CN110988363A - Detection reagent for antiviral protein MxA and preparation method thereof - Google Patents

Abstract

The invention discloses a latex enhanced immunoturbidimetric detection single reagent for antiviral protein MxA and a preparation method thereof. The invention adopts single reagent, does not need mixing, has high sensitivity and high detection speed, can directly measure various samples such as whole blood, serum, plasma and the like, and can be widely applied to various transmission or scattering analyzers including common biochemical analyzers, specific protein analyzers and the like.

Description

Detection reagent for antiviral protein MxA and preparation method thereof

Technical Field

The invention belongs to the field of medical immunodiagnosis, and particularly relates to preparation and application of a latex enhanced immunoturbidimetric detection single reagent for antiviral proteins MxA (MxA).

Background

Mx proteins are interferon-induced proteins with antiviral activity, and are widely present in vertebrates. The protein was first found in mice in 1963 and named Mx1, and the homologous protein was found in humans in 1985 and named MxA. The interferon is the first line of defense of human against virus infection, and can induce the mass expression of MxA after the human body is infected with virus, so that the measurement of the content of MxA in the human body can be used for detecting and diagnosing virus infection. Serum concentrations of MxA are generally below 50ng/mL in normal humans, and concentrations after viral infection are generally above 200 ng/mL. The detection of MxA can be combined with common bacterial infection indexes such as CRP, PCT and the like, is a test method for rapidly distinguishing infection types, and can better guide clinical medication

Common detection methods of MxA include an enzyme-linked immunosorbent assay, a colloidal gold method, a fluorescence chromatography method and a latex enhanced immunoturbidimetry. The latex enhanced immunoturbidimetry method is the most advanced method, the sensitivity is higher, the linear range is wider, and the operation is relatively simple and convenient. Latex enhanced immunoturbidimetry captures MxA in a test sample using MxA antibodies adsorbed on latex particles of several tens to several hundreds of nanometers in diameter, thereby forming cross-links between particles, changing the scattering or transmission absorbance (i.e., turbidity) of the solution. By measuring the change in the scattering power or transmission absorbance of the solution, the concentration of MxA in the test sample can be quantitatively calculated from the calibration curve. The test method can be applied to a general large-scale automatic biochemical analyzer and can also be applied to small-scale POCT (point of care testing) equipment such as a specific protein analyzer and the like, has strong applicability, and can well meet the use requirements from a large-scale three-hospitals to community primary hospitals in China.

The MxA latex enhanced turbidimetric immunoassay reagent on the market adopts a double reagent form of a diluent and a latex solution. In the test, the test sample must be diluted in a diluent and then mixed with a latex solution. On a common instrument platform of a third hospital, dilution and mixing operations can be completed by a large-scale full-automatic biochemical analyzer, but tests on a small-scale semi-automatic platform, particularly a POCT type portable platform (commonly used by hospitals below three levels and medical service units in counties and counties) need manual implementation of operators, additional operation steps are brought, more operation errors are introduced, and the precision of the tests is influenced.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the latex enhanced immunoturbidimetric detection reagent for the antiviral protein MxA (MxA), which has the advantages of single reagent, no need of mixed reagents, high sensitivity and high detection speed. The reagent can be used for testing on a large-scale automatic biochemical analyzer and can also be used for testing on small-scale POCT equipment such as a specific protein analyzer.

In the introduction of the background art, it is mentioned that the latex-enhanced immunodetection reagents commonly used in the market are in a two-reagent assay format. The diluent is often referred to as R1 and the latex solution is often referred to as R2. Pure latex solutions of antibody or antigen coated microspheres are thermodynamically unstable systems and the latex will spontaneously coagulate if no special treatment is applied. Therefore, in general, the latex solution (R2) in the detection reagent needs to be stabilized by optimizing the composition of the solution to prevent spontaneous aggregation of the latex during storage. Common optimization means include selecting different buffer systems, changing the surface charge of the microspheres, changing the ionic strength of the solution, adding stabilizers, etc. (j.a. Molina Bolivar et al,Journal ofMacromolecular Science Part C - Polymer Reviews,2005, 45:59-98). However, when the latex solution is used for the test, latex agglutination due to specific binding of antigen-antibody should not be prevented, which requires that the diluent (R1) can contribute to the formation of latex agglutination to some extent. Therefore, the typical diluent (R1) and the latex stock (R2) differ greatly in formulation. Mixing R1 and R2 directly generally results in latex instability. This is also the main reason why latex formulations are generally available on the market in the form of dual formulations.

In the present invention, in addition to conventional methods of stabilizing latex reagents, we also incorporate surfactants to optimize the reagent formulation to stabilize the latex solution. Surfactants can provide steric hindrance, ionic surfactants can also provide additional electrostatic repulsion, thereby preventing coagulation of the latex and stabilizing the latex reagents during storage. Meanwhile, by optimizing the reagent formula, when a sample containing the MxA contacts the latex reagent, the proper surfactant can promote the interaction of the MxA antigen and the MxA antibody, and the formation of specific latex agglutination is facilitated. We thus prepared the MxA latex reagent in a single reagent form. The reagent can be stored stably during storage, and can rapidly generate agglutination reaction when contacting with a sample, so that other diluents do not need to be mixed to promote the agglutination reaction. And the preferable type and concentration of the surfactant can effectively dissolve blood cells in blood, so that the detection reagent can directly measure a whole blood sample, and has the capability of simultaneously detecting whole blood, serum and plasma.

The detection reagent for the antiviral protein MxA (MxA) comprises MxA antibody sensitized latex particles, 4-hydroxyethyl piperazine ethanesulfonic acid, bovine serum albumin, sodium polyethylene sulfonate, dodecyl polyethylene glycol ether and Proclin300, wherein the ratio of the sodium polyethylene sulfonate to the dodecyl polyethylene glycol ether is 3: 1.

the concentration of the MxA antibody sensitized latex particles is 0.15 mg/mL.

The concentration of the 4-hydroxyethyl piperazine ethanesulfonic acid is 10 mM.

The concentration of the bovine serum albumin is 1 g/L.

The concentration of the sodium polyvinyl sulfonate is 3-6 g/L.

The concentration of the dodecyl polyglycol ether is 1-2 g/L.

The concentration of the Proclin300 is 1 g/L.

Preferably, the MxA antibody-sensitized latex particles are prepared as follows:

(1) preparation of activated latex particles: 0.18mL of carboxylated polystyrene microspheres with the particle size of 333nm and the concentration of 10mg/mL is taken, 3.6mL of 25mM MES with the pH value of 6.5 is added, the mixture is stirred for 10min, 7.04mg of EDC activator powder is added, the mixture is stirred at room temperature for 2.5h and then centrifuged at 12000rpm to collect precipitates, and 1.8mL of 25mM MES with the pH value of 6.5 is used for resuspension of the precipitates.

(2) Preparing sensitized latex particles: 0.18mL of 4.0mg/mL MxA polyclonal antibody was added to 1.8mL of 25mM MES, pH6.5, and the mixture was stirred for 1min to prepare an antibody solution to be conjugated. Quickly pouring the activated latex particles in the step (1) into the antibody solution to be coupled, and stirring for 2.5 h. And centrifuging at 12000rpm to collect the precipitate, resuspending 120mL of confining liquid, and stirring for 2.5h to obtain the single-reagent antiviral protein MxA (MxA) latex enhanced immunoturbidimetric reagent. The components of the sealing liquid are 10mM 4-hydroxyethyl piperazine ethanesulfonic acid, 1g/L bovine serum albumin, 3-6g/L sodium polyethylene sulfonate, 1-2g/L dodecyl polyglycol ether and 1g/L proclin300, the pH is =6.5, the ratio of the sodium polyethylene sulfonate to the dodecyl polyglycol ether is 3: 1.

the reagent detection method of the invention comprises the following steps:

when a sample is detected, the MxA antibody on the latex particles in the reagent provided by the invention and the MxA antigen in the sample generate antibody-antigen reaction, so that the latex is gradually condensed, and the turbidity of the test solution is changed. The change in turbidity can be measured by a transilluminator or scatterometer. The transmitted absorbance or scattered light intensity is linear with the concentration of MxA in the sample over a range of concentrations. The concentration of MxA in the sample to be tested can be quantitatively calculated by a calibration curve established by referring to the MxA calibrator.

The beneficial effects of the invention include:

1. the detection reagent has simple composition, only has single reagent, does not need to carry out the mixing operation of R1 and R2 during the test like the existing reagent in the market, and simplifies the operation procedure. Especially when using semi-automatic POCT equipment, for operating personnel bring very big facility, also better guarantee the accuracy of test simultaneously.

2. The reagent has the functions of hemolysis and detection, can directly test a whole blood sample, does not need the work of extracting serum and plasma, simplifies the operation and greatly shortens the whole test time including sample preparation.

3. The reagent reacts rapidly, and the reaction time is only 2 to 3 minutes. Compared with the existing double-reagent latex reagent, the detection time is 10 to 12 minutes, the speed is improved by nearly 4 times, so that the detection efficiency is greatly improved, and the rapid detection of an outpatient service is particularly facilitated.

4. The reagent has high sensitivity, and the blank limit is 5 ng/mL.

5. The reagent has good applicability and can be used on a transmission or scattering instrument. The device can be used on a full-automatic large-scale biochemical analyzer and also can be used on a small POCT device. Can simultaneously meet the detection requirements of large hospital clinical laboratories, hospital residences, small hospital outpatients and emergency departments.

Drawings

FIG. 1 is a calibration curve of POCT equipment in example 1

Detailed Description

The present invention will be described in further detail with reference to specific examples. The following examples are intended to further illustrate some, but not all, preferred embodiments of the present invention. Other embodiments of the invention based on the present invention, which can be made by a person skilled in the art without inventive step, belong to the scope of protection of the present invention.

Example 1

1) Preparation of latex reagents

Preparation methods 1 and 2 differ in the combination of surfactants.

The preparation method comprises the following steps: preparation of activated latex particles: 0.18mL of carboxylated polystyrene microspheres with the particle size of 333nm and the concentration of 10mg/mL is taken, 3.6mL of 25mM MES with the pH value of 6.5 is added, the mixture is stirred for 10min, 7.04mg of EDC activator powder is added, the mixture is stirred at room temperature for 2.5h and then centrifuged at 12000rpm to collect precipitates, and 1.8mL of 25mM MES with the pH value of 6.5 is used for resuspension of the precipitates.

Preparing sensitized latex particles: 0.18mL of 4.0mg/mL MxA polyclonal antibody was added to 1.8mL of 25mM MES, pH6.5, and the mixture was stirred for 1min to prepare an antibody solution to be conjugated. Quickly pouring the activated latex particles in the step (1) into the antibody solution to be coupled, and stirring for 2.5 h. And centrifuging at 12000rpm to collect the precipitate, resuspending 120mL of confining liquid, and stirring for 2.5h to obtain the single-reagent antiviral protein MxA (MxA) latex enhanced immunoturbidimetric reagent. The components of the sealing liquid are 10mM 4-hydroxyethyl piperazine ethanesulfonic acid, 1g/L bovine serum albumin, 3g/L sodium polyethylene sulfonate, 1g/L dodecyl polyglycol ether and 1g/L Proclin300, the pH =6.5, the ratio of the sodium polyethylene sulfonate to the dodecyl polyglycol ether is 3: 1.

the preparation method 2 comprises the following steps: preparation of activated latex particles: 0.18mL of carboxylated polystyrene microspheres with the particle size of 333nm and the concentration of 10mg/mL is taken, 3.6mL of 25mM MES with the pH value of 6.5 is added, the mixture is stirred for 10min, 7.04mg of EDC activator powder is added, the mixture is stirred at room temperature for 2.5h and then centrifuged at 12000rpm to collect precipitates, and 1.8mL of 25mM MES with the pH value of 6.5 is used for resuspension of the precipitates.

Preparing sensitized latex particles: 0.18mL of 4.0mg/mL MxA polyclonal antibody was added to 1.8mL of 25mM MES, pH6.5, and the mixture was stirred for 1min to prepare an antibody solution to be conjugated. Quickly pouring the activated latex particles in the step (1) into the antibody solution to be coupled, and stirring for 2.5 h. And centrifuging at 12000rpm to collect the precipitate, resuspending 120mL of confining liquid, and stirring for 2.5h to obtain the single-reagent antiviral protein MxA (MxA) latex enhanced immunoturbidimetric reagent. The blocking solution had a composition of 10mM 4-hydroxyethylpiperazine ethanesulfonic acid, 1g/L bovine serum albumin, 5g/L Triton X-100, and 1g/L Proclin300, pH = 6.5.

2) Preparation of calibrator

Commercially available MxA antigens were formulated into a series of calibrators with calibrant dilutions at concentrations of 0ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, and 500ng/mL, respectively.

3) Preparation of quality control product

A high concentration MxA sample (500 ng/mL) was diluted twice and twenty times with calibrator dilutions to 250ng/mL and 25ng/mL, respectively, as high and low value controls.

4) Calibration curves were established on POCT equipment.

6 MxA calibrators, each at a concentration of 0 to 500ng/mL, were reacted with the prepared MxA latex single reagent in a cuvette and tested using a POCT apparatus (in this example, a Laura electronic semi-automated specific protein analyzer EZ-400) to record the degree of reaction. The detection wavelength is 650nm, the scattered light intensity is detected, a two-point velocity method is adopted, the reaction time is 120s, and the difference value of the two-point reactivity is calculated. Calibration curves can be established based on the difference in concentration and reactivity. FIG. 1 is a calibration curve established for the single reagents of the latices obtained according to preparation methods 1 and 2 in example 1.

Example 2

Sensitivity of reagent (blank limit)

The reagents of example 1 were used, and a zero value calibrator was selected as a blank sample for testing and concentrations were tested on a POCT instrument. The test was repeated 20 times, and the average (X) and Standard Deviation (SD) of the test results of 20 times were calculated according to the standard curve established in example 1, and X +2SD was calculated as the sensitivity of the reagent. The test results are shown in table 1, and show that the sensitivity of the reagent obtained by the preparation method 1 is 5ng/mL, and the sensitivity obtained by the preparation method 2 is 15ng/mL, which indicates that the sensitivity of the reagent detection can be obviously improved after the combination of the surfactants is optimized. The clinical reference value for detecting the antiviral protein MxA (MxA) is about 50ng/mL, and the sensitivity of the MxA latex enhanced immunoturbidimetric single reagent prepared by the preparation method 1 in the invention is 10 times lower than the reference value, thereby completely meeting the use requirement.

TABLE 1 results of the sensitivity test of the reagent analysis of example 1

Example 1	Sensitivity of analysis	Sensitivity of analysis
			Test sequence number	Measurement results of preparation method 1 (ng/mL)	Preparation method 2 measurement results (ng/mL)
1	3.65	7.68
			2	3.76	6.66
3	1.26	12.43
			4	3.31	3.28
5	1.99	1.55
			6	3.6	4.45
7	3.67	3.57
			8	1.75	5.93
9	0.86	2.72
			10	0.99	8.69
11	3.91	8.3
			12	3.84	8.04
13	3.46	7.2
			14	0.81	11.71
15	3.84	2.23
			16	2.36	10.93
17	3.24	13.98
			18	1.92	5.88
19	2.51	14.71
			20	1.73	4
Mean value (X)	2.62	7.20
			SD	1.12	3.94
X+2SD	4.87	15.08

Example 3

Reproducibility of reagents

The control substance at a normal level with a MxA concentration of about 50ng/mL and the control substance at an abnormal level with a MxA concentration of about 200ng/mL were tested with the single reagent prepared in example 1, and each test was repeated 10 times to calculate the average (X) and Standard Deviation (SD) of the measured values. The Coefficient of Variation (CV) was calculated according to equation (1). The test results are shown in Table 2. From the measurements, coefficients of variation CV = 5.35% and 4.53% were calculated, meeting the technical requirement of reagent CV < 10%.

CV =（SD/X）×100% …………………………(1)

In the formula:

CV-coefficient of variation;

SD-Standard deviation;

x-the average of the measurements.

TABLE 2 results of repeatability measurements of example 1

Example 1	Repeatability of	Repeatability of
			Measurement of sequence number	Measurement result (50 ng/mL)	Measurement result (200 ng/mL)
1	49.13	198.10
			2	47.01	194.50
3	52.22	208.08
			4	52.38	193.05
5	52.25	193.45
			6	45.62	209.98
7	50.81	207.75
			8	51.91	213.87
9	49.80	197.47
			10	46.06	186.15
Mean value (X)	49.72	200.24
			SD	2.66	9.07
CV	5.35%	4.53%

Example 4

Comparison with Performance parameters of other reagents on the market

The comparison of the performance parameters of the single reagent of the invention and other MxA kits on the market is shown in Table 3. As can be seen from the table, the single reagent of the present invention is significantly superior to the comparative reagents on the market mainly in terms of the type of the sample to be tested, the amount and kind of reagents required, the sensitivity of the reagents, and the detection speed of the reagents.

TABLE 3 MxA reagent Performance parameter comparison

Manufacturer of the product	Example 1	Reagent of other company
			Test method	Nephelometry of nephelometry	Transmission turbidimetry
Type of sample	Fresh serum, plasma and whole blood	Fresh bloodClearing and regulating blood plasma
			Type of reagent	Liquid, polystyrene particles	Liquid, polystyrene particles
Amount of reagent	1	2
			Kind of reagent	Single reagent latex	Latex and auxiliary agent
Linear range	5-500ng/mL	50-500ng/mL
			Detection of elapsed time	2-3min	About 10min
Precision degree	≤10%	≤10%
			Accuracy of	≤15%	Is not shown
Difference between batches	≤10%	≤10%
			Whether or not to prepare diluent	Whether or not	Is that
Period of validity	12 months old	12 months old

Claims (1)

1. The single reagent for the latex-enhanced immunoturbidimetric assay of antiviral proteins MxA (MxA) is characterized by comprising 0.15mg/mL of MxA antibody sensitized latex particles, 10mM 4-hydroxyethylpiperazine ethanesulfonic acid, 1g/L of bovine serum albumin, 3-6g/L of sodium polyvinyl sulfonate, 1-2g/L of dodecyl polyethylene glycol ether and 1g/L of Proclin300, and the pH =6.5, wherein the ratio of the sodium polyvinyl sulfonate to the dodecyl polyethylene glycol ether is 3: 1;

the MxA antibody sensitized latex particle is prepared by the following method:

(1) preparation of activated latex particles: taking 0.18mL of carboxylated polystyrene microspheres with the particle size of 333nm and the concentration of 10mg/mL, adding 3.6mL of 25mM MES with the pH value of 6.5, stirring for 10min, adding 7.04mg of EDC activator powder, stirring for 2.5h at room temperature, centrifuging at 12000rpm, collecting precipitates, and re-suspending 1.8mL of 25mM MES with the pH value of 6.5;

(2) preparing sensitized latex particles: 0.18mL of MxA polyclonal antibody with the concentration of 4.0mg/mL is put into 1.8mL of 25mM MES with the pH value of 6.5, and the mixture is stirred for 1min to prepare an antibody solution to be coupled; quickly pouring the activated latex particles in the step (1) into an antibody solution to be coupled, and stirring for 2.5 h; centrifuging at 12000rpm, collecting precipitate, resuspending 120mL of confining liquid, and stirring for 2.5h to obtain single-reagent antiviral protein MxA (MxA) latex enhanced immunoturbidimetric reagent; the components of the sealing liquid are 10mM 4-hydroxyethyl piperazine ethanesulfonic acid, 1g/L bovine serum albumin, 3-6g/L sodium polyethylene sulfonate, 1-2g/L dodecyl polyglycol ether and 1g/L proclin300, the pH =6.5, the ratio of the sodium polyethylene sulfonate to the dodecyl polyglycol ether is 3: 1.

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