CN113125715A

CN113125715A - Human immunodeficiency virus antibody detection kit and application thereof

Info

Publication number: CN113125715A
Application number: CN202011642213.6A
Authority: CN
Inventors: 洪琳; 章春奇; 李建武; 王平平; 李临
Original assignee: Beyond Diagnostics Shanghai Co ltd; Chemclin Diagnostics Corp
Current assignee: Beyond Diagnostics Shanghai Co ltd; Chemclin Diagnostics Corp
Priority date: 2019-12-31
Filing date: 2020-12-31
Publication date: 2021-07-16
Anticipated expiration: 2040-12-31
Also published as: CN113125715B; CN116879550A

Abstract

The invention discloses a human immunodeficiency virus antibody detection kit, which comprises a reagent R1, wherein the reagent R1 comprises a first buffer solution and receptor particles suspended in the first buffer solution and capable of reacting with active oxygen to generate a chemiluminescent signal, and is characterized in that: the acceptor particle comprises a carrier, the interior of the carrier is filled with a luminescent composition, and the surface of the carrier is bonded with HIV antigen; the variation coefficient C.V value of the particle size distribution of the acceptor particles in the acceptor reagent is not less than 5% and not more than 20%; the sugar content per milligram mass of the acceptor particles is not higher than 25 μ g. The variation coefficient C.V value of the particle size distribution of the acceptor particles of the R1 reagent in the kit is not less than 5% and not more than 20%, and the sugar content in each milligram of the acceptor particles is not more than 25 mu g; furthermore, the R1 reagent can meet the commercial requirement of mass production, and has ultrahigh sensitivity and wide detection range.

Description

Human immunodeficiency virus antibody detection kit and application thereof

Technical Field

The technical scheme relates to the field of chemiluminescence detection, in particular to a human immunodeficiency virus antibody detection kit and application thereof.

Background

Immunoassays have evolved in many varieties over half a century. Depending on whether the substances to be tested are to be separated from the reaction system during the assay, heterogeneous (Heterogenous) immunoassays and Homogeneous (Homogeneous) immunoassays can be used. Heterogeneous immunoassay refers to the operation process of introducing a probe for labeling, wherein various related reagents are required to be separated after mixed reaction, and an object to be detected is separated from a reaction system and then detected, and is the mainstream method in the existing immunoassay. Such as enzyme-linked immunosorbent assay (ELISA method) and magnetic particle chemiluminescence method. Homogeneous immunoassay refers to direct measurement after mixing and reacting an analyte with a relevant reagent in a reaction system in the measurement process, and no redundant separation or cleaning step is needed. Up to now, various sensitive detection methods are applied to homogeneous immunoassays, such as optical detection methods, electrochemical detection methods, and the like.

Acquired immunodeficiency syndrome (AIDS) is a serious infectious disease which causes the death of infected persons by destroying the immune function of human bodies after HIV virus, namely human immunodeficiency virus (HIV for short) invades the human bodies, so that the human bodies generate various incurable infections and tumors. After HIV infection, the immune system is mainly damaged, T4 lymphocytes are damaged, the body resistance is reduced, severe infection and a few cancers are induced, and patients are susceptible to various rare diseases and die due to long-term consumption and general exhaustion. Therefore, the most important link in the prevention and treatment of acquired immunodeficiency syndrome is HIV detection.

Currently, the detection of HIV is as follows:

A. enzyme-linked immunosorbent assay (ELISA)

There are more than 8 kinds of ELISA methods currently used. Their specificity and sensitivity were over 99%.

B. Particle agglutination method (PA)

PA is a rapid and simple screening method. If the plants are positive, they should be confirmed by WB. PA does not need any special instrument, and the result can be distinguished by naked eyes. The whole process only needs 5 minutes. The disadvantages are false positives and high price.

C. Rapid reagent

a) Human Immunodeficiency Virus (HIV)1+2 type antibody diagnostic reagent (colloid selenium method)

The kit is only used for on-site primary screening and clinical emergency of a gratuitous blood donor, and a person who is detected to be positive needs to be further screened and confirmed.

b)InstantCHEK^TMHIVL +2 gold-labeled rapid diagnostic reagent

InstantCHEK^TMHIV1+2 is a rapid, simple and sensitive test for the detection of antibodies to the HIV viruses HIV-1 and HIV-2. The method is suitable for primary screening, and if the reagent is determined to be positive, another method such as ELISA or western blotting is required for detection.

HIV-antibody confirmation experiment

Immunoblot assay (WB), strip immunoassay (LIATEKHIV III), radioimmunoprecipitation assay (RIPA) and immunofluorescence assay (IFA). The confirmation test method commonly used in China is WB.

a) Immunoblotting experiment (western blot, WB)

The test method widely used for diagnosis of many infectious diseases is the first test method for confirming HIV antibodies in terms of HIV etiology diagnosis, and WB test results are often used as a "gold standard" for identifying the merits of other test methods.

The sensitivity of WB is generally not lower than that of the primary screening experiment, but the specificity is high, which is mainly based on the separation, concentration and purification of different antigen components of HIV, and can detect antibodies aiming at different antigen components, so that the accuracy of the primary screening experiment can be identified by a WB method. From the WB confirmation test results, although the primary screening test selects a reagent with better quality, such as the third-generation ELISA, false positives still occur, and accurate results can be obtained only by the confirmation test.

b) Immunofluorescence assay (IFA)

The IFA method is economical, simple and rapid, and has been recommended by the FDA for diagnosis of WB indeterminate samples. However, expensive fluorescence microscopes are required, well-trained technicians are required, observation and interpretation results are easily affected by subjective factors, the results are not suitable for long-term storage, and the IFA is not suitable for development and application in general laboratories.

The above methods have some difficulties which are difficult to overcome, and the chemiluminescence method has the advantages of high sensitivity, high specificity, wide linearity, rapidness, few influencing factors, accurate result and the like, and is the most widely applied disease detection method in recent years. The kit is gradually applied to HIV detection at present, is helpful for realizing early discovery, early diagnosis and early treatment of infection, and reduces false positive and missed detection rate.

The basic principle of light-activated chemiluminescence is a homogeneous immune response. It is based on two kinds of antigens or antibodies coated on the surfaces of particles, and immune complexes are formed in a liquid phase to draw the two kinds of particles close. Under the excitation of laser, the ionic oxygen transfer between particles occurs, and then high-level red light is generated, and the number of photons is converted into the concentration of target molecules through a single photon counter and mathematical fitting. When the sample does not contain the target molecules, immune complexes cannot be formed between the two particles, the distance between the two particles exceeds the ionic oxygen transmission range, the ionic oxygen is rapidly quenched in a liquid phase, and no high-energy level red light is generated during detection. The detection of HIV by light-activated chemiluminescence methods has the following problems which are difficult to solve at present: (1) the preparation process is complicated, and particularly, the preparation and modification processes of the microspheres are too complicated; (2) the false positive rate is high.

Therefore, there is a need to develop an HIV detection kit that can be mass-produced, has low cost, qualified quality, and stable performance, and can meet both the sensitivity requirement and the linear range requirement.

Disclosure of Invention

The invention aims to solve the technical problem of providing a human immunodeficiency virus antibody detection kit aiming at the defects of the prior art. When the kit is applied to homogeneous chemiluminescence analysis detection, the inventor of the application unexpectedly finds that the kit has ultrahigh sensitivity and wide detection range.

Based on the above, the present invention provides a human immunodeficiency virus antibody detection kit, which comprises a reagent R1, wherein the reagent R1 comprises a first buffer solution and receptor particles suspended therein, the receptor particles being capable of generating a chemiluminescent signal by the action of active oxygen, the receptor particles comprise a carrier, the interior of the carrier is filled with a luminescent composition, and the surface of the carrier is bonded with an HIV antigen; the variation coefficient C.V value of the particle size distribution of the acceptor particles in the acceptor reagent is not less than 5% and not more than 20%; the sugar content per milligram mass of the acceptor particles is not higher than 25 μ g.

The acceptor particles have a coefficient of variation of particle size distribution C.V value of no greater than 15% in the reagent R1.

The variation coefficient C.V value of the particle size distribution of the acceptor particles in the reagent R1 is not less than 8%.

The sugar content per milligram mass of the acceptor particles is not higher than 20 [ mu ] g.

The content of polysaccharide in the first buffer solution is 0.01-1 wt%, preferably 0.05-0.5 wt%.

The surface of the carrier is not coated with sugar molecules and is directly bonded with HIV antigens.

The surface of the carrier is provided with a bonding functional group for directly chemically bonding the HIV antigen on the surface of the carrier, wherein the bonding functional group is selected from at least one of amine group, amide group, hydroxyl group, aldehyde group, carboxyl group, epoxy group, maleimide group and sulfhydryl group; preferably selected from aldehyde groups, carboxyl groups, epoxy groups and maleimide groups.

The polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose, most preferably dextran or dextran derivatives. The sugar content can be detected by the anthrone method.

The kit also comprises a reagent R2, wherein the reagent R2 comprises a biotin-labeled HIV antigen.

The kit also comprises an Anti-HIV sample diluent.

The kit also includes an Anti-HIV negative control.

The kit also includes an Anti-HIV-1 positive control containing calf serum and HIV-1 positive serum.

The kit also includes an Anti-HIV-2 positive control containing calf serum and HIV-2 polyclonal antibodies.

The kit also includes an Anti-HIV weakly positive control containing calf serum and HIV-1 positive serum.

The invention has the beneficial effects that: the variation coefficient C.V value of the particle size distribution of the acceptor particles of the R1 reagent in the kit is not less than 5% and not more than 20%, and the sugar content in each milligram of the acceptor particles is not more than 25 mu g; furthermore, the R1 reagent can meet the commercial requirement of mass production, and has ultrahigh sensitivity and wide detection range. Meanwhile, the invention controls the sugar content in the receptor particles, reduces the influence of sugar on detection signals, improves the detection precision and reduces the production cost.

Drawings

FIG. 1 is a Gaussian distribution plot of the acceptor particles prepared in example 2.

FIG. 2 is a graph of a standard sugar content measurement.

Detailed Description

In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The practice of the invention is not limited to the following examples, and any variations and/or modifications made thereto are intended to fall within the scope of the invention.

Where a range of values is provided, it is understood that each intervening value, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Term (I)

The term "active oxygen" as used herein refers to a general term for a substance which is composed of oxygen, contains oxygen, and is active in nature, and is mainly an excited oxygen molecule, including superoxide anion (O) which is an electron reduction product of oxygen₂(-) and the two-electron reduction product hydrogen peroxide (H)₂O₂) The three-electron reduction product hydroxyl radical (. OH) and nitric oxide and singlet oxygen (1O)₂) And the like.

The term "acceptor particle" as used herein refers to a particle that contains a compound that reacts with reactive oxygen species to produce a detectable signal. The donor particles are induced by energy or an active compound to activate and release reactive oxygen species in a high energy state that are captured by the acceptor particles in close proximity, thereby transferring energy to activate the acceptor particles. In some embodiments of the present invention, the acceptor particle comprises a luminescent composition and a carrier, wherein the luminescent composition is filled in the carrier and/or coated on the surface of the carrier. The "carrier" according to the present invention is selected from the group consisting of tapes, sheets, rods, tubes, wells, microtiter plates, beads, particles and microspheres, which may be microspheres or microparticles known to those skilled in the art, which may be of any size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which may have any density, but preferably has a density close to that of water, preferably is capable of floating in water, and which are composed of a transparent, partially transparent or opaque material. The carrier may or may not have a charge, and when charged, is preferably a negative charge. The carrier may be a latex particle or other particle containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes.

In the present invention, the "luminescent composition", i.e. a compound referred to as a label, may undergo a chemical reaction in order to cause luminescence, for example by being converted into another compound formed in an electronically excited state. The excited state may be a singlet state or a triplet excited state. The excited state may relax to the ground state to emit light directly, or may return to the ground state itself by transferring excitation energy to an emission energy acceptor. In this process, the energy acceptor particle will be transitioned to an excited state to emit light.

The "variation coefficient C.V value of particle size distribution" described in the present invention refers to the variation coefficient of particle size in Gaussian distribution in the detection result of the nanometer particle size analyzer. The coefficient of variation is calculated as: C.V value (standard deviation SD/Mean) x 100%. The Standard Deviation (SD), also known as the Standard Deviation, describes the mean of the distances of the data from the mean (mean Deviation), which is the square of the Deviation and the root of the mean, expressed as a. The standard deviation is the arithmetic square root of the variance. The standard deviation reflects the degree of dispersion of a data set, and the smaller the standard deviation, the less the values deviate from the mean, and vice versa. The standard deviation σ is a distance between an inflection point (0.607 times the peak height) on the normal distribution curve and a vertical line between the peak height and the time axis, that is, a distance between two inflection points on the normal distribution curve is half. The peak width at half height (Wh/2) is the width of the peak at half the peak height, Wh/2 ═ 2.355 σ. The tangent is drawn by the inflection points on both sides of the normal distribution curve, the intercept at the base line is called the peak width or base line width, and W is 4 sigma or 1.699 Wh/2.

The term "test sample" as used herein refers to a mixture to be tested that contains or is suspected of containing a target molecule to be tested. The test sample that can be used in the present invention includes body fluids such as blood (which may be anticoagulated blood commonly seen in collected blood samples), plasma, serum, urine, semen, saliva, cell cultures, tissue extracts, and the like. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant material, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The sample to be tested can be diluted with a diluent as required before use. For example, to avoid the HOOK effect, the sample to be tested may be diluted with a diluent before the on-line detection and then detected on the detection instrument.

The term "target molecule to be detected" as used herein refers to a substance in a sample to be detected during detection. One or more substances having a specific binding affinity for the target molecule to be detected will be used for the detection of the target molecule. The target molecule to be detected may be a protein, a peptide, an antibody or a hapten which allows it to bind to an antibody. The target molecule to be detected may be a nucleic acid or oligonucleotide that binds to a complementary nucleic acid or oligonucleotide. The target molecule to be detected may be any other substance that can form a member of a specific binding pair. Other examples of typical target molecules to be detected include: drugs such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterial agents, antifungal agents, antiviral agents, purines, antitumor agents, amphetamines, heteroazoids, nucleic acids, and prostaglandins, and metabolites of any of these drugs; pesticides and metabolites thereof; and a receptor. Analytes also include cells, viruses, bacteria, and fungi.

The term "antibody" as used herein is used in the broadest sense and includes antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any case desired, the antibody may be further conjugated to other moieties, such as a member of a specific binding pair member, e.g., biotin or avidin (a member of a biotin-avidin specific binding pair member), and the like.

The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and that binds to the immune response product antibodies and sensitized lymphocytes in vitro and in vivo to produce an immune effect.

The term "binding" as used herein refers to direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to interactions such as salt and water bridges.

The term "specific binding" as used herein refers to the mutual discrimination and selective binding reaction between two substances, and is the conformation correspondence between the corresponding reactants in terms of the three-dimensional structure. Under the technical idea disclosed by the invention, the detection method of the specific binding reaction comprises but is not limited to the following steps: double antibody sandwich, competition, neutralization competition, indirect or capture.

Detailed description of the preferred embodiments

The present invention will be described in more detail with reference to examples.

The prior common sense is as follows: the more uniform the particle size of the microspheres, the better the performance of homogeneous chemiluminescent detection using the microspheres. Current research on microspheres employed in homogeneous chemiluminescence therefore tends to result in microspheres of more uniform particle size. After research, the inventor of the application finds that when the microspheres with uniform particle size are used for homogeneous chemiluminescence detection, the sensitivity and the detection range of the detection result are difficult to guarantee at the same time. However, by adopting the microspheres with proper particle size uniformity (for example, the variation coefficient of the particle size distribution of the microspheres is more than 5%), the sensitivity of the light-activated chemiluminescence detection can be ensured, and the detection range can be widened.

The inventor of the present invention controls the particle size distribution and sugar content of the receptor particles in the receptor reagent, i.e., R1 reagent, and further controls the amount of reporter molecules (e.g., antibody/antigen) on the surface of each receptor particle (small-particle size microspheres have a large specific surface area, large-particle size microspheres have a large amount of reporter molecules on the surface of unit mass microspheres, small-particle size microspheres have a small specific surface area, and small-unit mass microspheres have a small amount of reporter molecules on the surface of unit mass microspheres). The larger the variation coefficient of the particle size distribution of the receptor particles in the receptor reagent is, the higher the nonuniformity is, which means that the receptor particles with different sizes exist in a system, so that the high sensitivity and the wide detection range are realized.

In one aspect, the present invention provides a human immunodeficiency virus antibody detection kit, which includes a reagent R1, wherein the reagent R1 includes a first buffer solution and receptor particles suspended therein, which are capable of generating a chemiluminescent signal by the action of active oxygen, and the surface of the receptor particles is bound with HIV antigen, and is characterized in that: the variation coefficient C.V value of the particle size distribution of the acceptor particles in the acceptor reagent is not less than 5% and not more than 20%; the sugar content per milligram mass of the acceptor particles is not higher than 25 μ g.

The acceptor particle comprises a carrier, the interior of the carrier is filled with a luminescent composition, and the surface of the carrier is bonded with HIV antigen.

In some embodiments, the carrier surface is coated with polysaccharide molecules, and the HIV antigen is indirectly bound to the surface of the receptor particle by chemical bonding to the polysaccharide molecules.

The acceptor particle may have a coefficient of variation of particle size distribution C.V value of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% in the reagent R1.

Preferably, the acceptor particles have a coefficient of variation of particle size distribution C.V value in the reagent R1 of not more than 15%.

Preferably, the acceptor particles have a particle size distribution coefficient of variation C.V value of not less than 8% in the reagent R1.

Preferably the sugar content per milligram mass of the acceptor particles is not higher than 20 μ g; more preferably not higher than 10. mu.g.

Preferably, the content of the polysaccharide in the first buffer solution is 0.01-1 wt%, preferably 0.05-0.5 wt%.

In a preferred embodiment, the surface of the carrier is free of coated sugar molecules and is directly bound to the HIV antigen.

In the above examples, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose, most preferably dextran or dextran derivatives.

In the above examples, the sugar content was determined by the anthrone method.

The kit also comprises an Anti-HIV sample diluent.

The kit also includes an Anti-HIV negative control.

The kit comprises a plurality of reagent strips, wherein each reagent strip is provided with a plurality of reagent hole grooves for containing reagents, and at least one reagent hole groove is used for containing the reagent R1.

The invention also provides an application of the receptor reagent or the kit in a chemiluminescence analyzer.

The invention also provides an application of the receptor reagent or the kit in POCT detection. POCT refers to on-site rapid testing or clinical testing performed in the vicinity of the patient.

Example III

Example 1 preparation of acceptor particles a

1.1 Synthesis and characterization of the support

1) A100 mL three-necked flask was prepared, 40mmol of styrene, 3mmol of methacrylic acid and 10mL of water were added thereto, the mixture was stirred for 10min, and N was introduced thereinto₂30min；

2) 0.11g of ammonium persulfate and 0.2g of sodium chloride were weighed and dissolved in 40mL of water to prepare an aqueous solution. Adding the aqueous solution into the reaction system in the step 1), and continuously introducing N₂30min；

3) Heating the reaction system to 70 ℃ and reacting for 15 h;

4) the emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. Centrifuging, settling and cleaning the obtained emulsion for multiple times by using deionized water until the conductivity of the supernatant at the beginning of centrifugation is close to that of the deionized water, diluting the obtained emulsion by using water, and storing the diluted emulsion in an emulsion form;

1.2. landfill process of luminescent composition

1) A25 mL round-bottom flask was prepared, and 0.1g of a dimethylthiophene derivative and 0.1g of europium (III) complex (MTTA-EU) were added³⁺) 10mL of 95% ethanol, magnetically stirring, heating in a water bath to 70 ℃ to obtain a complex solution;

2) preparing a 100mL three-neck flask, adding 10mL 95% ethanol, 10mL water and 10mL carboxyl polystyrene latex microspheres with the concentration of 10% obtained in the step 1.1, magnetically stirring, and heating to 70 ℃ in a water bath;

3) slowly dripping the complex solution in the step 1) into the three-neck flask in the step 2), reacting at 70 ℃ for 2h, stopping stirring, and naturally cooling;

4) and centrifuging the emulsion for 1h at 30000G, and removing supernatant after centrifugation to obtain the carboxyl polystyrene microspheres embedded with the luminescent composition. The volume is determined by 20mM HEPES buffer solution to obtain the final concentration of 20 mg/mL.

1.3 coating of luminescent microspheres coupled with HIV antigen

1) The HIV antigen was dialyzed to 50mM MES buffer pH 5.0 and the concentration was found to be 1 mg/mL.

2) 0.5mL of carboxyl luminescent microspheres and 0.5mL of dialyzed HIV antigen were added to a 2mL centrifuge tube, mixed well, added with 100. mu.L of 10mg/mL EDAC solution (50mM MES buffer), and reacted at 2-8 ℃ for 4 hours.

3) After completion of the reaction, 0.5mL of 100mg/mL BSA solution (50mM MES buffer) was added and the reaction was carried out at 2-8 ℃ for 2 hours.

4) After completion of the reaction, the reaction mixture was centrifuged for 30min at 20000G, and the supernatant was discarded after centrifugation and resuspended in 50mM MES buffer. The centrifugal washing was repeated four times, and diluted to a final concentration of 100. mu.g/mL to obtain a receptor particle solution coupled with HIV antigen, to obtain receptor reagent A.

Example 2 preparation of acceptor particles b

2.1 preparation of polystyrene latex microspheres

1) A100 mL three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein, and 10mL of water were added thereto, and the mixture was stirred for 10min and then N was introduced thereinto₂30min；

3) Heating the reaction system to 70 ℃ and reacting for 15 h;

4) the emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. Washing the obtained emulsion with deionized water by secondary centrifugal sedimentation until the conductivity of the supernatant at the beginning of centrifugation is close to that of the deionized water, then diluting with water, and storing in an emulsion form;

2.2 landfill Process of luminescent compositions

2) preparing a 100mL three-neck flask, adding 10mL 95% ethanol, 10mL water and 10mL aldehyde polystyrene latex microspheres with the concentration of 10% obtained in the step 1.1, magnetically stirring, and heating to 70 ℃ in a water bath;

4) centrifuging the emulsion for 1h at 30000G, and removing supernatant after centrifugation to obtain the aldehyde polystyrene microspheres embedded with the luminescent composition.

2.3 surface coating of the acceptor particles with polysaccharides

1) Taking 50mg of aminodextran solid, putting the aminodextran solid in a 20mL round-bottom flask, adding 5mL of 50mM/pH 10 carbonate buffer solution, and stirring and dissolving the aminodextran solid at 30 ℃ in the dark;

2) adding 100mg of prepared aldehyde polystyrene microspheres in which the luminescent composition is buried into aminodextran solution, and stirring for 2 hours;

3) dissolving 10mg of sodium borohydride in 0.5mL of 50mM/pH 10 carbonate buffer solution, dropwise adding the solution into the reaction solution, and reacting overnight at 30 ℃ in a dark place;

4) after the reaction, the mixture 30000G was centrifuged, the supernatant was discarded, and 50mM/pH 10 carbonate buffer was added thereto for ultrasonic dispersion. After repeated centrifugal washing for three times, the solution is subjected to volume fixing by using 50mM/pH 10 carbonate buffer solution to ensure that the final concentration is 20 mg/mL;

5) adding 100mg aldehyde dextran solid into a 20mL round-bottom flask, adding 5mL 50mM/pH 10 carbonate buffer, and stirring and dissolving at 30 ℃ in the dark;

6) adding the microspheres into an aldehyde dextran solution and stirring for 2 hours;

7) dissolving 15mg of sodium borohydride in 0.5mL of 50mM/pH 10 carbonate buffer solution, dropwise adding the solution into the reaction solution, and reacting overnight at 30 ℃ in a dark place;

8) after the reaction, the mixture 30000G was centrifuged, the supernatant was discarded, and 50mM/pH 10 carbonate buffer was added thereto for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume was adjusted to 20mg/mL using 50mM/pH 10 carbonate buffer.

9) The average particle size of the microspheres in a Gaussian distribution measured by a nanometer particle size analyzer was 241.6nm, the coefficient of variation (C.V) was 12.90%, and the Gaussian distribution curve is shown in FIG. 1.

2.4 coupling Process of HIV antigens to receptor particles

1) HIV antigen i was dialyzed into 50mM CB buffer at pH 9.0 to a measured concentration of 1 mg/mL.

2) Adding 0.5mL of the acceptor particles obtained in the step 2.3 and 0.5mL of HIV antigen I into a 2mL centrifuge tube, uniformly mixing, adding 100 mu L of 10mg/mL NaBH₄The solution (50mM CB buffer) was reacted at 2-8 ℃ for 4 hours.

3) After completion of the reaction, 0.5mL of 100mg/mL BSA solution (50mM CB buffer) was added and the reaction was carried out at 2-8 ℃ for 2 hours.

4) After completion of the reaction, the reaction mixture was centrifuged at 30000G for 45min, and the supernatant was discarded after centrifugation and resuspended in 50mM MES buffer. The centrifugal washing was repeated four times, and diluted to a final concentration of 100. mu.g/mL to obtain a solution of HBsAg antigen I-conjugated receptor particles, giving receptor reagent B.

Example 3 detection of sugar content of microspheres

1) Pretreatment of microsphere samples:

the receptor reagent A containing 1mg of receptor microspheres a in example 1 and the receptor reagent B containing 1mg of receptor microspheres B in example 2 are respectively taken and centrifuged for 40min at 20000G, supernatant liquid is poured out and ultrasonically dispersed by purified water, and the centrifugation and dispersion are repeated for three times, and then the volume is increased to 1mg/mL by the purified water.

2) Preparing a glucose standard solution:

the 1mg/mL glucose stock solution was prepared as a standard solution at 0mg/mL, 0.025mg/mL, 0.05mg/mL, 0.075mg/mL, 0.10mg/mL, 0.15mg/mL with purified water.

3) Preparing an anthrone solution: the solution is prepared into 2mg/mL by 80 percent sulfuric acid solution (the solution is stable within 24 hours at room temperature, and the solution is prepared as before use).

4) 0.1mL of glucose standard solution with each concentration and a sample to be detected are respectively added into a centrifuge tube, and 1mL of anthrone test solution is respectively added into each tube.

5) Incubate at 85 ℃ for 30 min.

6) Centrifuging the sample reaction tube at 15000G for 40min, and sucking clear liquid from the bottom of the tube by a pipette tip to measure absorbance so as to avoid sucking suspended matters on the upper part.

7) The temperature was returned to room temperature and the absorbance at 620nm was measured (the measurement was preferably carried out within 2 h).

8) The relationship between the sugar content concentration and the absorbance of the standard solution is shown in table 1, the sugar content concentration of the standard solution is taken as the X value, the absorbance is taken as the Y value, one-time linear regression is carried out, the sugar content determination standard curve shown in figure 2 is obtained, and the sugar content concentration of the sample to be detected is determined on the basis of the sugar content determination standard curve.

TABLE 1

Serial number	Concentration mg/mL	Absorbance A	Absorbance B	Absorbance mean value
					1	0	0.0008	0.0008	0.0008
2	0.025	0.0880	0.0916	0.0898
					3	0.05	0.1547	0.1611	0.1579
4	0.075	0.2375	0.2471	0.2423
					5	0.1	0.3190	0.332	0.3255
6	0.15	0.4855	0.5053	0.4954

And (3) detection results:

the sugar content of the acceptor microsphere a in example 1 is not higher than 25 mug/mg microsphere;

the sugar content of acceptor microsphere b in example 2 was 60.5. mu.g/mg microsphere.

Practice ofExample 4 preparation of HIV detection kit and Performance test thereof

The kit consists of a reagent R1 (prepared in example 1), a reagent R2 (biotin labeled HIV antigen), and a sensitizing reagent R3 containing donor particles, an Anti-HIV negative control, an Anti-HIV-1 positive control, an Anti-HIV-2 positive control, an Anti-HIV weak positive control and an Anti-HIV sample diluent. The components are prepared respectively, the reagent R1 and the reagent R2 are combined into an Anti-HIV kit, and negative and positive controls, a weak positive control and a sample diluent are independently packaged respectively and then assembled into a kit. The preparation process is summarized as follows:

first reagent R1 (receptor particle coated HIV antigen): the receptor particles prepared in example 1 were added to the first buffer solution to prepare a solution having a concentration of 50. mu.g/mL.

The reagent R2 (biotin labeled HIV antigen): the treated HIV antigen and biotin are mixed uniformly according to a certain concentration and proportion to form a connector, and a certain amount of buffer solution is added after dialysis to prepare the HIV antigen-biotin conjugate.

A third Anti-HIV negative control: Anti-HIV qualitative reference dilution.

Fourth Anti-HIV-1 positive control: and diluting HIV-1 positive serum by using Anti-HIV qualitative reference substance diluent.

Fifthly, Anti-HIV-2 positive control: and diluting the HIV-2 polyclonal antibody by using Anti-HIV qualitative reference substance diluent.

Sixthly, Anti-HIV weak positive contrast: and diluting HIV-1 positive serum by using Anti-HIV qualitative reference substance diluent.

The kit can be used for qualitatively detecting the HIV (1+2 type) antibody of the human immunodeficiency virus, and the test is carried out by using an Anti-HIV weak positive control, an Anti-HIV negative control, an Anti-HIV-1 positive control and an Anti-HIV-2 positive control which are matched with each other, wherein 2 holes are added to the Anti-HIV weak positive control, 2 holes are added to the Anti-HIV negative control, the Anti-HIV-1 positive control and 1 hole is added to the Anti-HIV-2 positive control respectively. The reagents were allowed to equilibrate to ambient temperature before use.

Step 1: diluting the sample to be detected by 11 times by using Anti-HIV sample diluent, and fully and uniformly mixing (for example, adding 10 mu L of sample into 100 mu L of sample diluent);

step 2: adding 25 mu L of diluted sample and Anti-HIV negative control, Anti-HIV-1 positive control, Anti-HIV-2 positive control and Anti-HIV weak positive control into the reaction hole respectively;

and step 3: adding 25 mu L of reagent R1, 25 mu L of reagent R2 and 25 mu L of photosensitive reagent R3 into the reaction hole in sequence;

and 4, step 4: putting into LiCA500 full-automatic light-activated chemical luminescence detector produced by Boyang Biotechnology (Shanghai) Limited company, and automatically operating by the detector, wherein the specific steps are as follows:

A. vibration

B.37 ℃ incubation for 15min

C. Add photosensitizing reagent 175. mu.L automatically

D.37 ℃ incubation for 10min

E. Irradiating the micropores with laser light and calculating the quantity of photons emitted from each hole

F. And (3) calculating an S/CO value (the ratio of the light signal value of the sample to be detected to the Anti-HIV weak-positive control light signal) by software, and judging whether the sample is negative or positive.

[ method for determining effectiveness of kit ]

Anti-HIV weak positive control, Anti-HIV negative control, Anti-HIV-1 positive control, Anti-HIV-2 positive control are added in each test

The S/CO value of the Anti-HIV negative control is less than 0.6, the S/CO value of the Anti-HIV-1 positive control is more than or equal to 3, and the S/CO value of the Anti-HIV-2 positive control is more than or equal to 3.

[ METHOD FOR DETERMINING DETECTION RESULT IN KIT ]

S: the optical signal value of the sample to be detected;

CO: Anti-HIV weak positive control light signal value (CUT OFF reference);

the software automatically calculates the S/CO value, the sample to be detected is judged to be negative when the S/CO is less than 1, and the sample to be detected is judged to be positive when the S/CO is more than or equal to 1.

Through detection, the kit of the embodiment has the following good performances:

(1) negative reference product compliance rate: detecting with national reference substance, wherein the coincidence rate of 20 parts of HIV antibody negative reference substance is not less than 18 parts;

(2) positive reference compliance rate: the national reference substances are used for detection, 18 HIV-1 type antibody positive reference substances cannot generate false negative, and RLUP12 is more than or equal to RLUP 11; false negatives cannot occur in 2 HIV-2 type antibody positive samples;

(3) sensitivity: performing detection by using national reference substances, wherein 1 part of matrix serum is negative in 6 parts of sensitivity reference substance serum, and at least 3 parts of diluted serum are positive in 5 parts of diluted serum;

(4) precision: the detection is carried out by national reference products, C.V is less than or equal to 15 percent (n is 10);

(5) clinical sensitivity, clinical specificity: in the detection of 500 serum samples judged to be positive clinically, 499 samples are detected to be positive, the clinical sensitivity reaches 99.8%, in the detection of 600 serum samples judged to be negative clinically, 600 samples are detected to be negative, and the clinical specificity reaches 100%.

Example 5: effect of C.V value of acceptor particle on test results

5.1 preparation of acceptor particles with different C.V values

According to the method described in example 1, receptor particle solutions of coupled HIV antigens with different coefficients of variation in particle size distribution were obtained, specifically:

receptor particle 1: the average particle size of Gaussian distribution is 211.8nm, and the variation coefficient of the particle size distribution is C.V, which is 3.5%; nicomp distribution is unimodal.

Receptor particle 2: the average grain diameter of Gaussian distribution is 214.4nm, and the variation coefficient of grain diameter distribution is C.V value which is 5.0%; nicomp distribution is unimodal.

Receptor particle 3: the average grain diameter of Gaussian distribution is 212.1nm, and the variation coefficient of grain diameter distribution C.V is 6.9%; nicomp distribution is unimodal.

Receptor particle 4: the average grain diameter of Gaussian distribution is 213.9nm, and the variation coefficient of grain diameter distribution C.V is 8.3%; nicomp distribution is unimodal.

Receptor particle 5: the average grain diameter of Gaussian distribution is 212.4nm, and the variation coefficient of grain diameter distribution C.V is 14.8%; nicomp distribution is unimodal.

Receptor particle 6: the average particle size of Gaussian distribution is 211.2nm, and the variation coefficient of the particle size distribution is C.V, which is 20.0%; the Nicomp distribution is bimodal.

Receptor particle 7: the average grain diameter of Gaussian distribution is 210.8nm, and the variation coefficient of grain diameter distribution C.V is 30.3%; the Nicomp distribution is bimodal.

5.2 determination of detection Limit and HOOK samples with the kit

Defining the lowest detection limit as 0.5NCU, judging the detection capability of the quality control product of kangtiantan, and when the test signal of a certain experimental group sample is just greater than the CO signal, namely RLU (Cx) > RLU (C0), the test result is positive. The HOOK sample is defined as the sample with the concentration of the analyte higher than the concentration of the antigen antibody in the reagent, the test result has the risk of low false, and the test result is S/CO ═ RLU (Cx)/RLU (C0).

The experimental procedure was as follows:

1. taking a 0.5NCU kanchestan quality control sample as a detection limit sample, and judging and detecting capacity difference through S/CO value and negative and positive;

2. diluting the HOOK sample into a series of samples to be detected by 2-time gradient;

3. receptor particles with different particle sizes of C.V are coupled with HIV antigens to be prepared into a concentration of 20 mu g/mL, and the receptor particles are tested with the diluted kanchestan quality control product and the HOOK sample synchronously;

4. the effect of different particle sizes of C.V on the detection limit was compared to that of the HOOK samples.

The results are shown in table 2 below:

TABLE 2

As can be seen from table 2, when the variation coefficient (C.V value) of the particle size distribution of the acceptor particles is 5% or more and 20% or less, the kit containing the acceptor particles has both the ability to detect a low-concentration sample and the good anti-HOOK ability. When the variation coefficient of the particle size distribution of the receptor particles is more than or equal to 8% and not more than 15%, the kit containing the receptor particles has the capability of detecting low-concentration samples and has better HOOK resistance.

Example 6 influence of sugar content on assay results

The experimental steps are as follows:

1. selecting 10 tumor patient interference samples, and verifying the samples as HIV non-reactive samples through a confirmation reagent; and 20 HIV positive samples validated with validation reagents;

2. receptor particles with different sugar contents are coupled with HIV antigens to be prepared into a concentration of 20 mu g/mL, and the signal value is tested after the reaction with 30 samples;

3. after the S/CO value of each sample is calculated, when the S/CO is more than or equal to 1, the detection result is positive, otherwise, the detection result is negative;

4. and if the positive result appears in the tumor interference sample, the false positive result is judged, and if the negative result appears in the HIV positive sample, the false positive result is judged to be missed.

The results are shown in table 3 below:

TABLE 3

From the analysis of the results in the above table, it can be seen that when the sugar content of the acceptor particles is not higher than 25 μ g, the sensitivity is high, the detection capability of the low-value sample is high, and the interference of the false positive sample is small.

Example 7: detection of HIV antibody levels in samples from normal humans and patients suspected of being infected with HIV virus

First, clinical validation of HIV agent (using receptor agent a prepared in example 1):

1. randomly selecting 100 normal physical examination samples, 50 tumor patient samples and 50 samples suspected to be infected with HIV virus;

2. preparing a receptor reagent A into a concentration of 20 mu g/mL, reacting with 200 samples, and testing a signal value;

the results are shown in table 4 below:

TABLE 4

And (4) analyzing results: the coincidence rate of 150 negative samples is 100%, and false positives do not exist; the coincidence rate of 50 positive samples is 100%, and no missing detection exists.

Claims

1. A human immunodeficiency virus antibody detection kit comprising a reagent R1, said reagent R1 comprising a first buffer solution and, suspended therein, receptor particles capable of reacting with reactive oxygen species to produce a chemiluminescent signal, characterized in that: the acceptor particle comprises a carrier, the interior of the carrier is filled with a luminescent composition, and the surface of the carrier is bonded with HIV antigen; the variation coefficient C.V value of the particle size distribution of the acceptor particles in the acceptor reagent is not less than 5% and not more than 20%; the sugar content per milligram mass of the acceptor particles is not higher than 25 μ g.

2. The kit of claim 1, wherein: the acceptor particles have a coefficient of variation of particle size distribution C.V value of no greater than 15% in the reagent R1.

3. The kit according to any one of claims 1-2, characterized in that: the variation coefficient C.V value of the particle size distribution of the acceptor particles in the reagent R1 is not less than 8%.

4. The kit according to any one of claims 1 to 3, characterized in that: the sugar content per milligram mass of the acceptor particles is not higher than 20 [ mu ] g.

5. The kit according to any one of claims 1 to 4, characterized in that: the surface of the carrier is not coated with sugar molecules and is directly bonded with HIV antigens.

6. The kit according to any one of claims 2, wherein: the content of polysaccharide in the first buffer solution is 0.01-1 wt%, preferably 0.05-0.5 wt%.

7. The HIV antibody detection kit according to claim 5, wherein: the surface of the carrier is provided with a bonding functional group for directly chemically bonding the HIV antigen on the surface of the carrier, wherein the bonding functional group is selected from at least one of amine group, amide group, hydroxyl group, aldehyde group, carboxyl group, epoxy group, maleimide group and sulfhydryl group; preferably selected from aldehyde groups, carboxyl groups.

8. The HIV antibody detection kit according to any one of claims 1 to 4, wherein: the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably from dextran or dextran derivatives.

9. The kit according to any one of claims 1 to 8, wherein the kit further comprises reagent R2, reagent R2 comprising a biotin-labeled HIV antigen.

10. The kit of any one of claims 1 to 9, wherein the kit further comprises one or more of an Anti-HIV negative control, an Anti-HIV-1 positive control, an Anti-HIV-2 positive control, and an Anti-HIV weak positive control.

11. The kit according to any one of claims 1 to 10, wherein the kit comprises a plurality of reagent strips, each reagent strip is provided with a plurality of reagent wells for containing a reagent, and at least one of the reagent wells is used for containing the reagent R1.

12. The method of using the HIV antibody detection kit according to any one of claims 1 to 11, comprising the steps of diluting a sample to be tested with a diluent, adding a reagent R1 and a reagent R2 thereto, adding a photosensitive reagent thereto, irradiating with laser light, calculating the amount of photons, and judging whether the sample to be tested is HIV negative or positive based on the amount of photons.

13. A method of using the human immunodeficiency virus antibody detection kit of any one of claim 12, comprising the steps of:

step 1: diluting a sample to be detected by using Anti-HIV sample diluent, and fully and uniformly mixing;

step 2: adding the diluted sample and Anti-HIV negative control, Anti-HIV-1 positive control, Anti-HIV-2 positive control and Anti-HIV weak positive control into the reaction well respectively;

and step 3: sequentially adding a reagent R1, a reagent R2 and a photosensitive reagent into the reaction hole;

and 4, step 4: putting the reaction hole into a light-excited chemical luminescence detector, irradiating the reaction hole by laser, calculating the quantity of photons emitted by the reaction hole, and judging whether HIV is positive or negative according to the quantity of photons.

14. Use of the human immunodeficiency virus antibody detection kit according to any one of claims 1 to 11 in a chemiluminescence analyzer.