CN113219181A - Kit for quantitatively detecting serum amyloid A and preparation method thereof - Google Patents

Kit for quantitatively detecting serum amyloid A and preparation method thereof Download PDF

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CN113219181A
CN113219181A CN202110196269.1A CN202110196269A CN113219181A CN 113219181 A CN113219181 A CN 113219181A CN 202110196269 A CN202110196269 A CN 202110196269A CN 113219181 A CN113219181 A CN 113219181A
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kit
reagent
saa
buffer solution
buffer
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CN113219181B (en
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李云
刘霖
裴华
李元丽
李伯宏
陶静
刘雨薇
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Chongqing Zhongyuan Huiji Biotechnology Co Ltd
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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
    • 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
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Abstract

The invention discloses a kit for quantitatively detecting human serum amyloid A, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises 50-200mM of buffer solution, 0.1-0.5% of surfactant I, 0.1-0.5% of surfactant II, 0.1-0.5% of preservative, 0.1-0.8% of inorganic salt and 1-2% of coagulant; the reagent R2 comprises 50-100mM buffer solution, 1-5% protein protective agent, 0.1-0.5% preservative, 0.1-0.8% inorganic salt and latex particles coated with SAA monoclonal antibody. According to the invention, the benzoic acid and morpholine composition is added in the process of marking the latex microspheres, so that the marking efficiency of the latex microspheres and the antibody is increased, the HOOK resistance of the reagent in the detection of high-value positive samples is obviously improved, and the linear range of the reagent reaches 2-500 mg/L.

Description

Kit for quantitatively detecting serum amyloid A and preparation method thereof
Technical Field
The invention relates to the field of medical inspection, in particular to a kit for quantitatively detecting human serum amyloid A and a preparation method thereof.
Background
Serum Amyloid A (SAA) is an acute phase-reactive protein produced by hepatocytes and secreted into the serum, a heterogeneous class of proteins in the apolipoprotein family, which binds to plasma High Density Lipoprotein (HDL). The SAA content in a patient in an acute phase of inflammation or infection is rapidly increased within 48-72 hours and rapidly reduced in the recovery phase of the disease, and the SAA plays an important role in clinical application as a new detection index of the acute phase of inflammation or infection.
In normal human body, the content of SAA is below 10mg/L, and in the case of slight infection, the content of SAA in human body is increased and is in the range of 10-200 mg/L. However, for some patients with diseases, the content of SAA in vivo will increase significantly, for example, the content of SAA in severe children with hand, foot and mouth disease is as high as 600mg/L, and the extra high content of SAA in vivo can effectively reflect the severity of diseases, and is more beneficial for doctors to distinguish diseases.
At present, the detection method of SAA comprises an enzyme-linked immunosorbent assay, a latex enhanced immunoturbidimetry assay and the like, and enzyme-linked immunosorbent assay is not favorable for high-throughput detection due to the complicated process and long detection time. The latex enhanced immunoturbidimetry has the advantages of high detection speed and high accuracy, ensures the detection accuracy while realizing high-throughput detection, has gradually developed into an important clinical detection means, but the existing SAA latex enhanced immunoturbidimetry detection kit on the market has the defects of narrow detection linearity (the linear range of most reagents is below 200 mg/L), weak HOOK resistance and the like, and is not beneficial to disease diagnosis and judgment of different medical levels.
Disclosure of Invention
According to the above needs, the inventors have unexpectedly found that the labeling efficiency of the SAA antibody and latex can be significantly increased by adding the benzoic acid and morpholine composition in the process of labeling the latex microspheres of the SAA detection kit, and the detection linear range and the anti-HOOK capability of the reagent can be significantly improved by adjusting the reagent configuration.
The invention provides a kit for quantitatively detecting human Serum Amyloid A (SAA), which adopts the following technical means: a kit for quantitatively detecting human serum amyloid A is characterized by comprising a reagent R1 and a reagent R2;
the reagent R1 comprises the following components in percentage by weight: 50-200mM of buffer solution, 0.1-0.5% of surfactant I, 0.1-0.5% of surfactant II, 0.1-0.5% of preservative, 0.1-0.8% of inorganic salt and 1-2% of coagulant;
the reagent R2 comprises the following components in percentage by weight: 50-100mM of buffer solution, 1-5% of protein protective agent, 0.1-0.5% of preservative, 0.1-0.8% of inorganic salt and latex particles coated with SAA monoclonal antibody;
the preparation process of the latex particles coated with the SAA monoclonal antibody comprises the following steps: the SAA monoclonal antibody is coupled to latex particles in the presence of component a selected from at least one of morpholine or piperidine, preferably morpholine, and component B selected from at least one of benzoic acid or salicylic acid, preferably benzoic acid.
Preferably, in the reagent R1:
the buffer is selected from at least one of Tris, MOPS or phosphate buffer, preferably MOPS buffer; the surfactant I is selected from at least one of TritonX-100 or Tween-20, preferably Tween-20; the surfactant II is selected from at least one of Brii 58, Tween-80 or A90, preferably A90; the preservative is at least one selected from Proclin-300, sodium azide and gentamicin; the inorganic salt is selected from at least one of sodium chloride or potassium chloride; the coagulant is at least one selected from PEG6000 or PEG 8000.
Preferably, the pH of the reagent R1 is 6-8, preferably 6-6.5.
Preferably, in the reagent R2:
the buffer solution is selected from at least one of MOPS, Bicine or phosphate buffer solution, and is preferably Bicine buffer solution; the protein protective agent is selected from at least one of BSA, casein, sucrose or trehalose, and is preferably BSA; the preservative is at least one selected from Proclin-300, sodium azide and gentamicin; the inorganic salt is selected from at least one of sodium chloride or potassium chloride.
Preferably, the pH of the reagent R2 is 6-8, preferably 6.5-7.
Preferably, the size of the latex particles coated with the mouse anti-human SAA antibody is in the range of 100-200 nm.
A preparation method of the kit for quantitatively detecting the human serum amyloid A is characterized by comprising the following steps:
(1) preparation of reagent R1
The reagent R1 was formulated as follows:
50-200mM of buffer solution, 10.1-0.5% of surfactant, 0.1-0.5% of surfactant II, 0.1-0.5% of preservative, 0.1-0.8% of inorganic salt and 1-2% of coagulant;
the buffer is selected from at least one of Tris, MOPS or phosphate buffer, preferably MOPS buffer; the surfactant I is selected from at least one of Triton X-100 or Tween-20, preferably Tween-20; the surfactant II is selected from at least one of Brii 58, Tween-80 or A90, preferably A90; the preservative is at least one selected from Proclin-300, sodium azide and gentamicin; the inorganic salt is selected from at least one of sodium chloride or potassium chloride; the coagulant is selected from at least one of PEG6000 or PEG 8000;
(2) preparation of reagent R2
Preparation of SAA latex particles
Adding the latex microspheres into a marking buffer solution, and uniformly stirring; activating by adding an activating agent, and uniformly stirring; adding a SAA monoclonal antibody into the activated latex microsphere solution, simultaneously adding a composition of a component A and a component B, wherein the component A is selected from at least one of morpholine or piperidine, preferably morpholine, and the component B is selected from at least one of benzoic acid or salicylic acid, preferably benzoic acid, uniformly stirring, and then incubating for 6-14 h; centrifuging, and discarding the supernatant to obtain latex particles coated with the SAA monoclonal antibody;
preparation of R2 basic buffer solution
The R2 base buffer was formulated as follows:
50-100mM of buffer solution, 1-5% of protein protective agent, 0.1-0.5% of preservative and 0.1-0.8% of inorganic salt;
the buffer solution is selected from at least one of MOPS, Bicine or phosphate buffer solution, and is preferably Bicine buffer solution; the protein protective agent is selected from at least one of BSA, casein, sucrose or trehalose, and is preferably BSA; the preservative is at least one selected from Proclin-300, sodium azide and gentamicin; the inorganic salt is selected from at least one of sodium chloride or potassium chloride.
Preparation of reagent R2
Adding the SAA latex particles prepared in the step I into the R2 basic buffer solution prepared in the step II, dissolving, performing ultrasonic treatment, and incubating at 20-40 ℃ for 6-14h to obtain a reagent R2.
Preferably, in said step (i), a composition of 0.5-1% morpholine and 0.5-1% benzoic acid is added, respectively.
Preferably, in the step (r), the labeling buffer is at least one selected from phosphate buffer or Tris buffer, preferably phosphate buffer, the activator is EDC/NHS, and the mass ratio of the NHS to the EDC and the latex microspheres is: 1: 0.5-1.5: 800-1000, wherein the mass ratio of the SAA monoclonal antibody to the latex microsphere is 1: 350-400.
Preferably, in the step (iii), the content of the latex particles coated with the SAA monoclonal antibody in the R2 reagent is 0.05-0.1%.
The invention has the beneficial effects that: according to the invention, the labeling efficiency of the SAA antibody and the latex microspheres is increased by adding the benzoic acid and morpholine composition into the latex microsphere labeling solution, so that on one hand, the precision and accuracy of the kit detection are ensured, and on the other hand, the HOOK resistance (the HOOK resistance can reach 1043mg/L) of the kit in the detection of high-value positive samples is remarkably improved, so that the linear range of the reagent reaches 2-500 mg/L.
Drawings
FIG. 1 is a graph of the linear relationship between the theoretical concentration of a sample of SAA assignments and the assay values of a kit of group A provided in example 3 of the present invention;
FIG. 2 is a graph of the linear relationship between the theoretical concentration of a SAA-assigned sample and the measured values of a group B kit provided in example 3 of the present invention;
FIG. 3 is a graph of the linear relationship between the theoretical concentration of a sample assigned a SAA and the measured values of a kit in group C provided in example 3 of the present invention;
FIG. 4 is a graph of the linear relationship between the theoretical concentration of a SAA ultra-high assigned sample and the measured values of a group A kit provided in example 4 of the present invention;
FIG. 5 is a graph of the linear relationship between the theoretical concentration of a SAA ultra-high assigned sample and the measured values of a group B kit provided in example 4 of the present invention;
FIG. 6 is a graph of the linear relationship between the theoretical concentration of the SAA ultrahigh-assigned sample and the measured values of the group C kit provided in example 4 of the present invention.
FIG. 7 is a graph of the linear relationship between the theoretical concentration of the SAA ultrahigh-assigned sample and the measured values of the kit in group D provided in example 4 of the present invention.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following examples are included to more clearly and clearly illustrate the technical solutions of the present invention by way of illustration. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The specific embodiments of the present invention are merely illustrative of the invention and are not intended to limit the invention in any way.
Example 1 preparation of SAA assay kit
The amyloid A detection kit comprises a reagent R1 and a reagent R2 which are independent of each other.
1. Preparation of reagent R1
The preparation is carried out according to the following formula, and the mixture is fully stirred, evenly mixed and stored at the temperature of 2-8 ℃.
Reagent R1:
Figure BDA0002946765940000051
the rest of the solvent is purified water
2. Preparation of reagent R2
(1) Preparation of latex particles coated with mouse anti-human SAA monoclonal antibody
a. Adding 100ug 110nm latex microspheres into 20ml 100mM labeling buffer (phosphate buffer), and stirring;
b. adding 0.1ug NHS and 0.1ug EDC activated microspheres into the solution, and stirring at constant speed for 1 h;
c. adding 1ml of 10g/L mouse anti-human SAA monoclonal antibody into the activated latex microsphere solution, simultaneously adding 0.6% benzoic acid and 0.6% morpholine, stirring uniformly and incubating for 6 h;
d. and (3) centrifuging the crosslinked reagent for 25min at 12500rpm, discarding the supernatant, and retaining the labeled latex particles coated with the mouse anti-human SAA antibody.
(2) Preparation of R2 basic buffer
The preparation is carried out according to the following formula, and the mixture is fully stirred, evenly mixed and stored at the temperature of 2-8 ℃.
R2 base buffer:
Figure BDA0002946765940000052
Figure BDA0002946765940000061
the rest of the solvent is purified water
(3) Preparation of reagent R2
The latex particles coated with the mouse anti-human SAA antibody prepared in the above way are added into R2 basic buffer (the content of the latex particles coated with the SAA monoclonal antibody in the R2 buffer is 0.08%), dissolved, ultrasonically treated, and incubated at 37 ℃ overnight to obtain the R2 reagent.
Example 2 method of Using the kit
In this example, a fully automatic biochemical analyzer (Hitachi 7180) was used in combination with the kit of the present invention to perform sample detection.
1. Instrument parameter setting
Figure BDA0002946765940000062
2. Assay protocol
Figure BDA0002946765940000063
Figure BDA0002946765940000071
3. Computing method
And (3) using a multipoint nonlinear/semilogarithmic calibration mode, taking a spline function as a calculation mode, and making a metering/response curve according to the value of the calibrator and the absorbance change value, wherein the content of the SAA in the sample can be calculated on the dosage/response curve according to the absorbance change value.
The detection principle of the invention is that the content of human serum amyloid protein A (SAA) is measured by adopting a latex enhanced immunoturbidimetry method, the SAA in a sample is combined with an SAA antibody cross-linked on the surface of a latex microsphere, so that adjacent latex microspheres are cross-linked with each other, an agglutination reaction is generated to generate turbidity change, the turbidity is in direct proportion to the content of the SAA in the sample, the turbidity is measured at a certain wavelength, and the quantitative measurement of the SAA can be carried out through a calibration curve.
Example 3 kit Performance test
In order to verify all performances of the kit, 3 groups of kits are arranged for performance verification:
group A: the kit prepared in example 1 of the present invention.
Group B: the formula is as follows: reagent R1: 0.10% of fatty acid salt, 10mmol/L of glycine buffer solution, 0.01% of Tween20, 3.00% of sodium chloride, 0.01% of sodium azide, 0.01% of polyethylene glycol 2000, 0.20% of bovine serum albumin, and the solvent is purified water;
reagent R2: 10mmol/L glycine buffer solution, 0.01% Tween20, 3.00% sodium chloride, 0.01% sodium azide, 0.20% bovine serum albumin, and 0.10% latex microspheres coated with SAA monoclonal antibody;
preparing latex microspheres coated with SAA monoclonal antibody: adding 100ug of latex microspheres with the particle size of 50nm into 100ml of 10mmol/L glycine buffer solution, and uniformly stirring; adding 0.1ug NHS and 0.1ug EDC activated microspheres into the solution, and stirring at constant speed for 1 h; adding 1ml of 10g/L mouse anti-human SAA monoclonal antibody into the activated latex microsphere solution, stirring uniformly and incubating for 6 h; adding 1% protein protective agent BSA and 0.01% sodium azide, incubating for 1h, and aging at 37 ℃ for 3d to obtain the latex microsphere coated with the SAA monoclonal antibody.
Group C: the formula is as follows: reagent R1: 100mmol/L buffer solution, 180mmol/L potassium chloride, 20g/L polyethylene glycol-2000, 0.7g/L sodium azide, 30g/L bovine serum albumin, and the solvent is purified water;
reagent R2: 125mmol/L Tris buffer solution, 11g/L bovine serum albumin, 0.7g/L sodium azide and 4g/L latex coated anti-serum amyloid A antibody, wherein the solvent is purified water;
preparation of latex-coated anti-serum amyloid a antibody: firstly diluting polystyrene microspheres with the particle size of 120nm by using 50mmol/L MES buffer solution to obtain a solution containing the polystyrene microspheres with the mass concentration of 5%, then adding 0.5mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride into each milliliter of the solution, reacting for 1 hour at the temperature of 25 ℃, using a centrifugal machine, centrifuging for 30 minutes at the rotating speed of 20000rpm/min, discarding supernatant, diluting the precipitate by using 50mmol/L MES buffer solution, then using the centrifugal machine, centrifuging for 30 minutes at the rotating speed of 20000rpm/min, discarding supernatant, continuing to dilute the precipitate by using 50mmol/L MES buffer solution to ensure that the mass concentration of the polystyrene microspheres is 2.0%, then using the centrifugal machine, centrifuging for 30 minutes at the rotating speed of 20000rpm/min, discarding supernatant, retaining the precipitate, diluting the precipitate with 50mmol/L MES buffer solution, adding the same volume of MES buffer solution containing 8mg/ml anti-serum amyloid A antibody, stirring uniformly to make the final mass concentration of the polystyrene microsphere be 1%, reacting for 2 hours at 25 ℃, adding bovine serum albumin to make the final concentration of the bovine serum albumin be 20g/L, and sealing for 24 hours at 4 ℃ to prepare the latex-coated anti-serum amyloid A antibody.
(1) Accuracy verification
Three groups of kits are used for respectively testing the assignment samples of the Siemens SAA detection kit, 2 times of the tests are set, signals are read through a full-automatic biochemical analyzer (Hitachi 7180), and the relative deviation of the measured mean value and the target value is calculated for accuracy verification. The results are shown in the following table:
TABLE 1 accuracy verification
Figure BDA0002946765940000081
From the above experimental results, the relative deviations of the test value 1 and the target value 1 of the three-group kit were 0.11%, -3.82%, and 2.88%, respectively, and the relative deviations of the test value 2 and the target value 2 were 1.77%, 2.79%, and-1.97%, respectively. The quality control accuracy of the kit (group A) prepared in the embodiment 1 of the invention is better than that of the comparison kit-1 (group B) and that of the comparison kit-2 (group C).
(2) Precision verification
Selecting a low-value sample, a medium-value sample and a high-value sample of clinical SAA, testing the samples by using three groups of kits, respectively repeating the measurement for 10 times, reading signals by using a full-automatic biochemical analyzer (Hitachi 7180), respectively calculating a measurement mean value and a standard deviation, and calculating a variation coefficient to perform precision investigation. The results are shown in the following table:
TABLE 2 precision verification
Figure BDA0002946765940000091
From the above experimental results, the coefficient of variation of the three sets of kits in the detection of the low value sample is 1.86%, 6.82%, and 3.45%, the coefficient of variation of the median sample is 2.12%, 3.99%, and 4.01%, and the coefficient of variation of the high value sample is 1.34%, 9.18%, and 11.22, respectively, and the experimental results show that the three sets of kits have better precision in the detection of the low value sample and the median sample, and the coefficient of variation of the a set of kits is significantly lower than those of the B set and the C set in the detection of the high value sample, which indicates that the precision of the kit (a set) prepared in example 1 of the present invention is significantly better than that of the control kit-1 (B set) and the control kit-2 (C set) in the detection of the high value sample.
(3) Linear range verification
Selecting an assigned sample of a Siemens SAA detection kit (nephelometry) in a Siemens special protein instrument (BN II System), wherein the theoretical concentration value of a high-value sample is 500.18mg/L, the theoretical concentration value of a low-value sample is 2.01mg/L, then configuring concentration gradient samples according to a proportion by using the high-value sample and the low-value sample, respectively testing the samples by using three sets of kits, respectively repeating the measurement for 2 times for each sample, reading signals by a full-automatic biochemical analyzer (Hitachi 7180), and respectively calculating a measurement mean value to perform linear range investigation. The results are shown in the following table:
TABLE 3 Linear Range verification
Figure BDA0002946765940000101
From the above experimental results, it can be seen that the relative deviation between the detection value and the theoretical value of the kit (group A) prepared in example 1 of the present invention and the control kit-1 (group B) is smaller in the sample concentration range of 30-500mg/L, especially in the range of 200-500mg/L, the relative deviation between the detection value and the theoretical value is less than 5%. And when the sample concentration of the control kit-2 (group C) is more than 300mg/L, the relative deviation of the detection value and the theoretical value is more than 10 percent. Meanwhile, the detection results of the three groups of kits are subjected to correlation analysis with the theoretical value of the sample concentration (as shown in attached figures 1-3), the correlation between the detection values of the group A and the group B and the theoretical value is remarkably superior to that of the group C, and the correlation R between the detection values of the group A and the theoretical value is remarkably superior to that of the group C20.9999, group B R20.9996, group C R2Is 0.9704. The experimental result shows that the linear range of the kit prepared in the embodiment 1 of the invention is wider than that of the control kit-2, and particularly, the control kit-1 can not carry out accurate detection on a high-value sample with the concentration higher than 300 mg/L. According to the experimental result, the kit prepared by the embodiment of the invention is presumed to have better HOOK resistance.
Example 4 verification of HOOK resistance
In order to verify that the HOOK resistance of the reagent can be optimized by adding the composition of benzoic acid and morpholine in the process of marking the latex microspheres, 3 groups of kits are arranged for verification:
group A: the kit prepared in the invention example 1;
group B: the kit is different from the kit in the embodiment 1 only in that benzoic acid and morpholine are not added in the labeling process of the latex microspheres, and the other preparation methods are the same as the embodiment 1;
group C: the kit is different from the kit in the embodiment 1 only in that only morpholine is added in the labeling process of the latex microspheres, benzoic acid is not added, and the other preparation methods are the same as the embodiment 1;
group D: the kit is different from the kit in example 1 only in that only benzoic acid is added in the labeling process of the latex microspheres, no morpholine is added, and the rest preparation methods are the same as those in example 1.
The method comprises the steps of selecting a Siemens SAA detection kit (nephelometry) to assign values to ultra-high value samples in a Siemens special protein instrument (BN II System), enabling the sample concentration to be 1192.00mg/L, utilizing ultrapure water to dilute in proportion to prepare concentration gradient samples, using three groups of kits to test the samples respectively, repeatedly measuring each sample for 3 times, reading signals through a full-automatic biochemical analyzer (Hitachi 7180), and calculating a measured mean value and a relative deviation SD respectively to verify the HOOK resistance. The results are shown in the following table:
TABLE 4 HOOK-resistant capability verification of group A kit
Figure BDA0002946765940000111
From the above experimental results, the detection results of the kit of group a and the theoretical value of the sample concentration are compared and analyzed (as shown in fig. 4), and the results show that the kit (group a) prepared in example 1 of the present invention can achieve the anti-HOOK capability of 1043mg/L (the highest concentration corresponding to the mean value-3 SD > 500 is the anti-HOOK capability value of the reagent).
TABLE 5 verification of HOOK resistance of group B kit
Figure BDA0002946765940000121
According to the experimental results, the detection results of the B group kit are compared with the theoretical value of the sample concentration for analysis (as shown in figure 5), and the results show that the B group kit has poor HOOK resistance.
TABLE 6 HOOK-resistant capability verification of group C kit
Figure BDA0002946765940000122
According to the experimental results, the detection results of the group C kit are compared with the theoretical value of the sample concentration for analysis (as shown in figure 6), and the results show that the group C kit has poor HOOK resistance.
TABLE 7 HOOK-resistant capability verification of group D kits
Figure BDA0002946765940000131
According to the experimental results, the detection results of the group D kit are compared with the theoretical value of the sample concentration for analysis (as shown in figure 7), and the results show that the group D kit has poor HOOK resistance.
In conclusion, the benzoic acid and morpholine composition is introduced in the process of labeling the latex microspheres, so that the labeling efficiency of the latex microspheres can be effectively improved, and the subsequent detection performances of the reagent, including detection accuracy, sensitivity, linear range and HOOK resistance, are remarkably improved, and the reason is presumed to be that: the method is characterized in that only when amino on an antibody is combined with carboxyl on the microsphere in a correct direction, the antibody is marked effectively, benzoic acid is an organic matter containing carboxyl and can competitively react with the amino on the antibody in the marking process, morpholine can react with the carboxyl on the microsphere, morpholine can compete with the antibody for crosslinking the carboxyl on the microsphere by adding the morpholine in the marking process, the exposed amino on the antibody and the exposed carboxyl on the microsphere are controlled by controlling the concentration of benzoic acid and morpholine in a system, the binding sites of the antibody on the latex microsphere are effectively balanced, the antibody is guaranteed to be combined with the microsphere in a correct direction to the maximum extent, the marking efficiency of the antibody on the carboxyl microsphere is further improved, and the performance of a reagent is integrally improved.
Example 5
In this example, 4 sets of experiments were set up, wherein each set of experiments used a kit that differed from example 1 only in the labeling process of the latex microspheres:
group A: the kit is different from the kit in the embodiment 1 only in that 0.1 percent of benzoic acid and 0.1 percent of morpholine are added in the labeling process of the latex microspheres, and the other preparation methods are the same as the embodiment 1;
group B: the kit is different from the kit of example 1 only in that 0.5% benzoic acid and 0.5% morpholine are added during the labeling process of the latex microspheres, and the rest preparation method is the same as that of example 1.
Group C: the kit is different from the kit in example 1 only in that 1% of benzoic acid and 1% of morpholine are added in the process of marking the latex microspheres, and the rest preparation methods are the same as those in example 1.
Group D: the kit is different from the kit in example 1 only in that 2% of benzoic acid and 2% of morpholine are added in the process of marking the latex microspheres, and the rest preparation method is the same as that in example 1.
(1) Precision verification
Selecting clinical SAA median samples, testing the samples by using the five groups of kits, respectively repeating the measurement for 10 times, reading signals by a full-automatic biochemical analyzer (Hitachi 7180), respectively calculating a measurement mean value and a standard deviation, and calculating a variation coefficient to perform precision investigation. The results are shown in the following table: TABLE 8 verification of precision
Figure BDA0002946765940000141
The experimental result shows that 0.5-1% of benzoic acid and 0.5-1% of morpholine are added in the labeling process of the latex microspheres, which is helpful for improving the detection precision of the kit.
(2) Linear range verification
Selecting an assigned sample of a Siemens SAA detection kit (nephelometry) in a Siemens special protein instrument (BN II System), wherein the theoretical concentration value of a high-value sample is 500.18mg/L, the theoretical concentration value of a low-value sample is 2.01mg/L, then configuring concentration gradient samples according to a proportion by using the high-value sample and the low-value sample, respectively testing the samples by using four groups of kits, respectively repeatedly measuring each sample for 2 times, reading signals by a full-automatic biochemical analyzer (Hitachi 7180), and respectively calculating a measurement mean value to perform linear range investigation. The results are shown in the following table:
TABLE 9 Linear Range verification
Figure BDA0002946765940000142
From the above experimental results, it can be seen that the relative deviations of the detection values and the theoretical values of the group B kit and the group C kit are smaller in the sample concentration range of 30-500mg/L, and particularly in the range of 200-500mg/L, the relative deviations of the detection values and the theoretical values are smaller than 1%. And when the sample concentration of the kit in the group A and the kit in the group D are more than 300mg/L, the relative deviation of the detection value and the theoretical value is more than that of the kit in the group B and the kit in the group C, and the experimental result shows that when 0.5-1% of benzoic acid and 0.5-1% of morpholine composition are added in the labeling process of the latex microspheres, the improvement of the performance of the reagent is more facilitated.
Example 6
In this example, 5 sets of experiments were set up, wherein each set of experiments used a kit that differed from example 1 only in the labeling process of the latex microspheres:
group A: the kit is different from the kit in example 1 only in that 0.6% of morpholine and 0.6% of anthranilic acid are added in the labeling process of the latex microspheres, benzoic acid is not added, and the rest preparation methods are the same as those in example 1;
group B: the kit is different from the kit in the embodiment 1 only in that 0.6 percent of morpholine and 0.6 percent of salicylic acid are added in the labeling process of the latex microspheres, benzoic acid is not added, and the other preparation methods are the same as the embodiment 1;
group C: the kit is different from the kit in the embodiment 1 only in that 0.3 percent of piperidine and 0.6 percent of benzoic acid are added in the labeling process of the latex microspheres, no morpholine is added, and the other preparation methods are the same as the embodiment 1;
group D: the kit is different from the kit in the embodiment 1 only in that 0.6 percent of piperidine and 0.6 percent of salicylic acid are added in the labeling process of the latex microspheres, morpholine and benzoic acid are not added, and the other preparation methods are the same as the embodiment 1;
group E: the kit is different from the kit in example 1 only in that 0.6% of piperidine and 0.6% of anthranilic acid are added in the labeling process of the latex microspheres, morpholine and benzoic acid are not added, and the rest preparation methods are the same as those in example 1;
(1) precision verification
Selecting clinical SAA median samples, testing the samples by using the five groups of kits, respectively repeating the measurement for 10 times, reading signals by a full-automatic biochemical analyzer (Hitachi 7180), respectively calculating a measurement mean value and a standard deviation, and calculating a variation coefficient to perform precision investigation. The results are shown in the following table: TABLE 8 verification of precision
Figure BDA0002946765940000151
Figure BDA0002946765940000161
The experimental result shows that when the composition of benzoic acid and morpholine, morpholine and salicylic acid, piperidine and benzoic acid or piperidine and salicylic acid is added in the labeling process of the latex microspheres, the detection precision of the kit is improved.
(2) Linear range
Selecting an assigned sample of a Siemens SAA detection kit (nephelometry) in a Siemens special protein instrument (BN II System), wherein the theoretical concentration value of a high-value sample is 500.18mg/L, the theoretical concentration value of a low-value sample is 2.01mg/L, then configuring concentration gradient samples according to a proportion by using the high-value sample and the low-value sample, respectively testing the samples by using four groups of kits, respectively repeatedly measuring each sample for 2 times, reading signals by a full-automatic biochemical analyzer (Hitachi 7180), and respectively calculating a measurement mean value to perform linear range investigation. The results are shown in the following table:
TABLE 9 Linear Range verification
Figure BDA0002946765940000162
According to the experimental results, the relative deviation of the detection values and the theoretical values of the reagent kit in the B group, the C group and the D group is less than 10% in the range of the sample concentration of 2.01-500.18 mg/L, and the experimental results show that the reagent kit is beneficial to improving the performance of the reagent when the composition of benzoic acid and morpholine, morpholine and salicylic acid, piperidine and benzoic acid or piperidine and salicylic acid is added in the labeling process of the latex microspheres.
Although embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments without departing from the principles and spirit of the present invention.

Claims (10)

1. A kit for quantitatively detecting serum amyloid A is characterized by comprising a reagent R1 and a reagent R2;
the reagent R1 comprises the following components in percentage by weight: 50-200mM of buffer solution, 0.1-0.5% of surfactant I, 0.1-0.5% of surfactant II, 0.1-0.5% of preservative, 0.1-0.8% of inorganic salt and 1-2% of coagulant;
the reagent R2 comprises the following components in percentage by weight: 50-100mM of buffer solution, 1-5% of protein protective agent, 0.1-0.5% of preservative, 0.1-0.8% of inorganic salt and latex particles coated with SAA monoclonal antibody;
the preparation process of the latex particles coated with the SAA monoclonal antibody comprises the following steps: the SAA monoclonal antibody is coupled to latex particles in the presence of component a selected from at least one of morpholine or piperidine, preferably morpholine, and component B selected from at least one of benzoic acid or salicylic acid, preferably benzoic acid.
2. The kit for quantitative determination of serum amyloid A according to claim 1, wherein in said reagent R1:
the buffer is selected from at least one of Tris, MOPS or phosphate buffer, preferably MOPS buffer; the surfactant I is selected from at least one of Triton X-100 or Tween-20, preferably Tween-20; the surfactant II is selected from at least one of Brij 58, Tween-80 or A90, preferably A90; the preservative is at least one selected from Proclin-300, sodium azide and gentamicin; the inorganic salt is selected from at least one of sodium chloride or potassium chloride; the coagulant is at least one selected from PEG6000 or PEG 8000.
3. The kit for quantitative determination of serum amyloid A according to claim 2, wherein the pH of said reagent R1 is 6-8, preferably 6-6.5.
4. The kit for quantitative determination of serum amyloid A according to claim 1, wherein in said reagent R2:
the buffer solution is selected from at least one of MOPS, Bicine or phosphate buffer solution, and is preferably Bicine buffer solution; the protein protective agent is selected from at least one of BSA, casein, sucrose or trehalose, and is preferably BSA; the preservative is at least one selected from Proclin-300, sodium azide and gentamicin; the inorganic salt is selected from at least one of sodium chloride or potassium chloride.
5. The kit for quantitative determination of serum amyloid A according to claim 4, wherein said reagent R2 has a pH of 6-8, preferably 6.5-7.
6. The kit for quantitative determination of serum amyloid A according to claims 1-5, wherein the latex particles coated with mouse anti-human SAA antibody have a particle size in the range of 100-200 nm.
7. A method for preparing a kit for quantitative determination of serum amyloid A according to any one of claims 1 to 5, comprising the steps of:
(1) preparation of reagent R1
The reagent R1 was formulated as follows:
50-200mM of buffer solution, 0.1-0.5% of surfactant I, 0.1-0.5% of surfactant II, 0.1-0.5% of preservative, 0.1-0.8% of inorganic salt and 1-2% of coagulant;
the buffer is selected from at least one of Tris, MOPS or phosphate buffer, preferably MOPS buffer; the surfactant I is selected from at least one of TritonX-100 or Tween-20, preferably Tween-20; the surfactant II is selected from at least one of Brij 58, Tween-80 or A90, preferably A90; the preservative is at least one selected from Proclin-300, sodium azide and gentamicin; the inorganic salt is selected from at least one of sodium chloride or potassium chloride; the coagulant is selected from at least one of PEG6000 or PEG 8000;
(2) preparation of reagent R2
Preparation of SAA latex particles
Adding the latex microspheres into a marking buffer solution, and uniformly stirring; activating by adding an activating agent, and uniformly stirring; adding a SAA monoclonal antibody into the activated latex microsphere solution, simultaneously adding a composition of a component A and a component B, wherein the component A is selected from at least one of morpholine or piperidine, preferably morpholine, and the component B is selected from at least one of benzoic acid or salicylic acid, preferably benzoic acid, uniformly stirring, and then incubating for 6-14 h; centrifuging, and discarding the supernatant to obtain latex particles coated with the SAA monoclonal antibody;
preparation of R2 basic buffer solution
The R2 base buffer was formulated as follows:
50-100mM of buffer solution, 1-5% of protein protective agent, 0.1-0.5% of preservative and 0.1-0.8% of inorganic salt;
the buffer solution is selected from at least one of MOPS, Bicine or phosphate buffer solution, and is preferably Bicine buffer solution; the protein protective agent is selected from at least one of BSA, casein, sucrose or trehalose, and is preferably BSA; the preservative is at least one selected from Proclin-300, sodium azide and gentamicin; the inorganic salt is selected from at least one of sodium chloride or potassium chloride.
Preparation of reagent R2
Adding the SAA latex particles prepared in the step I into the R2 basic buffer solution prepared in the step II, dissolving, performing ultrasonic treatment, and incubating at 20-40 ℃ for 6-14h to obtain a reagent R2.
8. The method for preparing a kit for quantitatively detecting serum amyloid A according to claim 7, wherein in the step (i), the compositions of 0.5-1% morpholine and 0.5-1% benzoic acid are added respectively.
9. The method for preparing a kit for quantitatively detecting serum amyloid A according to claim 7, wherein in the step (r), the labeling buffer is at least one selected from phosphate buffer or Tris buffer, preferably phosphate buffer, the activator is EDC/NHS, and the mass ratio of the NHS to EDC and latex microspheres is: 1: 0.5-1.5: 800-1000, wherein the mass ratio of the SAA monoclonal antibody to the latex microsphere is 1: 350-400.
10. The method for preparing a kit for quantitative determination of Serum Amyloid A (SAA) according to claims 8-9, wherein in said step (iii), the content of latex particles coated with SAA monoclonal antibody in said R2 reagent is 0.05-0.1%.
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