CN112816698A

CN112816698A - Buffer reagent for improving detection clinical correlation and latex immunoturbidimetry kit

Info

Publication number: CN112816698A
Application number: CN201911132994.1A
Authority: CN
Inventors: 孙莹; 夏良雨
Original assignee: Guangdong Fapon Biotech Co Ltd
Current assignee: Guangdong Fapon Biotech Co Ltd
Priority date: 2019-11-18
Filing date: 2019-11-18
Publication date: 2021-05-18

Abstract

The invention relates to the field of immunodetection, and particularly provides a buffer reagent for improving clinical relevance of detection and a latex immunoturbidimetric kit. The buffer reagent for improving the detection clinical relevance comprises an electrolyte, wherein the electrolyte is choline chloride. The inventor finds that the addition of choline chloride in the buffer reagent for latex immunoturbidimetry can significantly improve the clinical relevance of the detection result and improve the detection accuracy. The buffer reagent is applied to latex immunoturbidimetry detection, is used as a first reagent in a detection reagent, and reacts with a second reagent containing antibody-coated latex particles, so that the clinical relevance of detection can be remarkably improved, and the effect of resisting interference of various interferents is also remarkable, thereby further improving the accuracy and the sensitivity of the detection kit.

Description

Buffer reagent for improving detection clinical correlation and latex immunoturbidimetry kit

Technical Field

The invention relates to the field of immunodetection, in particular to a buffer reagent and a latex immunoturbidimetry kit for improving clinical relevance of detection.

Background

The latex immunoturbidimetry performs an immunoreaction on an antigen to be detected in a sample and a specific antibody coated on latex microspheres in a detection reagent to form an immune complex, so that the latex microspheres are connected into a net shape, and the turbidity of a reaction solution system is increased. The turbidity is in positive correlation with the concentration of the antigen to be detected in the sample within a certain range, so the content of the antigen to be detected in the sample can be calculated by measuring the absorbance of a reaction system with a specific wavelength (570nm) and referring to a calibration curve. Compared with other detection methods, the latex immunoturbidimetry method has the advantages of large flux, short time consumption, capability of realizing automation, reduction of the influence of human factors on the detection result, and good sensitivity and accuracy. Therefore, the latex immunoturbidimetry has a good application prospect.

However, the latex immunoturbidimetry kits of various projects in the current market still have the problems of poor reagent specificity and anti-interference capability and the like, so that the accuracy of clinical detection results is influenced. Therefore, how to improve the clinical relevance value of the latex immunoturbidimetric detection reagent is further researched, which has important significance for the improvement of the immunodiagnosis method.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a buffer reagent for improving clinical relevance of detection and a latex immunoturbidimetric kit applying the buffer reagent, and aims to solve the problems that the clinical relevance of a detection result is poor, and the specificity, the sensitivity and the anti-interference capability are to be improved when a latex immunoturbidimetric method is adopted for detection in the prior art.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a buffer reagent for improving the clinical relevance of a test, the buffer reagent comprising an electrolyte, the electrolyte being choline chloride.

Further, the concentration of the choline chloride is 200-400 mmol/L;

preferably, the buffer reagent further comprises at least one of a buffer, a blocking agent, a surfactant, a sensitizer, a stabilizer, and a preservative.

Further, the buffer comprises HEPES buffer, PB buffer, PBS buffer, Tris buffer or phosphate buffer;

preferably, the blocking agent comprises at least one of RF and HAMA binding protein, murine IgG, murine IgM, and murine serum;

preferably, the surfactant comprises Brij35, tween 20 or triton X-100;

preferably, the sensitizer comprises polyethylene glycol 4000, polyethylene glycol 6000 or polyethylene glycol 8000;

preferably, the stabilizer comprises disodium ethylenediaminetetraacetate, BSA, or trehalose;

preferably, the preservative comprises Proclin300 or sodium azide.

Further, the buffer solution comprises HEPES buffer solution or PB buffer solution, the pH value of the buffer solution is preferably in the range of 6-8, and the concentration of the buffer solution is preferably 10-200 mmol/L;

preferably, the blocking agent comprises RF and HAMA binding protein, preferably the concentration of blocking agent is 4-6 g/L;

preferably, the RF and HAMA binding proteins include HIER-E-010;

preferably, the surfactant comprises Brij35, and the concentration of the surfactant is preferably 0.5-1.5 g/L;

preferably, the sensitizer comprises PEG6000, and the concentration of the sensitizer is preferably 15-25 g/L;

preferably, the stabilizer comprises disodium ethylene diamine tetraacetate, and the concentration of the stabilizer is preferably 0.5-1.5 g/L;

preferably, the concentration of the preservative is 0.5-1.5 g/L.

The buffer reagent is applied to a latex immunoturbidimetry method or the preparation of a latex immunoturbidimetry kit.

A latex immunoturbidimetry kit comprises a first reagent and a second reagent, wherein the first reagent is the buffer reagent.

Further, the second reagent comprises a buffer, a preservative, antibody-coated latex particles, a stabilizer, and an electrolyte;

preferably, the buffer comprises a HEPES buffer, the pH of the buffer is preferably in the range of 6-8, and the concentration of the buffer is preferably 30-70 mmol/L;

preferably, the concentration of the preservative is 0.5-1.5 g/L;

preferably, the stabilizing agent comprises BSA and trehalose, wherein the concentration of the BSA is preferably 4-6g/L, and the concentration of the trehalose is preferably 35-45 g/L;

preferably, the electrolyte comprises sodium chloride, and the concentration of the electrolyte is preferably 150-250 mmol/L.

Further, the antibody in the antibody-coated latex particle comprises a PG i antibody, a PG II antibody, or a CRP antibody;

preferably, the concentration of the antibody-coated latex particles is 2-3 g/L.

Further, the kit also comprises a calibrator and a quality control product.

Further, the diluent of the calibrator comprises a buffer solution, a surfactant, a preservative, an electrolyte and a stabilizer;

preferably, the surfactant comprises tween 20, and the concentration of the surfactant is preferably 0.5-1.5 g/L;

preferably, the concentration of the preservative comprises 0.5-1.5 g/L;

preferably, the electrolyte comprises sodium chloride, and the concentration of the electrolyte is preferably 150-250 mmol/L;

preferably, the stabilizing agent comprises BSA and disodium ethylene diamine tetraacetate, the concentration of the BSA is preferably 2-4g/L, and the concentration of the disodium ethylene diamine tetraacetate is preferably 0.5-1.5 g/L.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a buffer reagent for improving clinical relevance detection, which comprises an electrolyte, wherein the electrolyte is choline chloride. The inventor finds that the addition of choline chloride in the buffer reagent for latex immunoturbidimetry can significantly improve the clinical relevance of the detection result and improve the detection accuracy. In addition, the inventor proves through experiments that the buffer reagent provided by the invention has good universality, can improve the clinical relevance of detection in various detection items, and can be widely applied to various latex immunoturbidimetric detection items. The buffer reagent is applied to latex immunoturbidimetry detection, is used as a first reagent in a detection reagent, reacts with a second reagent containing antibody coated latex particles, can obviously improve the clinical relevance of detection, and has obvious effects on bilirubin interference resistance, vitamin C interference resistance, Intralipid fat emulsion interference resistance, hemoglobin interference resistance, ethylene diamine tetraacetic acid disodium interference resistance and heparin sodium interference resistance, thereby further improving the accuracy and sensitivity of the detection kit.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a calibration curve in example 3 of the present invention;

FIG. 2 is a linear correlation curve in example 3 of the present invention;

FIG. 3 is a graph showing the results of clinical correlations in example 4 of the present invention;

FIG. 4 is a graph showing the results of clinical correlation of 100mmol/L choline chloride buffer in example 5 of the present invention;

FIG. 5 is a graph showing the results of clinical correlation of 200mmol/L choline chloride buffer in example 5 of the present invention;

FIG. 6 is a graph showing the results of clinical correlation between 300mmol/L choline chloride buffer in example 5 of the present invention;

FIG. 7 is a graph showing the results of clinical correlation between 400mmol/L choline chloride buffer in example 5 of the present invention;

FIG. 8 is a graph showing the results of clinical correlation of 100mmol/L NaCl buffering agent in example 5 of the present invention;

FIG. 9 is a graph showing the results of clinical correlation of 200mmol/L NaCl buffering agent in example 5 of the present invention;

FIG. 10 is a graph showing the results of clinical correlation of 300mmol/L NaCl buffering agent in example 5 of the present invention;

FIG. 11 is a graph showing the results of clinical correlation between 400mmol/L NaCl buffering agent in example 5 of the present invention;

FIG. 12 is a graph showing the results of clinical correlation between 300mmol/L choline chloride buffer PG II in example 7 of the present invention;

FIG. 13 is a graph showing the results of clinical correlation between 300mmol/L NaCl buffering agent PG II in example 7 of the present invention;

FIG. 14 is a graph showing the results of clinical correlation between 300mmol/L potassium chloride buffer PG II in example 7 of the present invention;

FIG. 15 is a graph showing the results of clinical correlation between CRP as a 300mmol/L choline chloride buffering agent in example 7 of the present invention;

FIG. 16 is a graph showing the results of clinical correlation between CRP and 300mmol/L NaCl buffering agent in example 7 of the present invention;

FIG. 17 is a chart showing the results of clinical correlation between CRP as a 300mmol/L potassium chloride buffering agent in example 7 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

A buffer reagent for improving the clinical relevance of detection comprises an electrolyte, wherein the electrolyte is choline chloride.

The inventor finds that the addition of choline chloride in the buffer reagent for latex immunoturbidimetry can significantly improve the clinical relevance of the detection result and improve the detection accuracy. In addition, the inventor proves through experiments that the buffer reagent provided by the invention has good universality, can improve the clinical relevance of detection in various detection items, and can be widely applied to various latex immunoturbidimetric detection items. The buffer reagent is applied to latex immunoturbidimetry detection, is used as a first reagent in a detection reagent, reacts with a second reagent containing antibody coated latex particles, can obviously improve the clinical relevance of detection, and has obvious effects on bilirubin interference resistance, vitamin C interference resistance, Intralipid fat emulsion interference resistance, hemoglobin interference resistance, ethylene diamine tetraacetic acid disodium interference resistance and heparin sodium interference resistance, thereby further improving the accuracy and sensitivity of the detection kit.

In a preferred embodiment, the concentration of choline chloride is 200-400 mmol/L. The inventor finds that the detection effect is better when the concentration of the choline chloride is in the range of 200-400mmol/L, the clinical relevance is remarkable, and the concentration of the choline chloride is typically, but not limited to, 200mmol/L, 250mmol/L, 300mmol/L, 350mmol/L or 400 mmol/L.

In a preferred embodiment, the buffering agent comprises at least one of a buffer, a blocking agent, a surfactant, a sensitizer, a stabilizer, and a preservative in addition to the electrolyte choline chloride.

In preferred embodiments, the buffer comprises HEPES buffer, PB buffer, PBs buffer, Tris buffer, or phosphate buffer.

In preferred embodiments, the blocking agent comprises at least one of RF (rheumatoid factor) and HAMA (heterophilic antibody) binding protein, murine IgG, murine IgM, and murine serum.

In a preferred embodiment, the surfactant comprises Brij35, Tween 20, or Triton X-100.

In preferred embodiments, the sensitizer comprises polyethylene glycol 4000, polyethylene glycol 6000 or polyethylene glycol 8000.

In a preferred embodiment, the stabilizer comprises disodium ethylenediaminetetraacetate, BSA or trehalose.

In a preferred embodiment, the preservative comprises Proclin300 or sodium azide.

In the invention, other components of the buffer reagent are optimized, for example, the selection of the blocking agent solves the technical problem of poor RF and HAMA interference resistance in latex immunoturbidimetric detection in the prior art, thereby further improving the accuracy and sensitivity of the detection kit; the matching of the components such as the electrolyte, the surfactant, the sensitizer and the like ensures that the buffer reagent has good detection specificity, high sensitivity and strong anti-interference capability, and can accurately and quickly obtain the content of the antigen to be detected.

In a preferred embodiment, the buffer comprises a HEPES buffer or PB buffer, the pH of the buffer preferably ranges from 6 to 8, and the concentration of the buffer preferably ranges from 10 to 200 mmol/L; the blocking agent preferably comprises RF and HAMA binding protein, and the concentration of the blocking agent is preferably 4-6g/L, wherein the RF and HAMA binding protein comprises HIER-E-010; the surfactant preferably comprises Brij35, and the concentration of the surfactant is preferably 0.5-1.5 g/L; the sensitizer preferably comprises PEG6000, and the concentration of the sensitizer is preferably 15-25 g/L; the stabilizer comprises disodium ethylene diamine tetraacetate, and the concentration of the stabilizer is preferably 0.5-1.5 g/L; the concentration of the preservative is preferably 0.5 to 1.5 g/L. The inventor finds that the combination of the selection and the proportion of the components leads the clinical relevance of latex immunoturbidimetry detection to be better, and the accuracy and the sensitivity to be improved.

It should be noted that HIER-E-010 is a blocking agent sold under the trade name HIER-E-010 by Fipeng Bio Inc.

In a more preferred embodiment, the buffering agent comprises: 100mmol/L HEPES buffer solution, 5g/L HIER-E-010, 300mmol/L choline chloride, 1g/L Brij35, 20g/L PEG6000, 1g/L disodium ethylene diamine tetraacetate and 1g/L Proclin 300. The inventor finds that the detection effect of the formula is obvious through tests, and researches find that the use of choline chloride can obviously improve the specificity of the detection kit and has good clinical relevance with commercially available kits; meanwhile, the detection kit has wide linear range and good accuracy, for example, in the PG I detection project, the lower detection limit can reach 2ng/ml, the linear range is 2-200ng/ml, and the basis is provided for accurate clinical diagnosis. The PEG6000 and Brij35 have synergistic effect, so that the detection kit has high precision, and the repeatability and accuracy of a single measurement result can be ensured. The use of HIER-E-010 makes the detection kit have better anti-RF and HAMA interference ability, and can fully inhibit the interference effect of high-concentration RF, HAMA and other interferents. In addition, the proportion of the components is scientifically and reasonably limited, so that the components and the proportion thereof act together to obviously improve the detection performance.

The invention provides application of the buffer reagent in a latex immunoturbidimetry method or preparation of a latex immunoturbidimetry kit.

A latex immunoturbidimetry kit comprises a first reagent (R1) and a second reagent (R2), wherein the first reagent is a buffer reagent provided by the invention.

The kit adopts the buffer reagent provided by the invention as the first reagent, so that the clinical relevance is obviously improved, and the overall performance of the kit is effectively improved.

In a preferred embodiment, the second reagent comprises a buffer, a preservative, antibody-coated latex particles, a stabilizer, and an electrolyte.

In a preferred embodiment, the buffer comprises a HEPES buffer, the pH of the buffer is preferably in the range of 6-8, the concentration of the buffer is preferably 30-70 mmol/L; the concentration of the preservative is preferably 0.5-1.5 g/L; the stabilizing agent preferably comprises BSA and trehalose, wherein the concentration of the BSA is preferably 4-6g/L, and the concentration of the trehalose is preferably 35-45 g/L; the electrolyte preferably comprises sodium chloride, and the concentration of the electrolyte is preferably 150-250 mmol/L.

In a preferred embodiment, the antibody in the antibody-coated latex particle comprises antibodies PG I (pepsinogen I), PG II (pepsinogen II) or CRP (C-reactive protein), and the concentration of the antibody-coated latex particle is preferably 2-3 g/L. The antibody of the present invention may be an antibody commonly used in the art, and is not particularly limited, but is preferably a PG i antibody, a PG II antibody, or a CRP antibody, and more preferably a PG i antibody. More preferably, the PG I antibody comprises two PG I monoclonal antibodies (antibody source: Fipeng organism; antibody cargo: MABM-PG I-1-5 and MABM-PG I-2-24), which can effectively improve the sensitivity and specificity of detection.

In a preferred embodiment, the second reagent comprises: 50mmol/L HEPES buffer, 5g/L BSA, 200mmol/L NaCl, 40g/L trehalose, 2.5g/L PG I antibody coated latex particles and 1g/L Proclin 300. The inventor finds that the addition of the electrolyte sodium chloride, the stabilizing agent BSA and the trehalose in the second reagent can effectively improve the stability of the second reagent, prolong the effective period of the reagent and simultaneously facilitate the improvement of the accuracy of PG I detection. The proportion of each component in the second reagent is obtained by scientific and reasonable screening, so that the performance of the detection kit is obviously improved under the combined action of the components in the proportion.

In a more preferred embodiment, the latex particles coated with PG I antibody in the second reagent are prepared as follows: and activating the polystyrene latex microspheres by using EDC and NHS, adding the PG I antibody for incubation, and centrifuging to obtain latex particles coated by the PG I antibody.

For example, the preparation of the second reagent comprises:

(1) activation of microspheres: adding 0.1ml of a 200nm diameter polystyrene latex microsphere solution (10% concentration) into 4.5ml of 0.01M MES (pH6.0) buffer solution, adding 0.05ml of EDC (carbodiimide, 10mg/ml concentration) and 0.05ml of NHS (N-hydroxysuccinimide, 20mg/ml concentration), and activating at room temperature for 20 min;

(2) coupling of pepsinogen i antibodies: adding 0.5mg of antibody (two pepsinogen I monoclonal antibodies are mixed at a ratio of 1: 1, the monoclonal antibodies are from Fipeng organisms, the antibody cargo numbers are MABM-PG I-1-5 and MABM-PG I-2-24) into the activated solution, uniformly mixing for 2-4h at room temperature, centrifuging at 14000rpm for 30min, discarding the supernatant, uniformly mixing the precipitate with 5ml of second reagent (latex particles without antibody coating), and then ultrasonically dispersing microspheres to obtain a PG I antibody marked latex microsphere solution with the concentration of 0.1mg/ml, namely the second reagent.

In a preferred embodiment, the kit further comprises a calibrator and a quality control.

In a preferred embodiment, the dilution of the calibrator comprises buffers, surfactants, preservatives, electrolytes and stabilizers.

In a preferred embodiment, the buffer in the dilution of the calibrator comprises HEPES buffer, the pH of the buffer is preferably in the range of 6-8, and the concentration of the buffer is preferably in the range of 30-70 mmol/L; the surfactant preferably comprises Tween 20, and the concentration of the surfactant is preferably 0.5-1.5 g/L; the concentration of the preservative preferably comprises 0.5-1.5 g/L; the electrolyte preferably comprises sodium chloride, and the concentration of the electrolyte is preferably 150-250 mmol/L; the stabilizer preferably comprises BSA and disodium ethylene diamine tetraacetate, the concentration of the BSA is preferably 2-4g/L, and the concentration of the disodium ethylene diamine tetraacetate is preferably 0.5-1.5 g/L.

In a more preferred embodiment, the dilution of the calibrator comprises: 50mmol/L HEPES buffer solution, 200mmol/L sodium chloride, 3g/L BSA, 1g/L disodium ethylenediamine tetraacetic acid, 1g/L Tween 20 and 1g/L Proclin 300. The inventor finds that the effect of preparing the calibration curve is best by limiting the specific components and the proportion of the diluent, the system stability is good, the influence on detection is small, and the linear range of the calibration curve is wide.

In a preferred embodiment, the ratio of the volume amounts of the sample to be detected, the first reagent and the second reagent is (5.5-7.5): (140-160): 50.

the invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Example 1 buffer reagents to improve the detection of clinical relevance

A buffer reagent for improving detection clinical relevance comprises the following specific components in the following table 1:

TABLE 1

Example 2 immunoturbidimetric assay kit for PG I latex

A latex immunoturbidimetric assay kit comprising a first reagent (buffer reagent in example 1), a second reagent (Table 2 below), and a PG I calibrator (dilution of calibrator is Table 3):

TABLE 2

TABLE 3

The gradient concentrations of the PG I calibrator were: 0ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 100ng/ml and 200 ng/ml.

The preparation of the second reagent comprises:

(1) activation of microspheres: adding 0.1ml of a 200nm diameter polystyrene latex microsphere solution (microspheres are purchased from JSR, concentration 10%) into 4.5ml of 0.01M MES (pH6.0) buffer, adding 0.05ml of EDC (carbodiimide, concentration 10mg/ml) and 0.05ml of NHS (N-hydroxysuccinimide, concentration 20mg/ml), and activating at room temperature for 20 min;

(2) coupling of pepsinogen i antibodies: and adding 0.5mg of antibody (two pepsinogen I monoclonal antibodies are mixed in a ratio of 1: 1, the monoclonal antibodies are from Fipeng organisms, and the products are MABM-PGI-1-5 and MABM-PGI-2-24) into the activated solution, uniformly mixing for 2-4h at room temperature, centrifuging at 14000rpm for 30min, discarding the supernatant, uniformly mixing precipitates with 5ml of a second reagent (latex particles without antibody coating), and then ultrasonically dispersing microspheres to obtain a pepsinogen I antibody marked latex microsphere solution with the concentration of 0.1mg/ml, namely the second reagent.

EXAMPLE 3 determination of Linear Range

Serum collected by a clinical standard method by using the detection kit in example 2 is used as a sample to be detected, and the reaction system is shown in the following table 4:

TABLE 4

Adding into a cuvette	Blank tube	Calibration tube	Sample tube
				First reagent (μ L)	150	150	150
Distilled water (mu L)	6.5	-	-
				Calibrator (mu L)	-	6.5	-
Sample to be tested (μ L)	-	-	6.5
				Second reagent (μ L)	50	50	50

Mixing, incubating at 37 deg.C for 0.5 min and 5 min, reading absorbance A1 and A2 at 570nm, and calculating Δ A ═ A2-A1; wherein the values of the blank tube were used to prepare a calibration curve as the 0 value of the calibration curve.

The utilization concentration is as follows: calibrators of 0ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 100ng/ml and 200ng/ml gave calibration curves (results are shown in FIG. 1). And substituting the absorbance difference value of the sample to be detected into a calibration curve to obtain the content of PG I in the sample to be detected.

Reference value ranges are shown in table 5:

TABLE 5

Serum pepsinogen results	PG positive degree result judgment
		1 is more than or equal to 70ng/mL or 1/2 is more than 3.0	Negative (-)
1 < 70ng/mL and 1/2 < 3.0	Positive (+) mild atrophy
		1 < 50ng/mL and 1/2 < 3.0	Positive (++) moderate atrophy
1 < 30ng/mL and 1/2 < 2.0	Positive (+++) severe atrophy

The PG I calibrator sample with the concentration of 110ng/mL is diluted into gradient concentration, each concentration is repeatedly measured for 3 times by using the kit provided by the invention, the average value is taken, the linear correlation coefficient is calculated with the theoretical value, the detection result is shown in Table 6, and the linear correlation is shown in FIG. 2. The corresponding linear correlation equation is shown in fig. 2, and is: 1.0221x-2.8234, correlation coefficient R²The result shows that the kit performs linear detection when the kit is 0.9976The detection range is 2-200ng/mL, and the lower detection limit can reach 2 ng/mL.

TABLE 6PG I antigen gradient concentration test results

In addition, the sensitivity and precision of the kit provided by the invention are tested, and clinical samples of 2ng/ml (low value 1), 6ng/ml (low value 2) and 64ng/ml (high value 1) are tested, and the results are shown in the following table 7:

TABLE 7

Example 4 detection of clinical relevance

The detection kit of example 2 was subjected to a sample alignment test with the PG I detection reagent of Abbott corporation (kit A), and the results of Table 8 were measured, as shown in FIG. 3, and the correlation coefficient R was determined²0.9914, good correlation, can be used for clinical diagnosis.

TABLE 8

Example 5 reagent composition optimization

The buffer reagent in example 1 (the first reagent of the reagent kit in example 2) and the content and the type of the electrolyte are optimized, different choline chloride and different sodium chloride are selected as the electrolyte in the buffer reagent (the specific ratio is shown in table 9), and other components and the types of the detection reagent kit are selectedThe mixture ratio is inconvenient, the clinical relevance of the detection and the control Yapei reagent is shown in a table 10, and the statistical results of the components 1 to 8 are respectively shown in fig. 4 to 11. Analysis of the data gave the optimum salt composition and formulation with concentration component 3, i.e. the clinical relevance R when the electrolyte used in the buffer reagent was choline chloride at a concentration of 300mM²The highest is 0.9822. The use of choline chloride in buffer reagents resulted in better results of clinical relevance of the reagents compared to sodium chloride, which may be analyzed as: (1) choline chloride can block complement interference in a sample; (2) different salts in the system can lead the surfaces of the antibody, the antigen and the matrix protein to carry different electric charge quantities, and the choline chloride can better change the electric charge quantities carried by the surfaces of the antigen, the antibody and the matrix protein, improve the surface charge threshold of the protein combined with the antibody and lead the hybrid protein not to be combined with the antibody easily.

TABLE 9

Watch 10

In a similar manner, the other components and the mixture ratio are not changed, the type and the concentration of the blocking agent in the buffer reagent are optimized to obtain 5 groups of formula components in table 11, and the anti-RF interference performance is detected, and the result is shown in table 12.

TABLE 11

TABLE 12

Referring to the above method for optimizing the types and contents of electrolytes and blocking agents in the buffer reagent, similar experiments were also performed on other components in the buffer reagent in this example to obtain the components and the formula provided by the present invention.

Example 6 interference rejection detection

In this embodiment, the anti-interference capability of the kit in embodiment 2 on hemoglobin, bilirubin, chyle, ascorbic acid, and RF is detected, and the results are as follows: hemoglobin < 1000mg/dL (Table 15); bilirubin is less than or equal to 20mg/dL (Table 14); chyle ≤ 1000mg/dL (table 13); ascorbic acid < 50mg/dL (Table 16); the samples with RF less than or equal to 800IU/mL (Table 17) have no significant interference on PG I measurement, and the relative deviation is less than or equal to +/-10%.

TABLE 13 Lipemia interference

TABLE 14 bilirubin interference

TABLE 15 hemoglobin interference

TABLE 16 ascorbic acid interference

TABLE 17

Example 7 application of buffer reagents to clinical relevance assays for other assay items

(1) Detection reagent for pepsinogen II (latex enhanced turbidimetric immunoassay)

Pepsinogen II detection reagent R2(pH 7.5): 20mM Hepes, 150mM NaCl, 0.5% BSA, 4% sucrose, 0.1% Proclin300 and 0.1% pepsinogen II monoclonal antibody sensitized nanometer latex particles.

A comparison of R1 for the pepsinogen II assay reagents for different salt types and concentrations, the compositional formula is shown in Table 18, the results are shown in Table 19, and FIGS. 12-14:

TABLE 18 optimization of salt concentration and type in PG II reagent R1

Table 19 optimization of salt concentration and type in PG II reagent R1

And (4) conclusion: the use of choline chloride as the electrolyte in R1 has a higher clinical relevance than sodium chloride and potassium chloride in PGII agents.

(2) Detection reagent for CRP (latex enhanced immunoturbidimetry)

CRP detection reagent R2(ph 7.5): 20mM Hepes, 150mM NaCl, 0.5% BSA, 4% sucrose, 0.1% Proclin300, 0.1% CRP monoclonal antibody sensitized nano latex particles.

The results are shown in Table 21 and FIGS. 15-17, comparing R1 for CRP test reagents of different salt types and concentrations, with the formula shown in Table 20:

TABLE 20 optimization of salt concentration and type in CRP reagent R1

TABLE 21 optimization of salt concentration and type in CRP reagent R1

And (4) conclusion: among CRP agents, choline chloride, which is used as an electrolyte in R1, has a higher clinical relevance than sodium chloride and potassium chloride.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

1. A buffer reagent for improving the clinical relevance of a test, wherein the buffer reagent comprises an electrolyte, and the electrolyte is choline chloride.

2. The buffer reagent of claim 1, wherein the concentration of choline chloride is 200-400 mmol/L;

3. The buffered reagent of claim 2, wherein the buffer comprises a HEPES buffer, a PB buffer, a PBS buffer, a Tris buffer, or a phosphate buffer;

preferably, the surfactant comprises Brij35, tween 20 or triton X-100;

preferably, the preservative comprises Proclin300 or sodium azide.

4. The buffer reagent according to claim 3, wherein the buffer comprises HEPES buffer or PB buffer, the pH of the buffer is preferably in the range of 6-8, and the concentration of the buffer is preferably 10-200 mmol/L;

preferably, the RF and HAMA binding proteins include HIER-E-010;

preferably, the concentration of the preservative is 0.5-1.5 g/L.

5. Use of a buffer reagent according to any one of claims 1 to 4 in latex immunoturbidimetry or in the preparation of a latex immunoturbidimetry kit.

6. A latex immunoturbidimetric kit comprising a first reagent and a second reagent, the first reagent being a buffer reagent according to any of claims 1 to 4.

7. The kit of claim 6, wherein the second reagent comprises a buffer, a preservative, antibody-coated latex particles, a stabilizer, and an electrolyte;

preferably, the concentration of the preservative is 0.5-1.5 g/L;

8. The kit of claim 6, wherein the antibody-coated latex particle antibody comprises a PG I antibody, a PG II antibody or a CRP antibody;

9. The kit of any one of claims 6 to 8, wherein the kit further comprises a calibrator and a quality control.

10. The kit of claim 9, wherein the dilution of the calibrator comprises buffers, surfactants, preservatives, electrolytes, and stabilizers;

preferably, the concentration of the preservative comprises 0.5-1.5 g/L;