JP2011016773A - Anti-polyhistidine-tag antibody and method for avoiding nonspecific reaction by using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an agent for avoiding a nonspecific reaction, readily and effectively inhibiting the nonspecific reaction accompanied with measurement in an immunity measuring method, and providing more effective nonspecific reaction-inhibiting effects, and to provide a measuring method in which the nonspecific reaction is inhibited.SOLUTION: The method for avoiding the nonspecific reaction includes allowing an antibody having the nonspecific factor activity-inhibiting abilities, and unreactive with an analyte, especially an antibody against a polyhistidine-tag, of the agent for avoiding the nonspecific reaction for removing the influence caused by the specific interference by a heterophile antibody, for example, a human anti-mouse antibody (HAMA), to coexist in the reaction system in a method for determining the analyte by using the immunity measuring method.

Description

The present invention relates to a method for avoiding a non-specific reaction for suppressing a non-specific reaction caused by a heterophilic antibody such as a human anti-mouse antibody (HAMA) which is an obstacle for accurately detecting and quantifying an analyte in an immunoassay. And a non-specific reaction avoiding agent.

In general, an immunoassay method using a mouse monoclonal antibody is used to measure an analyte (measurement target) contained in a specimen such as blood or urine. However, in an immunoassay based on the specific binding of an antigen-antibody reaction, the reliability of the measurement value is often impaired due to a non-specific reaction other than the original specific antigen-antibody reaction. It recognized.

This phenomenon is caused when components other than the antigen contained in the specimen react with the labeled antibody. A heterophilic antibody such as a human anti-mouse antibody (hereinafter referred to as HAMA) contained in the specimen to be tested binds to a solid phase, for example, in a normal sandwich ELISA measurement, even in the absence of an analyte. Non-specific cross-linking between the detected antibody and the labeled detected antibody occurs, resulting in a false positive signal. While the automation of testing has progressed and rapid measurement has become possible, false reactions such as HAMA have increased, and this non-specific reaction is often overlooked. In this way, a non-specific reaction is observed in the immunoassay system, and a sample that is originally negative may be determined to be positive by reacting with a sample that does not contain the target analyte (Non-Patent Document 1, (Refer nonpatent literature 2.).

One of the reasons for the presence of HAMA in human-derived specimens is that it occurs because large amounts of mouse monoclonal antibodies are administered to patients as a treatment. In vivo administration of mouse antibodies produces HAMA, and raising the immune response against foreign antigens is a problem. In recent antibody drugs, chimeric antibodies fused with mouse-derived antigen-binding sites and human-derived constant regions, and patients with HAMA, although the appearance of HAMA in vivo has decreased due to advances in humanized antibody production technology Continues to grow and the problem cannot be completely ignored.

If the binding between the monoclonal antibody used for the measurement and HAMA is not prevented, an inaccurate measurement result may cause a serious problem such as a diagnostic error. Even if a monoclonal antibody having excellent specificity is used in an actual measurement system, it is a big problem that its performance cannot be fully utilized (see Patent Document 1).

Various attempts have been made in the past to obtain a correct measurement value that avoids the non-specific reaction described above.
For example, the sample to be measured is generally pretreated by heating or using an appropriate reagent, and various animal sera, immunoglobulin fractions, albumin, skim milk, surfactants, etc. are added to the measurement system. I have been. Further, by using an antibody fragment such as Fab or F (ab ′) ₂ for a specific reaction, a nonspecific reaction caused by the Fc portion of the antibody is also avoided (Patent Documents 2 and 3). reference).
In addition, a monoclonal antibody that has a different reaction specificity from the monoclonal antibody used in the measurement system and does not inhibit the reaction related to the measurement system is also added to the measurement system. For example, the use of aggregates derived from monoclonal or polyclonal antibodies has been proposed. The aggregate may be a homopolymer of the antibody or a heteropolymer of an antibody fragment or a protein such as albumin or a polysaccharide macromolecule such as dextran.

In addition, in order to suppress non-specific reactions, the monoclonal antibody used in the specific reaction was prepared by heat treatment, etc. The original antibody specific activity has been lost, but the non-specific reaction suppression activity is retained. A monoclonal antibody-derived substance is disclosed (see Patent Document 4).
However, although these methods have some effects on suppressing nonspecific reactions, the effects are still insufficient for some specimens, and some of the target antigen-antibody reactions may be partially inhibited. It was not always satisfactory.

Japanese Patent No. 2109086 JP 54-119292 A Japanese Patent Laid-Open No. 04-221762 Japanese Patent No. 2561134

Clin. Chem. 34/1, 261-264 (1988) Clin. Chem. 35/1, 146-151 (1989)

The present invention has been made against the background of such prior art problems. That is, an object of the present invention is to provide a non-specific reaction excellent in easily and effectively suppressing non-specific reactions associated with measurement in order to realize accurate detection and quantification of trace components in a sample in an immunoassay. The object is to provide an avoidance method and a non-specific reaction avoidance agent.

As a result of diligent studies to solve the above problems, the present inventors have found that an antibody against 6 to 18 continuous polyhistidine tags or a fragment protein thereof has an effect of suppressing nonspecific reaction by HAMA, The present invention has been completed.

A histidine tag consisting of 6 to 18 consecutive histidine residues is one of the most commonly used carrier proteins, and is expressed by being fused to the N-terminus or C-terminus of the target polypeptide. Using the property that histidine specifically coordinates and binds to transition metals such as nickel ions, nickel from a culture supernatant or lysate expressing histidine-tagged polypeptide in E. coli, yeast, animal cells, etc. -It is known that purification is possible using chelate chromatography columns.
Further, in order to detect and quantify the expression of the histidine fusion polypeptide quickly and specifically, it is measured by Western blot, ELISA or the like using a polyclonal antibody or a monoclonal antibody specific for the histidine tag portion. The base sequence of the anti-polyhistidine tag antibody gene has not been clarified.

The present inventors have prepared hybridoma cells that produce anti-polyhistidine tag antibodies, and have been able to obtain antibody genes from these cells. Surprisingly, when the non-specific reaction suppression effect by HAMA was evaluated for this antibody, the anti-polyhistidine-tagged antibody was affected by non-specific interference in the method of quantification using the immunoassay of the antigen. An advantageous effect of avoiding non-specific reaction, which can be expected to be significantly removed, can be obtained more than expected by those skilled in the art.

The present invention includes the following inventions in order to solve the above problems.
That is, the present invention is a protein having a binding property to a polyhistidine tag, specifically, a protein constituting an antibody or an antibody fragment. As an example thereof, protein fragments encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 1 which is DNA encoding the variable region of the heavy chain and SEQ ID NO: 2 which is DNA encoding the variable region of the light chain, these It is characterized by being an antibody molecule comprising the above protein fragment, or an antibody fragment comprising these protein fragments. In the case of an antibody molecule, its subclass is not particularly limited, but is preferably an antibody classified as IgG1. In the case of an antibody fragment, specifically, Fab, F (ab ′) ₂ , scFv and the like can be mentioned. The polyhistidine tag includes 6 to 18 consecutive histidine residues.

As another aspect of the present invention, in the method for quantifying an analyte using an immunoassay method, the above-described protein is used as a nonspecific reaction avoiding agent capable of removing the influence of nonspecific interference. The non-specific reaction avoiding method is characterized in that an antibody against a polyhistidine tag, for example, having the ability to inhibit non-specific factor activity and not reacting with an analyte, for example, is allowed to coexist in the reaction system.

That is, the present invention has the following configuration.
[Item 1] A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 1 and / or SEQ ID NO: 2.
[Item 2] An antibody comprising the polypeptide according to item 1.
[Claim 3] An antibody fragment comprising the polypeptide of Item 1.
[Item 4] The antibody fragment according to item 3, wherein the antibody fragment is Fab, F (ab ′) ₂ or scFv.
[Item 5] In the method for quantifying an analyte using an immunoassay, the method is a nonspecific reaction avoidance method for removing the influence of nonspecific interference, wherein the substance according to any one of Items 1 to 4 is reacted. A non-specific reaction avoidance method including a step of coexisting in a system
[Item 6] The nonspecific reaction avoidance method according to Item 5, wherein the immunoassay is a sandwich method.
[Item 7] A nonspecific reaction avoiding agent comprising the substance according to any one of Items 1 to 4.
[Item 8] An immunoassay kit comprising the nonspecific reaction avoiding agent according to Item 7.

According to the present invention, nonspecific reactions caused by heterophilic antibodies can be eliminated in immunoassays, so that specificity is improved and false positives are reduced, enabling highly reliable immunoassays in fields such as clinical tests. It becomes.

It is a figure which shows the result confirmed about the reactivity with the antigen of the anti- His-Tag antibody acquired by recombining to the CHO-K1 cell. It is a figure which shows the result measured about the HAMA avoidance ability of an anti- His-Tag antibody.

Hereinafter, the present invention will be described in detail. However, the scope of the present invention is not limited to these descriptions, and modifications other than the following examples can be made as appropriate without departing from the spirit of the present invention.

One of the embodiments of the present invention is a polypeptide encoded by the base sequences described in SEQ ID NOs: 1 and 2 (these polypeptides have the amino acid sequences described in SEQ ID NO: 3 and SEQ ID NO: 4, respectively), Alternatively, it is a substance such as an antibody molecule or antibody fragment containing them.
These substances have binding properties to the polyhistidine tag, and specifically constitute antibodies or antibody fragments. The polyhistidine tag of the antigen is exemplified by a tag consisting of 6 to 18 consecutive histidine residues. Preferably, it consists of 6 consecutive histidine residues and may be fused to the N-terminus and / or C-terminus of other proteins.

The base sequence of SEQ ID NO: 1 is DNA encoding the variable region of the heavy chain of an antibody against polyhistidine. The base sequence of SEQ ID NO: 2 is DNA encoding the variable region of the light chain of the antibody against polyhistidine.

The antibody molecule comprising the polypeptide encoded by the nucleotide sequences set forth in SEQ ID NOs: 1 and 2 is not particularly limited in its subclass, but is preferably an antibody classified as IgG1.
Specific examples of the antibody fragment containing the polypeptide encoded by the base sequences described in SEQ ID NOs: 1 and 2 include Fab, F (ab ′) 2, scFv and the like.

The polypeptide, antibody or antibody fragment of the present invention is produced by a method known to those skilled in the art, and may be an antibody or antibody fragment produced recombinantly, for example, by genetic engineering, and is an antibody against a polyhistidine tag. It also includes the case where an antibody is obtained by linking a gene to an expression vector and transfecting a mammalian cell. The antibody gene may be an antibody fragment (Fab fragment, F (ab ′) ₂ fragment, Fc fragment), each single chain antibody, or a single chain antibody (scFv). Two vectors containing separate heavy and light chains may be used, or a single vector in which the antibody heavy and light chains are connected in tandem.
Fab and F (ab ′) ₂ may be obtained by expressing the full length of the antibody and then decomposing with a proteolytic enzyme such as pepsin, trypsin or papain.

Mammalian cells (host cells) to be transfected are not particularly limited. For example, Chinese hamster ovary cells (for example, CHO-K1 cells, available from: ATCC CCL-61, RIKEN BioResource Center RCB0285, RIKEN BioResource Center RCB0403, etc.), human embryonic kidney cells (eg, HEK293 cells, source: ATCC CRL-1573, RIKEN Bioresource Center RCB1637, RIKEN Bioresource Center RCB2202, etc.), African green monkey kidney cells (eg, COS-7 cells, suppliers: eg, ATCC CRL-1651, RIKEN BioResource Center RCB0539, etc.), human cervical cancer cells (eg, HeLa) Cells, for example: ATCC CCL-2, ATCC CCL-2.2, RIKEN BioResource Center RCB0007, RIKEN BioResource Center RCB0191), mouse myeloma cells (for example, NS0 cells, sources: for example, RIKEN) Examples include various mammalian cells such as BioResource Center RCB0213).

Another embodiment of the present invention is a method for avoiding a non-specific reaction that eliminates the influence of non-specific interference in a method for quantifying an analyte using an immunoassay method, the method according to SEQ ID NOS: 1 and 2. A non-specific reaction avoidance method comprising a step of coexisting a polypeptide encoded by a base sequence or a substance such as an antibody molecule or antibody fragment containing them in a reaction system.

An immunoassay is a method for measuring a target analyte by utilizing the high specificity of binding between antigens and antibodies. In order to dramatically increase the sensitivity of this immunoassay, labeling methods such as radioactive compounds, fluorescent substances, enzymes, and metals can be combined to measure trace amounts of substances. They are called radioimmunoassay (RIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), etc. according to the labeling method.

The method of using the above-mentioned substance as a nonspecific reaction avoiding agent is not particularly limited, but the nonspecific reaction avoiding agent in an appropriate buffer in advance before the specific immune reaction step performed in the immunoassay. And a specimen are contacted in the same immune reaction and incubated for an appropriate time (for example, about 1 minute to 2 hours).
During this time, a substance that causes a nonspecific reaction (nonspecific reaction substance) in the specimen reacts with the nonspecific reaction avoiding agent, and the nonspecific reaction activity in the specific immune reaction step is lost. In the specific immune reaction step, a mixture of the sample that has been incubated and the nonspecific reaction avoiding agent may be used as it is.

A simpler method of using the present invention as a non-specific reaction avoiding agent of the present invention is a method of performing a specific immune reaction in the presence of a non-specific reaction avoiding agent.
For example, in the case of the most commonly used positive sandwich method, a nonspecific reaction avoiding agent is simply added to the buffer of the first immune reaction step in which the immobilized antibody and the antigen in the sample are reacted. The desired effect can be obtained without requiring any special process.
Furthermore, if it is also added to the buffer solution of the second immune reaction step in which the antigen immobilized through the specific reaction site of the antibody reacts with the labeled antibody, non-specificity that was not completely absorbed in the first immune reaction step Since the reactant is also absorbed, the measurement reliability can be further enhanced.
Usually, a sufficient effect can be obtained if it is added to the buffer solution of the first immune reaction step.

In the one-step sandwich method, a nonspecific reaction can be avoided by adding a nonspecific reaction avoiding agent to the buffer of the immune reaction process and causing the reaction.
The nonspecific reaction avoiding agent of the present invention can also increase the binding force with a substance that causes a nonspecific reaction by allowing at least two kinds of antibodies having different subclasses to coexist.
In addition, if an excessive amount is added, a substance that causes a nonspecific reaction preferentially reacts with the nonspecific reaction avoiding agent, so that the nonspecific reaction is avoided.

Another embodiment of the present invention is a non-specific reaction avoidance agent comprising a substance encoded by the nucleotide sequences set forth in SEQ ID NOs: 1 and 2, or an antibody molecule or antibody fragment containing them.
Moreover, it is an immunoassay kit containing this nonspecific reaction avoidance agent.

The nonspecific reaction avoiding agent of the present invention is used by adding to an appropriate buffer in advance before the immune reaction step, or by adding to a buffer for the immune reaction step.
The concentration of the non-specific reaction avoiding agent in the system for reacting with the non-specific reactant is preferably 0.1 to 500 μg / ml, more preferably 1 to 50 μg / ml. The buffer used may be a known appropriate buffer used for normal immune reactions. Further, it can be used together with an auxiliary agent usually added to the buffer, for example, a reaction accelerator, a detergent or a stabilizer. Furthermore, it can also be used by mixing with another nonspecific reaction avoiding agent.

The nonspecific reaction avoiding agent of the present invention may be used alone as a component reagent of a kit, or may be a component of another component reagent. However, it is preferable to add it as one component of the constituent reagent in consideration of the effect of suppressing the nonspecific reaction without increasing the measurement operation.
The non-specific reaction avoiding agent of the present invention is preferably added to a buffer solution or a labeled antibody solution for reacting a specimen with a solid-phased antibody to form a kit constituent reagent. As the medium for suspending the nonspecific reaction avoiding agent, various buffer solutions according to the type of the substance to be measured, the measurement principle used, and the measurement method are used. Any buffer solution may be used as long as it has an ion concentration or pH that does not inactivate the measurement substance and does not inhibit the antigen-antibody reaction. For example, phosphate buffer (10 to 100 mM; pH 6 ~ 8) or Tris-hydrochloric acid (50 mM / NaCl 100 mM; pH 7-8) or the like can be used.

Examples of reaction accelerators include dextran sulfate or polyethylene glycol. Examples of detergents include Triton X-100 and Tween 20. Examples of stabilizers include proteins such as albumin, skim milk, and gelatin, sodium azide, thimerosal, and caisson. Preservatives such as CG can be mentioned.
As the labeled antibody, an antibody to which a substance that produces a signal that can be quantified by any means such as an enzyme such as horseradish peroxidase or alkaline phosphatase, a radioactive substance, or a fluorescent substance can be typically used. When these constituent reagents are freeze-dried products, they can be added to the reconstitution liquid.

Examples of the specimen used in the immunoassay using the nonspecific reaction avoiding agent of the present invention include body fluids such as serum, plasma, spinal fluid and saliva, urine, fecal extract and the like.
There are no particular restrictions on the analyte that can be measured, but substances used for clinical examination, for example, in vivo substances contained in body fluids and disease markers such as human immunoglobulin, human albumin, human fibrinogen, β ₂ -Microglobulin, ferritin, C-reactive protein, α-fetoprotein, carcinoembryonic antigen, CA19-9, basic fetoprotein, tissue polypeptide antigen, immunosuppressive acidic protein, CA-50, pancreatic carcinoembryonic antigen, sialyl Le ^x- i antigen, SCC antigen, CA15-3, CA72-4, sialyl Tn antigen, CA125, NCC-ST-439, gamma-seminoprotein, prostate specific antigen, thyroid stimulating hormone, neuron specific enolase, hepatitis virus antigen, Human chorionic gonadotropin, human placental lactogen, a For example.
Examples of immunoassay include radioimmunoassay (RIA), enzyme immunoassay (EIA), and fluorescent immunoassay (FIA). Furthermore, chemiluminescence, latex agglutination, etc. included. The sandwich method is preferably used in most of the measurement kits by RIA, EIA, etc., and includes a positive sandwich method, a one-step sandwich method, a solid phase sandwich method, and the like. More preferably, a solid phase sandwich method is mentioned. In the sandwich method, an antigen-specific unlabeled antibody (primary antibody) bound to a solid phase is reacted with an antigen, and then a labeled secondary antibody is bound to a complex of the antigen and the primary antibody to detect the antigen. Method, and generally high antigen specificity is an advantage.

Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited thereto.

6x histidine tag (histidine tag consisting of 6 consecutive histidine residues) fused sarcosine-degrading enzyme is used as an antigen to immunize Balb / cA mice, and mouse B cells are removed and fused with mouse myeloma cells SP2 / 0. Thereafter, hybridoma cells producing a monoclonal antibody specific for the 6 × histidine tag were screened by ELISA. In addition, Freund's complete adjuvant (manufactured by DIFCO, product number: 0638-60-7) was used for the first immunization, and Freund's incomplete adjuvant (manufactured by DIFCO, product number: 0639-60-6) was used for the second and subsequent boosters. . The obtained hybridoma cells were cultured in CD Hybridoma Medium (GIBCO, product number: 11279-023), and total RNA was extracted from 1 × 10 ⁶ cells using RNeasy Plus Mini Kit (product of QIAGEN, product number: 74134). Messenger RNA was extracted with Poly (A) Purist mRNA Purification Kits (Ambion, product number: AM1916). Further, single-stranded cDNA was synthesized using RiverTra Ace-α- (registered trademark; manufactured by Toyobo Co., Ltd., product number: FSK-101).
Using Mouse Ig-Primer Set (manufactured by Novagen, product number: 69831-3), the heavy chain and the light chain were PCR amplified with KOD-Plus- (manufactured by Toyobo, product number: KOD-201), and then the CMV promoter was EF The sequence was confirmed by cloning into the restriction enzyme XbaI-NotI sites of the pCI-neo plasmid (Promega, product number: E1841) changed to the −1α promoter. The base sequence of DNA encoding the variable region of the heavy chain is shown in SEQ ID NO: 1, and the base sequence of DNA encoding the variable region of the light chain is shown in SEQ ID NO: 2, respectively. These polypeptides have the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Conversion to an amino acid sequence was performed using Genetyx (Geneticx), which is sequence analysis software. Thereby, the gene sequence of this antibody was identified.

The expression vector prepared in Example 1 was transfected into CHO-K1 cells (RIKEN BioResource Center RCB0285) using Lipofectamine LTX (manufactured by Invitrogen, product number: 15338-100). After culturing for 24 hours, the culture supernatant was collected, and the reactivity with 6 × histidine tag antigen was confirmed by ELISA measurement.
Specifically, culture supernatants diluted to 10 ng / ml, 50 ng / ml, and 100 ng / ml, respectively, on an ELISA plate on which an antigen having a concentration of 10 μg / ml was immobilized (mouse antibody was used as the antibody concentration in the supernatant). (Measured by sandwich ELISA.) 50 μl was added and incubated at 35 ° C. for 2 hours. Next, 50 μl of anti-mouse antibody-HRP (manufactured by DAKO), which was washed 3 times with PBS-T (10 mM phosphate buffered saline containing 0.1% Tween 20) and diluted 4000 times, was added at 35 ° C. for 1 hour. After the incubation, the plate was further washed three times with PBS-T and reacted with TMB + (3,3 ′, 5,5′-tetramethylbenzidine; manufactured by DAKO) for 5 minutes. After stopping the reaction by adding 50 μl of 1N sulfuric acid, the absorbance was measured with a plate reader (product name: SPECTRA, manufactured by TECAN Australia) (main wavelength: 450 nm, subwavelength: 620 nm). As a standard, an antibody obtained from a mouse hybridoma cell that produces an antibody specific for a polyhistidine tag was used.
The results are shown in FIG.

As a result, it was confirmed that the antibody was reactive with the 6 × histidine tag antigen in the same manner as the antibody derived from the hybridoma cell, and it was proved that the obtained antibody gene was the target antibody gene.

An anti-polyhistidine tag antibody expression vector was transfected into CHO-K1 cells (RIKEN BioResource Center RCB0285) using Lipofectamine LTX (manufactured by Invitrogen, product number: 15338-100), cultured for 1 day, and then 400 μg / ml. A cell population obtained by performing drug selection culture for 2 weeks in Ham's F-12 medium (manufactured by Nacalai Tesque, product number: 17458-65) containing G418 disulfate solution (manufactured by Nacalai Tesque, product number: 16513-84) By cloning by the limiting dilution method, a single recombinant CHO-K1 cell producing an antibody specific for the polyhistidine tag was constructed. Confirm that the effect of HAMA can be suppressed by positive sandwich ELISA using antibodies purified by HiTrap MabSelect SuRe (manufactured by GE Healthcare, product number: 11-0034-93), which is a protein A column. did.
MAK33-IgG1 (manufactured by Roche, product number: 1200941), mouse IgG (manufactured by CHEMICON, product number: PP54), Heterophilic Blocking Reagent 1 (hereinafter referred to as HBR) (manufactured by SCANTIBODIES LABORATORY, INC., Product number: 3KC534-073 The HAMA interference removal effect was measured using five types of antibodies obtained from mouse hybridoma cells producing a specific antibody and recombinant CHO-K1 cells. As HAMA serum, HAMA serum type 1 (Roche, product number: 1767275) was used.
More specifically, an Anti-hCEA antibody (manufactured by Medix Biochemica) diluted 2000 times with 50 mM carbonate buffer (pH 9.6) was added to a 96-well ELISA plate (manufactured by Sumitomo Bakelite, product number: MS-8896F) at 50 μl / well. And then solidified. In addition, antibody solutions for HAMA suppression diluted with 0.1% BSA + PBS solution were prepared so that the anti-polyhistidine tag antibody concentrations were 0.5 μg / ml and 1 μg / ml. Next, 50 μl of the HAMA serum was mixed with 50 μl of the antibody solution, each 50 μl / well was dispensed into the 96-well ELISA plate, and incubated at room temperature for 1 hour. Then, after washing 3 times with PBS-T and sufficiently removing water, Anti-hCEA antibody labeled with peroxidase was diluted 4000 times with 0.1% BSA + PBS solution, added 50 μl / well, and incubated at room temperature for 1 hour did. After washing 4 times with PBS-T and draining water, 50 μl / well of TMB + color developing solution (manufactured by DAKO) was dispensed, reacted at room temperature for 5 minutes, and stopped with 1N sulfuric acid. The HAMA avoidance rate was calculated by measurement with a plate reader (product name: SPECTRA, manufactured by TECAN Austria) (main wavelength: 450 nm, subwavelength: 620 nm).
The results are shown in FIG.

With the anti-polyhistidine tag antibodies derived from hybridoma and recombinant CHO-K1 cells, almost the same HAMA avoidance effect was confirmed. A mouse IgG antibody (manufactured by CHEMICON, product number: PP54), which is a normal anti-mouse antibody, has a HAMA avoidance effect of 1 μg / ml of 61%, whereas the anti-polyhistidine tag antibody derived from recombinant CHO-K1 cells HAMA avoidance rate is as high as 72%, compared with 69% of commercially available HAMA avoidant MAK33-IgG1 (Roche, product number: 1200941) and 68% of HBR (SCANTIBODIES LABORATORY, INC., Product number: 3KC534-075) However, an excellent result was obtained that there was an equivalent avoidance effect. It was confirmed that the HAMA avoidance effect of the anti-polyhistidine tag antibody is extremely effective. Since the antibody used for the evaluation is the same mouse antibody and the constant region is considered to be the same, the difference in the variable region including the CDR region (complementary determining region) contributing to the reactivity with the antigen is avoided by HAMA. It is suggested that it affects the effect.

According to the present invention, non-specific reactions can be eliminated in immunoassays, and therefore, non-specific reactions caused by non-specific factors can be suppressed for biological samples containing non-specific factors, and antigens can be accurately measured, Reduction of false positives and improvement of specificity are achieved, and more reliable clinical testing is possible, which is expected to make a significant contribution to the industry.

Claims (8)

A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 1 and / or SEQ ID NO: 2. An antibody comprising the polypeptide of claim 1. An antibody fragment comprising the polypeptide of claim 1. The antibody fragment according to claim 3, wherein the antibody fragment is Fab, F (ab ') ₂ or scFv. A method for quantifying an analyte using an immunoassay method, a nonspecific reaction avoidance method for removing the influence of nonspecific interference, wherein the substance according to any one of claims 1 to 4 is contained in a reaction system. A non-specific reaction avoidance method including a step of coexistence. 6. The nonspecific reaction avoidance method according to claim 5, wherein the immunoassay is a sandwich method. The nonspecific reaction avoidance agent containing the substance in any one of Claims 1-4. An immunoassay kit comprising the nonspecific reaction avoiding agent according to claim 7.