ratio of the surface area of the microspheres to its own volume, it is easy to cause a small amount of antibody coating per unit volume. This results in an excess of antigen and the inability to detect analytes with high concentrations. If the particle size is too small, the ratio of the surface area of the latex to its own volume will be large, making agglutination less likely to occur, resulting in slow changes in absorbance and reduced detection sensitivity of the reagent.

Contents of the invention

The purpose of the present invention is to provide a latex immunoturbidimetric SAA detection reagent with high detection sensitivity, wide reportable range, good stability, and can effectively inhibit the interference of rheumatoid factor and its preparation method.

The present invention provides the following technical solutions:

An SAA detection reagent and a preparation method thereof, including reagent 1 and reagent 2;

The components and concentrations of Reagent 1 are as follows:

Acetate buffer: 100-300mM

Sodium chloride: 10-20g/L

Magnesium sulfate: 10-20g/L

Polyethylene glycol disuccinic acid: 2-4g/L

Isopropyl lauroyl sarcosine: 1-5g/L

Sodium oxodeoxycholate: 0.1-0.5g/L

Sodium deoxycholate: 1-5g/L

Sodium azide: 0.1g/L

The solvent is purified water, and the pH of reagent 1 is 5.5 to 6.0;

The components and concentrations of Reagent 2 are as follows:

Piperazine-1,4-diethanesulfonic acid buffer: 50-200mM

Rabbit anti-human SAA antibody coated latex particles: 1-3%

Sodium starch octenylsuccinate: 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 reagent 2 is 6.5 to 7.5.

Preferably, the rabbit anti-human SAA antibody-coated latex particles in Reagent 2 are a mixture of two latex particles, with the larger diameter being 180 nm to 200 nm; the smaller diameter latex particles being 30 nm to 40 nm;

Preferably, the number of small-diameter latex particles:the number of large-diameter latex particles=1:7.

The preparation method of SAA detection reagent is as follows:

S1, preparation of reagent 1:

According to the component amounts of reagent 1, add sodium chloride, magnesium sulfate, polyethylene glycol disuccinic acid, lauroyl sarcosine isopropyl ester, sodium taurodeoxycholate, sodium deoxycholate, and sodium azide in sequence. Add the acetic acid buffer solution to the acetic acid buffer and mix thoroughly, then add the solvent water. After continuing to stir and mix, adjust the pH of the solution to 5.5 to 6.0, then filter the solution using a microporous membrane. The resulting filtrate is reagent 1;

S2, preparation of reagent 2:

S21. Add sodium starch octenylsuccinate, polyvinylpyrrolidone, sodium alginate, and sodium azide to the piperazine-1,4-diethanesulfonic acid buffer solution in sequence, stir evenly, and then add small-diameter SAA antibodies. After ultrasonically vibrating the latex particles for 30-60 minutes, add solvent water, continue stirring for 30-60 minutes, filter with a microporous membrane, and save the filtrate.

S22. Add an equal volume of large-diameter SAA antibody latex particles dropwise into the S21 solution. After ultrasonic vibration for 2-4 hours, mix evenly to obtain Mixed Solution 1; then drop an equal volume of large-diameter SAA antibody latex particles into the above Mixed Solution 1. SAA antibody latex particles are vibrated ultrasonically for 2-4 hours until all are mixed evenly to obtain mixture 2; then drop in a large-diameter SAA antibody latex particle liquid of the same volume as the above mixture 2 and vibrate ultrasonically for 2-4 hours. Until everything is mixed evenly, the reagent 2 solution is obtained.

Preferably, the test conditions for the detection reagent to measure SAA in the sample are as follows: temperature: 37°C; light diameter of the cuvette: 1.0 cm. The detection main wavelength is 546nm and the secondary wavelength is 700nm.

Preferably, the reagent method for measuring SAA in a sample is as follows: mix the sample with reagent 1, incubate it at 37°C for 5 minutes, then add reagent 2, mix, incubate at 37°C for 30 seconds, read the absorbance A1, and continue incubating for 4 minutes and 30 seconds. Seconds, read the absorbance A2; ΔA = A2-A1. The sample dosage is 2 μl, the reagent 1 dosage is 200 μl, and the reagent 2 dosage is 40 μl.

Beneficial effects of the present invention:

(1) The combined application of sodium deoxycholate, sodium deoxycholate and lauroyl sarcosine isopropyl ester helps to dissociate the non-specific binding between rheumatoid factor and protein and weaken the influence of rheumatoid factor on the test results. interference.

(2) The combined application of sodium chloride and magnesium sulfate helps to promote and maintain the stability of the sample and reaction system and prevent system turbidity.

(3) The rabbit anti-human SAA antibody-coated latex particles in Reagent 2 are a mixture of two large and small latex particles. This can make the reagent have low blank absorbance, high sensitivity, and a wide reporting range; and use the equal-volume incremental method for mixing. Operation, the two latex particles can be evenly distributed, and the detection stability of the reagent is good.

(4) The combined application of sodium starch octenylsuccinate, polyvinylpyrrolidone and sodium alginate in reagent 2 helps to promote and maintain the stability of reagent 2 and improve the stability of the reagent.

Detailed ways

Example 1:

A SAA detection reagent and its preparation method, including reagent 1 and reagent 2; the components and concentration of reagent 1 are as follows:

Acetate buffer: 100mM; sodium chloride: 10g/L; magnesium sulfate: 10g/L; polyethylene glycol disuccinic acid: 2g/L; lauroyl sarcosine isopropyl ester: 1g/L; bovine deoxycholate Sodium phosphate: 0.1g/L; sodium deoxycholate: 1g/L; sodium azide: 0.1g/L; the solvent is purified water, the pH of reagent 1 is 5.5; the components and concentration of reagent 2 are as follows: piperazine -1,4-diethylsulfonic acid buffer: 50mM; rabbit anti-human SAA antibody-coated latex particles: 1%; sodium starch octenylsuccinate: 1g/L; polyvinylpyrrolidone: 2g/L; sodium alginate : 2g/L; sodium azide: 0.1g/L; the solvent is purified water, and the pH of reagent 2 is 6.5. The rabbit anti-human SAA antibody-coated latex particles in Reagent 2 are a mixture of two latex particles. The larger diameter particle is 180nm; the smaller diameter latex particle is 30nm; the number of small diameter latex particles: large diameter Number of latex particles = 1:7.

The preparation method of SAA detection reagent is as follows:

S1, preparation of reagent 1:

According to the component amounts of reagent 1, add sodium chloride, magnesium sulfate, polyethylene glycol disuccinic acid, lauroyl sarcosine isopropyl ester, sodium taurodeoxycholate, sodium deoxycholate, and sodium azide in sequence. Add the solution to the acetic acid buffer and mix thoroughly, add the solvent water, continue stirring and mix, adjust the pH of the solution to 5.5, filter the solution using a microporous membrane, and the resulting filtrate is reagent 1;

S2, preparation of reagent 2:

S21. Add sodium starch octenylsuccinate, polyvinylpyrrolidone, sodium alginate, and sodium azide to the piperazine-1,4-diethanesulfonic acid buffer solution in sequence, stir evenly, and then add small-diameter SAA antibodies. After ultrasonically vibrating the latex particles for 30 minutes, add solvent water, continue stirring for 30 minutes, then filter with a microporous membrane, and save the filtrate.

S22. Add an equal volume of large-diameter SAA antibody latex particles dropwise into the S21 solution. After ultrasonic vibration for 2 hours, mix evenly to obtain Mixed Solution 1; then drop an equal volume of large-diameter SAA Antibody latex particles as the above Mixed Solution 1. The latex particles were vibrated ultrasonically for 2 hours until all were mixed evenly, to obtain Mixed Solution 2; then a large-diameter SAA antibody latex particle liquid of the same volume as the above Mixed Solution 2 was dropped, and ultrasonically vibrated for 2 hours until all were mixed evenly, i.e. Obtain reagent 2 solution.

The test conditions for the detection reagent to determine SAA in the sample are as follows: temperature: 37°C; light diameter of the cuvette: 1.0cm. The detection main wavelength is 546nm and the secondary wavelength is 700nm. The method for reagent determination of SAA in a sample is as follows: mix the sample with reagent 1 and incubate it at 37°C for 5 minutes, then add reagent 2, mix well, incubate at 37°C for 30 seconds, read the absorbance A1, continue to incubate for 4 minutes and 30 seconds, and read Absorbance A2; ΔA=A2-A1. The sample dosage is 2 μl, the reagent 1 dosage is 200 μl, and the reagent 2 dosage is 40 μl.

Example 2:

A SAA detection reagent and its preparation method, including reagent 1 and reagent 2; the components and concentration of reagent 1 are as follows:

Acetate buffer: 300mM; sodium chloride: 20g/L; magnesium sulfate: 20g/L; polyethylene glycol disuccinic acid: 4g/L; lauroyl sarcosine isopropyl ester: 5g/L; bovine deoxycholate Sodium acid acid: 0.5g/L; sodium deoxycholate: 5g/L; sodium azide: 0.1g/L; the solvent is purified water, the pH of reagent 1 is 6.0; the components and concentration of reagent 2 are as follows: piperazine -1,4-diethylsulfonic acid buffer: 200mM; rabbit anti-human SAA antibody-coated latex particles: 3%; sodium starch octenylsuccinate: 5g/L; polyvinylpyrrolidone: 5g/L; sodium alginate : 5g/L; sodium azide: 0.1g/L; the solvent is purified water, and the pH of reagent 2 is 7.5. The rabbit anti-human SAA antibody-coated latex particles in Reagent 2 are a mixture of two latex particles, the larger diameter is 200nm; the smaller diameter latex particles are 40nm; the number of small diameter latex particles: large diameter Number of latex particles = 1:7.

The preparation method of SAA detection reagent is as follows:

S1, preparation of reagent 1:

According to the component amounts of reagent 1, add sodium chloride, magnesium sulfate, polyethylene glycol disuccinic acid, lauroyl sarcosine isopropyl ester, sodium taurodeoxycholate, sodium deoxycholate, and sodium azide in sequence. Add the acetic acid buffer solution to the acetic acid buffer and mix thoroughly, then add the solvent water. After continuing to stir and mix, adjust the pH of the solution to 6.0 and filter the solution using a microporous membrane. The resulting filtrate is reagent 1;

S2, preparation of reagent 2:

S21. Add sodium starch octenylsuccinate, polyvinylpyrrolidone, sodium alginate, and sodium azide to the piperazine-1,4-diethanesulfonic acid buffer solution in sequence, stir evenly, and then add small-diameter SAA antibodies. After ultrasonically vibrating the latex particles for 60 minutes, add solvent water, continue stirring for 60 minutes, then filter with a microporous membrane, and save the filtrate.

S22. Add an equal volume of large-diameter SAA antibody latex particles dropwise into the S21 solution. After ultrasonic vibration for 4 hours, mix evenly to obtain Mixed Solution 1; then drop an equal volume of large-diameter SAA Antibody latex particles as the above Mixed Solution 1. The latex particles were vibrated ultrasonically for 4 hours until all were mixed evenly, to obtain Mixed Solution 2; then a large-diameter SAA antibody latex particle liquid of the same volume as the above Mixed Solution 2 was dripped in, and ultrasonically vibrated for 4 hours until all were mixed evenly, i.e. Obtain reagent 2 solution.

The test conditions for the detection reagent to determine SAA in the sample are as follows: temperature: 37°C; light diameter of the cuvette: 1.0cm. The detection main wavelength is 546nm and the secondary wavelength is 700nm. The method for reagent determination of SAA in a sample is as follows: mix the sample with reagent 1 and incubate it at 37°C for 5 minutes, then add reagent 2, mix well, incubate at 37°C for 30 seconds, read the absorbance A1, continue to incubate for 4 minutes and 30 seconds, and read Absorbance A2; ΔA=A2-A1. The sample dosage is 2 μl, the reagent 1 dosage is 200 μl, and the reagent 2 dosage is 40 μl.

Example 3:

A SAA detection reagent and its preparation method, including reagent 1 and reagent 2; the components and concentration of reagent 1 are as follows: acetate buffer: 200mM; sodium chloride: 15g/L; magnesium sulfate: 15g/L; polyethylene glycol Alcohol disuccinic acid: 3g/L; Lauroylsarcosine isopropyl ester: 3g/L; Sodium taurodeoxycholate: 0.3g/L; Sodium deoxycholate: 3g/L; Sodium azide: 0.1g /L; the solvent is purified water, the pH of reagent 1 is 5.7; the components and concentration of reagent 2 are as follows: piperazine-1,4-diethylsulfonic acid buffer: 125mM; rabbit anti-human SAA antibody-coated latex particles: 2%; sodium starch octenylsuccinate: 3g/L; polyvinylpyrrolidone: 3.5g/L; sodium alginate: 3.5g/L; sodium azide: 0.1g/L; solvent is purified water, reagent 2 The pH is 7. The rabbit anti-human SAA antibody-coated latex particles in Reagent 2 are a mixture of two latex particles, the larger diameter is 190nm; the smaller diameter latex particles are 35nm; the number of small diameter latex particles: large diameter Number of latex particles = 1:7.

The preparation method of SAA detection reagent is as follows:

S1, preparation of reagent 1:

According to the component amounts of reagent 1, add sodium chloride, magnesium sulfate, polyethylene glycol disuccinic acid, lauroyl sarcosine isopropyl ester, sodium taurodeoxycholate, sodium deoxycholate, and sodium azide in sequence. Add the solution to the acetic acid buffer and mix thoroughly, add the solvent water, continue stirring and mix, adjust the pH of the solution to 5.7, filter the solution using a microporous membrane, and the resulting filtrate is reagent 1;

S2, preparation of reagent 2:

S21. Add sodium starch octenylsuccinate, polyvinylpyrrolidone, sodium alginate, and sodium azide to the piperazine-1,4-diethanesulfonic acid buffer solution in sequence, stir evenly, and then add small-diameter SAA antibodies. After ultrasonically vibrating the latex particles for 45 minutes, add solvent water, continue stirring for 45 minutes, then filter with a microporous membrane, and save the filtrate.

S22. Add an equal volume of large-diameter SAA antibody latex particles dropwise into the S21 solution. After ultrasonic vibration for 3 hours, mix evenly to obtain Mixed Solution 1; then drop an equal volume of large-diameter SAA Antibody latex particles as the above Mixed Solution 1. The latex particles were vibrated ultrasonically for 3 hours until all were mixed evenly, to obtain Mixed Solution 2; then a large-diameter SAA antibody latex particle liquid of the same volume as the above Mixed Solution 2 was dropped, and ultrasonically vibrated for 3 hours until all were mixed evenly, i.e. Obtain reagent 2 solution.

The test conditions for the detection reagent to determine SAA in the sample are as follows: temperature: 37°C; light diameter of the cuvette: 1.0cm. The detection main wavelength is 546nm and the secondary wavelength is 700nm. The method for reagent determination of SAA in a sample is as follows: mix the sample with reagent 1 and incubate it at 37°C for 5 minutes, then add reagent 2, mix well, incubate at 37°C for 30 seconds, read the absorbance A1, continue to incubate for 4 minutes and 30 seconds, and read Absorbance A2; ΔA=A2-A1. The sample dosage is 2 μl, the reagent 1 dosage is 200 μl, and the reagent 2 dosage is 40 μl.

The performance of the kits obtained in Examples 1 to 3 of the present invention will be described below in conjunction with tables.

1. Blank absorbance

Distilled water was used to test the reagent, and the results showed that the blank absorbance of the reagent of the technology of the present invention was significantly lower than that of the conventional commercial reagent, indicating that the blank of the reagent of the technology of the present invention was lower.

2. Analytical sensitivity test

Test the reagent with a sample with a concentration of 20 mg/L, repeat twice, record the absorbance change produced under the specified parameters of the reagent, and calculate the average value of the difference. The results show that when the same sample is tested, the absorbance difference tested by the technical reagent of the present invention is significantly greater than that of conventional commercially available technical reagents, indicating that the analytical sensitivity of the technical reagent of the present invention is better.

3. HOOK effect evaluation

Dissolve the standard substance in physiological saline and add it to the high-value sample, and prepare high-value samples with four different concentrations: linear range high value, linear range high value 1.25 times, linear range high value 1.5 times, linear range high value 2 times, etc. , repeat the measurement on the above samples, and the concentration at which the absorbance decreases is the hook effect inflection point value. Based on this, the highest point concentration of the hook effect of the reagent is determined. The results show that the concentration at which absorbance decreases in commercially available conventional technical reagents is less than that of the technical reagents of the present invention, indicating that the reportable range of the technical reagents of the present invention is wider.

4. Precision test

Under repeatability conditions, use low-value quality control products (30.7mg/L) and high-value quality control products (80.6mg/L) to test the technical reagents of the present invention. Repeat the test 10 times and calculate the average value of the measured values. and standard deviation(s). Calculate the coefficient of variation (CV) according to the following formula.

The results show that the coefficients of variation of the detection reagent of the present invention when testing low- and high-value quality control products are 1.63% and 1.00% respectively, indicating that the detection stability of the reagent of the present invention is good.

From the above results, it can be seen that the SAA detection reagent of the present invention has the advantages of low blank absorbance, high sensitivity, wide reportable range, and good test stability.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still describe the foregoing embodiments. Modify the technical solution, or make equivalent replacements for some of its technical features. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims (3)

1. A serum amyloid A detection reagent, characterized in that: it includes reagent 1 and reagent 2; The components and concentrations of Reagent 1 are as follows: Acetate buffer: 100-300mM Sodium chloride: 10-20g/L Magnesium sulfate: 10-20g/L Polyethylene glycol disuccinic acid: 2-4g/L Isopropyl lauroyl sarcosine: 1-5g/L Sodium oxodeoxycholate: 0.1-0.5g/L Sodium deoxycholate: 1-5g/L Sodium azide: 0.1g/L The solvent is purified water, and the pH of reagent 1 is 5.5 to 6.0; The components and concentrations of Reagent 2 are as follows: Piperazine-1,4-diethanesulfonic acid buffer: 50-200mM Rabbit anti-human serum amyloid A antibody coated latex particles: 1-3% Sodium starch octenylsuccinate: 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 reagent 2 is 6.5 to 7.5; The latex particles coated with rabbit anti-human serum amyloid A antibody in Reagent 2 are a mixture of two latex particles. The larger diameter particle size is 180nm ~ 200nm; the smaller diameter latex particle size is 30nm ~ 40nm. 2. A serum amyloid A detection reagent according to claim 1, characterized in that: the number of small-diameter latex particles: the number of large-diameter latex particles=1:7. 3. A kind of serum amyloid A detection reagent preparation method according to claim 1, characterized in that, specifically as follows: S1, preparation of reagent 1: According to the component amounts of reagent 1, add sodium chloride, magnesium sulfate, polyethylene glycol disuccinic acid, lauroyl sarcosine isopropyl ester, sodium taurodeoxycholate, sodium deoxycholate, and sodium azide in sequence. Add the solution to the acetic acid buffer and mix thoroughly, add the solvent water, continue to stir and mix, adjust the pH of the solution to 5.5~6.0, filter the solution using a microporous membrane, and the resulting filtrate is reagent 1; S2, preparation of reagent 2: S21. Add sodium starch octenylsuccinate, polyvinylpyrrolidone, sodium alginate, and sodium azide to the piperazine-1,4-diethanesulfonic acid buffer solution in sequence, stir evenly, and then add small-diameter serum starch Protein A antibody latex particles, after ultrasonic vibration for 30-60 minutes, add solvent water, continue stirring for 30-60 minutes, then filter with a microporous filter membrane, and save the filtrate; S22. Add an equal volume of large-diameter serum amyloid A antibody latex particles dropwise into the S21 solution. After ultrasonic vibration for 2-4 hours, mix well to obtain Mixed Solution 1; then drop in an equal volume of the above Mixed Solution 1. The large-diameter serum amyloid A antibody latex particles are vibrated ultrasonically for 2-4 hours until all are mixed evenly to obtain Mixture 2; then drop in the large-diameter SAA antibody latex particles of the same volume as the above-mentioned Mixture 2. , ultrasonic vibration for 2-4 hours until everything is mixed evenly, and the reagent 2 solution is obtained.