CN109406796B - Rheumatoid factor detection kit and detection method thereof - Google Patents
Rheumatoid factor detection kit and detection method thereof Download PDFInfo
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- CN109406796B CN109406796B CN201811507591.6A CN201811507591A CN109406796B CN 109406796 B CN109406796 B CN 109406796B CN 201811507591 A CN201811507591 A CN 201811507591A CN 109406796 B CN109406796 B CN 109406796B
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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
- G01N33/6854—Immunoglobulins
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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
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
Abstract
The invention discloses a rheumatoid factor detection kit and a preparation method thereof. The kit comprises a reagent R1, a reagent R2 and a reagent R3; the reagent R1 contains magnetic particles coated with rabbit IgG; the reagent R2 contains anti-human IgM/IgG/IgA antibody, anti-human IgM antibody, anti-human IgG antibody or anti-human IgA antibody marker; the reagent R3 is prepared by mixing a buffer system, inorganic salt, carbohydrate, protein, colorant, nonionic surfactant and preservative according to a certain proportion. The rheumatoid factor detection kit has the advantages of high sensitivity, wide linear range and strong anti-interference capability, and can safely and quickly detect the rheumatoid factor.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a rheumatoid factor detection kit and a detection method thereof.
Background
Rheumatoid Factor (RF) is an anti-denatured IgG autoimmune antibody that binds to the Fc region of human and various animal IgG molecules. RF has five kinds of IgM, IgG, IgA, IgE and IgD, wherein IgM is the main, IgG and IgA can be seen, and IgE and IgD are rare. Rheumatoid factor and human denatured IgG form immune complex, activate complement, and cause tissue damage. RF is detectable in the serum of most Rheumatoid Arthritis (RA) patients and is one of the important clinical criteria for diagnosing RA. Although the detection of RF can not directly obtain the result of disease diagnosis, and the conclusion of disease diagnosis can be obtained only by synthesizing various indexes such as clinical symptoms and the like, the detection of RF can be used as an important reference index and has important significance for the later-stage RA diagnosis.
At present, most of rheumatoid factors adopt a latex agglutination method and a nephelometry method, but the two methods cannot perform typing detection on RF and have low sensitivity. With the clinical approval of RF typing detection, RF ELISA products are available on the market both domestically and abroad, and the indirect method principle is mostly adopted. The prior chemiluminescence platform has fewer products, but compared with ELISA with complex operation, the chemiluminescence method has the advantages of relatively simple operation, high sensitivity and wide detection range. So the trend is to use chemiluminescence method to detect rheumatoid factor.
Chemiluminescence immunoassay is a non-radioactive immunoassay technique that has developed very rapidly worldwide in recent years. The detection principle is that the luminescent substance is used as a signal amplification system and the immunological binding is directly measured by virtue of the luminous intensity. The method has high sensitivity and wide detection range, and is an important development direction for immunological detection.
Disclosure of Invention
In order to solve the problems of the prior art, the invention provides a rheumatoid factor detection kit and a detection method thereof.
A rheumatoid factor detection kit comprises a reagent R1, a reagent R2 and a reagent R3; the reagent R1 contains magnetic particles coated with rabbit IgG; the reagent R2 contains one or more markers of anti-human IgM antibody, anti-human IgG antibody and anti-human IgA antibody; the reagent R3 is prepared by mixing a buffer system, inorganic salt, carbohydrate, protein substances, a coloring agent, disodium ethylene diamine tetraacetate, a nonionic surfactant and a preservative according to a certain proportion.
In a preferred embodiment, the label is labeled with one of horseradish peroxidase, alkaline phosphatase, an acridinium ester or an acridinium ester derivative.
In a preferred embodiment, the reagent R3 is composed of the following components in the following concentrations: a buffer system with a pH value of 6-8 and a concentration of 10-50 mM; 5-50 g/L of inorganic salt; 5-200 g/L of a saccharide; 5-100 g/L of protein substances; 0.05-0.5 g/L of colorant; 8-50 mM disodium ethylene diamine tetraacetate; 0.01-10 mL/L of nonionic surfactant and 0.1-10 mL/L of preservative.
In another preferred embodiment, the magnetic particles in the reagent R1 are paramagnetic particles, and the magnetic particles are selected from one of avidin magnetic particles, tosyl magnetic particles, and carboxyl magnetic particles; the reagent R1 also comprises a buffer system, inorganic salt, carbohydrate, protein, nonionic surfactant, polyalcohol and preservative; the R2 also includes buffer systems, inorganic salts, carbohydrates, proteinaceous materials, colorants, nonionic surfactants, polyols, and preservatives.
In another embodiment, a kit for detecting rheumatoid factor, the kit comprises a reagent R1, a reagent R2 and a reagent R3;
the reagent R1 is composed of the following components in concentration:
0.2-2 mg/mL of magnetic particles coated with rabbit IgG; a buffer system with a pH value of 6-8.5 and a concentration of 10-200 mM; 4-50 g/L of inorganic salt; 2-300 g/L of a saccharide; 5-160 g/L of protein substances; 0.01-10 mL/L of nonionic surfactant; 5-50 mL/L of polyhydric alcohol and 0.1-10 mL/L of preservative;
the reagent R2 is composed of the following components in concentration:
0.03-4 mu g/mL of a marker of a mixture of anti-human IgM antibody, anti-human IgG antibody and anti-human IgA antibody; a buffer system with a pH value of 5.5-8.5 and a concentration of 10-200 mM; 4-50 g/L of inorganic salt; 2-300 g/L of a saccharide; 5-160 g/L of protein substances; 0.05-0.5 g/L of colorant; 0.01-10 mL/L of nonionic surfactant; 5-50 mL/L of polyhydric alcohol and 0.1-10 mL/L of preservative;
the reagent R3 is composed of the following components in concentration:
a buffer system with a pH value of 6-8 and a concentration of 10-50 mM; 5-50 g/L of inorganic salt; 5-200 g/L of a saccharide; 5-100 g/L of protein substances; 0.05-0.5 g/L of colorant; 8-50 mmol/L disodium ethylene diamine tetraacetate; 0.01-10 mL/L of nonionic surfactant and 0.1-10 mL/L of preservative.
In a preferred embodiment, the buffer system is a phosphate buffer system, a Tris buffer system, a Mes buffer system or a Hepes buffer system; the inorganic salt is sodium chloride; the saccharide is selected from one or more of mannitol, trehalose, glucose, fructose, sucrose, lactose and galactose; the protein substance is selected from one or more of casein and bovine serum albumin; the colorant is fruit green pigment, sunset yellow, amaranth or lemon yellow; the non-ionic surface agent is Tween-20, Tween-80, Span-60, Triton X-45, Triton X-100 or Triton X-305; the polyol is glycerol; the preservative is KroVinn series preservative, ProClin series preservative or sodium azide.
In another preferred embodiment, the kit further comprises a calibrator; the calibrator consists of the following components in concentration: 0.1-500 AU of rheumatoid factor; a buffer system with a pH value of 6-8 and a concentration of 0-50 mM; 4-20 g/L of inorganic salt; 1-200 g/L of a saccharide; 5-150 g/L of protein substances; 0.20g/L of a colorant; 0.01-10 mL/L of nonionic surfactant; 5-50 mL/L of polyhydric alcohol and 0.1-10 mL/L of preservative.
A method for detecting rheumatoid factors by using the rheumatoid factor detection kit comprises the following steps:
firstly, a sample is uniformly mixed with a reagent R1 and a reagent R3, the mixture is fully combined, and then the sample is washed for a plurality of times under the action of a magnetic field,
then, the reagent R2 is added, after sufficient binding, unbound substances are washed away, and then the luminescent substrate is added,
then, the absorbance after the luminescence reaction was measured.
In a preferred embodiment, the volume ratio of the sample to the reagents R1 and R3 is 1: 4-6: 8-12.
In a further preferred embodiment, the volume ratio of the sample, reagent R1, reagent R3 is 1:5: 10.
The present invention will be explained in detail below.
According to the kit for detecting the rheumatoid factor, a full-automatic chemiluminescence magnetic particle immunoassay method is adopted for detecting the rheumatoid factor; the invention also employs reagent R3. In the preservation process, the reagent R1 and the reagent R2 are easy to deteriorate or have short preservation time; therefore, additives such as stabilizers and preservatives are often added into the reagent R1 and the reagent R2 to keep the reagent R1 and the reagent R2 in good test activity during detection, but the addition of the additives excessively affects the accuracy of the detection result; in addition, due to the limited capacity of the kit, the test reagents cannot be placed in the kit as much as possible; therefore, the reagent R3 is added to the method to solve the problem.
The inventor finds that if the reagent R3 is independently prepared, the sample, the reagent R1 and the reagent R3 are mixed according to a proper proportion during testing, so that the problem of short storage time of the detection reagent can be effectively solved, and meanwhile, the detection reagent can be ensured to have an optimal buffer solution system during detection, and the detection sensitivity and accuracy are improved. In addition, the inventor unexpectedly finds that the separately configured reagent R3 also effectively reduces the cross reaction in the rheumatoid factor detection process, namely when the conventional rheumatoid detection kit is used, if the sample is directly detected, the detection process is easily interfered by complement in the sample, and the detection accuracy is influenced; to avoid complement interference in the sample, the sample is often heat treated prior to detection. The rheumatoid detection kit is added with the reagent R3, and after the reagent R3 is added into a sample, the sample does not need heat treatment, and the interference of complement can be avoided by direct test. Therefore, the kit improves the detection specificity due to the addition of the reagent R3, and has stronger anti-interference capability.
In the invention, the rabbit IgG coated on 1mg of magnetic particles in the reagent R1 is preferably 0.3-60 mu g. When the reagent R2 contains a marker of a mixture of anti-human IgM antibody, anti-human IgG antibody and anti-human IgA antibody, the mass ratio of the anti-human IgM antibody, the anti-human IgG antibody and the anti-human IgA antibody in the mixture is preferably 6-12: 0.5-2.
In conclusion, the kit has the advantages of high sensitivity, wide linear range, strong anti-interference capability, safety, quick detection and the like.
Detailed Description
In order to make the technical solutions in the present application better understood, the present invention will be further described with reference to the following examples, and it is obvious that the described examples are only a part of the examples of the present application, but not all of the examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Definition of
Buffer system
The buffer system of the invention is a mixed solution composed of weak acid and salt thereof, weak base and salt thereof, and can offset and reduce the influence of external strong acid or strong base on the pH value of the solution to a certain extent, thereby keeping the pH value of the solution relatively stable. The buffer system comprises a phosphoric acid buffer system, a Tris buffer system, a Mes buffer system or a Hepes buffer system. The phosphoric acid buffer system comprises NaH2PO4And Na2HPO4、NaH2PO4And K2HPO4、KH2PO4And Na2HPO4、KH2PO4And K2HPO4. When the buffer system used is a phosphoric acid buffer system, the concentration of the buffer system is measured as phosphate radical concentration.
Inorganic salt
The inorganic salt functions in the buffer of the present invention to provide an electrolyte. In the invention, the inorganic salt is preferably sodium salt or potassium salt; more preferably, the inorganic salt of the present invention is sodium chloride.
Saccharide material
The function of the carbohydrate in the buffer solution is stabilizing function, and the carbohydrate commonly used in the buffer solution is mannitol, trehalose, glucose, fructose, sucrose, lactose and galactose.
Protein substance
The protein substance has a stabilizing effect in the buffer solution, and the protein substance is casein and/or bovine serum albumin.
Coloring agent
Any substance that can cause a substance to develop a desired color for a design is referred to as a colorant. It may be organic or inorganic, and may be natural or synthetic. Colorants, also known as food colors, are substances that are used to impart color to and improve the color of food, primarily for the purpose of coloring food. At present, more than 60 kinds of food coloring agents are commonly used in the world, and 46 kinds of food coloring agents are allowed to be used in China and are divided into food synthetic coloring agents and food natural coloring agents according to the sources and the properties of the food synthetic coloring agents and the food natural coloring agents. The colorants of the present invention are identification. Common colorants include fruit green pigment, amaranth, carmine, lemon yellow, sunset yellow, indigo, brilliant blue, red, allura red, and the like.
Nonionic surfactant
Nonionic surfactants are those which do not ionize in aqueous solution and whose hydrophilic groups are composed predominantly of a certain number of oxygen-containing groups, typically ether and hydroxyl groups. Because the ionic surfactant is not in an ionic state in the solution, the ionic surfactant has high stability, is not easily influenced by the existence of strong electrolyte inorganic salts, is not easily influenced by pH value, and has good compatibility with other types of surfactants. The nonionic surfactant of the present invention is used to promote solubilization and enhance immune response. The nonionic surfactant of the present invention is preferably Tween-20, Tween-80, Span-60, Triton X-45, Triton X-100 or Triton X-305.
Preservative
Preservatives are a class of food additives that inhibit microbial activity and prevent spoilage of food products. The preservative in the invention has the function of corrosion prevention. The preservative is KroVinn series preservative, ProClin series preservative or sodium azide. The KroVinn series antiseptic is preferably KroVin 100, KroVin 400, KroVin 500 or KroVin 750; the ProClin series preservative is preferably ProClin50, ProClin150, ProClin200, ProClin300, ProClin950 or ProClin 5000.
Taking a detection kit of rheumatoid factor (IgM) as an example,
the preparation method of the kit comprises the following steps:
1) reagent R1-magnetic particle coated rabbit IgG, the specific process is as follows:
antibody biotinylation: taking a proper amount of rabbit IgG, adding a proper amount of biotin ester, uniformly mixing and reacting on a mixing instrument for 0.5-3 hours at room temperature, then adding a proper amount of Tris aqueous solution, uniformly mixing and reacting on the mixing instrument for 10-30 minutes at room temperature, dialyzing the mixed solution with a phosphate buffer solution, and dialyzing to obtain biotinylated rabbit IgG;
coating antibody: mixing a proper amount of biotinylated rabbit IgG and streptavidin magnetic particles, uniformly mixing the mixture on a mixing instrument at room temperature for reaction for 0.5-3 hours, washing the coated magnetic particles with a magnetic particle protection buffer solution, and then suspending the magnetic particles again to 0.2-2 mg/mL to obtain a reagent R1. (the coating amount of biotinylated rabbit IgG is 0.3-60 mu g/mg). The formula of the magnetic particle protection buffer solution is as follows:
table 1 shows the formulation of the magnetic particle protection buffer
| Specific components | Concentration of |
| Buffer system | 10～200mM(pH6～8.5) |
| Inorganic salt | 4～50g/L |
| Saccharide material | 2～300g/L |
| Protein substance | 5～160g/L |
| Nonionic surfactant | 0.01～10mL/L |
| Polyhydric alcohols | 5～50mL/L |
| Preservative | 0.1～10mL/L |
2) Acridinium ester labeled anti-human IgM antibody
And (2) adding a proper amount of acridine ester into a proper amount of anti-human IgM antibody, uniformly mixing and reacting for 0.5-5 hours on a mixing instrument at room temperature in a dark place, then adding a proper amount of lysine aqueous solution, uniformly mixing and reacting for 10-30 minutes on the mixing instrument at room temperature in the dark place, and purifying the mixed solution by a desalting column to obtain the acridine ester labeled anti-human IgM antibody. Diluting the obtained product to 0.03-4 mu g/mL by using a marker protection buffer solution to obtain an immune marker, namely a reagent R2. The components of the marker protection buffer are shown in table 2:
table 2 shows the composition of the marker protection buffer of the present invention
| Specific components | Concentration of |
| Buffer system | 10～200mM(pH5.5～8.5) |
| Inorganic salt | 4～50g/L |
| Saccharide material | 2～300g/L |
| Protein substance | 5～160g/L |
| Coloring agent | 0.05～0.5g/L |
| Nonionic surfactant | 0.01～10mL/L |
| Polyhydric alcohols | 5～50mL/L |
| Preservative | 0.1～10mL/L |
3) Preparation of reagent R3
The reagent R3 according to the invention was prepared as shown in table 3:
table 3 shows the formulation of the reagent R3 according to the invention
| Specific components | Concentration of |
| Buffer system | 10～50mM(pH6～8) |
| Inorganic salt | 5～50g/L |
| Saccharide material | 5～200g/L |
| Protein substance | 5～100g/L |
| Coloring agent | 0.05～0.5g/L |
| Ethylenediaminetetraacetic acid disodium salt | 8～50mM |
| Nonionic surfactant | 0.01～10mL/L |
| Preservative | 0.1～10mL/L |
4) Preparation of calibrator
The calibrator of the invention comprises two points of calibrator 1 and calibrator 2, and a proper amount of rheumatoid factor is respectively diluted to 30AU/mL and 300AU/mL by calibrator diluent. The components of the standard dilution are shown in the table.
Table 4 shows the composition of the dilution of the calibrator of the invention
| Specific components | Concentration of |
| Buffer system | 0～50mM(pH6～8) |
| Inorganic salt | 4～20g/L |
| Saccharide material | 1～200g/L |
| Protein substance | 5～150g/L |
| Coloring agent | 0.20g/L |
| Nonionic surfactant | 0.01～10mL/L |
| Polyhydric alcohols | 5～50mL/L |
| Preservative | 0.1～10mL/L |
Secondly, the detection method of the concentration of the rheumatoid factor in human serum (blood plasma) comprises the following steps:
the whole detection is carried out on a full-automatic chemiluminescence apparatus.
First, a sample is mixed with rabbit IgG-coated magnetic microparticles (reagent R1) and an assay buffer (reagent R3) to react, and if the sample contains rheumatoid factor, an antibody-antigen complex is formed. The magnetic particles in the mixed solution are adsorbed under the action of the magnetic field, and the unbound substances are washed and removed by multiple times of washing. A label of anti-human IgM antibody (reagent R2) is then added, at which point an antigen-antibody-secondary antibody sandwich complex will be formed.
Then, the magnetic particles in the mixed solution are adsorbed by the magnetic field, and the unbound substances are washed and removed by a plurality of times of washing. Thereafter, the immunoassay substrate solution 1 and the immunoassay substrate solution 2 were added, and the chemiluminescence photon intensity (RLU) thereof was measured. The intensity of the photons produced is proportional to the concentration of rheumatoid factor (IgM) in the sample. And then calculating the concentration of the rheumatoid factor corresponding to the photon intensity according to a standard curve.
In the following examples, the specific components and formulations of reagent R1, reagent R2, and calibrator in the kit are shown in tables 5-7, respectively.
Table 5 shows the ingredients and specific contents of the reagent R1
Table 6 shows the ingredients and specific contents of the reagent R2
| Anti-human IgM labeled with acridinium ester | 1μg/mL |
| Phosphoric acid buffer system | 20mM,ph6.5 |
| Sodium chloride | 35g/L |
| Casein protein | 10g/L |
| Bovine serum albumin | 10g/L |
| Trehalose | 20g/L |
| Fruit green pigment | 0.1g/L |
| TX-100 | 3mL/L |
| Glycerol | 20mL/L |
| Biological preservative PC-300 | 3mL/L |
Table 7 Components of calibrator dilutions
| Rheumatoid factor | 30AU/mL and 100AU/mL |
| Phosphoric acid buffer system | 30Mm,ph7.5 |
| Sodium chloride | 10g/L |
| Bovine serum albumin | 100g/L |
| Trehalose | 67g/L |
| Fruit green pigment | 0.20g/L |
| TX-100 | 6.8mL/L |
| Glycerol | 24mL/L |
| Biological preservative PC-950 | 5.5mL/L |
Example 1
The components of the reagent R3 in the kit of this example are shown in the following table:
table 8 shows the composition of the reagent R3 in example 1
When the kit prepared by using the reagent R3 in example 1 is used for sample detection, the detection CV value is less than 3%. See in particular the table below.
Table 9 shows CV values obtained by repeating the same samples 10 times
The kits of the comparative examples described below, both reagent R1 and reagent R2, differ only by reagent R3.
Comparative example 1
The components of the reagent R3 in the kit of this comparative example are shown in the following table:
table 10 shows the composition of the reagent R3 in comparative example 1
Comparative example 2
The components of the reagent R3 in the kit of this comparative example are shown in the following table:
table 11 shows the composition of the reagent R3 in comparative example 2
| Phosphoric acid buffer system | 25mM,ph7.5 |
| Sodium chloride | 20g/L |
| Glucose | 220g/L |
| Casein protein | 2.5g/L |
| Fruit green pigment | 0.3g/L |
| Ethylenediaminetetraacetic acid disodium salt | 30mM |
| Tween-20 | 2mL/L |
| Biological preservative PC-950 | 6mL/L |
Comparative example 3
The components of the reagent R3 in the kit of this comparative example are shown in the following table:
table 12 shows the composition of the reagent R3 in comparative example 3
| Phosphoric acid buffer system | 25mM,ph7.5 |
| Sodium chloride | 20g/L |
| Glucose | 85g/L |
| Casein protein | 10g/L |
| Fruit green pigment | 0.3g/L |
| Tween-20 | 2mL/L |
| Biological preservative PC-950 | 6mL/L |
Example 2
In this example, for the detection of a sample having a known concentration, the detection reagent R1 and the detection reagent R2 are the same, and the reagent R3 is the same as in example 1, the reagent R3 is not used, and a physiological saline solution is used instead of the reagent R3. The detection method is the same as the detection method of the concentration of the rheumatoid factor in human serum (blood plasma). The results are shown in the following table:
TABLE 13 chemiluminescent photon intensities measured for known samples
As can be seen from Table 13, the kit added with the reagent R3 has high detection sensitivity and high signal-to-multiple ratio among concentrations; the detection sensitivity is low and the signal multiple ratio among various concentrations is low without adding the reagent R3; the use of physiological saline instead of the reagent R3 has low detection sensitivity, and is not favorable for the detection of low-concentration samples.
Example 3
The reagent kit for testing of this example was the same as the reagent R1 and the reagent R2, and the reagent R3 was the reagent R3 used in example 1 and comparative example 1, respectively. Then, samples with known concentrations are detected, and the detection results are shown in the following table:
TABLE 14 photon intensities detected for different reagent R3 formulations on known samples
As can be seen from the above table, although the concentration of the sample was detected using the kit of comparative example 1, the background thereof was low, the specific signal detected using the kit of comparative example 1 was much lower than that of example 1, resulting in lower sensitivity than that of example 1. Therefore, the reagent R3 of the present invention must be properly compounded to provide a high sensitivity of the kit.
Example 4
The kits of example 1 and comparative example 2 were used for the kits of this example, respectively. Then, samples with known concentrations are detected, and the detection results are shown in the following table:
TABLE 15 photon intensities detected for known samples for different reagent R3 formulations
As can be seen from the table, if the contents of the components of the reagent R3 in the kit are individually out of the proper range, the detection accuracy may be affected, and the detection sensitivity is low. Therefore, the kit can be detected with good accuracy and high sensitivity only in the components and proper value range of the reagent R3 provided by the invention.
Example 5
And (3) testing the anti-interference capability of the reagent R3 on the rheumatoid factor sample.
Clinical samples were tested using the kits of example 1 and comparative example 3, respectively, and then the concentration of rheumatoid factor RF in the samples was calculated from the photon intensities obtained by the tests. When the kit in example 1 was used, the sample was subjected to heat treatment and non-heat treatment; the results are shown in the following table:
TABLE 16 determination of RF concentration in samples with kits containing different reagents R3
As can be seen from Table 16, when the kit was used, no interference of complement in the sample could be avoided when the reagent R3 having an inappropriate composition was added or not. Only when the reagent R3 with proper component composition is used in the kit, complement interference can be avoided, and the test specificity and the test accuracy of the kit are improved. By using the kit, the sample can be directly tested without heating, and the testing process is simple.
Example 6
The invention relates to a detection kit for rheumatoid factors (IgM/IgG/IgA):
the reagents R1 and R3 in the kit are the same as in example 1, the marker and the protection buffer solution in the reagent R2 are the same as in example 1, and the marker is a mixture of an anti-human IgM antibody, an anti-human IgG antibody and an anti-human IgA antibody according to the mass ratio of 8:1: 1. The kit of the embodiment is used for detecting samples with known concentrations, and the detection result is high in accuracy and good in stability.
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Claims (9)
1. Use of a kit comprising disodium edetate for reducing interference caused by the presence of complement in a sample in rheumatoid factor detection; wherein the kit comprises a reagent R1, a reagent R2 and a reagent R3; the reagent R1 contains magnetic particles coated with rabbit IgG; the reagent R2 contains one or more markers of anti-human IgM antibody, anti-human IgG antibody and anti-human IgA antibody; the reagent R3 is composed of the following components in concentration: a phosphate buffer system with a pH value of 6-8 and a concentration of 10-50 mM; 5-50 g/L of sodium chloride; 5-200 g/L glucose; 5-100 g/L casein; 0.05-0.5 g/L of colorant; 8-50 mM disodium ethylene diamine tetraacetate; 0.01-10 mL/L of Tween-20 and 0.1-10 mL/L of preservative.
2. Use according to claim 1, characterized in that: the marker is marked with one of horseradish peroxidase, alkaline phosphatase, acridinium ester or acridinium ester derivatives.
3. Use according to any one of claims 1 to 2, characterized in that: the magnetic particles in the reagent R1 are paramagnetic particles, and the magnetic particles are selected from one of avidin magnetic particles, tosyl magnetic particles and carboxyl magnetic particles; the reagent R1 also comprises a buffer system, inorganic salt, carbohydrate, protein, nonionic surfactant, polyalcohol and preservative; the R2 also includes buffer systems, inorganic salts, carbohydrates, proteinaceous materials, colorants, nonionic surfactants, polyols, and preservatives.
4. Use according to claim 1, characterized in that: the reagent R1 is composed of the following components in concentration:
0.2-2 mg/mL of magnetic particles coated with rabbit IgG; a buffer system with a pH value of 6-8.5 and a concentration of 10-200 mM; 4-50 g/L of inorganic salt; 2-300 g/L of a saccharide; 5-160 g/L of protein substances; 0.01-10 mL/L of nonionic surfactant; 5-50 mL/L of polyhydric alcohol and 0.1-10 mL/L of preservative;
the reagent R2 is composed of the following components in concentration:
0.03-4 mu g/mL of a marker of a mixture of anti-human IgM antibody, anti-human IgG antibody and anti-human IgA antibody; a buffer system with a pH value of 5.5-8.5 and a concentration of 10-200 mM; 4-50 g/L of inorganic salt; 2-300 g/L of a saccharide; 5-160 g/L of protein substances; 0.05-0.5 g/L of colorant; 0.01-10 mL/L of nonionic surfactant; 5-50 mL/L of polyhydric alcohol and 0.1-10 mL/L of preservative.
5. Use according to claim 1, characterized in that: the kit further comprises a calibrator; the calibrator consists of the following components in concentration: 0.1-500 AU of rheumatoid factor; a buffer system with a pH value of 6-8 and a concentration of 0-50 mM; 4-20 g/L of inorganic salt; 1-200 g/L of a saccharide; 5-150 g/L of protein substances; 0.20g/L of a colorant; 0.01-10 mL/L of nonionic surfactant; 5-50 mL/L of polyhydric alcohol and 0.1-10 mL/L of preservative.
6. A rheumatoid factor detection kit is characterized in that: the kit comprises a reagent R1, a reagent R2 and a reagent R3;
the reagent R1 contains magnetic particles coated with rabbit IgG;
the reagent R2 contains one or more markers of anti-human IgM antibody, anti-human IgG antibody and anti-human IgA antibody;
the reagent R3 is composed of the following components in concentration:
a 25mM phosphate buffer system having a pH of 7.5; 20g/L of sodium chloride; 85g/L glucose; 10g/L casein; 0.3g/L of fruit green pigment; 30mM disodium edetate; 2mL/L of Tween-20 and 6mL/L of PC950 as a preservative.
7. The rheumatoid factor detection kit according to claim 6, wherein: the kit comprises a reagent R1, a reagent R2 and a reagent R3;
the reagent R1 is composed of the following components in concentration:
0.2-2 mg/mL of magnetic particles coated with rabbit IgG; a buffer system with a pH value of 6-8.5 and a concentration of 10-200 mM; 4-50 g/L of inorganic salt; 2-300 g/L of a saccharide; 5-160 g/L of protein substances; 0.01-10 mL/L of nonionic surfactant; 5-50 mL/L of polyhydric alcohol and 0.1-10 mL/L of preservative;
the reagent R2 is composed of the following components in concentration:
0.03-4 mu g/mL of a marker of a mixture of anti-human IgM antibody, anti-human IgG antibody and anti-human IgA antibody; a buffer system with a pH value of 5.5-8.5 and a concentration of 10-200 mM; 4-50 g/L of inorganic salt; 2-300 g/L of a saccharide; 5-160 g/L of protein substances; 0.05-0.5 g/L of colorant; 0.01-10 mL/L of nonionic surfactant; 5-50 mL/L of polyhydric alcohol and 0.1-10 mL/L of preservative.
8. The rheumatoid factor detection kit according to claim 7, wherein: the buffer system is a phosphoric acid buffer system, a Tris buffer system, a Mes buffer system or a Hepes buffer system; the inorganic salt is sodium chloride; the saccharide is selected from one or more of mannitol, trehalose, glucose, fructose, sucrose, lactose and galactose; the protein substance is selected from one or more of casein and bovine serum albumin; the colorant is fruit green pigment, sunset yellow, amaranth or lemon yellow; the non-ionic surface agent is Tween-20, Tween-80, Span-60, Triton X-45, Triton X-100 or Triton X-305; the polyol is glycerol; the preservative is KroVinn series preservative, ProClin series preservative or sodium azide.
9. The rheumatoid factor detection kit according to any one of claims 6 to 8, wherein: the kit further comprises a calibrator; the calibrator consists of the following components in concentration: 0.1-500 AU of rheumatoid factor; a buffer system with a pH value of 6-8 and a concentration of 0-50 mM; 4-20 g/L of inorganic salt; 1-200 g/L of a saccharide; 5-150 g/L of protein substances; 0.20g/L of a colorant; 0.01-10 mL/L of nonionic surfactant; 5-50 mL/L of polyhydric alcohol and 0.1-10 mL/L of preservative.
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| CN104914244A (en) * | 2015-05-27 | 2015-09-16 | 山东博科生物产业有限公司 | Kit for combined detection of hepatitis C virus antigen and antibody through chemiluminescence |
| CN105911285A (en) * | 2016-05-27 | 2016-08-31 | 安徽伊普诺康生物技术股份有限公司 | Kit for determining rheumatoid factors |
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| CN108196069B (en) * | 2018-02-01 | 2020-06-09 | 深圳德睿生物科技有限公司 | Hepatitis C virus antibody detection reagent containing recombinant fusion antigens A and B, application of hepatitis C virus antibody detection reagent and recombinant fusion antigens A and B |
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