CN104360056B - A kind of method that promotes the sensitivity of latex enhancing immune turbidimetry - Google Patents

Abstract

The invention relates to the field of in vitro diagnostic reagents, in particular to a method for improving the sensitivity of latex-enhanced immune turbidimetry. The invention provides a method for improving the sensitivity of latex-enhanced immune turbidimetry, which includes the following steps: 1) selecting large-diameter latex particles to react with antibodies, and performing antibody coating; 2) cleaning and sealing the coated latex After the step, disperse in the buffer solution to prepare reagent 2 of the latex enhanced immune turbidimetric assay kit; 3) configure the reagent 1 of the latex enhanced immune turbidimetric assay kit; 4) set parameters on the automatic biochemical analyzer; 5 ) to run the biochemical analyzer, after calibration with a sample of known concentration, detect the sample to be tested to obtain the absorbance change value, and calculate the content of the sample to be tested according to the calibration curve. The method for improving the sensitivity of latex-enhanced immune turbidimetry provided by the present invention greatly improves the analytical sensitivity and precision of reagents without increasing the detection cost, and has good application prospects.

Description

A method for improving the sensitivity of latex-enhanced immunoturbidimetry

technical field

The invention relates to the field of in vitro diagnostic reagents, in particular to a method for improving the sensitivity of latex-enhanced immune turbidimetry.

Background technique

When light passes through a turbid medium solution, due to the presence of turbid particles in the solution, part of the light is absorbed, and the amount absorbed is proportional to the amount of turbid particles. This method of measuring light absorption is called turbidimetry. This method was earlier reported by Schultre and Schuick in 1959 and applied to the formation of complexes after the binding of plasma proteins and antibodies, resulting in changes in turbidity, and then the transmission turbidimetry. Generally, the transmission turbidimetry of antibody to antigen quantification is used, called For immunoturbidimetry. The principle is to use the specific combination of antigen and antibody to form a complex, and to quantify the antigen or antibody by measuring the amount of complex formation.

However, in the classic immunoturbidimetric method, a small amount of small antigen-antibody complexes is extremely difficult to form turbidity, unless it is left for a long time; if a larger complex is formed, the amount of antigen and antibody is also large, which obviously does not meet the requirement of microquantification. requirements. Therefore, the latex-enhanced immunoturbidimetric assay (PETIA), which is now widely used in biochemical analyzers, was developed: an improved immunoturbidimetric assay based on polyclonal antibodies, using genetic engineering methods to combine antibodies with latex particles. When combined, an antigen-antibody-latex particle complex is formed, which enhances the reaction absorbance. The reaction solution is turbidized at a certain wavelength, and compared with the same treated standard solution, the antigen content in the sample is calculated. Using a biochemical analyzer for turbidimetric determination, the entire analysis process only takes a few minutes. Compared with the traditional immunoturbidimetric method, this method has higher sensitivity.

With the development of laboratory medicine, more and more low-concentration detection substances are detected, and the concentration of detection substances is decreasing from the μg/mL level to the ng/mL level. The sensitivity requirements for diagnostic reagents are also getting higher and higher. For traditional For the latex-enhanced immunoturbidimetric method, it is necessary to further improve the detection sensitivity to meet the detection requirements. The traditional methods to improve the detection sensitivity include using antibodies with higher affinity, using larger particles, and latex particles with better stability. Choose a better buffer system to promote the antigen-antibody reaction, but these will increase the cost of the reagent. How to improve the analytical sensitivity and precision of the analyte at the ng/mL level without increasing the cost has always been a part of reagent development. big challenge.

Contents of the invention

In view of the shortcomings of the prior art described above, the purpose of the present invention is to not only detect the generation of milk antibody antigen complexes, but also detect the reduction of unbound latex particles through the selection of the reaction wavelength, so as to improve the analytical sensitivity and precision of the detected substances, It is used to solve the problems in the prior art.

In order to achieve the above purpose and other related purposes, the first aspect of the present invention provides a method for improving the sensitivity of latex-enhanced immune turbidimetry, comprising the following steps:

1) Select latex particles with a large particle size to react with the antibody for antibody coating. The particle size of the latex particles with a large particle size is 0.2-1.0 μm;

2) After the coated latex is washed and sealed, it is dispersed in the buffer solution to prepare the reagent 2 of the latex-enhanced immune turbidimetric assay kit;

3) A buffer solution for promoting the reaction of antigen and antibody is configured to prepare the reagent 1 of the latex-enhanced immune turbidimetric assay kit;

4) On the fully automatic biochemical analyzer, set the parameters, wherein the main wavelength is set to 300-500nm, the secondary wavelength is set to 500-800nm, and the reaction direction is the negative direction;

5) Run the biochemical analyzer, and after calibration with a sample of known concentration, detect the sample to be tested to obtain the absorbance change value, and calculate the content of the sample to be tested according to the calibration curve.

Preferably, in the step 2, the buffer is Tris-HCl buffer with a concentration of 50 mM, wherein the concentration of bovine serum albumin (BSA) is 5 g/L, and the concentration of sodium azide is 1 g/L.

Preferably, in the step 3, the buffer is Tris-HCl buffer, its formula is: Tris50mmol/L, the concentration of bovine serum albumin BSA is 3g/L, the concentration of sodium azide is 1g/L, the concentration of PEG6000 The concentration is 50g/L, stachyose 0.8-3g/L; alum 0.1-1g/L; sodium fructose diphosphate 0.8-3g/L; sodium hexametaphosphate 0.05-0.5g/L, pH=7.0-7.4.

Preferably, the method for improving the sensitivity of latex-enhanced immunoturbidimetry is not aimed at the diagnosis and treatment of diseases.

More preferably, the direct purpose of measuring the content of the analyte is not to obtain diagnostic results or health conditions, but only to obtain information as an intermediate result from the human or animal body, or only to the tissues and body fluids that have been separated from the human or animal body. or excreta are processed or tested to obtain information as an intermediate result.

The second aspect of the present invention provides the application of the method for improving the sensitivity of latex-enhanced immunoturbidimetry in the field of biological detection.

The third aspect of the present invention provides a high-sensitivity latex-enhanced immune turbidimetric assay kit, including reagent 1 and reagent 2, the reagent 2 is a dispersion of latex particles coated with antibodies, and the particle size of the latex particles is 0.2 -1.0 μm, the reagent 1 is a buffer to promote the reaction of antigen and antibody.

Preferably, the formula of the reagent 1 is: Tris50mmol/L, the concentration of bovine serum albumin BSA is 3g/L, the concentration of sodium azide is 1g/L, the concentration of PEG6000 is 50g/L, stachyose 0.8- 3g/L; alum 0.1-1g/L; sodium fructose diphosphate 0.8-3g/L; sodium hexametaphosphate 0.05-0.5g/L.

Preferably, in the reagent 1, the pH value is neutral.

Preferably, in the reagent 2, the coated antibody corresponds to the detection substance.

More preferred is a D-dimer antibody.

Preferably, in the reagent 2, the concentration of Tris-HCl buffer in the dispersion is 50 mM, the concentration of bovine serum albumin BSA is 5 g/L, and the concentration of sodium azide is 1 g/L.

The fourth aspect of the present invention provides the preparation method of the high-sensitivity latex-enhanced immune turbidimetric assay kit, comprising the following steps:

1. Select latex particles with a large particle size to react with the antibody for antibody coating, and the particle size of the latex particles is 0.2-1.0 μm;

2. Disperse the coated latex in a specific buffer solution after washing and sealing steps to prepare reagent 2 of the latex-enhanced immune turbidimetric assay kit;

3. Prepare the reagent 1 of the latex-enhanced immune turbidimetric assay kit with a buffer solution for promoting the reaction of the antigen and the antibody. The fifth aspect of the present invention provides the use of the high-sensitivity latex-enhanced immunoturbidimetric assay kit in the field of biological detection.

The present invention is based on the basic principle of latex-enhanced immune turbidimetry: that is, the latex coated with antibodies reacts with the antigen to form a larger latex-antigen-antibody complex, which causes an increase in turbidity and enhances the reaction absorbance. Test at the same place, compare with the standard solution treated in the same way, and calculate the antigen content in the specimen. The inventors believe that when the particle size of the coated antibody latex is large, the latex particle itself has a certain turbidity and has a large absorption at a specific wavelength, and when the coated antibody latex and the antigen form a larger latex antigen After the antibody complex, the unreacted coated antibody latex particles also decrease. According to the Mie scattering theory, microspheres with different diameters have different light reflection capabilities for fixed wavelengths, so the absorbance of the entire reaction solution increases at certain wavelengths, but The absorbance will decrease at certain wavelengths. By measuring the absorbance change values of the rising wavelength and the falling wavelength and summing them up, the analytical sensitivity can be increased by more than 1 times.

Compared with the prior art, the present invention has the following advantages:

1. Greatly improve the analytical sensitivity and precision of reagents: the inventors of the present invention have found that through a special buffer solution that promotes the antigen-antibody reaction, and further utilize the large particle size coated latex microspheres to reduce the absorbance change and latex antigen-antibody The change in absorbance caused by the increase of the complex (the change in absorbance caused by the decrease of the latex microspheres coated with large particle size is often greater than the change in absorbance caused by the increase in the latex antigen-antibody complex), after the addition of the two absorbance changes, the analytical sensitivity of the reagent The increase is doubled or more, and at the same time, the increase in analytical sensitivity also brings about an increase in precision.

2. Can not increase or reduce cost: firstly, the present invention is only the change of the measurement method, which can be easily realized by modifying the parameters on the automatic biochemical analyzer, so the cost will not be increased; secondly, due to the large particle size latex microsphere The antibody required for coating is often less than the same amount of small-diameter latex microspheres (smaller surface area), and the concentration of latex required by the present invention is smaller, so the present invention can often reduce the cost of reagents.

Description of drawings

Fig. 1 is the reaction full-wavelength scanning kinetic experiment result of the detection kit prepared in Example 1.

2 is a calibration curve diagram of the detection kit prepared in Example 1.

detailed description

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention; in the description and claims of the present invention, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural forms.

When the examples give numerical ranges, it should be understood that unless otherwise specified in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the embodiments of the present invention can also be used Any methods, apparatus and materials of the prior art similar or equivalent to the practice of the present invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field conventional technology.这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULARCLONING：ALABORATORYMANUAL，Secondedition，ColdSpringHarborLaboratoryPress，1989andThirdedition，2001；Ausubel等，CURRENTPROTOCOLSINMOLECULARBIOLOGY，JohnWiley&Sons，NewYork，1987andperiodicupdates；theseriesMETHODSINENZYMOLOGY，AcademicPress，SanDiego；Wolffe，CHROMATINSTRUCTUREANDFUNCTION， Thirdedition, Academic Press, San Diego, 1998; METHODSINENZYMOLOGY, Vol. 304, Chromatin (P.M. Wassarman and A.P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODSINMOLECULARBIOLOGY, Vol. Totowa, 1999 et al.

It should be specified that the raw materials used in the present invention can be commercially available products substantially, wherein Tris is purchased from Sigma company; Added in the form, purchased from Japan JSR company; D-dimer antibody and D-dimer were purchased from Finland hytest company.

Example 1

1. Preparation of Quantitative Detection Latex Enhanced Immunoturbidimetric Kit

Preparation of Reagent 1:

Prepared from Tris-HCl buffer solution with a concentration of 50mM, 3g bovine serum albumin BSA, 1g sodium azide, 2.2g stachyose, 0.6g alum, 1.9g sodium fructose diphosphate, 0.3g sodium hexametaphosphate and 50g PEG6000 , the concentration of Tris-HCl buffer in final reagent 1 is 50mM, the concentration of bovine serum albumin BSA is 3g/L, the concentration of sodium azide is 1g/L, the concentration of PEG6000 is 50g/L, stachyose 2.2g /L, alum 0.6g/L, sodium fructose diphosphate 1.9g/L, sodium hexametaphosphate 0.3g/L;

Weigh 6.06g of tris (Tris), 3g of BSA, 50g of PEG6000, 2.2g of stachyose, 0.6g of alum, 1.9g of sodium fructose diphosphate, 0.3g of sodium hexametaphosphate, and 1g of sodium azide in 0.8L In deionized water, adjust the pH to 7.0 with HCl, and dilute to 1 L to obtain reagent 1.

Preparation of Reagent 2:

Prepared from 50mM Tris-HCl buffer, 5g bovine serum albumin BSA, 1g sodium azide and sensitized polystyrene latex particles coated with mouse anti-human D-dimer antibody, final reagent 2 The concentration of Tris-HCl buffer in the medium is 50mM, the concentration of bovine serum albumin BSA is 5g/L, and the concentration of sodium azide is 1g/L;

①Activation and washing of milk particles:

Take 100 μL of carboxylated polystyrene latex particle solution with a mass volume ratio of 10%, add 10 μL of EDAC solution with a mass concentration of 1% to it, place it in a shaker at 37°C for 0.5 to 1 hour, and then centrifuge at a speed of 12000rmp Separation for 30 min, then discard the supernatant, wash three times with a glycine buffer solution with a concentration of 50 mM and a pH of 7.2, and finally disperse the precipitate in 1 L of a glycine buffer solution with a concentration of 50 mM and a pH of 7.2 to make the final solution aggregate The concentration of styrene latex particles (in terms of mass volume ratio) is 0.5-1%.

② Antibody purification:

Add the mouse anti-human D-dimer antibody into the dialysis bag, dialyze in 100mM PBS buffer solution with pH 7.2 for 48 hours, change the buffer solution 3 times during the dialysis process, and obtain the purified mouse anti-human D-dimer antibody. Dimeric antibody.

③ Coupling of antibody latex particles:

Mix the carboxylated polystyrene latex particle solution activated in step ① with the antibody purified in step ②, mix well, and incubate on a shaker at 37°C for 2 hours to obtain the antibody-latex particle complex; then mix the antibody-latex particle The complex was centrifuged at 12000rpm for 30min, discarded the supernatant, washed twice with PBS buffer solution with a concentration of 100mM and a pH of 7.2, and finally added 10ml of a Tris-HCl buffer solution with a concentration of 50mM and a pH of 7.0, stirred and mixed Evenly. Wherein the Tris-HCl damping solution of 10ml contains the sodium azide of the bovine serum albumin BSA of 0.5mg and 1mg; The final concentration is 0.05-0.20%.

Preparation of calibrator:

It is prepared by PBS buffer solution with a concentration of 100mM, 3g bovine serum albumin BSA, 1g sodium azide and D-dimer, the concentration of PBS buffer solution in the final calibrator is 100mM, and the concentration of bovine serum albumin BSA is 3g/L, the concentration of sodium azide is 1g/L;

Weigh 35.61Na ₂ HPO ₄ 2H ₂ O, 3g bovine serum albumin BSA, 1g sodium azide and dissolve in 0.8L deionized water, adjust the pH to 7.2, dilute to 1L with distilled water, dissolve D-dimer In the above-prepared solution, prepare solutions with D-dimer concentrations of 0, 0.5, 1, 2, 4, and 8 ng/mL, respectively, to obtain calibrator.

2. The full-wavelength scanning kinetic experiment of the reaction of the detection kit

Mix the calibrator and reagent 1 evenly, incubate at 37°C for 5min, then add reagent 2, incubate at 37°C for 10S, use a UV spectrophotometer to scan the reaction solution at full wavelength (for the first time), react at 37°C for 4min for 50S, and use a UV spectrophotometer Scan the reaction solution at full wavelength (for the second time), and calculate the absorbance change value ΔA=A2-A1; the wavelength range of full-wavelength scanning is 200-800nm. Table 1 shows the experimental results of full-wavelength scanning kinetics.

Table 1

It can be seen that the decrease in absorbance of the reaction solution at 340nm is greater than the increase in absorbance at 600nm. If the absorbance changes at the two wavelengths are added together, the analytical sensitivity of the reagent can be greatly improved.

3. Quantitative Detection of Latex Enhanced Immunoturbidimetric Kit

Testing instrument: OLYMPUSAU640 automatic biochemical analyzer

The parameters set according to the present invention are: detection method: END. measurement wavelength: main wavelength 340nm, negative wavelength 600nm; sample volume: 25μL; reagent 1: 240μL; reagent 2: 80μL; calibration method: multi-point calibration; .

Preparation of Reagent 1:

Prepared from Tris-HCl buffer solution with a concentration of 50mM, 3g bovine serum albumin BSA, 1g sodium azide, 2.2g stachyose, 0.6g alum, 1.9g sodium fructose diphosphate, 0.3g sodium hexametaphosphate and 50g PEG6000 , the concentration of Tris-HCl buffer in final reagent 1 is 50mM, the concentration of bovine serum albumin BSA is 3g/L, the concentration of sodium azide is 1g/L, the concentration of PEG6000 is 50g/L, stachyose 2.2g /L, alum 0.6g/L, sodium fructose diphosphate 1.9g/L, sodium hexametaphosphate 0.3g/L;

Weigh 6.06g of tris (Tris), 3g of BSA, 50g of PEG6000, 2.2g of stachyose, 0.6g of alum, 1.9g of sodium fructose diphosphate, 0.3g of sodium hexametaphosphate, and 1g of sodium azide in 0.8L In deionized water, adjust the pH to 7.0 with HCl, and dilute to 1 L to obtain reagent 1.

Preparation of Reagent 2:

Prepared from 50mM Tris-HCl buffer, 5g bovine serum albumin BSA, 1g sodium azide and sensitized polystyrene latex particles coated with mouse anti-human D-dimer antibody, final reagent 2 The concentration of Tris-HCl buffer in the medium is 50mM, the concentration of bovine serum albumin BSA is 5g/L, and the concentration of sodium azide is 1g/L;

The comparison parameters are: detection method: END. measurement wavelength: dominant wavelength 600nm, no negative wavelength; sample volume: 25 μL; reagent 1: 240 μL; reagent 2: 80 μL; calibration method: multi-point calibration; reaction direction: positive.

Preparation of comparative reagent 1:

Prepared from Tris-HCl buffer with a concentration of 50mM, 3g bovine serum albumin BSA, 1g sodium azide and 50g PEG6000, the concentration of Tris-HCl buffer in final reagent 1 is 50mM, and the concentration of bovine serum albumin BSA is 3g /L, the concentration of sodium azide is 1g/L, and the concentration of PEG6000 is 50g/L;

Weigh 6.06g of tris (Tris), 3g of BSA, 50g of PEG6000, 1g of sodium azide and dissolve in 0.8L of deionized water, adjust the pH to 7.0 with HCl, and dilute to 1L to obtain reagent 1.

Reagent 2 is the same as above.

Mix the calibrator and reagent 1 evenly, incubate at 37°C for 5 minutes, add reagent 2, incubate at 37°C for 10 seconds, read the absorbance value A1, and read the absorbance value A2 after reacting for 4 minutes and 50 seconds, and calculate the absorbance change value △A=A2-A1; Then take the △A value as the ordinate, and the corresponding concentration of the calibrator as the abscissa, and draw a calibration curve, as shown in Figure 2.

4. Comparison experiment of analytical sensitivity and precision of latex-enhanced immunoturbidimetric kit

According to the calibration curve, the analytical sensitivity results of the calculated reagents are shown in Table 2. Table shows the analytical sensitivity of the present invention setting parameters and contrast parameters.

Table 2

In the precision experiment, the test was repeated 10 times with high, middle and low value samples, and the precision of the reagent was characterized by the imprecision of the test results. See Table 3 for the results.

table 3

The analytical sensitivity and precision of the reagent with the parameter of the present invention are obviously better than those of the comparative parameter, but the cost does not increase.

To sum up, the present invention effectively overcomes various shortcomings in the prior art and has high industrial application value.

The above-mentioned embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical ideas disclosed in the present invention should still be covered by the claims of the present invention.

Claims (4)

1. A method for promoting the sensitivity of latex-enhanced immune turbidimetry, comprising the steps of: 1) Select latex particles with a large particle size to react with the antibody for antibody coating, the antibody is a D-dimer antibody, and the latex particles with a large particle size have a particle size of 0.2-1.0 μm; 2) After the coated latex is washed and sealed, it is dispersed in a buffer to prepare the reagent 2 of the latex-enhanced immune turbidimetric assay kit; in step 2, the buffer is a Tris-HCl buffer with a concentration of 50 mM , wherein the concentration of bovine serum albumin BSA is 5g/L, and the concentration of sodium azide is 1g/L; 3) A buffer solution for promoting the reaction of antigen and antibody is configured to prepare latex-enhanced immune turbidimetry kit reagent 1; in step 3, the buffer solution is Tris-HCl buffer solution with a concentration of 50 mM, wherein bovine serum albumin BSA The concentration of sodium azide is 3g/L, the concentration of sodium azide is 1g/L, the concentration of PEG6000 is 50g/L, stachyose 0.8-3g/L; alum 0.1-1g/L; sodium fructose diphosphate 0.8-3g/L ; Sodium hexametaphosphate 0.05-0.5g/L; 4) On the fully automatic biochemical analyzer, set the parameters, wherein the main wavelength is set to 300-500nm, the secondary wavelength is set to 500-800nm, and the reaction direction is the negative direction; 5) Run the biochemical analyzer, and after calibration with a sample of known concentration, detect the sample to be tested to obtain the absorbance change value, and calculate the content of the sample to be tested according to the calibration curve. 2. The method for improving the sensitivity of latex-enhanced immune turbidimetry as claimed in claim 1, characterized in that the method for improving the sensitivity of latex-enhanced immune turbidimetry is not for the purpose of diagnosis and treatment of diseases. 3. the method for promoting the sensitivity of latex-enhanced immune turbidimetry as claimed in claim 2 is characterized in that the direct purpose of the measurement of the content of the analyte is not to obtain diagnostic results or health conditions, but to obtain as The method of obtaining the information of the intermediate result, or simply processing or detecting the tissue, body fluid or excrement that has been separated from the human or animal body to obtain the information of the intermediate result. 4. Use of the method for improving the sensitivity of latex-enhanced immunoturbidimetry according to any one of claims 1-3 in the field of biological detection.