JP2024026911A - Method for measuring target antigen, and insoluble particles and target antigen measurement kit used therein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an insoluble particle that demonstrates excellent agglutination capabilities in an antigen-antibody reaction.

SOLUTION: An insoluble particle includes a granular carrier such as a latex particle, and an antibody such as IgG, carried on the granular carrier and including an Fc region having a specific amino acid sequence, wherein the C-terminal amino acid of the heavy chain of the antibody is lysine. There are also provided a target antigen measurement kit including the insoluble particle or an antibody and a granular carrier for preparing the insoluble particle, as well as a method for measuring a target antigen using the insoluble particle.

SELECTED DRAWING: None

COPYRIGHT: (C)2024,JPO&INPIT

Description

The present invention relates to a method for measuring a target antigen, insoluble particles used therefor, and a kit for measuring the target antigen.

As a method for measuring a target antigen using an immune reaction, there is a method using a particulate carrier such as latex. A particulate carrier to which an antibody specific to a target antigen is bound causes an antigen-antibody reaction when the target antigen is present. Then, due to their specificity and affinity, they bind to each other by bridging the target antigens and aggregate. The presence or absence of the target antigen and the amount present are measured based on the presence or absence of the generated aggregate and the degree of aggregation.

Patent Document 1 discloses a peptide that contains a specific amino acid sequence and exhibits a specific adsorption function to a solid phase on the surface of a hydrophilized resin. Methods of binding antibodies have been proposed.

Patent No. 5553336

In the method of Patent Document 1, since an amino acid sequence that does not originally exist is added to the antibody, there is a possibility that the antibody will have undesirable characteristics such as a non-specific reaction and a decrease in the expression level of the antibody. One of the objects of the present invention is to provide a method for measuring a target antigen that does not cause such problems, and also to provide insoluble particles that can be used in such a measuring method.

Usually, the C-terminal lysine of the heavy chain of an antibody is removed by post-translational modification by carboxypeptidase, so the C-terminal lysine of the heavy chain of commonly used antibodies is removed. The present inventors have demonstrated that the presence of lysine at the C-terminus of the antibody heavy chain increases the amount of antibody binding to insoluble particles, resulting in superior aggregation ability of insoluble particles when an antigen-antibody reaction occurs. This discovery led to the completion of the present invention.

One aspect of the present invention is an insoluble particle containing a particulate carrier and an antibody against a target antigen supported on the particulate carrier, wherein the C-terminal amino acid of the heavy chain of the antibody is lysine.

The insoluble particles may be latex particles.

The antibody can be an IgG. Further, the Fc region of IgG may be a polypeptide having an amino acid sequence having 90% or more identity with any one of SEQ ID NOs: 1 to 13, and the C-terminal amino acid is lysine; The Fc region of may be a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 13.

Another aspect of the present invention is a kit for measuring a target antigen, which includes the above-mentioned insoluble particles, or includes an antibody and a particulate carrier for preparing the above-mentioned insoluble particles.

The above kit can be a kit for qualitatively evaluating the presence or absence of a target antigen in a test sample, or for quantitatively evaluating the concentration of a target antigen in a test sample.

Another aspect of the present invention is a method for measuring a target antigen using the above-mentioned insoluble particles.

The measurement method may include the steps of contacting the insoluble particles with a test sample that may contain a target antigen, and measuring an agglutination reaction of the insoluble particles due to an antigen-antibody reaction between the antibody and the target antigen. .

The above measurement method can be a method for qualitatively evaluating the presence or absence of a target antigen in a test sample, or a method for quantitatively evaluating the concentration of a target antigen in a test sample.

According to the present invention, insoluble particles with excellent aggregation ability when an antigen-antibody reaction occurs can be provided. By using insoluble particles with excellent aggregation ability, highly sensitive detection of target antigens can be expected.

It is a graph showing the results of agglutination reaction of anti-IgE antibody-sensitized latex.

Embodiments of the present invention will be described in detail below.

The insoluble particles according to one embodiment contain a particulate carrier and an antibody against a target antigen supported on the particulate carrier, and the C-terminal amino acid of the heavy chain of the antibody is lysine. Antibodies in which the C-terminal amino acid of the heavy chain is lysine have a high ability to bind to particulate carriers. Furthermore, insoluble particles containing an antibody in which the C-terminal amino acid of the heavy chain is lysine have excellent aggregation ability when an antigen-antibody reaction occurs.

Target antigens include, for example, protein markers such as CRP (C-reactive protein), prostate-specific antigen, ferritin, β-2 microglobulin, myoglobin, hemoglobin, albumin, and creatinine; immunoglobulins such as IgG, IgE, IgA, and IgM; Various tumor markers, lipoproteins such as LDL, HDL, and TG, influenza A virus, influenza B virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, norovirus, adenovirus, astrovirus, HAV, HBs, HCV, Viral antigens such as HIV and EBV, bacterial antigens such as Chlamydia trachomatis, streptococcus, Bordetella pertussis, Helicobacter pylori, Leptospira, Treponema pallidum, Toxoplasma gondii, Borrelia, Legionella, anthrax, MRSA, etc. Toxins produced, mycoplasma lipid antigens, peptide hormones such as human chorionic gonadotropin, steroids such as steroid hormones, physiologically active amines such as epinephrine and morphine, vitamins such as vitamin B, prostaglandins, and antibiotics such as tetracycline. Examples include, but are not limited to, substances, pesticides, environmental hormones, etc.

Examples of the particulate carrier include particles such as latex particles, ceramic particles, alumina particles, silica-alumina particles, and carbon black particles. Among these particles, latex particles are preferred. Examples of the material of the latex include polystyrene, divinylbenzene, etc., and polystyrene is preferable. The average particle size of the particulate carrier can be 0.1 to 5 μm. Note that the particle size of the granular carrier can be measured by dynamic light scattering. In this specification, "average particle size" refers to the particle size when the integrated value from small particle sizes reaches 50% of the total in a volume-based particle size distribution curve obtained by dynamic light scattering. (median diameter).

The antibody is not particularly limited as long as it can specifically bind to the target antigen, and may be, for example, IgG. IgG is composed of two heavy chains (H chains) and two light chains (L chains). The heavy chain is composed of, in order from the N-terminus, a variable region (VH), a first constant region (CH1), a second constant region (CH2), and a third constant region (CH3). The light chain is composed of a variable region (VL) and a constant region (CL) in order from the N-terminus. The portion composed of CH2 and CH3 is the Fc region. One heavy chain and one light chain are linked by a disulfide bond between cysteine residues present in CH1 and cysteine residues present in CL. Further, heavy chains are bonded to each other by disulfide bonds between cysteine residues present in the hinge region located between CH1 and CH2. The antibody may be a fragment of IgG in which a heavy chain is present, or it may be rIgG (reduced IgG) composed of one heavy chain and one light chain, or it may be a fragment of IgG that has a heavy chain. It may be a fragment consisting of a single heavy chain, or a fragment consisting of a single heavy chain. In an antibody having two heavy chains, the C-terminus of at least one heavy chain may be lysine, and the C-terminus of both heavy chains may be lysine.

When the antibody is IgG, its Fc region is a polypeptide having an amino acid sequence having 90% or more identity with any one of SEQ ID NOs: 1 to 13, and whose C-terminal amino acid is lysine. More preferably, it is a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 13. A polypeptide having an amino acid sequence that has 90% or more identity with any one of SEQ ID NOs: 1 to 13 and whose C-terminal amino acid is lysine preferably has an effector function. A polypeptide in which the effector function of the Fc region is maintained also has a high binding efficiency to insoluble particles.

Here, SEQ ID NO: 1 is Protein Accession No. P01857 (human Ig gamma-1 C region), SEQ ID NO: 2 is Protein Accession No. P01859 (human Ig gamma-2 C region), and SEQ ID NO: 3 is is Protein Accession No. P01860 (human Ig gamma-3 C region), SEQ ID NO: 4 is Protein Accession No. P01861 (human Ig gamma-4 C region), and SEQ ID NO: 5 is Protein Accession No. P01868 (mouse Ig gamma-1 C region), SEQ ID NO. 6 is Protein Accession No. P01863 (mouse Ig gamma-2a C region), and SEQ ID NO. 7 is Protein Accession No. P01867 (mouse Ig gamma- 2b C region), SEQ ID NO: 8 is Protein Accession No. P03987 (mouse Ig gamma-3 C region), and SEQ ID NO: 9 is Protein Accession No. P20759 (rat Ig gamma-1 C region). , SEQ ID NO: 10 is Protein Accession No. P20760 (rat Ig gamma-2a C region), SEQ ID NO: 11 is Protein Accession No. P20761 (rat Ig gamma-2b C region), and SEQ ID NO: 12 is Protein Accession No. P20762 (rat Ig gamma-2c C region), and SEQ ID NO: 13 is Protein Accession No. P01870 (rabbit Ig gamma C region).

Antibodies in which the C-terminal amino acid of the heavy chain is lysine can be produced using genetically modified plants. Antibodies produced using genetically modified plants have the characteristic that the C-terminal lysine of the heavy chain is unlikely to be deleted. Such antibodies can be produced using a plant transient expression system, and for example, the method described in Japanese Patent No. 5015012 can be used. Antibodies produced by such a method are not susceptible to post-translational modification by carboxypeptidase, so it is easy to obtain antibodies in which the C-terminal lysine of the heavy chain is maintained. Furthermore, an antibody in which the C-terminal amino acid of the heavy chain is lysine can also be obtained as a genetically recombinant antibody using mammalian cells deficient in carboxypeptidase as an expression host. Examples of mammalian cells in this case include, but are not limited to, CHO (Chinese Hamster Ovary) cells and HEK293 (Human Embryonic Kidney cells 293) cells. Furthermore, regarding methods for obtaining genetic recombinants, there are transient expression systems that use plasmid vectors or viral vectors that lack autonomous replication ability, and semi-transient expression systems that use episomal vectors that are endowed with nuclear import signals and have autonomous replication ability. -stable expression systems, stable expression systems in which a target gene is inserted into the genome of an expression host, etc., and are not particularly limited.

The particulate carrier and the antibody whose C-terminal amino acid of the heavy chain is lysine can be bound by common techniques such as physical adsorption and chemical bonding.

A method for measuring a target antigen according to one embodiment uses insoluble particles that contain a particulate carrier and an antibody against the target antigen supported on the particulate carrier, and in which the C-terminal amino acid of the heavy chain of the antibody is lysine. Such a measurement method can be performed to qualitatively evaluate the presence or absence of a target antigen in a test sample, or to quantitatively evaluate the concentration of a target antigen in a test sample.

In one embodiment, the above measurement method comprises a particulate carrier and an antibody against the target antigen supported on the particulate carrier, and the insoluble particles in which the C-terminal amino acid of the heavy chain of the antibody is lysine and the target antigen. and a step of measuring an agglutination reaction of the insoluble particles due to an antigen-antibody reaction between the antibody and the target antigen. The above-mentioned insoluble particles may be prepared already, or may be prepared by binding a particulate carrier to an antibody whose C-terminal amino acid of the heavy chain is lysine at the time of measurement.

Mixing a test sample with a suspension containing a granular carrier and insoluble particles that contain an antibody against a target antigen supported on the granular carrier, and in which the C-terminal amino acid of the heavy chain of the antibody is lysine. Then, the insoluble particles can be brought into contact with the test sample that may contain the target antigen. When the two are brought into contact, the insoluble particles aggregate due to the interaction between the target antigen contained in the test sample and the antibody contained in the insoluble particles, and the absorbance of the suspension changes. The amount of change (end point method) or rate of change (rate method) in this absorbance is measured. For the measurement, turbidimetry or colorimetry is preferably used. For example, by irradiating light from the outside of the cell with light in the range from visible light to near-infrared light, usually 300 nm to 1000 nm, preferably 500 nm to 900 nm, and detecting changes in absorbance or intensity changes in scattered light, the aggregation reaction of the insoluble particles is carried out. is measured.

The time for performing the aggregation reaction can be 1 minute to 30 minutes, preferably 1 minute to 10 minutes, but is not limited thereto. The temperature at which the aggregation reaction is carried out can be from 35°C to 40°C, and can also be from 36 to 38°C, but is not limited thereto.

A plurality of standard samples containing the target antigen to be measured at various known concentrations are prepared, and the amount or rate of change in absorbance of these samples is measured by the above method. A calibration curve is drawn by plotting the concentration of the antigen to be measured in the standard sample on the horizontal axis and the amount or rate of change in the measured absorbance on the vertical axis. By measuring the amount or rate of change in absorbance of an unknown test sample using the same method and applying the measurement results to the above-mentioned calibration curve, the target antigen in the test sample can be quantitatively evaluated. Further, by setting a threshold value for the amount of change or rate of change in absorbance in advance, it is possible to qualitatively evaluate that the target antigen is present in the test sample when the threshold value is exceeded.

The test sample is not particularly limited as long as it can contain the target antigen, but includes body fluids such as blood, serum, plasma, urine, feces, saliva, tissue fluid, spinal fluid, swab fluid, etc., or diluted products thereof, Blood, serum, plasma, urine, stool, spinal fluid or dilutions thereof are preferred.

A kit for measuring a target antigen according to one embodiment includes a particulate carrier and an antibody against the target antigen supported on the particulate carrier, and includes insoluble particles in which the C-terminal amino acid of the heavy chain of the antibody is lysine. . The target antigen measurement kit can be used to qualitatively evaluate the presence or absence of a target antigen in a test sample, or to quantitatively evaluate the concentration of a target antigen in a test sample. can be used in the method for measuring the target antigen described above.

In one embodiment, the target antigen measurement kit includes an antibody for preparing the above-mentioned insoluble particles and a particulate carrier. In the kit for measuring a target antigen according to this embodiment, the insoluble particles are prepared by binding an antibody and a particulate carrier at the time of measurement.

The target antigen measurement kit may further include a positive control or a target antigen for use in preparing a calibration curve, a buffer for diluting the test sample, and a buffer for mixing the insoluble particles and the test sample. , a buffer for binding the antibody and the particulate carrier, and the like.

1. Preparation of anti-IgE antibody An antibody whose heavy chain constant region consists of SEQ ID NO: 5 was prepared by the following method.

1.1. Preparation of anti-IgE antibody lacking C-terminal lysine of heavy chain (Anti-IgE Ab-K0) Anti-IgE antibody-producing hybridomas were intraperitoneally administered to mice, and ascitic fluid was collected several to several tens of days later. Impurities were removed by subjecting the ascites to Protein A column chromatography.

1.2. Preparation of an anti-IgE antibody (Anti-IgE Ab-K67) lacking the C-terminal lysine of the heavy chain It was prepared using a plant transient expression system described in Japanese Patent No. 5015012. The light chain of the anti-IgE antibody was cloned into a tobacco mosaic virus (TMV) vector (Icon Genetics), and each heavy chain (including CH3-deficient or CH2-deficient) was cloned into a potato virus X (PVX) vector (Icon Genetics). was cloned into (manufactured by). After each of these vectors was introduced into separate Agrobacterium cells, transformed and cultured, a mixture of the culture solutions was infiltrated into Nicotiana benthamiana leaves, and harvested in about one week. The harvested leaves (infected leaves) were frozen.

200 g of frozen infected leaves were weighed out and ground with a cutter mixer. 500 mL of an extraction solution (100mM Tris, 250mM NaCl, 40mM sodium ascorbate) was added to the ground material, and the infected leaves were ground repeatedly. The supernatant was collected by centrifugation. The collected liquid was concentrated by ammonium sulfate precipitation, and then filtered through a 0.22 μm filter. Pure substances were removed by subjecting the filtrate to Protein A column chromatography.

2. Analysis of anti-IgE antibody heavy chain C-terminal lysine The purified anti-IgE antibody was subjected to reductive alkylation and enzymatic digestion (Trpsin/Lys-C), and the amino acid sequence of the protein was analyzed by LC-MS. The anti-IgE antibody (Anti-IgE Ab-K0) prepared by the mouse ascites method completely lacked lysine at the C-terminus of the heavy chain. On the other hand, only 33% of the entire heavy chain C-terminal lysine of the anti-IgE antibody (Anti-IgE Ab-K67) prepared using a plant transient expression system was observed to be deleted.

3. Latex agglutination test of anti-IgE antibody The binding efficiency of the obtained antibody to latex was evaluated according to the following procedure.

(1) Preparation of reagent A reagent for measurement by immunoagglutination method was prepared as follows using an anti-IgE antibody.
i) Sensitized particles carrying 0.1 mg of antibody per 1 mL of polystyrene latex suspension are suspended in a buffer solution (glycine, pH 7.3) to a concentration of 0.1% to prepare a latex suspension. did.
ii) A buffer solution (glycine, pH 8.3) was prepared.

(2) Measurement using an automatic analyzer Automatic measurements were performed using an end point method using a 7180 model automatic analyzer manufactured by Hitachi. The antigen (IgE) at each concentration was measured using the reagent prepared as described above. 140 μL of the buffer solution prepared in (1) above was added to 3.5 μL of the IgE solution, and the mixture was stirred and mixed at 37°C. After standing for 5 minutes, 70 μL of latex suspension was added, and the mixture was further stirred and mixed at 37°C. The agglutination reaction for about 5 minutes was measured as a change in absorbance.

Table 1 shows the results when the preparation conditions (amounts of antibody and latex) were varied.

As is clear from the results shown in Table 1, the antibody that is not deficient in the C-terminal lysine of the antibody heavy chain (Anti-IgE Ab-K67) is different from the antibody that is deficient in the C-terminal lysine (Anti-IgE Ab- It was confirmed that it binds to latex more efficiently than K0).

Figure 1 shows the results of evaluating the degree of agglutination due to reaction with antigen using sensitized latex of Anti-IgE Ab-K0 and Anti-IgE Ab-K67 prepared under the same conditions (preparation conditions 1 and 2 in Table 1, respectively). Shown below. As is clear from the results shown in Figure 1, the latex carrying an antibody (Anti-IgE Ab-K67) that does not lack the C-terminal lysine of the antibody heavy chain does not support the antibody that lacks the C-terminal lysine (Anti-IgE Ab-K67). -IgE Ab-K0), it was confirmed that the degree of aggregation was greater than that of latex supporting IgE Ab-K0), and that highly sensitive measurement was possible.

Claims (10)

An insoluble particle comprising a particulate carrier and an antibody against a target antigen supported on the particulate carrier, wherein the C-terminal amino acid of the heavy chain of the antibody is lysine. Insoluble particles according to claim 1, wherein the particulate carrier is a latex particle. The insoluble particle according to claim 1 or 2, wherein the antibody is IgG. The insoluble particle according to claim 3, wherein the IgG Fc region has an amino acid sequence having 90% or more identity with any one of SEQ ID NOS: 1 to 13, and the C-terminal amino acid is lysine. . The insoluble particle according to claim 3, wherein the IgG Fc region consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 13. A method for measuring a target antigen, comprising the insoluble particles according to any one of claims 1 to 5, or comprising an antibody and a particulate carrier for preparing the insoluble particles according to any one of claims 1 to 5. kit. The kit according to claim 6, which is used for qualitatively evaluating the presence or absence of a target antigen in a test sample, or for quantitatively evaluating the concentration of a target antigen in a test sample. A method for measuring a target antigen using the insoluble particles according to any one of claims 1 to 5. A step of contacting the insoluble particles according to any one of claims 1 to 5 with a test sample that may contain a target antigen, and measuring an agglutination reaction of the insoluble particles due to an antigen-antibody reaction between the antibody and the target antigen. process,
A method for measuring a target antigen, including: 10. The method according to claim 8 or 9, for qualitatively evaluating the presence or absence of a target antigen in a test sample, or quantitatively evaluating the concentration of a target antigen in a test sample.