CN111458497A

CN111458497A - Dispersing method of coupled antibody latex and immune turbidimetry kit

Info

Publication number: CN111458497A
Application number: CN202010207554.4A
Authority: CN
Inventors: 柳建敏; 裘艳群
Original assignee: Ningbo Haiyi Biotechnology Co ltd
Current assignee: Ningbo Haiyi Biotechnology Co ltd
Priority date: 2020-03-23
Filing date: 2020-03-23
Publication date: 2020-07-28

Abstract

The invention belongs to the technical field of biological detection, and relates to a dispersion method of coupled antibody latex and an immunoturbidimetric kit. The dispersion method of the coupled antibody latex comprises the following steps: dispersing the coupling antibody latex particles prepared in the early stage into a diluent to obtain a coupling antibody latex suspension; and (3) performing ultrasonic treatment on the coupled antibody latex suspension in an ultrasonic cell disruption instrument for 3-5min, and performing circulating homogenization treatment by a high-pressure homogenizer for 1-5 times to obtain a uniform coupled antibody latex solution.

Description

Dispersing method of coupled antibody latex and immune turbidimetry kit

Technical Field

The invention belongs to the technical field of biological detection, and relates to a dispersion method of coupled antibody latex and an immunoturbidimetric kit.

Background

The method mainly adopts an immunological method, mainly enzyme-linked immunosorbent E L ISA, radioimmunoassay RIA, colloidal gold chromatography and the like, and the E L ISA is clinically used for more than twenty years, but still has the defects of poor quantification, long operation time, low automation degree, high RIA sensitivity, instability, poor repeatability and risk of radioactive pollution.

However, the latex enhanced immunoturbidimetry has some defects in the production process, the production period is long, and the requirements on instruments and equipment are high, so that the production cost is high.

The production process of the reagent 2 of the common latex enhanced turbidimetric immunoassay kit comprises the following steps:

(1) activating the latex carrier: adding the polymer latex carrier into a buffer solution, and stirring T1 to enable the functional group to be unfolded and activated;

(2) lengthening functional groups: adding a proper amount of chemical cross-linking agent, and stirring T2 to lengthen the functional group arm on the latex carrier;

(3) coupling antibody: adding a proper amount of target antibody, and stirring T3 to covalently bond the antibody and the functional group on the latex carrier;

(4) and (3) sealing: adding a proper amount of confining liquid, and stirring T4 to couple the redundant functional groups of the unconjugated antibody on the latex carrier with the confining liquid;

(5) centrifuging: setting proper centrifugal parameters, centrifuging the sealed latex carrier solution by T5, removing supernatant, and precipitating to obtain antibody-coupled latex carrier particles;

(6) resuspending: adding a proper amount of diluent of the reagent 2 into the precipitate, performing ultrasonic treatment T6, and scattering the precipitate to obtain a uniform solution.

The traditional resuspension uses ultrasonic treatment, the size of an ultrasonic cell crusher is fixed, the volume for treating latex particle diluent is very limited, the step can be completed only through repeated operation when the yield is increased, the working procedure time is prolonged in multiples, and the production scale and the production efficiency are limited. And the ultrasonic treatment can also increase the temperature of the solution, thereby affecting the performance of the latex, so the ultrasonic treatment also needs ice bath treatment, and the actual operation is not automatic. Meanwhile, the use process of the ultrasonic wave can generate larger noise, so that operators are uncomfortable.

In order to shorten the production period, the resuspension step can adopt other modes to replace ultrasonic treatment, thereby effectively solving the defects caused by single ultrasonic treatment.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the method for dispersing the coupled antibody latex, and the coupled antibody latex suspension is homogenized by using the high-pressure homogenizer, so that the process time is greatly shortened and the production efficiency is improved on the premise of ensuring the dispersion quality.

The first aspect of the present invention provides a dispersion method of a conjugated antibody latex, the dispersion method comprising the steps of:

dispersing the coupling antibody latex particles prepared in the early stage into a diluent to obtain a coupling antibody latex suspension;

and (3) circularly homogenizing the coupling antibody latex suspension by a high-pressure homogenizer for 1-5 times to obtain a uniform coupling antibody latex solution.

Preferably, the preparation method of the conjugated antibody latex particles comprises the following steps:

adding a buffer solution into the polymer latex; then adding a chemical cross-linking agent to activate the polymer latex; adding antibody protein and stirring for reaction; finally adding a sealing liquid for sealing, and obtaining a precipitate after sealing reaction and centrifugation, namely the coupled antibody latex particle.

Preferably, the polymer latex is polystyrene latex and/or polyacrylate latex.

Preferably, the average particle diameter of the conjugated antibody latex particles in the homogeneous conjugated antibody latex solution is 50 to 150 nm.

Preferably, the high-pressure homogenizer is operated at a pressure of 600-1000bar and a flow rate of 3-15L/h.

Preferably, the conjugated antibody latex suspension is subjected to shearing treatment for 10-12min before high-pressure homogenization treatment.

Preferably, the shear rate is 450-1000 rmp/min.

Preferably, the coupled antibody latex suspension is subjected to ultrasonic treatment in an ultrasonic cell disruptor for 3-5min before the high-pressure homogenization treatment.

The second aspect of the present invention provides a conjugated antibody latex solution obtained by the dispersion method of the first aspect of the present invention.

In a second aspect of the present invention, there is provided an immunoturbidimetric kit for detecting a biological sample, the kit comprising a conjugated antibody latex solution according to the second aspect of the present invention.

Immunoturbidimetric kits for the detection of biological samples generally comprise two groups of reagents: reagent 1 and reagent 2, reagent 2 comprises the conjugated antibody latex solution of the second aspect of the invention, and further comprises some inorganic salts, stabilizers, preservatives and other components.

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

(1) the high-pressure homogenizer is adopted to replace an ultrasonic cell disruptor to disperse the coupled antibody latex suspension, and has the characteristics of large handling capacity, short dispersing time and the like, and particularly shows the advantages of more remarkable time shortening and cost reduction when the handling capacity is larger, compared with the characteristics of fixed size and limited sample handling capacity of the ultrasonic cell disruptor;

(2) before the coupled antibody latex suspension is subjected to high-pressure homogenization treatment, shearing treatment is carried out, and the high-pressure homogenization dispersion effect is greatly improved through shearing pre-dispersion;

(3) the invention adopts the ultrasonic cell disruption instrument and the high-pressure homogenizer to be used together, and has shorter dispersion time on the premise of ensuring the dispersion effect compared with the high-pressure homogenizer which is used separately and combined with ultrasonic treatment and shearing treatment, and can greatly reduce the dispersion time of the suspension, thereby shortening the production period and reducing the production cost.

Detailed Description

Hereinafter, embodiments will be described in detail with respect to the dispersion method of the conjugated antibody latex of the present invention, however, these embodiments are exemplary and the present disclosure is not limited thereto.

In some embodiments of the present invention, the method for dispersing the conjugated antibody latex comprises the following steps:

The preparation method of the coupled antibody latex particles prepared in the early stage comprises the following steps: adding the polymer latex into a buffer solution, and stirring to enable the functional group of the polymer latex to be unfolded and activated; then adding a proper amount of chemical cross-linking agent to lengthen the functional group arm on the polymer latex; adding antibody protein, stirring and reacting to covalently combine the antibody protein with functional groups on the polymer latex carrier; finally adding a sealing liquid for sealing, and obtaining a precipitate after sealing reaction and centrifugation, namely the coupled antibody latex particle.

The polymer latex can be polystyrene latex and/or polyacrylate latex, the buffer can be one or more of boric acid buffer, MES buffer, PBS buffer and HEPES buffer, and the pH value of the buffer is 5.0-9.0.

The chemical crosslinker can be 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) alone, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in combination with N-hydroxysuccinimide (NHS), or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in combination with N-hydroxythiosuccinimide (Sulfo-NHS).

The antibody protein may be a monoclonal antibody or a polyclonal antibody such as a troponin I antibody, a procalcitonin antibody, a retinol-binding protein antibody, a C-reactive protein antibody, a phosphatidyl proteoglycan antibody, a cystatin C antibody, a serum amyloid antibody, an β 2-microglobulin antibody, or a hemoglobin antibody.

The blocking solution can be a buffer solution containing bovine serum or horse serum or sheep serum, so that redundant functional groups of unconjugated antibodies on the polymer latex carrier are coupled with the blocking solution.

The latex particles form a large aggregate precipitate due to the interaction of surface charges of the latex particles in the high-speed centrifugation process, and a suspension needs to be dispersed after the latex particles are diluted by adding a diluent.

The diluent can be one or more of HEPES buffer solution, MES buffer solution and PBS buffer solution, and has a pH value of 5.0-9.0.

The invention adopts a high-pressure homogenizer to disperse the suspension, the high-pressure homogenizer is operated under the pressure of 600-1000bar and the flow rate of 3-15L/h to obtain the coupled antibody latex solution with uniform distribution, and the average particle size of the coupled antibody latex particles in the coupled antibody latex solution can be controlled between 50nm and 150 nm.

In other embodiments of the present invention, the method for dispersing the conjugated antibody latex comprises the following steps:

shearing the coupled antibody latex suspension for 10-12min, and circularly homogenizing for 1-5 times by a high-pressure homogenizer to obtain a uniform coupled antibody latex solution.

The suspension containing the large-block sediment needs to be sheared before entering the high-pressure homogenizer, so that the large-block sediment is primarily dispersed, and the improvement of the dispersion uniformity of the high-pressure homogenizer is facilitated.

However, the means for shearing and dispersing the material are all shearing treatment, and examples of the shearing treatment means include a magnetic stirrer, a paddle stirrer and the like.

The shearing time and the shearing speed are not enough, so that the dispersion effect of a high-pressure homogenizer is influenced, the coupled antibody latex particles are not uniformly dispersed, and large aggregated particles exist, so that the test result of the kit is influenced; however, the shearing speed is too high, the temperature of the material rises, the activity of the antibody on the surface of the latex particles is affected, and the production efficiency is reduced if the shearing treatment time is too long. Therefore, the shearing speed of the invention is preferably 450-1000rmp/min, and the shearing time is preferably 10-12 min. The selection of a shear rate of 450-.

After the shearing treatment of the latex suspension of the coupled antibody, homogenizing the latex suspension by a high-pressure homogenizer, preferably operating the high-pressure homogenizer at the pressure of 700-900bar and the flow rate of 3-15L/h, wherein the circulation time is preferably 2-4 times.

and (3) performing ultrasonic treatment on the coupled antibody latex suspension in an ultrasonic cell disruption instrument for 3-5min, and performing circulating homogenization treatment by a high-pressure homogenizer for 1-5 times to obtain a uniform coupled antibody latex solution.

The invention adopts the combination of the ultrasonic cell disruptor and the high-pressure homogenizer, which can greatly reduce the dispersion time of the suspension, thereby shortening the production period.

The ultrasonic treatment can be carried out directly at room temperature when the time is short, or can be carried out at 0-10 ℃.

After the ultrasonic treatment of the coupled antibody latex suspension, the coupled antibody latex suspension is homogenized by a high-pressure homogenizer, the high-pressure homogenizer is preferably operated under the pressure of 700-900bar and the flow rate of 3-15L/h, and the circulation time is preferably 2-4 times.

The technical solution of the present invention is further described below by means of specific examples. The raw materials used in the examples of the present invention are those commonly used in the art, and the methods used in the examples are those conventional in the art, unless otherwise specified.

Example 1

Preparation of latex particles of conjugated antibodies:

diluting the polystyrene latex particles to a final concentration of 1% by using 50mM boric acid buffer solution with pH of 9.0, and reacting for 5min at room temperature; adding EDC, reacting at room temperature for 30min to make EDC and polystyrene latex 150 μ g: 1 mg; adding retinol binding protein polyclonal antibody, mixing retinol binding protein polyclonal antibody and polystyrene latex 50 μ g: 1mg, stirring, and reacting at room temperature for 1 h; adding Bovine Serum Albumin (BSA) to make the final concentration of the BSA 1%, and sealing for 2h at room temperature; the above-mentioned blocked solution was equally divided into 15 equal parts, and the supernatants were centrifuged at 10 ℃ and 10,000rpm, respectively, to obtain latex particles of retinol-binding protein-conjugated polyclonal antibody (hereinafter referred to as conjugated antibody latex particles).

15 equal parts of coupled antibody latex particles are respectively dissolved in HEPES buffer solution to obtain coupled antibody latex suspension liquid, the volume is 200m L, the mass concentration is 2% (w/v), the 15 equal parts of suspension liquid are respectively dispersed by different dispersion processes and parameters, and the dispersion processes are respectively marked as methods 1 to 15, and the specific steps are as follows:

the method 1 adopts the traditional ultrasonic dispersion, the actual dispersion time is 20min, the ultrasonic pause time is added, the total ultrasonic consumption time is 26.7min, and the parameters of an ultrasonic cell disruptor (brand: sonic, model: VCX750) are shown in Table 1:

TABLE 1

Item	Require that
		Tip diameter	13mm
Amplitude (Amp)	60％
		ON/OFF	6sec/2sec
Temperature of	Ice bath
		Concentration of latex	2％

Method 2 before high pressure homogenization, the raw materials are firstly sheared at a shearing speed of 450rmp/min for 10min, and then high pressure homogenization is carried out, wherein the parameters of a high pressure homogenizer are shown in the following table 2:

TABLE 2

Item	Require that
		Pressure intensity	600bar
Speed of rotation	7.5L/h
		Concentration of latex	2％
Number of dispersions	3

Method 3 shearing treatment is carried out for 10min at a shearing speed of 450rmp/min before high-pressure homogenization, and then high-pressure homogenization is carried out, wherein the parameters of a high-pressure homogenizer are shown in the following table 3:

TABLE 3

Item	Require that
		Pressure intensity	800bar
Speed of rotation	7.5L/h
		Concentration of latex	2％
Number of dispersions	3

Method 4 shearing treatment is carried out for 10min at a shearing speed of 450rmp/min before high-pressure homogenization, and then high-pressure homogenization is carried out, wherein the parameters of a high-pressure homogenizer are shown in the following table 4:

TABLE 4

Method 5 shearing treatment is carried out for 10min at a shearing speed of 450rmp/min before high-pressure homogenization, and then high-pressure homogenization is carried out, wherein the parameters of a high-pressure homogenizer are shown in the following table 5:

TABLE 5

Item	Require that
		Pressure intensity	800bar
Speed of rotation	7.5L/h
		Concentration of latex	2％
Number of dispersions	4

Method 6 shearing treatment is carried out for 10min at a shearing speed of 450rmp/min before high-pressure homogenization, and then high-pressure homogenization is carried out, wherein the parameters of a high-pressure homogenizer are shown in the following table 6:

TABLE 6

Item	Require that
		Pressure intensity	800bar
Speed of rotation	7.5L/h
		Concentration of latex	2％
Number of dispersions	2

Method 7 shearing treatment is carried out for 10min at a shearing speed of 450rmp/min before high-pressure homogenization, and then high-pressure homogenization is carried out, wherein the parameters of a high-pressure homogenizer are shown in the following table 7:

TABLE 7

Item	Require that
		Pressure intensity	800bar
Speed of rotation	15L/h
		Concentration of latex	2％
Number of dispersions	3

In the method 8, before high-pressure homogenization, shearing treatment is carried out for 12min at a shearing speed of 450rmp/min, and then high-pressure homogenization is carried out, wherein the parameters of a high-pressure homogenizer are shown in the table 3.

Method 9 high pressure homogenization was used without shearing, and the high pressure homogenizer parameters are as shown in table 3.

The method 10 comprises shearing at a shearing speed of 450rmp/min for 6min before high-pressure homogenizing, and then high-pressure homogenizing, wherein the parameters of the high-pressure homogenizer are shown in Table 3.

In the method 11, before the high-pressure homogenization treatment, the shearing treatment is carried out for 6min at the shearing speed of 450rmp/min, and then the high-pressure homogenization treatment is carried out, wherein the parameters of the high-pressure homogenizer are shown in the table 5.

The method 12 comprises performing ultrasonic treatment in an ultrasonic cell disruptor for 1min before high-pressure homogenization treatment, adding ultrasonic dwell time until the total ultrasonic time is 1.33min, and performing high-pressure homogenization treatment with the parameters of the ultrasonic cell disruptor shown in Table 1 and the parameters of the high-pressure homogenizer shown in Table 3.

The method 13 comprises performing ultrasonic treatment in an ultrasonic cell disruptor for 1min before high pressure homogenization treatment, adding ultrasonic dwell time for 1.33min, and performing high pressure homogenization treatment with the parameters of the ultrasonic cell disruptor shown in Table 1 and the parameters of the high pressure homogenizer shown in Table 5.

The method 14 comprises performing ultrasonic treatment in an ultrasonic cell disruptor for 3min before high-pressure homogenization treatment, adding ultrasonic dwell time, wherein the total ultrasonic time is 4min, and performing high-pressure homogenization treatment, wherein the parameters of the ultrasonic cell disruptor are shown in Table 1, and the parameters of the high-pressure homogenizer are shown in Table 3.

The method 15 comprises performing ultrasonic treatment in an ultrasonic cell disruptor for 5min before high-pressure homogenization treatment, adding ultrasonic dwell time until the total ultrasonic time is 6.67min, and performing high-pressure homogenization treatment with the parameters of the ultrasonic cell disruptor shown in Table 1 and the parameters of the high-pressure homogenizer shown in Table 3.

The latex solutions of the retinol binding protein-coupled polyclonal antibody obtained by the above methods 1-15 were diluted to 0.2% (w/v) with HEPES buffer.

The latex solution of the coupled retinol binding protein polyclonal antibody obtained after dilution in the method 1-15 uses the same set of retinol binding protein standard sample to carry out calibration test, and respectively measures the same sample,

calibration: and (3) putting the coated latex solution of the coupled retinol binding protein polyclonal antibody obtained by the method 1-15, the retinol binding protein reagent 1 matched with the kit and a retinol binding protein calibrator into a biochemical analyzer for calibration.

And (3) sample measurement: after the calibration is finished, the samples with the numbers of 1-10 are pushed into a biochemical analyzer, and 15 calibrated retinol binding protein calibration channels are selected for simultaneous sample measurement.

Analytical data are shown in tables 8 and 9 below:

TABLE 8 analysis of solution calibration data by methods 1-15

Remarking: in table 8, method i represents method 1-method 14; the background value represents the self-absorbance value of the latex solution of the coupled retinol binding protein polyclonal antibody; C6/C5 indicates the absorbance ratio corresponding to the concentration of the 6 th standard to the concentration of the 5 th standard.

TABLE 9 methods 1-15 concentration values (mg/L) of solutions for determination of samples 1-10

As can be seen from the data results of table 8 and table 9:

a. comparing methods 2, 3, 4, 5 and 6, wherein the method 2 adopts a pressure of 600bar, the pressure value is small, the solution of the method 2 cannot achieve a good dispersion effect, the linear ductility difference caused by the agglutination still exists among particles, and the deviation of the measurement result of the high-concentration sample is large; the method 4 adopts 1000bar pressure, and the pressure value is too large, so that the prepared latex particles are too dispersed, the particles are likely to be damaged, and the calibration signal is low; the method 5 has overlarge dispersion times, increases the time and increases the production period; method 6, when the dispersion times are insufficient, the latex particles are not sufficiently dispersed, the linear ductility is poor due to agglutination, and the high-value sample cannot be accurately measured; comparing method 3 with method 7, it can be seen that slowing or speeding up the flow rate has little effect on the results and that the flow rate can be adjusted according to actual operating requirements.

b. Comparing methods 3, 8, 9, 10 and 11, it can be known that the dispersion effect can be greatly improved by performing the shearing treatment for 10min before the high-pressure homogenization treatment, and the result is not greatly affected by continuously increasing the shearing treatment time to 12min, but the time is increased; when the shearing time is only 6min, the solution is not fully pre-dispersed before the high-pressure homogenizing treatment, the final dispersing effect of the solution is poor, and the high-pressure homogenizer is damaged.

c. Comparing methods 3, 12, 13, 14 and 15, it can be seen that the reaction time can be greatly shortened on the premise of ensuring the dispersion quality by combining the ultrasonic treatment and the high-pressure homogenization treatment, the ultrasonic treatment can be carried out for 3 minutes before the high-pressure homogenization treatment, and the ultrasonic treatment time is continuously increased without great influence on the result, but the time is increased.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

Claims

1. A method for dispersing a latex of a conjugated antibody, comprising the steps of:

2. The dispersion method according to claim 1, wherein the pre-prepared conjugated antibody latex particles are prepared by a method comprising the steps of:

adding a buffer solution into the polymer latex for activation; then adding a chemical cross-linking agent; adding antibody protein and stirring for reaction; finally adding a sealing liquid for sealing, and obtaining a precipitate after sealing reaction and centrifugation, namely the coupled antibody latex particle.

3. The dispersion method according to claim 2, wherein the polymer latex is polystyrene latex and/or polyacrylate latex.

4. The dispersion method according to claim 1, wherein the average particle diameter of the conjugated antibody latex particles in the homogeneous conjugated antibody latex solution is 50 to 150 nm.

5. The dispersion method according to claim 1, wherein the high-pressure homogenizer is operated at a pressure of 600-1000bar and a flow rate of 3-15L/h.

6. The dispersion method according to claim 1, wherein the conjugated antibody latex suspension is subjected to a shearing treatment for 10 to 12min before the high-pressure homogenization treatment.

7. The dispersion method according to claim 6, wherein the shear rate is 450-1000 rmp/min.

8. The dispersion method according to claim 1, wherein the coupled antibody latex suspension is sonicated in an ultrasonic cell disruptor for 3-5min before the high pressure homogenization treatment.

9. A conjugated antibody latex solution obtained by the dispersion method according to claim 1.

10. An immunoturbidimetric kit for detecting a biological sample, comprising the conjugated antibody latex solution of claim 9.