CN114502957A

CN114502957A - Kit and method for detecting HIV antibodies

Info

Publication number: CN114502957A
Application number: CN201980101119.9A
Authority: CN
Inventors: 李可; 张裕平
Original assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Current assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Priority date: 2019-10-14
Filing date: 2019-10-14
Publication date: 2022-05-13
Also published as: WO2021072593A1

Abstract

The application provides a kit for detecting HIV antibodies, which comprises a fusion protein of a labeling enzyme and HIV antigenic protein, wherein the antigenic protein contains one or more of GP41 antigen, GP36 antigen and GP120 antigen, or more than two fusion antigens thereof. The application also provides a corresponding method for detecting antibodies generated in a sample after HIV infection.

Description

Kit and method for detecting HIV antibodies

Technical Field

The embodiment of the application relates to the field of Human Immunodeficiency Virus (HIV) antibody detection, in particular to an enzyme marker used in HIV antibody immunodetection, especially a double-antigen sandwich method or a capture method.

Background

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), belongs to the family of retroviridae, the lentivirus genus, the primate Immunodeficiency subgenus. The transmission pathways of HIV mainly include blood and contaminated blood products, sexual contact and maternal-fetal transmission before, during and after pregnancy.

When HIV infection occurs, the human immune system produces corresponding antibodies to various proteins of the HIV virus. Among them, envelope proteins are most immunogenic, usually produce antibodies at the earliest stage after infection, and remain throughout the disease stage, so antibody detection of HIV infection is usually targeted against antibodies to HIV envelope proteins.

The HIV immune detection method comprises enzyme-linked immunosorbent assay, chemiluminescence immunoassay, gold labeling, fluorescence immunoassay, etc. Among them, the enzyme immunoassay techniques such as enzyme-linked immunosorbent assay and enzymatic chemiluminescence immunoassay are common HIV immunoassay methods using enzyme for labeling as a reporter molecule, in a reaction mode of a double-antigen sandwich method, a conjugate of the enzyme for labeling in the enzyme immunoassay with an antigen substance, an antibody to be detected present in a sample, and an antigen substance coated on a solid support form a sandwich structure, and a qualitative or quantitative detection result of HIV is obtained by analyzing the enzyme for labeling in the sandwich structure. In the reaction mode of the capture method, a conjugate of a labeling enzyme in an enzyme immunoassay and an antigen substance, an antibody to be detected existing in a sample, and anti-human IgG and anti-human IgM antibodies coated on a solid support form a complex, and the labeling enzyme in the complex is analyzed, so that the qualitative or quantitative detection result of HIV is obtained.

Generally, in HIV enzyme immunoassays based on the double antigen sandwich method or the capture method, a chemically activated cross-linking method is used to link a labeling enzyme to an antigenic substance to form a cross-linked substance or conjugate. However, this connection process has the disadvantage of being complex to operate and difficult to control; in addition, such linkage results in a heterogeneous molecular molar ratio of enzyme to antigen, resulting in a product that is a mixture of enzyme and antigen cross-linkers at different molecular molar ratios. Therefore, the mixture of the enzyme and the antigen cross-linking substance prepared by the chemical activation cross-linking method is applied to the detection of HIV antibody, which causes the problems of difficult production control, large batch difference of detection results and the like of the syphilis detection kit.

Disclosure of Invention

In order to solve the above problems, the present embodiments provide an immunoassay kit for HIV antibodies, comprising a fusion protein of a labeling enzyme and an HIV antigenic protein.

As used in the examples herein, "antigenic protein" or "antigenic substance" refers to a protein that is immunoreactive and that is useful in the immunological detection of HIV; it may be one HIV antigen or a fragment thereof, or a fusion antigen of two or more HIV antigens or fragments thereof (or a chimeric protein of two or more HIV antigens or fragments thereof).

In the present examples, the antigenic protein comprises one or more of GP41 antigen, GP36 antigen and GP120 antigen, or comprises two or more fusion antigens of GP41 antigen, GP36 antigen and GP120 antigen.

As used in the examples herein, "HIV antigen" refers to a substance that is immunoreactive and can be used in the immunodetection of HIV, and is selected from the group consisting of conserved proteins of HIV or fragments thereof. Preferably, the HIV antigen of the present embodiments may be an envelope protein of HIV having high immunoreactivity, for example, one or more of GP41, GP36 antigen, and GP120 antigen. In the present examples, the HIV antigen may be present in one or more copies.

In some embodiments, the HIV antigenic proteins of the present application contain a GP41 antigen and a GP36 antigen, or a GP41 antigen, a GP36 antigen and a GP120 antigen.

In some embodiments, the HIV antigenic proteins of the present application are chimeric proteins comprising multiple HIV antigens, for example, a chimeric protein comprising a GP41 antigen and a GP36 antigen, or a chimeric protein comprising a GP41 antigen and a GP120 antigen, or a chimeric protein comprising a GP36 antigen and a GP120 antigen, or a chimeric protein comprising a GP41 antigen, a GP36 antigen and a GP120 antigen.

In the case where the antigenic protein contains two or more HIV antigens, these HIV antigens may be present in any order. In exemplary embodiments, the antigenic protein of the present application may contain, in order from N-terminus to C-terminus, GP41 antigen and GP36 antigen; alternatively, GP36 antigen GP41 antigen may be contained in sequence from N-terminus to C-terminus.

In particular embodiments, the HIV antigenic proteins of the present application contain, in order from N-terminus to C-terminus, GP41 antigen, GP36 antigen and GP120 antigen; or GP41 antigen, GP120 antigen and GP36 antigen; or GP36 antigen, GP41 antigen and GP120 antigen; or GP36 antigen, GP120 antigen and GP41 antigen; or GP120 antigen, GP41 antigen and GP36 antigen; or GP120 antigen, GP36 antigen and GP41 antigen.

In the present embodiment, the HIV antigens may be linked directly or may be linked via a Linker, as long as the HIV antigens are linked while their respective structures and activities are not affected. In the examples of this application, flexible linker, e.g. flexible (Gly)₄Ser) _nGGGS, GGSGGGSG, and the like.

In the present examples, "enzyme" and "labeling enzyme" are used interchangeably to refer to an enzyme used in an enzyme immunoassay. For example, it may be an enzyme used in enzyme-linked immunosorbent assay and enzymatic chemiluminescent immunoassay.

In some embodiments, the enzyme of the present application may be alkaline phosphatase (EC 3.1.3.1), which may catalyze the hydrolysis of phosphate group-containing chromogenic and chemiluminescent substrates such as nitrobenzene phosphate (PNP), sodium beta-glycerophosphate, naphthyl phosphate, 3- (2-spiroadamantane) -4-methoxy-4- (3-phosphoryl) -phenyl-1, 2-dioxetane (AMPPD), and the like.

The alkaline phosphatase of the embodiments of the present application may be naturally occurring, artificially synthesized, or produced by genetic engineering. In addition, the alkaline phosphatase of the embodiments of the present application may be a modified alkaline phosphatase such as a surface glycosylation treatment or a deglycosylation treatment.

The source of alkaline phosphatase is not particularly limited in the examples of the present application as long as the enzyme immunoassay can be achieved. Exemplary alkaline phosphatases can be derived from bacteria, such as e.coli; mammals, such as cattle (e.g., Genebank: AF052227.1 (sources htHIVs:// www.ncbi.nlm.nih.gov /)) or humans (e.g., Genebank: M12551.1); shrimp; but is not limited thereto.

In some embodiments, the enzyme of the present application may be horseradish peroxidase (EC 1.11.1.7), which is ferriporphyrin prosthetic, catalyzes the polymerization of phenol, aniline, and its substitutes in the presence of hydrogen peroxide, and is widely distributed in the plant kingdom, with the highest content in horseradish.

The horseradish peroxidase of the embodiments of the present application may be naturally occurring, artificially synthesized, or produced by genetic engineering. In addition, the horseradish peroxidase of the present examples may be modified.

In some embodiments, the enzymes of the present application also include mutants thereof. Mutants of the enzymes of the examples of the present application have greater than 80%, alternatively greater than 85%, greater than 90%, greater than 95%, greater than 98% or greater than 99% sequence homology to the wild type. Exemplary alkaline phosphatase mutants can be GeneBank: m29670.1 (sources htHIVs:// www.ncbi.nlm.nih.gov /), but the embodiments of the present application are not so limited. An exemplary horseradish peroxidase mutant can be GnenBank: XM _018585035.1, but the embodiments of the present application are not limited thereto.

Determination of sequence homology, with the wild-type sequence acting as a reference sequence, the sequence to be tested is compared with the reference sequence. Sequence homology of the test sequence to the reference sequence is then calculated using a sequence comparison algorithm. Two examples of algorithms suitable for determining sequence homology are the BLAST and BLAST2.0 algorithms described in Altschul et al (1977) Nuc.acids Res.25: 3389-. Software for performing BLAST analysis is publicly available through NCBI.

In the present embodiment, the enzyme may be fused to any position of the antigenic protein. For example, the enzyme may be fused to the N-terminus of an HIV antigenic protein; alternatively, the enzyme may be fused to the C-terminus of the antigenic protein; alternatively, the enzyme may be fused between any two adjacent antigens in the antigenic protein.

In some embodiments, the enzyme is fused to the N-terminus of GP41 antigen and GP36 antigen, respectively.

In some embodiments, the enzyme is fused to the C-terminus of GP41 antigen and GP36 antigen, respectively.

In some embodiments, the fusion protein of an HIV antigenic protein and an enzyme comprises, in order from N-terminus to C-terminus, a GP41 antigen, an enzyme, and a GP36 antigen.

In some embodiments, the fusion protein of an HIV antigenic protein and an enzyme comprises, in order from N-terminus to C-terminus, the enzyme, GP41 antigen, GP36 antigen, and GP120 antigen.

In some embodiments, the fusion protein of an HIV antigenic protein and an enzyme comprises, in order from N-terminus to C-terminus, a GP41 antigen, a GP36 antigen, a GP120 antigen, and an enzyme.

In some embodiments, the fusion protein of an HIV antigenic protein and an enzyme comprises, in order from N-terminus to C-terminus, a GP41 antigen, an enzyme, a GP36 antigen, and a GP120 antigen.

In specific embodiments, alkaline phosphatase is fused to the N-terminus of GP41 antigen and GP36 antigen, respectively; or alkaline phosphatase fused to the C-terminus of GP41 antigen and GP36 antigen, respectively; or alkaline phosphatase fused between the GP41 antigen and the GP36 antigen; or horseradish peroxidase is respectively fused at the N ends of GP41 antigen and GP36 antigen; or horseradish peroxidase is respectively fused at the C ends of GP41 antigen and GP36 antigen; or horseradish peroxidase is fused between the GP41 antigen and the GP36 antigen.

In some embodiments, the immunodetection kit for Human Immunodeficiency Virus (HIV) antibodies may comprise at least one of a fusion protein of a labeling enzyme with GP36, a fusion protein of a labeling enzyme with GP41 antigen, and a fusion protein of a labeling enzyme with GP120 antigen. In a specific embodiment, the immunoassay kit comprises a fusion protein of a labeling enzyme and GP36 and a fusion protein of a labeling enzyme and GP41 antigen, such as a fusion protein of alkaline phosphatase and GP36 and alkaline phosphatase and GP41 antigen or a fusion protein of horseradish peroxidase and GP36 and a fusion protein of horseradish peroxidase and GP41 antigen.

In the examples of the present application, the enzyme may be conjugated to an antibodyThe antigenic proteins can be directly connected or can be connected through a Linker, so long as the respective structures and activities of the antigenic proteins are not affected while the ligase and the antigenic proteins are connected. In the examples of this application, flexible linker, e.g. flexible (Gly)₄Ser) _nGGGS, GGSGGGSG, and the like.

In the present embodiment, "fusion protein" refers to a fusion protein of an enzyme and an antigenic protein. For example, it can be expressed as enzyme + GP41+ GP36+ GP120, and in this case, it refers to a fusion protein in which the enzyme, GP41 antigen, GP36 antigen and GP120 antigen are present in this order from N-terminus to C-terminus.

The fusion protein of the enzyme and the HIV antigenic protein can be prepared by the conventional recombinant expression technology. In the embodiment of the present application, the recombinant expression technology may be a prokaryotic expression technology, such as an escherichia coli expression technology; and eukaryotic expression techniques such as yeast expression techniques and insect cell expression techniques, among others.

It will be appreciated by those skilled in the art that the kits of the embodiments herein may also include other reagents or components for determining HIV antibodies based on a double antigen sandwich or capture method, for example, a solid support comprising HIV antigenic proteins coated thereon (the HIV antigenic proteins coated on the solid support and the HIV antigenic proteins in the fusion protein are capable of binding to the same HIV antibody in a sample) or a solid support coated with anti-human IgM antibodies and anti-human IgG antibodies; a calibrator for plotting a standard curve; a quality control material for quality control; a substrate solution for carrying out a chemiluminescent reaction; and/or wash buffers and sample dilutions, etc.

In some embodiments, the kit further comprises a solid support coated with a fusion antigen comprising GP41 antigen and GP36 antigen; or comprises a solid support coated with GP41 antigen and a solid support coated with GP36 antigen.

In some embodiments, the kit further comprises a solid support coated with a fusion antigen comprising GP41 antigen, GP36 antigen, and GP120 antigen; or comprises a solid phase support coated with GP41 antigen, a solid phase support coated with GP36 antigen and a solid phase support coated with GP120 antigen.

In another aspect, the embodiments herein also relate to the use of a fusion protein of an enzyme and an HIV antigenic protein for the preparation of an immunoassay kit for the detection of HIV. The immunoassay kit comprises:

a first reagent comprising a solid phase coating having a first ligand coated thereon, the first ligand being capable of binding to an HIV antibody in a sample;

a second reagent comprising an enzyme label which is a second ligand fused to an enzyme, wherein the second ligand is an HIV antigenic protein and is capable of binding to an HIV antibody to which the first ligand binds.

In a specific embodiment, HIV is detected based on a double antigen sandwich or capture method.

In one variation of the embodiments herein, there is provided an immunoassay kit for the detection of HIV, comprising:

a first reagent comprising a solid phase coating, said solid phase coating being a solid support coated with an HIV antigenic protein (first ligand).

A second reagent comprising an enzyme label which is an HIV antigenic protein (second ligand) fused with an enzyme, wherein the HIV antigenic protein coated on the solid support and the HIV antigenic protein in the fusion protein are capable of binding to the same HIV antibody in the sample; and

the specification describes detection based on the double antigen sandwich method.

It will be appreciated by those skilled in the art that the type of HIV antigenic protein coated on the solid support in the first reagent and the type of HIV antigenic protein fused to the fusion protein in the second reagent are the same, but the source or the manner or order of attachment between the antigens may be the same or different. For example, the HIV antigenic protein coated on the solid support may be a fusion antigen of GP41, GP36 and GP120 linked in sequence, and the HIV antigenic protein fused in the fusion protein in the second agent may be a fusion antigen of GP41, GP120 and GP36 linked in sequence.

Furthermore, in the present embodiment, the HIV antigenic protein coated on the solid support may be an HIV antigenic protein as described above, or an HIV antigenic protein linked in other ways, such as chemically linked.

In the present embodiment, HIV antibodies refer to antibodies produced in a subject following HIV infection, such as Anti-HIV IgG antibodies and Anti-HIV IgM antibodies.

In another variation of the embodiments herein, there is provided an immunoassay kit for detecting HIV, comprising:

a first reagent comprising a solid phase coating, said solid phase coating being a solid phase support coated with anti-human IgM antibody and anti-human IgG antibody (first ligand).

A second reagent comprising an enzyme label which is a fusion protein of an enzyme and an HIV antigenic protein (second ligand); and

the specification describes detection based on a capture method.

As used in the examples herein, "solid support" refers to a solid surface to which an antigen or antibody can be attached. The solid phase support used in the present application is not particularly limited, and commercially available solid phase supports and any solid phase support that can be used for immunoassay can be used in the present application. Exemplary solid supports may be magnetic beads (e.g., superparamagnetic microspheres), microplate, plastic plates, plastic tubes, latex beads, agarose beads, glass, nitrocellulose membranes, nylon membranes, silica plates, or microchips, but the embodiments are not limited thereto.

In the present examples, the solid phase coating may be present in a conventional diluent containing protein and surfactant and having buffering capacity.

In the present examples, the enzyme label may be present in a conventional diluent containing protein and surfactant and having buffering capacity.

In the present example, the fusion protein can be present in the second agent at a concentration of, for example, about 100ng/mL to 500 ng/mL.

In particular embodiments, the kits of the present application may further comprise a third reagent comprising a blocking agent and a surfactant. For example, the blocking agent is selected from one or more of the group consisting of: skimmed milk powder, BSA, gelatin, serum, casein, ovalbumin, animal IgG, and surfactant (e.g., Tween-20, Tween-80, TritonX-100, etc.).

In particular embodiments, the kits of the present application may further comprise a fourth reagent comprising a reducing agent. For example, the reducing agent is selected from one or more of the group consisting of: DTT, beta-mercaptoethanol.

In the examples of the present application, the blocking agent and the surfactant are soluble in a conventional diluent having a buffering capacity; the reducing agent is soluble in conventional diluents having buffering capacity.

In some embodiments, the kit may further comprise a reaction substrate for the labeling enzyme, preferably the reaction substrate is 3- (2-helical adamantane) -4-methoxy-4- (3-phospholyl) -phenyl-1, 2-dioxetane.

Unless specifically stated otherwise, the terms "first", "second", "third", and "fourth", etc. in the embodiments of the present application are used only to distinguish a plurality of similar elements, and are not intended to indicate any difference in importance or order between the elements.

The embodiment of the application also relates to a method for detecting HIV antibodies generated after human immunodeficiency virus HIV is infected in a sample by using the fusion protein of the enzyme and the HIV antigenic protein, which comprises the following steps:

mixing the sample with the solid support coated with the first ligand such that the first ligand coated on the solid support is substantially bound to the antibodies to HIV in the sample;

washing the mixture to remove unbound substances;

adding an enzyme marker with a second ligand into the cleaned mixture, and uniformly mixing to ensure that the second ligand in the enzyme marker is combined with the HIV antibody combined on the solid support to form a sandwich compound, wherein the enzyme marker is a fusion protein of a marking enzyme and HIV antigenic protein;

washing the sandwich composite to remove unbound substances;

and adding a chemiluminescence substrate into the washed sandwich compound, and detecting the number of photons generated by the reaction to obtain a chemiluminescence signal value of the sample.

In the examples of the present application, the first ligand is an HIV antigenic protein, or an anti-human IgG antibody and an anti-human IgM antibody.

The embodiment of the application obtains the fusion protein of the marking enzyme and the HIV antigenic protein through recombinant expression, and applies the fusion protein to the HIV immunoassay kit based on a double-antigen sandwich and/or capture method mode. It should be noted that:

by replacing the linker of enzyme and antigen substance generated by chemical activation crosslinking with the fusion protein, the fusion protein of the labeling enzyme and the HIV antigenic protein in the kit has uniform molar ratio of the labeling enzyme to the HIV antigenic protein, thereby avoiding the problem of large batch difference of kit production and detection results.

On the other hand, the kit of the embodiment of the present application shows better discrimination of negative and positive samples and has better sample coincidence rate, compared to the conventional double antigen sandwich method or capture method kit.

In yet another aspect, kits containing the fusion proteins of the embodiments avoid disruption of antigen or enzyme activity by chemically activated cross-linking methods.

In another aspect, the embodiment of the present application saves the steps required for performing the chemical crosslinking reaction, so that the operation is more convenient and faster.

Detailed Description

The technical means in the embodiments of the present application are described below clearly and completely, and it is obvious that the described embodiments are only a part of the embodiments of the present application, not all embodiments. All other embodiments obtained by a person of ordinary skill in the art without any inventive work based on the embodiments in the present application are within the scope of protection of the present application.

As described above, the fusion protein of the enzyme and the HIV antigenic protein in the present examples can be produced by recombinant expression techniques.

An exemplary recombinant expression method may comprise the steps of:

i) a host cell transfected or transformed with a nucleic acid molecule or expression vector;

ii) culturing the host cell under conditions to express the fusion protein; and

iii) isolating the fusion protein.

In the above step, the nucleic acid molecule encodes the above fusion protein; and expression vectors for producing fusion proteins of the enzyme with antigenic proteins of HIV by recombinant expression, comprising a nucleic acid molecule encoding the above fusion protein operably linked to expression control sequences, and additionally comprising genetic elements for maintenance and propagation in the respective host cell, such as an origin of replication and/or a selectable marker gene.

Preparation of fusion proteins

Production of fusion proteins using E.coli expression systems:

the first step is as follows: and (4) confirming the target gene. According to the gene sequences and amino acid sequences of two proteins to be fused, a proper linker is added between the two proteins, for example, the linker is GGGGS, GGSGGGSG or AEAAAKA, and a His tag is added at the C end of the fusion protein for purification, so that the target gene sequence is preliminarily determined. And finally determining a target gene sequence suitable for the Escherichia coli expression strain BL21(DE3) through codon optimization.

The second step: and (5) constructing a vector. And selecting a proper enzyme cutting site, and integrating the target gene into a proper expression vector pET 28.

The third step: transformation and screening of positive clone strains. The expression vector containing the target gene is transferred into escherichia coli by electric transformation or heat shock transformation. And (4) adding proper antibiotics according to the reporter gene on the expression vector, and screening out the positive bacterial strain.

The fourth step: and (3) expressing and purifying the protein.

HIV antigen nucleic acid sequence information: GP41, GeneBank: x83215.1; GP36, GeneBank: u67387.1; GP120, GeneBank: y14420.1 (sources htHIVs:// www.ncbi.nlm.nih.gov /).

Reagent preparation method

First reagent Ra:

3.5mL of magnetic beads coated with GP41 and GP36 are measured by a pipettor or a measuring cylinder and added into a magnetic bead coated tube to replace supernatant, namely, the supernatant is sucked away after magnetic separation, then an equal volume (3.5mL) of magnetic bead coated diluent is added, and the mixture is uniformly mixed; uniformly mixing the magnetic beads, and adding the mixture into a solution preparation bottle filled with 96.5mL of magnetic bead coated substance diluent; stirring until the magnetic bead suspension is completely mixed to prepare a first reagent Ra, wherein the concentration of the magnetic beads coated with the HIV antigenic protein is 0.6 mg/mL; the magnetic bead coating diluent is a conventional diluent with buffering capacity and contains protein and a surfactant.

A second reagent Rb:

measuring 99mL of tracer diluent by using a proper measuring cylinder, adding the tracer diluent into a solution preparation bottle, and measuring 1mL of 'fusion protein of enzyme and HIV antigenic protein' or 'conjugate of chemically-linked enzyme and HIV antigenic protein' by using a liquid transfer device, wherein the fusion protein of enzyme and HIV antigenic protein 'or the conjugate of chemically-linked enzyme and HIV antigenic protein' is used as an enzyme marker and added into the tracer diluent; stirring the solution by a stirrer to fully dissolve and uniformly mix the solution; then filtering the prepared solution by using a proper filter with the pore diameter of 0.22 mu m, and collecting filtrate to prepare a tracer Rb, wherein the concentration of the enzyme marker is 300 ng/mL; the tracer diluent is a conventional diluent with buffering capacity and contains protein and surfactant.

A third reagent Rc:

a diluent with buffering capacity, and containing BSA, Tween-20 and skimmed milk powder.

Fourth reagent Rd:

a diluent having buffering capacity and containing BSA and DTT.

Double-antigen sandwich detection method

The first step is as follows: adding the sample, the fourth reagent and the first reagent into the reaction tube, and incubating for 10 minutes at 37 ℃ so that the HIV antigen coated on the solid phase of the magnetic beads is fully combined with Anti-HIV IgG and Anti-HIV IgM antibodies in the sample; after the incubation is completed, the magnetic bead solid phase is placed in a magnetic field to be attracted, the substances bound on the magnetic bead solid phase are retained, and other unbound substances are washed and removed.

The second step is that: adding the third reagent and the second reagent into the reaction tube, and uniformly mixing; after incubation at 37 ℃ for 10 minutes, the HIV antigen on the enzyme label binds to the Anti-HIV IgG and Anti-HIV IgM antibodies captured on the magnetic beads to form a sandwich complex. After incubation in the reaction tube is complete, the complex is attracted by the magnetic field and other unbound material is washed away.

The third step: a chemiluminescent substrate is added to the reaction tube to produce chemiluminescence. And measuring the number of photons generated by the reaction through a photomultiplier to obtain a chemiluminescence signal value of the sample.

In the examples of the present application, COI (Cutoff index) is the ratio of the chemiluminescence signal value (RLU) of the measurement sample to the threshold value (Cutoff value), wherein COI ≧ 1 indicates that the measurement sample is a positive sample, and COI < 1 indicates that the measurement sample is a negative sample. For qualitative detection methods, the threshold (cutoff) is the cut-off that determines whether the test result is positive or negative.

In the embodiment of the present application, the negative coincidence rate refers to a ratio of the number of samples determined to be negative obtained by using the test method of the embodiment of the present application to negative samples actually participating in evaluation, and the positive coincidence rate refers to a ratio of the number of samples determined to be positive obtained by using the test method of the embodiment of the present application to positive samples actually participating in evaluation; the true negative and positive results of the sample are from hospital diagnostic results.

Example 1

To compare the HIV detection effect of the fusion protein of the enzyme and the HIV antigenic protein of the present example with respect to the conjugate of the enzyme and the antigenic protein produced by chemical crosslinking, the following two reagent combinations were prepared.

Combination 1: using the above described E.coli expression system, a second reagent Rb was prepared by fusing alkaline phosphatase with GP41 and GP36 as HIV antigenic proteins, respectively, i.e., the second reagent Rb comprises a fusion protein of alkaline phosphatase and GP41 and a fusion protein of alkaline phosphatase and GP36, wherein alkaline phosphatase (Genebank: AF052227.1 (source htHIVs:// www.ncbi.nlm.nih.gov /)) in the second reagent Rb is fused to the N-terminus of GP41 and GP36 antigenic proteins, respectively, and prepared by the above "reagent preparation method".

And (3) combination 2: a second reagent Rb was formulated using a conjugate of alkaline phosphatase and GP41 and a conjugate of alkaline phosphatase and GP36, respectively, prepared by chemical ligation, the remainder being identical to combination 1.

Next, using combinations 1 and 2, 500 diagnosed HIV negative samples and 500 positive samples from the general hospital were tested according to the "double antigen sandwich test method" described above, respectively.

The results are shown in table 1 below.

TABLE 1

Reagent combination	Combination 1	Combination 2
Negative sample 1(COI)	0.12	0.11
Negative sample 2(COI)	0.16	0.17
Positive sample 1(COI)	4.60	3.20
Positive sample 2(COI)	12.16	8.67
Positive sample 3(COI)	70.68	62.36
Negative coincidence rate (500 cases)	99.6％	99.4％
Positive coincidence rate (500 cases)	100％	100％

As can be seen from Table 1, the negative coincidence rate of the combination 1 is 99.6%, the positive coincidence rate is 100%, which is superior to the conventional double-antigen sandwich method using chemical ligation (combination 2: negative coincidence rate 99.4%, positive coincidence rate 100%); furthermore, as can be seen from the COI values, combination 1 showed better negative/positive sample discrimination than combination 2. The above results show that: the use of the alkaline phosphatase-GP 41 fusion protein and the alkaline phosphatase-GP 36 fusion protein can avoid the damage of the preparation process of chemical bond connection to the activity of antigen or alkaline phosphatase in the conventional double antigen sandwich method. In the examples of the present application, the alkaline phosphatase and the antigen in the fusion protein of alkaline phosphatase-GP 41 and the fusion protein of alkaline phosphatase-GP 36 both retained higher activity.

Example 2

To compare the HIV detection effect of the fusion protein of the enzyme with the HIV antigenic protein of the present examples against the conjugate of the enzyme with the antigenic protein produced by chemical cross-linking, the following two reagent combinations were prepared:

and (3) combination: a second reagent Rb is prepared by utilizing an escherichia coli expression system and fusing GP41 and GP36 serving as HIV antigenic proteins and horseradish peroxidase respectively, namely the second reagent Rb comprises a fusion protein of the horseradish peroxidase and GP41 and a fusion protein of the horseradish peroxidase and GP36, wherein the horseradish peroxidase (GeneBank: KU504630.1 (from htHIVs:// www.ncbi.nlm.nih.gov /)) in the second reagent Rb is fused at the N ends of the GP41 and GP36 antigenic proteins respectively, and the reagent is prepared according to the reagent preparation method.

And (4) combination: a second reagent Rb was prepared using a conjugate of horseradish peroxidase and GP41 and a conjugate of horseradish peroxidase and GP36, respectively, prepared by chemical ligation, the remainder being identical to combination 1.

Next, 500 HIV negative samples and 500 positive samples of example 1 were tested according to the above-described "double antigen sandwich assay" using combinations 3 and 4, respectively. The results are shown in table 2 below.

TABLE 2

Reagent combination	Combination 3	Combination 4
Negative sample 1(COI)	0.22	0.20
Negative sample 2(COI)	0.20	0.24
Positive sample 1(COI)	3.79	2.89
Positive sample 2(COI)	10.41	7.80
Positive sample 3(COI)	55.92	47.49
Negative coincidence rate (500 cases)	99.6％	99.4％
Positive coincidence rate (500 cases)	100％	100％

As can be seen from Table 2, the negative coincidence rate of the combination 3 is 99.6%, the positive coincidence rate is 100%, which is superior to the conventional double-antigen sandwich method using chemical ligation (combination 4: negative coincidence rate 99.4%, positive coincidence rate 100%); furthermore, as can be seen from the COI values, combination 3 showed better negative/positive sample discrimination than combination 4. The above results show that: the use of the horseradish peroxidase-GP 41 fusion protein and the horseradish peroxidase-GP 36 fusion protein can avoid the damage of the preparation process to the activity of the antigen or the horseradish peroxidase by using a chemical bond connection method in the conventional double-antigen sandwich method; in this example, the horseradish peroxidase and antigen in the horseradish peroxidase-GP 41 fusion protein and the horseradish peroxidase-GP 36 fusion protein all maintained high activity.

Example 3

To examine the effect of the fusion site of the enzyme on the fusion proteins of the examples of the present application, the following three reagent combinations were prepared:

combination 1: the second reagent Rb is prepared by using an escherichia coli expression system and fusing GP41 and GP36 as HIV antigenic proteins and alkaline phosphatase respectively, namely the second reagent Rb comprises a fusion protein of the alkaline phosphatase and GP41 and a fusion protein of the alkaline phosphatase and GP36, wherein the alkaline phosphatase in the second reagent Rb is fused at the N end of the GP41 and GP36 antigenic proteins respectively, and the reagent Rb is prepared according to the reagent preparation method.

And (4) combination 5: in the fusion protein of the second agent Rb, alkaline phosphatase was fused to the C-terminal of GP41 and GP36, respectively, and the rest was the same as in combination 1.

And (4) combination 6: the fusion protein of the second agent Rb, which uses the GP41+ GP36 chimeric protein as an antigenic protein to fuse with alkaline phosphatase, is prepared by the method of reagent preparation, wherein the alkaline phosphatase is fused between GP41 and GP 36.

Next, using combinations 1, 5 and 6, 500 HIV negative and 500 positive samples from the confirmed diagnosis in example 1 were tested according to the "double antigen sandwich test method" described above, respectively, and the results are shown in table 3 below.

TABLE 3

Reagent combination	Combination 1	Combination 5	Combination 6
Negative sample 1(COI)	0.12	0.18	0.25
Negative sample 2(COI)	0.16	0.14	0.18
Positive sample 1(COI)	4.60	4.78	4.36
Positive sample 2(COI)	12.16	12.22	11.12
Positive sample 3(COI)	70.68	67.48	64.12
Negative coincidence rate (500 cases)	99.6％	99.6％	99.4％
Positive coincidence rate (500 cases)	100％	100％	100％

As can be seen from table 3, when alkaline phosphatase was fused to the N-terminus (combination 1) and C-terminus (combination 5) of GP41 and GP36, respectively, or fused to the middle (combination 6) of the GP41+ GP36 chimeric protein, both the negative and positive match rates of the samples were high. Furthermore, as can be seen from table 3, the fusion of the enzyme in the middle of the HIV antigenic protein was slightly less effective than the fusion of the enzyme at the N-and C-termini of the HIV antigenic protein. Although not wishing to be bound by theory, the inventors believe that in the case of fusing the enzyme in the middle of the HIV antigenic protein, the active center of the enzyme is partially blocked by the other proteins.

Example 4

and (3) combination: and (2) configuring a second reagent Rb by using an escherichia coli expression system and using GP41 and GP36 as HIV antigenic proteins respectively, wherein horseradish peroxidase in the second reagent Rb is fused at the N ends of GP41 and GP36 antigenic proteins respectively, and the reagent Rb is prepared according to the reagent preparation method.

And (3) combination 7: in the fusion protein of the second reagent Rb, horseradish peroxidase was fused to the C-terminal of GP41 and GP36, respectively, and the rest was the same as that of combination 3.

And (4) combination 8: the antigen protein is GP41+ GP36 chimeric protein, and horseradish peroxidase in the fusion protein of the second reagent Rb is fused between GP41 and GP36, and the preparation method is carried out according to the above reagent preparation method.

Next, using combinations 3, 7 and 8, 500 HIV negative and 500 positive samples from the confirmed diagnosis in example 1 were tested according to the "double antigen sandwich test method" described above, respectively, and the results are shown in table 4 below.

TABLE 4

Reagent combination	Combination 3	Combination 7	Combination 8
Negative sample 1(COI)	0.22	0.18	0.24
Negative sample 2(COI)	0.20	0.17	0.22
Positive sample 1(COI)	3.79	3.98	3.88
Positive sample 2(COI)	10.41	8.99	8.58
Positive sample 3(COI)	55.92	58.42	59.19
Negative coincidence rate (500 cases)	99.6％	99.4％	99.2％
Positive coincidence rate (500 cases)	100％	100％	100％

As shown in Table 4, horseradish peroxidase fused to the N-terminus (combination 3) and C-terminus (combination 7) of GP41 and GP36, respectively, or fused to the middle of the GP41+ GP36 chimeric protein (combination 8) can achieve higher negative and positive coincidence rates of the samples. Furthermore, as can be seen from table 4, the fusion of the enzyme in the middle of the HIV antigenic protein is slightly less effective than the fusion of the enzyme in the N and C terminals of the HIV antigenic protein, and although not wishing to be bound by theory, the inventors believe that in the case of the fusion of the enzyme in the middle of the HIV antigenic protein, the active center of the enzyme is partially blocked by other proteins.

Example 5

To examine the effect of different fusion protein combinations on the detection effect of the examples of the present application, the following three reagent combinations were prepared:

combination 1: a second reagent Rb is prepared by utilizing an escherichia coli expression system and fusing GP41 and GP36 serving as HIV antigenic proteins and alkaline phosphatase respectively, namely the second reagent Rb comprises a fusion protein of the alkaline phosphatase and GP41 and a fusion protein of the alkaline phosphatase and GP36, wherein the alkaline phosphatase in the second reagent Rb is fused at the N end of the GP41 and GP36 antigenic proteins respectively, and the second reagent Rb is prepared according to the reagent preparation method.

Combination 9: the second agent Rb comprising a fusion protein of alkaline phosphatase and GP41, a fusion protein of alkaline phosphatase and GP36 and a fusion protein of alkaline phosphatase and GP120 was prepared by fusing GP41, GP120 and GP36 as HIV antigenic proteins and alkaline phosphatase, respectively, wherein the alkaline phosphatase in the second agent Rb is fused to the N-terminus of GP41, GP120 and GP36 antigenic proteins, respectively, and the rest is the same as in combination 1.

Combination 10: the second reagent Rb is prepared by fusing GP41, GP120 and GP36 as HIV antigenic proteins and horseradish peroxidase, respectively, i.e., the second reagent Rb comprises a fusion protein of horseradish peroxidase and GP41, a fusion protein of horseradish peroxidase and GP36 and a fusion protein of horseradish peroxidase and GP120, wherein the horseradish peroxidase in the second reagent Rb is fused at the N-terminus of GP41, GP120 and GP36 antigenic proteins, respectively, and the rest is the same as in combination 1.

Next, using combinations 1, 9 and 10, 500 HIV negative and 500 positive samples from the confirmed diagnosis in example 1 were tested according to the "double antigen sandwich test method" described above, respectively, and the results are shown in table 5 below.

TABLE 5

Reagent combination	Combination 1	Combination 9	Assembly 10
Negative sample 1(COI)	0.12	0.21	0.16
Negative sample 2(COI)	0.16	0.12	0.14
Positive sample 1(COI)	4.60	4.80	3.72
Positive sample 2(COI)	12.16	12.75	10.42
Positive sample 3(COI)	70.68	72.15	59.79
Negative coincidence rate (500 cases)	99.6％	99.6％	99.2％
Positive coincidence rate (500 cases)	100％	100％	100％

As can be seen from Table 5, the combination of the fusion proteins of the enzyme and different antigens can achieve better negative and positive matching rates of the sample.

Example 6

To examine the effect of HIV detection using the enzyme mutants, the following reagent combinations were prepared:

combination 1: a second reagent Rb is prepared by using an Escherichia coli expression system and using GP41 and GP36 as HIV antigenic proteins respectively, wherein wild type alkaline phosphatase (Genebank: AF052227.1 (source htHIVs:// www.ncbi.nlm.nih.gov /)) in the second reagent Rb is fused at the N ends of the GP41 and GP36 antigenic proteins respectively, and the reagent Rb is prepared according to the reagent preparation method.

Combination 11: mutant alkaline phosphatase (GeneBank: M29670.1 (origin htHIVs:// www.ncbi.nlm.nih.gov /)) was used, and the remainder was the same as in combination 1.

Next, 500 HIV negative samples and 500 positive samples of example 1 were tested according to the above-mentioned "double antigen sandwich assay" using combinations 1 and 11, respectively, and the results are shown in Table 6.

TABLE 6

Reagent combination	Combination 1	Combination 11
Negative sample 1(COI)	0.12	0.17
Negative sample 2(COI)	0.16	0.22
Positive sample 1(COI)	4.60	8.45
Positive sample 2(COI)	12.16	25.36
Positive sample 3(COI)	70.68	148.25
Negative coincidence rate (500 cases)	99.6％	99.6％
Positive coincidence rate (500 cases)	100％	100％

As is clear from Table 6, both of the wild type alkaline phosphatase and the mutant alkaline phosphatase were able to achieve high negative and positive match rates. In addition, the detection region was better graduated when the mutant alkaline phosphatase was used than when the wild-type alkaline phosphatase was used.

Example 7

In order to examine the influence of different expression techniques on the HIV detection effect of the examples of the present application, eukaryotic expression systems were further used to prepare fusion proteins of alkaline phosphatase and GP41 and fusion proteins of alkaline phosphatase and GP 36.

The specific method for producing the fusion protein by the eukaryotic expression system comprises the following steps:

the first step is as follows: and (4) confirming the target gene. Adding a nucleotide sequence capable of coding a linker short peptide between the two gene sequences of the two proteins to be fused, and adding a His tag at the C end of the fusion protein for purification, and primarily determining the target gene sequence. And finally determining a target gene sequence suitable for the pichia pastoris expression strain through codon optimization. In the examples of the present application, the number of amino acids in the linker short peptide is more than 8, especially more than 10, and may be, for example, (Gly)₄Ser) ₃Or AEAAAKEAAAKA, the Pichia strain can be methanol-inducible Pichia pastoris (X33).

The second step is that: and (3) constructing a vector. Selecting proper enzyme cutting sites, and integrating the target gene into proper expression vector pPICZ alpha A or pPIC 9K.

The third step: transformation and screening of positive clone strains. The expression vector containing the target gene is transferred into pichia pastoris cells or 293 cells through electrotransformation. Adding proper antibiotics according to the reporter gene on the expression vector, screening out positive strains,

the fourth step: and (3) expressing and purifying the protein.

The preparation according to the reagent preparation method respectively obtains the combination 12 (pichia pastoris) and the combination 13(293 cells).

Next, using combinations 1, 12 and 13, 500 HIV negative samples and 500 positive samples in example 1 were tested according to the above "double antigen sandwich assay" and the results are shown in table 7.

TABLE 7

Reagent combination	Combination 1	Combination 12	Combination 13
Negative sample 1(COI)	0.12	0.14	0.11
Negative sample 2(COI)	0.16	0.19	0.15
Positive sample 1(COI)	4.60	5.20	4.25
Positive sample 2(COI)	12.16	13.59	11.29
Positive sample 3(COI)	70.68	80.59	71.48
Negative coincidence rate (500 cases)	99.6％	99.4％	99.4％
Positive coincidence rate (500 cases)	100％	100％	100％

As can be seen from Table 7, the fusion protein prepared by using the prokaryotic expression system and the eukaryotic expression system can achieve better negative coincidence rate and positive coincidence rate of the sample.

Claims

A kit for detecting HIV antibodies, comprising:

a fusion protein of a labeling enzyme and an HIV antigenic protein;

wherein the antigenic protein comprises one or more of GP41 antigen, GP36 antigen and GP120 antigen, or a fusion antigen of two or more thereof.
The kit of claim 1, wherein the kit comprises a fusion protein of the enzyme for labeling and GP41 antigen and a fusion protein of the enzyme for labeling and GP36 antigen.
The kit of claim 2, wherein the kit further comprises a fusion protein of the labeling enzyme and GP120 antigen.
The kit according to claim 1, wherein the HIV antigenic protein comprises a fusion antigen of GP41 antigen and GP36 antigen or a fusion antigen of GP41 antigen, GP36 antigen and GP120 antigen.
The kit according to any one of claims 1 to 4, wherein the marker is fused to the N-terminus or C-terminus of the antigenic protein with an enzyme.
The kit according to any one of claims 1 to 5, wherein the labeling enzyme is alkaline phosphatase or horseradish peroxidase.
The kit of claim 6, wherein the alkaline phosphatase is fused to the antigenic protein at the N-terminus or C-terminus; alternatively, the horseradish peroxidase is fused to the N-terminus or C-terminus of the antigenic protein.
The kit according to claim 7, wherein the fusion protein is alkaline phosphatase, GP41 antigen and GP36 antigen in sequence from N-terminus to C-terminus; or GP41 antigen, GP36 antigen and alkaline phosphatase; or the fusion protein is composed of horseradish peroxidase, GP41 antigen and GP36 antigen in sequence from the N end to the C end; or GP41 antigen, GP36 antigen and horseradish peroxidase.
The kit according to any one of claims 1 to 8, wherein the kit further comprises:

a solid support coated with HIV antigenic proteins, wherein the antigenic proteins coated on the solid support comprise one or more of GP41 antigen, GP36 antigen and GP120 antigen, or two or more fusion antigens thereof, and the HIV antigenic proteins coated on the solid support and the HIV antigenic proteins in the fusion proteins can be combined with the same IgG antibody or the same IgM antibody in a sample; and/or

Solid supports coated with anti-human IgG and anti-human IgM antibodies.
The kit according to any one of claims 2 to 4, wherein the kit further comprises:

a solid support coated with a fusion antigen comprising GP41 antigen and GP36 antigen; or

A solid support coated with GP41 antigen and a solid support coated with GP36 antigen.
The kit of any one of claims 1 to 10, further comprising a blocking agent; preferably, the blocking agent is selected from one or more of the group consisting of: skimmed milk powder, BSA, gelatin, serum, casein, ovalbumin, animal IgG and surfactant.
The kit of any one of claims 1 to 11, further comprising a reducing agent; preferably, the reducing agent is selected from one or more of the group consisting of: DTT, beta-mercaptoethanol.
The kit of any one of claims 1 to 12, wherein the kit further comprises a reaction substrate for the labelling enzyme, preferably the reaction substrate is 3- (2-helical adamantane) -4-methoxy-4- (3-phospholyl) -phenyl-1, 2-dioxetane.
The kit of any one of claims 1 to 13, wherein the fusion protein is produced via the following expression techniques: prokaryotic expression technology or eukaryotic expression technology.
A method for detecting antibodies produced in a sample following infection with HIV comprising the steps of:

mixing the sample with the solid support coated with the first ligand such that the first ligand coated on the solid support is substantially bound to the antibodies to HIV in the sample;

washing the mixture to remove unbound substances;

adding an enzyme marker with a second ligand into the cleaned mixture, and uniformly mixing to ensure that the second ligand in the enzyme marker is combined with the HIV antibody combined on the solid support to form a sandwich compound, wherein the enzyme marker is a fusion protein of a marking enzyme and HIV antigenic protein;

washing the sandwich composite to remove unbound substances;

and adding a chemiluminescence substrate into the washed sandwich compound, and detecting the number of photons generated by the reaction to obtain a chemiluminescence signal value of the sample.
The method of claim 15, wherein said first ligand is an HIV antigenic protein, or an anti-human IgG antibody and an anti-human IgM antibody.