CN115453108A - Sealing stage treatment agent for solid phase carrier in immunodiagnostic reagent, application and product - Google Patents

Sealing stage treatment agent for solid phase carrier in immunodiagnostic reagent, application and product Download PDF

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CN115453108A
CN115453108A CN202211074307.7A CN202211074307A CN115453108A CN 115453108 A CN115453108 A CN 115453108A CN 202211074307 A CN202211074307 A CN 202211074307A CN 115453108 A CN115453108 A CN 115453108A
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virus
treatment agent
buffer
blocking
solid phase
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潘少丽
程珍珠
章朦
于秀玲
武云波
池朗山
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Guangdong Fapon Biotech Co Ltd
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Guangdong Fapon Biotech Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/186—Hepatitis C; Hepatitis NANB

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Communicable Diseases (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to the field of immunodiagnosis, and particularly provides a treatment agent for a solid phase carrier in an immunodiagnosis reagent at a sealing stage, application and a product. The treatment agent for the solid phase carrier in the immunodiagnostic reagent can reduce non-specific reaction in the immunodiagnostic process and obviously reduce the false positive detection rate in the immunodiagnostic detection after the solid phase carrier is sealed.

Description

Treatment agent for solid phase carrier in immunodiagnostic reagent at closed stage, application and product
Technical Field
The invention relates to the field of immunodiagnosis, in particular to a solid phase carrier sealing-stage treating agent for an immunodiagnostic reagent, application and a product.
Background
In immunodiagnostic analysis, a blocking reagent is usually used to block sites on a solid phase carrier which are not coated with a specific detection object, so as to reduce or eliminate a detectable signal caused by non-specific binding between the non-coated sites on the solid phase carrier and a substance containing a detectable label, thereby reducing a background value and a false positive rate and improving the detection specificity. In the blocking reagent, after being coated with a solid phase carrier, irrelevant proteins (also called inert proteins and blocking proteins) are usually utilized to block uncoated sites, and the most commonly used blocking agents are bovine serum albumin, calf serum, gelatin, casein, low fat/removed milk powder and the like. Blocking is essential for most immunoassays, especially for indirect assays, where the indirect methodology itself is highly non-specific.
In addition, in practical applications, although blocking proteins can block solid phase carriers, phenomena such as individual false positives still occur, and how to reduce the false positive rate is a problem to be solved in the prior art.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first object of the present invention is to provide a blocking-stage treating agent for solid phase carriers in an immunodiagnostic reagent.
The second object of the present invention is to provide the use of the blocking-stage treatment agent in immunoturbidimetry, ELISA, chemiluminescence immunoassay, and immunochromatography.
The third purpose of the invention is to provide an immunodiagnostic kit which has good accuracy and low false positive detection rate.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a blocking stage treatment agent for a solid phase carrier in an immunodiagnostic reagent comprising at least one anionic surfactant.
Further, the concentration of the anionic surfactant is 0.005 to 0.5w/v%, preferably 0.01 to 0.4w/v%, and more preferably 0.15 to 0.25w/v%;
optionally, the anionic surfactant is selected from sulfate type anionic surfactants, carboxylate type anionic surfactants;
optionally, the anionic surfactant is selected from fatty alcohol sulfates, N-acylamino carboxylates;
optionally, the anionic surfactant is selected from lauryl sulfate, lauroyl-N-methylaminoacetate, N-oleoyl polycarboxylic acid amino acid salt;
optionally, the anionic surfactant is selected from sodium lauroyl-N-methyl aminoacetate, sodium dodecyl sulfate.
Further, the closed-stage treatment agent further comprises a buffer agent;
optionally, the buffering agent is selected from histidine buffer, glycine buffer, borax buffer, disodium hydrogen phosphate-citric acid buffer, phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, barbital buffer, ammonia-ammonium chloride buffer, ADA buffer, PIPES buffer, MOPSO buffer, BES buffer, MOPS buffer, TES buffer, HEPES buffer, tris buffer, EPPS buffer, tricine buffer, bicine buffer, TAPS buffer, BIS-Tris buffer, HEPES buffer, MES buffer, or TEA buffer;
optionally, the concentration of the buffer reagent is 5-100mM, more preferably 5-50mM, still more preferably 10-25mM;
optionally, the working pH of the blocking stage treatment agent is 7-8.5, more preferably 7-8, still more preferably 7.2-7.6;
optionally, the seal stage treatment agent further comprises at least one metal salt;
alternatively, the metal salt is selected from sodium chloride, potassium chloride;
alternatively, the concentration of the metal salt is 100-500mM, preferably 150-300mM.
Further, the closed-stage treatment agent further comprises at least one sugar;
optionally, the sugar is selected from sucrose, dextran, mannitol, or pullulan;
alternatively, the concentration of the sugar is 1 to 10w/v%, preferably 2.5 to 8w/v%, and more preferably 5w/v%.
Further, the blocking stage treatment agent also comprises at least one blocking protein in a sufficient amount,
optionally, the blocking protein is selected from casein, BSA, gelatin, hydrolyzed gelatin, tryptone, fish gelatin, calf serum, low-fat milk powder, skimmed milk powder;
alternatively, blocking proteins are used at concentrations such as 0.05-5w/v% BSA, 5-30v/v% calf serum, 0.5-2w/v% fish gelatin, 0.1-3w/v% casein, 0.5-4w/v% tryptone.
Further, the sealing-stage treatment agent further comprises at least one preservative;
optionally, the preservative is selected from ProClin300, sodium azide;
alternatively, the concentration of the preservative is 0.1 to 3v/v%, preferably 0.1 to 0.5v/v%, and more preferably 0.1v/v%.
Further, the material of the solid phase carrier is, for example, plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon;
alternatively, the type of solid support is, for example, a microparticle, a bead, a test tube, a microtiter plate, a cuvette, a membrane, a scaffold molecule, a filter paper, a disc, or a chip;
alternatively, the solid support is for example a magnetic particle, a microtiter plate, a PVDF membrane.
The blocking stage treatment agent is applied to immunoturbidimetry, ELISA, chemiluminescence immunoassay or immunochromatography.
An immunodiagnostic kit comprising the blocking stage treatment agent.
Further, the immunodiagnostic includes detection of antibodies to infectious pathogens;
alternatively, the infectious pathogen comprises a virus, bacterium, fungus, chlamydia, mycoplasma or parasite;
alternatively, the infectious pathogen includes an aids virus, a hepatitis a virus, a hepatitis b virus, a hepatitis c virus, a hepatitis d virus, a hepatitis e virus, a hepatitis g virus, a rubella virus, a human cytomegalovirus, a herpes simplex virus type 1, a herpes simplex virus type 2, a rabies virus, a human T-lymphocyte leukemia virus, a dengue virus, a human papilloma virus, a west nile virus, a forest encephalitis virus, a measles virus, an influenza virus, a parainfluenza virus, a varicella virus, an echovirus, a coxsackie virus, a japanese encephalitis virus, a coxsackie virus, an EB virus, a mumps virus, a treponema pallidum, a borrelia trachoma, chlamydia pneumoniae, a psittaci chlamydia, a ureaplasma urealyticum, a mycoplasma pneumoniae, a mycobacterium tuberculosis, a helicobacter pylori, a gonococcus, a plasmodium, a trypanosoma cruzi, a toxoplasma gondii.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a blocking stage treating agent for a solid phase carrier in an immunodiagnostic reagent, which comprises at least one anionic surfactant. The inventor unexpectedly finds that the false positive rate in immunoassay can be effectively reduced by the existence of the anionic surfactant in the solid phase carrier sealing process.
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 carried out according to 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 blocking stage treatment agent for a solid phase carrier in an immunodiagnostic reagent comprising at least one anionic surfactant.
The inventor finds that the false positive rate in immunoassay can be effectively reduced by the existence of the anionic surfactant in the solid phase carrier blocking process. According to research and analysis, some false positives in immunoassay are related to blocking protein, the blocking protein can be positively charged in a pH environment close to neutral, an anionic surfactant with negative charges is added to neutralize the positive charges of the blocking protein, and nonspecific binding caused by charge action is blocked under the interaction, so that the generation of false positives in the detection process is reduced.
The anionic surfactant is a partially negatively charged surfactant that functions as a surface active agent after ionized in water. The anionic surfactants mainly comprise sulfate (R-OSO) type (R-OSO) according to the hydrophilic group 3 - M + ) Sulfonate type (R-SO) 3 - M + ) Phosphate ester (salt) type (R-OPO) 3 - M + ) Of the carboxylate type (R-COO) - M + ) R is a hydrocarbon group, M + Is a cation, typically in the form of sodium, potassium, lithium, ammonium, calcium, triethanolamine, etc. Exemplary sulfate ester type anionic surfactants include fatty alcohol sulfates, sulfates of unsaturated alcohols such as fatty alcohol polyoxyethylene ether sulfates and the like. Exemplary sulfonate anionic surfactants include alkyl sulfonates such as sodium dodecyl sulfate, alkyl benzene sulfonates such as sodium linear and branched alkyl benzene sulfonates, alkenyl sulfonates, succinate sulfonates and the like. Exemplary phosphate ester-type anionic surfactants include alkyl phosphate monoesters, alkyl phosphate diesters, fatty alcohol-polyoxyethylene ether phosphate monoesters, fatty alcohol-polyoxyethylene ether phosphate diesters, alkylphenol polyoxyethylene ether monoesters, alkylphenol polyoxyethylene ether diesters, and the like. Exemplary carboxylate type anionic surfactants include higher fatty acid salts such as soap, N-acylamino carboxylates, alkyl ether carboxylates such as fatty alcohol polyoxyethylene ether carboxylates, and stearoyl lactylates such as stearoyl lactylate.
In alternative embodiments, the anionic surfactant is a fatty alcohol sulfate such as lauryl sulfate (i.e., lauryl sulfate); n-acyl amino carboxylates (reaction products of fatty acid chlorides with amino acids) such as lauroyl sarcosinate (i.e. lauroyl-N-methylaminoacetate), N-oleoyl polycarboxylic acid amino salts.
In alternative embodiments, salt forms include cations such as sodium, potassium, lithium, ammonium, calcium, triethanolamine, and the like.
In an alternative embodiment, the concentration of anionic surfactant is 0.005-0.5w/v%, preferably 0.01-0.4w/v%, and more preferably 0.15-0.25w/v%.
In the present invention, "w/v%" means that a certain substance is contained in a mass g per 100mL of the blocking stage treating agent, and for example, the concentration of the anionic surfactant is 0.005 to 0.5w/v%, which means that the content of the anionic surfactant is 0.005 to 0.5g per 100mL of the blocking stage treating agent.
In the present invention, "v/v%" means that a certain substance is contained in a volume mL per 100mL of the blocking stage treating agent.
In some embodiments, the anionic surfactant comprises sodium lauroyl-N-methylaminoacetate and/or sodium lauryl sulfate, wherein the anionic surfactant is sodium lauroyl-N-methylaminoacetate, or, when both are sodium lauroyl-N-methylaminoacetate and sodium lauryl sulfate, the concentration of sodium lauroyl-N-methylaminoacetate is preferably 0.01 to 0.4w/v%, for example 0.02w/v%; when the anionic surfactant is sodium lauryl sulfate, or sodium lauroyl-N-methylaminoacetate and sodium lauryl sulfate, the concentration of sodium lauryl sulfate is preferably 0.01 to 0.4w/v%, for example, 0.02w/v%. Each of the concentrations of sodium lauroyl-N-methylaminoacetate and sodium lauryl sulfate may independently be, but is not limited to, 0.01w/v%, 0.02w/v%, 0.05w/v%, 0.1w/v%, 0.17w/v%, 0.2w/v%, 0.25w/v%, 0.27w/v%, 0.3w/v%, 0.32w/v%, 0.35w/v%, 0.38w/v%, or 0.4w/v%.
In alternative embodiments, the blocking stage treatment agent further comprises a buffering agent that maintains the blocking stage treatment agent within a desired pH buffering range, and there are a variety of buffer pairs available for some buffers, for example, borax buffers including borax-boric acid buffers, borax-calcium chloride buffers; for example, the phosphate buffer is primarily a hydrogen phosphate-dihydrogen phosphate buffer pair. Examples of the buffer agent include, but are not limited to, histidine buffer, glycine buffer, borax buffer, disodium hydrogen phosphate-citric acid buffer, phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, barbital buffer, ammonia-ammonium chloride buffer, ADA buffer, PIPES buffer, MOPSO buffer, BES buffer, MOPS buffer, TES buffer, HEPES buffer, tris buffer, EPPS buffer, tricine buffer, bicine buffer, TAPS buffer, BIS-Tris buffer, HEPES buffer, MES buffer, and TEA buffer.
In alternative embodiments, the buffer should be present in an amount sufficient to achieve its intended function, i.e. to maintain the desired pH, and the concentration of the buffering agent is, for example, 5-100mM, more preferably 5-50mM, still more preferably 10-25mM.
In alternative embodiments, the working pH of the blocking stage treatment agent is from 7 to 8.5, further from 7 to 8, still further preferably from 7.2 to 7.6, and may be, but is not limited to, 7, 7.2, 7.3, 7.4, 7.6, 8, 8.3, or 8.5.
In an alternative embodiment, the blocking stage treatment agent further comprises at least one metal salt, in an alternative embodiment, the metal salt is selected from sodium chloride, potassium chloride, but is not limited thereto.
In an alternative embodiment, the concentration of the metal salt is, for example, 100 to 500mM, preferably 150 to 300mM. The concentration of the metal salt may be, but is not limited to, 100mM, 200mM, 300mM, 400mM, or 500mM.
In an alternative embodiment, the blocking stage treatment agent further comprises at least one sugar that acts as a viscosity modifier, primarily to protect the protein adsorbed by the solid support, acting as a protectant. In alternative embodiments, the sugar is selected from sucrose, dextran, mannitol, or pullulan, but is not limited thereto.
In alternative embodiments, the concentration of the sugar is preferably 1-10w/v%, more preferably 5w/v%, and the concentration of the sugar may be, but is not limited to, 1w/v%, 2w/v%, 3w/v%, 4w/v%, 5w/v%, 6w/v%, 7w/v%, 8w/v%, 9w/v%, or 10w/v%.
In an alternative embodiment, the blocking stage treatment agent further comprises at least one blocking protein in a sufficient amount, by "sufficient amount" of blocking protein is meant that the binding sites on the solid support are capable of being completely blocked. In an alternative embodiment, the blocking protein is selected from the group consisting of casein, BSA, gelatin, hydrolyzed gelatin, tryptone, fish gelatin, calf serum, low fat milk powder, skimmed milk powder, but is not limited thereto.
In alternative embodiments, blocking proteins are used at concentrations such as 0.05-5w/v% BSA, 5-30v/v% calf serum, 0.5-2w/v% fish gelatin, 0.1-3w/v% casein or 0.5-4w/v% tryptone.
In alternative embodiments, reducing agents may be selectively added to open disulfide bonds of antigens to fully expose epitopes according to specific diagnostic procedures, and the reducing agents are commonly used in the art and are not particularly limited, for example, β -mercaptoethanol (BME), dithiothreitol (DTT), tris- (2-formylethyl) phosphine hydrochloride (TCEP-HCl), or the like.
In an alternative embodiment, the blocking stage treatment agent further comprises at least one preservative which can inhibit microbial growth in the blocking stage treatment agent, which can be, but is not limited to ProClin300, sodium azide. In an alternative embodiment, the concentration of the preservative is 0.1-3v/v%, and may be, but is not limited to, 0.1v/v%, 0.3v/v%, 0.5v/v%, 0.7v/v%, 0.8v/v%, or 1v/v%, preferably 0.1-0.5v/v%, and more preferably 0.1v/v%.
In alternative embodiments, the solid support is made of a material such as plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon; in alternative embodiments, the type of solid support is, for example, a microparticle, bead, test tube, microtiter plate, cuvette, membrane, scaffold molecule, filter paper, disk, or chip; in some embodiments, the solid support is, for example, a magnetic microparticle, a microtiter plate, a PVDF membrane.
Since the blocking-stage treatment agent provided by the present invention can solve the problem of false positive caused by non-specific binding reaction caused by blocking protein in the existing blocking treatment, the blocking-stage treatment agent provided by the present invention can be applied to solid phase carriers used in detection means such as indirect agglutination, ELISA or chemiluminescence, for example, latex, magnetic beads, polystyrene (ELISA plate, cuvette, etc.), and the like, because the blocking-stage treatment agent provided by the present invention relates to the operation of blocking solid phase carriers in immunodiagnosis.
The blocking stage treatment agent is applied to immunoturbidimetry, ELISA, chemiluminescence immunoassay or immunochromatography ELISA.
An immunodiagnostic kit comprising a closed-phase treatment agent as provided by the invention. The accuracy of the immunodiagnosis reagent in the kit is obviously improved, and the false positive detection rate is reduced.
In alternative embodiments, immunodiagnostics include detection of antibodies to infectious pathogens;
in alternative embodiments, the infectious pathogen comprises a virus, bacterium, fungus, chlamydia, mycoplasma or parasite;
in alternative embodiments, the infectious pathogen comprises hiv, hepatitis a virus, hepatitis b virus, hepatitis c virus, hepatitis d virus, hepatitis e virus, hepatitis g virus, rubella virus, human cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2, rabies virus, human T-lymphoblastic leukemia virus, dengue virus, human papilloma virus, west nile virus, forest encephalitis virus, measles virus, influenza virus, parainfluenza virus, varicella virus, echoviruses, coxsackie virus, encephalitis b virus, coxsackie virus, EB virus, mumps virus, treponema pallidum, borrelia trachomatis, chlamydia pneumoniae, chlamydia psittaci, ureaplasma urealyticum, mycoplasma pneumoniae, mycobacterium tuberculosis, helicobacter pylori, gonococcus, plasmodium, trypanosoma cruzi, toxoplasma gondii.
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.
Preparation of a blocking stage treating agent base liquid 1: disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (10mM), naCl (150mM), tryptone (10 g/L), sucrose (50 g/L) and Procline (300 ml/1000ml) were added, and the pH was adjusted to 7.4.
Preparation of the blocking stage treating agent base liquid 2: tris-HCl buffer solution 25mM, KCl 200mM, BSA 10g/L, trehalose 80g/L and Procline300 ml/1000ml, after mixing, the volume is determined to be 1L, the mixture is subpackaged for 4 degrees for storage, and the pH is adjusted to 7.2.
Preparation of the blocking stage treatment agent base liquid 3: boric acid-sodium borate buffer 50mM, naCl 300mM, casein 5g/L, dextran 25g/L, procline300 ml/1000ml, pH adjusted to 7.6.
Example 1
Sodium lauroyl-N-methylaminoacetate was added to the base solution 1 at a concentration of 0.02w/v%.
Example 2
Sodium lauryl sulfate was added to the base solution 1 at a concentration of 0.02w/v%.
Example 3
Sodium lauryl sulfate and sodium lauroyl-N-methylaminoacetate were added to the base solution 1 at a concentration of 0.02w/v%.
Example 4
Sodium lauroyl-N-methylaminoacetate was added to the base solution 2 at a concentration of 0.4w/v%.
Example 5
Sodium lauroyl-N-methylaminoacetate was added to the base solution 3 at a concentration of 0.01w/v%.
Example 6
Sodium lauryl sulfate was added to the base liquid 2 at a concentration of 0.4w/v%.
Example 7
Sodium lauryl sulfate was added to the base solution 3 at a concentration of 0.01w/v%.
Comparative example 1
On the basis of the base liquid 1, a nonionic surfactant TWEEN-20 was added at a concentration of 0.02w/v%.
Comparative example 2
On the basis of the base solution 1, a non-ionic surfactant Tritonx-100 with the concentration of 0.02w/v% is added.
Comparative example 3
On the basis of the base liquid 1, a nonionic surfactant BRIJ-35 was added at a concentration of 0.02w/v%.
Comparative example 4
Base liquid 1, no surfactant was added.
Comparative example 5
Base liquid 2, without any surfactant added.
Comparative example 6
Base liquid 3, without any added surfactant.
Test examples
Take the HCV enzyme immunoassay project as an example
1. CBS with pH 9.6 of 50mM CB and 150mM NaCl is used as a coating solution, and SDS (sodium dodecyl sulfate) with the weight ratio of 1; adding HCV antigen into the coating solution according to a certain proportion, uniformly mixing, and directly coating at 4 ℃ for 20-24 hours in a manner of coating 100 mu l per hole;
2. and (3) sealing: taking out the coated plate, standing at room temperature, washing the plate with washing solution for 2 times, and sealing after drying; add 200. Mu.l of blocking stage treatment per well for 2 hours at 37 ℃ (or overnight at 4 ℃). Spin-drying in a drying room with humidity less than 30% or an electronic drying oven for 24 hours for later use;
3. preparing an enzyme-labeled secondary antibody working solution: diluting the antihuman IgG-HRP with an enzyme diluent according to the recommended titer, and uniformly mixing for later use;
4. reaction mode and reaction time: reacting 100 mu l of sample diluent and 10 mu l of sample to be detected in a 37 ℃ incubator for 60min, washing the plate for 5 times, beating to dry, adding 100 mu l of enzyme-labeled secondary antibody working solution, reacting at 37 ℃ for 30min, washing the plate for 5 times, adding 50 mu l of each of color developing agents A and B, developing for 30min, adding 50 mu l of stop solution, and reading within 10min by using a dual-wavelength reading value of 450nm-630 nm.
The sample diluent components contained 10mM PB, pH7.4, 10% NBS,0.75% Casein-2Na, 500mM NaCl,0.2% TritonX-100 and 0.1% Procline300.
The enzyme diluent component contained 10mM PB, pH7.4, 150mM NaCl,30% NBS,0.2% Casein-2Na, 5% Tween-20 and 0.05% thimerosal.
In the above test, the blocking-stage treatment agents in examples 1 to 3 and comparative examples 1 to 4 were used to perform the test on 10 positive samples, 5 negative samples and 1358 clinical samples, and the test results of the positive samples and the suspected false positive samples were verified by the sandwich method and the RIBA method, and the results are shown in table 1 below:
TABLE 1
Figure BDA0003830953710000121
From the results in the table, it can be seen that, by comparison, the anionic surfactant sodium lauroyl-N-methylaminoacetate has the best effect and better specificity than other conditions when used as a blocking stage treatment agent, and the effect of the anionic surfactant sodium dodecyl sulfate is also better than the effects of the other three nonionic surfactants, so that the anionic surfactant can further eliminate false positives in the blocking stage treatment agent, and the detection result has higher specificity. The false positive rate of the base liquid in the treatment agent in the sealing stage is close to that of the treatment agent in the sealing stage by adding the nonionic surfactant, which indicates that more false positive specimens cannot be eliminated by adding the nonionic surfactant.
False positive samples fed back on the market are collected and detected by using examples 1 and 2, examples 4-7 and comparative examples 4-6 respectively, and the positive detection rate is checked, and the results are shown in the following table 2:
TABLE 2
Figure BDA0003830953710000122
Figure BDA0003830953710000131
By contrast, anionic surfactants can further eliminate false positives in the blocking stage treatment.
The sealing stage treatment agents in example 1 and comparative example 1 were used to prepare sealing plates, to prepare kits, and the results of the comparison of the national standard disks are shown in the following table 3:
TABLE 3
Figure BDA0003830953710000132
Figure BDA0003830953710000141
Note: 0.5NCU/ml is a kantchestan standard product; "CV" is the result of three replicate wells of the same serum; "L1-L4" is the result of a gradient dilution of serum in a serum pan.
By way of comparison: the kit prepared by adding the sodium lauroyl-N-methylamino acetate into the closed-stage treating agent has better effect than the kit prepared by adding the TWEEN-20 into the closed-stage treating agent, and is characterized in that the sensitivity and the specificity of the sodium lauroyl-N-methylamino acetate represented by an anionic surfactant are better than those of the TWEEN-20.
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 (10)

1. Use of lauroyl-N-methylaminoacetate in the preparation of a treatment agent for the blocking phase of a solid support in an immunodiagnostic reagent comprising the detection of antibodies against infectious pathogens.
2. Use according to claim 1, wherein the solid support material is plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon.
3. Use according to claim 2, wherein the solid support material is a plastic.
4. Use according to claim 1, wherein the type of solid support is a microparticle, bead, test tube, microtiter plate, cuvette, membrane, scaffold molecule, filter paper, disc or chip.
5. Use according to claim 1 or claim 4, wherein the solid support is of the type of a microtiter plate.
6. Use according to claim 1, wherein the infectious pathogen comprises a virus, a bacterium, a fungus, a chlamydia, a mycoplasma or a parasite.
7. The use of claim 1 or 6, wherein the infectious agent comprises hepatitis C virus.
8. Use according to claim 1, wherein the lauroyl-N-methylaminoacetate is sodium lauroyl-N-methylaminoacetate.
9. An immunodiagnostic kit comprising the closed-phase treatment agent of any one of claims 1 to 8.
10. The kit of claim 9, wherein the kit is for detecting antibodies to hepatitis c virus.
CN202211074307.7A 2019-12-11 2019-12-11 Sealing stage treatment agent for solid phase carrier in immunodiagnostic reagent, application and product Pending CN115453108A (en)

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