CN113311169A - Kit for determining immunoglobulin G4 and preparation method thereof - Google Patents

Kit for determining immunoglobulin G4 and preparation method thereof Download PDF

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CN113311169A
CN113311169A CN202110588168.9A CN202110588168A CN113311169A CN 113311169 A CN113311169 A CN 113311169A CN 202110588168 A CN202110588168 A CN 202110588168A CN 113311169 A CN113311169 A CN 113311169A
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reagent
kit
buffer
immunoglobulin
monoclonal antibody
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温路新
刘霖
蒋会会
刘雨薇
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Chongqing Zhongyuan Huiji Biotechnology Co Ltd
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Abstract

The invention discloses a kit for determining immunoglobulin G4, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises 50-100mol/L buffer solution, 0.1-1mol/L inorganic salt, 10-40G/L accelerator, 2-10G/L surfactant and 0.5-1.5G/L preservative; the reagent R2 includes: 10-80mmol/L buffer solution, 0.1-1mol/L inorganic salt, 20-80g/L blocking protein and 30-80ml/L latex microspheres coated with IgG4 monoclonal antibody, wherein the particle size of the latex microspheres is 20-50nm, and the preparation process of the latex microspheres coated with IgG4 monoclonal antibody comprises the following steps: after the latex microsphere blocking was complete, 0.2-0.5mg of IgG4 monoclonal antibody was added back thereto. According to the invention, by selecting the microspheres with extremely low particle size and adding a trace amount of antibody in the preparation process of the latex microspheres coated with the IgG4 monoclonal antibody, the labeling efficiency of the latex microspheres and the antibody is increased, the HOOK resistance of the reagent is obviously improved, and the linear range of the reagent reaches 0.04-7.0 g/L.

Description

Kit for determining immunoglobulin G4 and preparation method thereof
Technical Field
The invention relates to the field of medical inspection, in particular to a kit for determining immunoglobulin G4 and a preparation method thereof.
Background
Immunoglobulin G (Ig G) is an immunoglobulin produced by plasma cells in the highest amount in blood, and accounts for about 75.0% to 80.0% of the total amount of immunoglobulin, and plays an important role in both cellular and humoral immunity. IgG is divided into different subtypes due to different sequences of heavy chain constant domains, such as four subtypes of IgGl, IgG2, IgG3 and IgG4, wherein IgG4(immunoglobulin G4, IgG4) belongs to the subtype with the lowest content in IgG, and accounts for about 0.8-11.7% of the total IgG content in serum, and the normal content of serum IgG4 in human is 0.08-1.4G/L, the average is 0.56G/L, and the half-life period is 21 days.
Immunoglobulin G4-related disease (IgG4-related disease, IgG4-RD) is a newly discovered systemic autoimmune disease characterized by alterations in one or more organ focally disseminated inflammatory cell infiltration and/or organ enlargement, manifested by elevated serum IgG4 levels, and typical histopathological alterations-lymphoid plasma cell infiltration, basket interstitial fibrosis, and obliterative phlebitis. IgG4-RD may affect a single organ or multiple organs simultaneously or sequentially, thus presenting distinct clinical syndromes: mainly including autoimmune pancreatitis (AIP), Mikulicz Disease (MD), retroperitoneal fibrosis (RPF), sclerosing cholangitis, mesangial lesion, tubulointerstitial nephritis (TIN), and the like.
Currently, the detection means of IgG4 are mainly: enzyme-linked immunosorbent assay (ELISA), which is not yet used clinically on a large scale because it requires manual manipulation and is time-consuming, and immunoturbidimetric assay. The R2 reagent is prepared by using antiserum or polyclonal antibody directly by a common turbidimetric method, but a large amount of nonspecific protein exists in the antiserum or polyclonal antibody, the differences among four subtypes of IgGl, IgG2, IgG3 and IgG4 are small, cross reaction easily occurs by using polyclonal antibody, and IgG4 is difficult to be specifically identified.
Disclosure of Invention
The invention provides a kit for quantifying immunoglobulin G4, which is characterized in that latex microspheres with extremely low particle sizes are selected, and the labeling process of the latex microspheres is optimized as follows: the method for adding a trace amount of IgG4 monoclonal antibody back in the process of labeling the latex microspheres increases the labeling efficiency of the IgG4 monoclonal antibody and the latex microspheres, and obviously improves the detection performance of the reagent, particularly the HOOK resistance of the reagent.
In order to achieve the purpose, the invention adopts the following technical means: a kit for the determination of immunoglobulin G4, comprising reagent R2, characterized in that: the reagent R2 comprises 10-80mmol/L buffer solution, 0.1-1mol/L inorganic salt, 20-80g/L blocking protein and 30-80ml/L latex microspheres coated with IgG4 monoclonal antibodies, and the particle size of the latex microspheres is 20-50 nm.
Preferably, the reagent R1 is further included, and the reagent R1 includes: 50-100mol/L buffer solution, 0.1-1mol/L inorganic salt, 10-40g/L accelerator, 2-10g/L surfactant and 0.5-1.5g/L preservative.
Preferably, in the reagent R2: the buffer is selected from at least one of MOPS, Tris, boric acid buffer or phosphate buffer, preferably boric acid buffer or Tris buffer; the inorganic salt is selected from at least one of sodium chloride or potassium chloride; the blocking protein is at least one of BSA or casein.
Preferably, in the reagent R1: the buffer is selected from at least one of MOPS, sodium citrate buffer, Tris or MES, and the inorganic salt is selected from at least one of sodium chloride or potassium chloride; the accelerator is selected from at least one of PEG8000, PEG6000 or PEG 20000; the surfactant is selected from at least one of TX-100, Brij58, A90 or Tween-20; the preservative is selected from at least one of sodium azide or PC 3000.
Preferably, the pH of the reagent R1 is 6.0-8.0; the PH of the reagent R2 is 6.0-8.0.
A method for preparing the kit for detecting immunoglobulin G4 according to any one of the above, comprising the steps of:
(1) preparation of reagent R1
The reagent R1 was formulated as follows:
50-100mol/L buffer solution, 0.1-1mol/L inorganic salt, 10-40g/L accelerator, 2-10g/L surfactant and 0.5-1.5g/L preservative;
the buffer is selected from at least one of MOPS, sodium citrate buffer, Tris or MES; the inorganic salt is selected from at least one of sodium chloride or potassium chloride; the accelerator is selected from at least one of PEG8000, PEG6000 or PEG 20000; the surfactant is selected from at least one of TX-100, Brij58, A90 or Tween-20; the preservative is selected from at least one of sodium azide or PC 3000;
(2) preparation of reagent R2
Adding the latex microspheres into a marking buffer solution, and uniformly stirring; then adding an NHS/EDC activating agent for activation, and uniformly stirring; adding an IgG4 monoclonal antibody into the activated latex microsphere solution, stirring uniformly, and then incubating for 6-14 h; adding 1% of blocking protein, incubating for 1h, adding IgG4 monoclonal antibody back to the prepared reagent, mixing, and aging in an oven for 3 days.
The buffer is selected from at least one of MOPS, Tris, boric acid buffer or phosphate buffer; preferably a boric acid buffer or a Tris buffer; the inorganic salt is selected from at least one of sodium chloride or potassium chloride; the blocking protein is at least one of BSA or casein.
Preferably, the latex microspheres have a particle size of 20-50 nm.
Preferably, in the step (2), the IgG4 monoclonal antibody is added back to the prepared reagent in an amount of 0.2-0.5 mg.
Preferably, the activator adopts EDC/NHS, and the mass ratio of the NHS to the EDC and the latex microspheres is as follows: 1: 20-40, wherein the mass ratio of the IgG4 monoclonal antibody to the latex microspheres is 1: 0.5-1.
Preferably, the content of the latex particles coated with the IgG4 monoclonal antibody in the reagent R2 is 20-30%.
The invention has the beneficial effects that:
(1) according to the invention, by selecting the microspheres with extremely low particle size and adding a trace amount of antibody in the preparation process of the latex microspheres coated with the IgG4 monoclonal antibody, the labeling efficiency of the latex microspheres and the antibody is increased, the detection performance of the kit is obviously improved, the HOOK resistance of the reagent is obviously improved, and the linear range of the reagent reaches 0.04-7.0 g/L;
(2) the conventional centrifugation step is omitted in the preparation process of the latex reagent, so that the preparation process of the kit is greatly shortened, and the preparation process is simplified;
(3) according to the invention, by selecting the mode of labeling the low-particle-size microspheres with the IgG4 monoclonal antibody, the sample is not required to be diluted additionally in the detection process, the sample pretreatment steps are reduced, and the detection flow of the sample is simplified on the basis of ensuring the detection precision and accuracy.
Drawings
FIG. 1 is a graph of the linear relationship between the theoretical concentration of assigned sample IgG4 and the measured values of group A kit, provided in example 3 of the present invention;
FIG. 2 is a graph of the linear relationship between the theoretical concentration of assigned sample IgG4 and the measured values of group B kits, provided in example 3 of the present invention;
FIG. 3 is a graph of the linear relationship between the theoretical concentration of assigned sample IgG4 and the assay value of group C kit, provided in example 3 of the present invention;
FIG. 4 is a graph of the linear relationship between the theoretical concentration of a sample with ultrahigh assigned value of IgG4 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 an ultra-high valued sample of IgG4 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 a sample with ultrahigh assigned value of IgG4 and the measured values of a 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 a sample with ultrahigh assigned value of IgG4 and the measured values of a group D kit, provided in example 4 of the present invention;
FIG. 8 is a graph of the linear relationship between the theoretical concentration of a sample of ultra-high assigned IgG4 values and the values measured with a group E kit, provided in example 4 of the present invention;
FIG. 9 is a graph of the linear relationship between the theoretical concentration of a super-high assigned sample of IgG4 and the measured values of a group A kit, provided in example 5 of the present invention;
FIG. 10 is a graph of the linear relationship between the theoretical concentration of a super-high assigned sample of IgG4 and the measured values of a group B kit, provided in example 5 of the present invention;
FIG. 11 is a graph of the linear relationship between the theoretical concentration of a sample of ultra-high assigned IgG4 value and the measured values of a group C kit, provided in example 5 of the present invention;
FIG. 12 is a graph of the linear relationship between the theoretical concentration of a sample of ultra-high assigned IgG4 value and the assay values of a set D kit, provided in example 5 of the present invention;
FIG. 13 is a graph of the linear relationship between the theoretical concentration of a super-high valued sample of IgG4 and the measured values of a group E kit, provided in example 5 of the present invention;
FIG. 14 is a graph of the linear relationship between the theoretical concentration of a super-high assigned sample of IgG4 and the measured values of a group A kit, provided in example 6 of the present invention;
FIG. 15 is a graph of the linear relationship between the theoretical concentration of a super-high assigned sample of IgG4 and the measured values of a group B kit, provided in example 6 of the present invention;
FIG. 16 is a graph of the linear relationship between the theoretical concentration of a sample with an ultrahigh assigned value of IgG4 and the measured values of a group C kit, provided in example 6 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 IgG4 test kit
The immunoglobulin G4(IgG4) 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 BDA0003084097490000051
2. preparation of reagent R2
b. Adding 100 mu L of 40nm (solid content is 10%) latex microspheres into 2ml of 30mM Tris marking buffer solution, and uniformly stirring;
c. adding 0.3mg of NHS and 0.5mg of EDC activated microspheres into the solution, and stirring at a constant speed for 0.5 h;
d. adding 150.0 mu L of 10g/L mouse anti-human IgG4 monoclonal antibody into the activated latex microsphere solution, stirring uniformly, and then incubating for 6 h;
e. adding 1% blocking protein BSA, incubating for 1h, adding 20ul of 10g/L mouse anti-human IgG4 monoclonal antibody into the prepared reagent, mixing, and aging in an oven at 37 ℃ for 3 days.
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 BDA0003084097490000061
2. Assay protocol
Figure BDA0003084097490000062
3. Computing method
IgG4 in a serum sample can be combined with anti-IgG 4 antibody in a reagent to form an antigen-antibody-microsphere complex, certain turbidity is generated, the turbidity is proportional to the content of the antigen when certain antibody exists, the turbidity is measured at certain wavelength, and the IgG4 can be quantitatively measured through a multi-point calibration curve.
Complement IgG4(g/L) ═ CS × Δ AT/Δ AS (g/L) in the sample
(wherein. DELTA.AT represents the absorbance of the sample tube AS compared with the absorbance of the blank tube,. DELTA.AS represents the absorbance of the calibration tube AS compared with the absorbance of the blank tube, and the concentration of IgG4 in the CS calibration solution)
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 the invention example 1;
group B: the kit prepared by the method described in example 1 of patent CN 112198319A;
group C: immunoglobulin G subtype 4 detection kit (latex enhanced immunoturbidimetry-Borong).
(1) Accuracy verification
Three groups of kits are used for respectively carrying out accuracy tests on immunoglobulin G4 determination kit (nephelometry, Siemens) assignment samples, 2 times of the accuracy tests are set, signals are read through a full-automatic biochemical analyzer (Hitachi 7180), and the relative deviation of the determination mean value and the target value is calculated for accuracy verification. The results are shown in the following table:
TABLE 1 accuracy verification
Figure BDA0003084097490000071
From the above experimental results, the relative deviations of the test value 1 and the target value 1 of the three sets of kits are 1.36%, 3.18% and 4.09%, respectively, and the relative deviations of the test value 2 and the target value 2 are 0.91%, 3.41% and 4.77%, respectively. The detection 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 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 IgG4, 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 BDA0003084097490000081
From the above experimental results, the coefficient of variation of the three sets of kits in the detection of the low value sample is 1.57%, 4.97% and 3.35% respectively, the coefficient of variation in the detection of the median sample is 3.35%, 2.35% and 1.49% respectively, and the coefficient of variation in the detection of the high value sample is 0.84%, 4.88% and 8.29% respectively, and the experimental results show that the three sets of kits all have better precision in the detection of the median sample, while in the detection of the low value sample, the CV value of the a set of kits is significantly lower than that of the B set of kits, and in the detection of the high value sample, the CV value of the a set of kits is significantly lower than that of the B set and the C set, which indicates that the kit (a set) prepared in example 1 of the present invention has better precision than that of the control kit-1 (B set) and the control kit-2 (C set) in the detection of the low value sample and the high value sample.
(3) Linear range verification
An immunoglobulin G4 determination kit (nephelometry, Siemens, Commodity number) is selected to assign a value to an ultrahigh-value sample in a full-automatic biochemical analyzer (Hitachi 7180), wherein the theoretical concentration value of the high-value sample is 7.12G/L, then the high-value sample and ultrapure water are utilized to prepare concentration gradient samples according to a proportion, three groups of kits are used for respectively testing the samples, each sample is repeatedly determined for 2 times, signals are read through the full-automatic biochemical analyzer (Hitachi 7180), and the determination mean values are respectively calculated for linear range investigation. The results are shown in the following table:
TABLE 3 Linear Range verification
Figure BDA0003084097490000091
From the aboveAs shown by experimental results, the kit (group A) prepared in example 1 of the invention has small relative deviation of the detection value and the theoretical value in the sample concentration range of 0.04-7.12g/L, and particularly has relative deviation of less than 1% in the detection value and the theoretical value in the range of 5.34-7.12 g/L. When the sample concentration of the control kit-1 (group B) and the control kit-2 (group C) is more than 5.34g/L, the relative deviation of the detection value and the theoretical value is more than 20 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 the attached figures 1-3), the correlation between the detection value of the group A and the theoretical value is obviously superior to that between the detection value of the group B and the detection value of the group C, and the correlation R between the detection value of the group A and the theoretical value is obviously superior to that between the detection value of the group C and the group B20.9999, group B R20.9795, group C R2Is 0.9586. 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-1 and the control kit-2, and particularly, the two groups of control kits cannot carry out accurate detection on high-value samples with the concentration higher than 5.34 g/L. According to the experimental result, the HOOK effect appears in the two groups of control kits in the measuring process, so that the measured value of the two groups of control kits for the high-value sample is not accurate.
EXAMPLE 4 Effect of Back-adding antibodies on the anti-HOOK Effect of reagents
In order to verify the latex microsphere labeling process, the anti-HOOK capacity of the reagent can be effectively optimized by adding the IgG4 monoclonal antibody, and 5 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 example 1 only in that the IgG4 monoclonal antibody is not added back in the labeling process of the latex microspheres, and the rest preparation methods are the same as those in example 1;
group C: the kit is different from the kit in the embodiment 1 only in that 10ul 10g/L IgG4 monoclonal antibody is added back in the labeling process of the latex microspheres, 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 50ul 10g/L IgG4 monoclonal antibody is added back in the labeling process of the latex microspheres, and the other preparation methods are the same as the embodiment 1;
group E: the kit is different from the kit in the embodiment 1 only in that 100ul 10g/L IgG4 monoclonal antibody is added back in the labeling process of the latex microspheres, and the other preparation methods are the same as the embodiment 1;
an immunoglobulin G4 determination kit (nephelometry, Siemens) is selected to assign values to ultra-high value samples in a full-automatic biochemical analyzer (Hitachi 7180), the concentration of the samples is 27.62G/L, each concentration gradient sample is prepared by diluting ultrapure water in proportion, three groups of kits are used for respectively testing the samples, each sample is repeatedly determined for 3 times, signals are read by the full-automatic biochemical analyzer (Hitachi 7180), and the determination mean value and the relative deviation SD are respectively calculated to verify the HOOK resistance. The results are shown in the following table:
TABLE 4 verification of HOOK resistance of kit
Figure BDA0003084097490000101
According to the experimental results, the detection results of the five groups of kits are compared and analyzed with the theoretical value of the sample concentration (as shown in the attached figures 4-8), and the results show that the kit (group A) prepared in the embodiment 1 of the invention can achieve the HOOK resistance of 24.17g/L (the highest concentration corresponding to the measurement mean value-3 SD of more than 7.00 is the HOOK resistance value of the reagent); the kit in the group B has lower HOOK resistance; the HOOK resistance of the group C kit is 17.26 g/L; the HOOK resistance of the kit in the group D is 20.72 g/L; the HOOK resistance of the kit in the group E was 13.81 g/L.
In conclusion, the invention can effectively improve the anti-HOOK capacity of the reagent by adding a proper amount of antibody in the labeling process of the latex microspheres, and the anti-HOOK capacity of the reagent is the best when the antibody is added in an amount of 0.2-0.5mg of IgG4 monoclonal antibody.
Example 5 Effect of latex microsphere particle size on the HOOK Effect of Agents
In order to verify the influence of the particle size of the latex microspheres on the HOOK resistance of the reagent, 5 groups of kits are arranged for verification:
group A: the kit is different from the kit in the embodiment 1 only in that latex microspheres with the particle size of 10nm are selected, and the other preparation methods are the same as those in the embodiment 1;
group B: the kit is different from the kit in the embodiment 1 only in that the latex microspheres with the particle size of 30nm are selected, and the other preparation methods are the same as those in the embodiment 1;
group C: the kit is different from the kit in the embodiment 1 only in that latex microspheres with the particle size of 50nm are selected, and the other preparation methods are the same as those in the embodiment 1;
group D: the kit is different from the kit in the embodiment 1 only in that latex microspheres with the particle size of 100nm are selected, and the other preparation methods are the same as those in the embodiment 1;
group E: the kit is different from the kit in the embodiment 1 only in that latex microspheres with the particle size of 150nm are selected, and the other preparation methods are the same as the embodiment 1.
An immunoglobulin G4 determination kit (nephelometry, Siemens) is selected to assign values to ultra-high value samples in a full-automatic biochemical analyzer (Hitachi 7180), the concentration of the samples is 27.62G/L, each concentration gradient sample is prepared by diluting ultrapure water in proportion, three groups of kits are used for respectively testing the samples, each sample is repeatedly determined for 3 times, signals are read by the full-automatic biochemical analyzer (Hitachi 7180), and the determination mean value and the relative deviation SD are respectively calculated to verify the HOOK resistance. The results are shown in the following table:
TABLE 5 verification of HOOK resistance of kit
Figure BDA0003084097490000111
Figure BDA0003084097490000121
Comparing the detection results of the five groups of kits with the theoretical value of the sample concentration according to the experimental results (as shown in figures 9-13), wherein the results show that the kit of group A has low HOOK resistance (the highest concentration corresponding to the mean value-3 SD is greater than 7.00, which is the HOOK resistance value of the reagent); the HOOK resistance of the kit in the group B is 20.72 g/L; the HOOK resistance of the group C kit is 24.17 g/L; the HOOK resistance of the kit in the group D is 17.26 g/L; the kit of group E had low HOOK resistance.
In conclusion, the invention can effectively improve the HOOK resistance of the detection reagent by selecting the microspheres with low particle size, and when the latex microspheres with the particle size of 20-50nm are selected, the HOOK resistance of the kit is the highest.
Example 6 Effect of IgG4 monoclonal antibody labeling of latex microspheres on the anti-HOOK ability of reagents
In order to verify the influence of IgG4 monoclonal antibody labeled latex microspheres on the HOOK resistance of the reagent, 3 groups of kits are arranged for verification:
group A: the kit is different from the kit in the embodiment 1 only in that a rabbit anti-human immunoglobulin G4 monoclonal antibody is selected
Group B: the kit is different from the kit in the embodiment 1 only in that a goat anti-human immunoglobulin G4 polyclonal antibody is selected;
group C: the kit is different from the kit in the embodiment 1 only in that a rabbit anti-human immunoglobulin G4 polyclonal antibody is selected;
an immunoglobulin G4 determination kit (nephelometry, Siemens) is selected to assign values to ultra-high value samples in a full-automatic biochemical analyzer (Hitachi 7180), the concentration of the samples is 27.62G/L, each concentration gradient sample is prepared by diluting ultrapure water in proportion, three groups of kits are used for respectively testing the samples, each sample is repeatedly determined for 3 times, signals are read by the full-automatic biochemical analyzer (Hitachi 7180), and the determination mean value and the relative deviation SD are respectively calculated to verify the HOOK resistance. The results are shown in the following table:
TABLE 6 verification of HOOK resistance of kit
Figure BDA0003084097490000131
Comparing the detection results of the three groups of kits with the theoretical value of the sample concentration according to the experimental results (as shown in the attached figures 14-16), wherein the results show that the HOOK resistance of the kit in the group A is 20.72g/L (the highest concentration corresponding to the mean value-3 SD is greater than 7.00, which is the HOOK resistance value of the reagent); the HOOK resistance of the kit in the group B is 13.81 g/L; the HOOK resistance of the group C kit was 13.81 g/L.
In conclusion, the anti-HOOK capacity of the detection reagent can be effectively improved by selecting the IgG4 monoclonal antibody to mark the microspheres with low particle size. Meanwhile, the sample does not need to be additionally diluted in the detection process, so that the sample pretreatment steps are reduced, and the detection flow of the sample is simplified on the basis of ensuring the detection precision and accuracy.
Example 7
In this example, 4 sets of experiments were set up, wherein each set of experiments used a kit different from example 1 only in the kind of surfactant in the reagent R1, and the remaining kits were prepared in the same manner as in example 1. The five kits are simultaneously adopted for detection, and the detection results are shown in the following table:
TABLE 7
Figure BDA0003084097490000132
Note: in the reagent R1 in the group A in the embodiment, the concentration is 2g/L Brij 58; group B is 2 g/LTX-100; group C is 2g/LA 90; group D was 2g/L TWEEN-20, and the remaining components and preparation process were the same as in example 1.
Experimental results show that when the 4 surfactants are added into the reagent R1, the detection precision of the reagent is high, and particularly when Tween-20 is added, the precision reaches 1.65% at most.
Example 8
In this example, 5 sets of experiments were set up, wherein each set of experiments was carried out using a kit differing from example 1 only in the concentration of the surfactant (TWEEN-20) in the reagent R1, and the remaining kits were prepared in the same manner as in example 1. The five kits are simultaneously adopted for detection, and the detection results are shown in the following table:
TABLE 8
Figure BDA0003084097490000141
Note: in the embodiment, the concentration of TWEEN-20 in the reagent R1 in the group A is 1 g/L; the group B is 2 g/L; group c is 5 g/L; the group D is 10 g/L; the E group is 20g/L, and the rest components and the preparation process are the same as those of the example 1.
The experiment result shows that when the concentration range of the surfactant TWEEN-20 in the reagent R1 is 2-10g/L, the detection precision of the kit is higher.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not 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 the determination of immunoglobulin G4, comprising reagent R2, characterized in that: the reagent R2 comprises 10-80mmol/L buffer solution, 0.1-1mol/L inorganic salt, 20-80g/L blocking protein and 30-80ml/L latex microspheres coated with IgG4 monoclonal antibodies, and the particle size of the latex microspheres is 20-50 nm.
2. The kit for detecting immunoglobulin G4 according to claim 1, wherein: also included is reagent R1, the reagent R1 including: 50-100mol/L buffer solution, 0.1-1mol/L inorganic salt, 10-40g/L accelerator, 2-10g/L surfactant and 0.5-1.5g/L preservative.
3. The kit for detecting immunoglobulin G4 of claim 1, wherein in the reagent R2: the buffer is selected from at least one of MOPS, Tris, boric acid buffer or phosphate buffer, preferably boric acid buffer or Tris buffer; the inorganic salt is selected from at least one of sodium chloride or potassium chloride; the blocking protein is at least one of BSA or casein.
4. The kit for detecting immunoglobulin G4 according to claim 2, wherein in the reagent R1: the buffer is selected from at least one of MOPS, sodium citrate buffer, Tris or MES, and the inorganic salt is selected from at least one of sodium chloride or potassium chloride; the accelerator is selected from at least one of PEG8000, PEG6000 or PEG 20000; the surfactant is selected from at least one of TX-100, Brij58, A90 or Tween-20; the preservative is selected from at least one of sodium azide or PC 3000.
5. The kit for detecting immunoglobulin G4 of claim 2, wherein the reagent R1 has a pH of 6.0-8.0; the PH of the reagent R2 is 6.0-8.0.
6. A method for preparing the kit for measuring immunoglobulin G4 according to any one of claims 1-5, comprising the steps of:
(1) preparation of reagent R1
The reagent R1 was formulated as follows:
50-100mol/L buffer solution, 0.1-1mol/L inorganic salt, 10-40g/L accelerator, 2-10g/L surfactant and 0.5-1.5g/L preservative;
the buffer is selected from at least one of MOPS, sodium citrate buffer, Tris or MES; the inorganic salt is selected from at least one of sodium chloride or potassium chloride; the accelerator is selected from at least one of PEG8000, PEG6000 or PEG 20000; the surfactant is selected from at least one of TX-100, Brij58, A90 or Tween-20; the preservative is selected from at least one of sodium azide or PC 3000;
(2) preparation of reagent R2
Adding the latex microspheres into a marking buffer solution, and uniformly stirring; then adding an NHS/EDC activating agent for activation, and uniformly stirring; adding an IgG4 monoclonal antibody into the activated latex microsphere solution, stirring uniformly, and then incubating for 6-14 h; adding 1% of blocking protein, incubating for 1h, adding IgG4 monoclonal antibody back to the prepared reagent, mixing, and aging in an oven for 3 days.
The buffer is selected from at least one of MOPS, Tris, boric acid buffer or phosphate buffer; preferably a boric acid buffer or a Tris buffer; the inorganic salt is selected from at least one of sodium chloride or potassium chloride; the blocking protein is at least one of BSA or casein.
7. The method for preparing the kit for detecting the immunoglobulin G4 of claim 6, wherein the particle size of the latex microspheres is 20-50 nm.
8. The method for preparing a kit for measuring immunoglobulin G4 according to claim 7, wherein the IgG4 monoclonal antibody is added back to the prepared reagent in the step (2) in an amount of 0.2 to 0.5 mg.
9. The method for preparing the kit for detecting the immunoglobulin G4 according to claim 8, wherein the activator is EDC/NHS, and the mass ratio of the NHS to the EDC and the latex microspheres is: 1: 20-40, wherein the mass ratio of the IgG4 monoclonal antibody to the latex microspheres is 1: 0.5-1.
10. The method for preparing the kit for measuring immunoglobulin G4 of claims 6-9, wherein the content of latex particles coated with IgG4 monoclonal antibody in the reagent R2 is 20-30%.
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CN114295833A (en) * 2021-12-30 2022-04-08 安徽大千生物工程有限公司 Latex-enhanced immunoturbidimetry kit for quantitatively detecting IgG4 and preparation and detection methods thereof

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