JP2010254663A - Anti-koddna polymerase antibody and method for inhibiting non-specific reaction by using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a non-specific reaction inhibitor effective for easily and effectively inhibiting a non-specific reaction accompanying to an immunoassay method and attaining better assayed result, and to provide an assaying method inhibiting non-specific reactions. <P>SOLUTION: There are provided the non-specific reaction inhibitor to eliminate influence of non-specific interference with a heterophile antibody such as human anti-mouse antibody (HAMA) in quantitative determination of an analyte by immunoassay, and the method for inhibiting non-specific reactions characterized by coexistence of an antibody having a function to inhibit non-specific factor activity and non-reactive with the analyte, especially an antibody to a DNA polymerase and derived from Thermococcus kodakaraensis KOD1 strain in the reaction system. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a heterophilic antibody such as a human anti-mouse antibody (hereinafter sometimes referred to as “HAMA”) which is an obstacle for accurately detecting and quantifying an analyte (measurement target) used in an immunoassay. The present invention relates to a non-specific reaction suppression method and a non-specific reaction inhibitor for suppressing a non-specific reaction due to.

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

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, contained in the specimen to be tested labeled the antibody bound to the solid phase, for example in a normal sandwich ELISA measurement, despite the absence of analyte. Non-specific cross-linking with the detected antibody occurs resulting in a false positive signal. Thus, a non-specific reaction was observed in the immunoassay system, and a sample that was originally negative was sometimes determined to be positive by reacting with a sample that did not contain the target analyte. (See Non-Patent Document 1) 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 has often been overlooked.

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 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 and Non-Patent Documents 1 and 2).

Various attempts have been made in the past in order to obtain a correct measurement value that suppresses the above-described non-specific reaction.
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. In addition, the use of Fab or F (ab ') ₂ antibody fragments, such as the specific reaction, is also made possible to suppress the non-specific reaction caused by the Fc portion of antibodies (see Patent Documents 2 and 3) .
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 Publication No. 8-23560 JP 54-119292 A Japanese Patent Laid-Open No. 04-221762 Japanese Patent No. 2561134 Japanese Patent No. 3132624 Japanese Patent No. 3968606

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. It is in providing the suppression method and its nonspecific reaction inhibitor.

The inventors of the present invention have made extensive studies to solve the above problems. As a result, antibodies against a DNA polymerase derived from a hyperthermophilic archaeon, particularly a DNA polymerase derived from Thermococcus kodakaraensis strain KOD1. The present invention has been completed by finding that an antibody against the protein (hereinafter sometimes referred to as “KOD DNA polymerase”) or a fragment protein thereof has an effect of suppressing non-specific reaction by HAMA.

The DNA polymerase belonging to the family B (also called α type) derived from a hyperthermophilic archaeon is a thermostable DNA polymerase derived from Thermococcus kodakaraensis KOD1 strain (formerly Pyrococcus sp. KOD1). (Patent Document 5) is known.
These DNA polymerases belonging to Family B have 3′-5 ′ exonuclease activity (Proof reading activity), and the accuracy at the time of nucleic acid incorporation is Family A such as Taq DNA polymerase (also called pol I type). It has the characteristic that it is excellent compared with the DNA polymerase which belongs to.
The KOD DNA polymerase is used in a hot start PCR (polymerase chain reaction) method capable of detecting and quantifying a small amount of nucleic acid quickly and specifically. Two types of monoclonals specific for KOD DNA polymerase are used for hot start PCR. An antibody is used (see Patent Document 6 and Non-Patent Document 2).
The base sequences of these antibody genes are disclosed, for example, in Genbank nucleotide sequence databases (accession number AB017091, AB017092).

The present inventors have obtained an antibody gene from a hybridoma cell (3G8) that produces an anti-KOD DNA polymerase antibody, and have the same nucleotide sequence as that of a known antibody gene present in the database Genbank nucleotide sequence databanks (accession number AB017091, AB017092). It was possible to obtain antibody genes having novel base sequences that differed in part. Surprisingly, the anti-KOD DNA polymerase antibody expressed by this antibody gene was evaluated for its inhibitory effect on the non-specific reaction by HAMA. The anti-KOD DNA polymerase antibody remarkably eliminates the influence of such interference, and an advantageous non-specific reaction suppression effect that can be expected by those skilled in the art was obtained.

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.
[Claim 2] A polypeptide encoded by the base sequence set forth in SEQ ID NO: 2.
[Item 3] An antibody molecule comprising the polypeptide of Item 1 or 2.
[Item 4] An antibody fragment comprising the polypeptide according to item 1 or 2.
[Item 5] The antibody fragment according to item 4, wherein the antibody fragment is Fab, F (ab ′) ₂ or scFv.
[Item 6] A nonspecific reaction suppression method for removing the influence of nonspecific interference in a method for quantifying an analyte using an immunoassay method, wherein the substance according to any one of Items 1 to 5 is reacted A non-specific reaction suppression method comprising a step of coexisting in a system.
[Item 7] The nonspecific reaction suppression method according to Item 6, wherein the immunoassay is a sandwich method.
[Item 8] A nonspecific reaction inhibitor comprising the substance according to any one of Items 1 to 5.
[Item 9] An immunoassay kit comprising the nonspecific reaction inhibitor according to Item 8.

According to the present invention, non-specific reactions caused by heterophilic antibodies can be suppressed in immunoassays, so that specificity can be improved and false positives can be reduced, enabling highly reliable immunoassay in fields such as clinical examinations. It becomes.

It is a figure which shows the result of having compared the KOD3G8 antibody light chain gene on the database with the light chain gene of the acquired antibody of the present invention. It is a figure which shows the result of having compared the KOD3G8 antibody heavy chain gene on a database with the heavy chain gene of the acquired antibody of this invention. It is a figure which shows the analytical curve obtained as a result of confirming about the reactivity with the antigen of the acquired KOD3G8 antibody. It is a figure which shows the result measured about the HAMA suppression ability of KOD3G8 antibody. It is a figure which shows the result measured about the HAMA suppression ability of recombinant CHO-K1 cell origin KOD3G8 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.

The present invention includes a protein having a binding property to a DNA polymerase derived from a hyperthermophilic archaeon, and specifically includes a protein constituting an antibody or an antibody fragment.

The hyperthermophilic archaeon is a bacterium belonging to archaea, and those belonging to the genus Thermococcus and Pyrococcus are exemplified. Specific examples include Thermococcus kodakaraensis, Thermococcus litoralis, Pyrococcus furiosus, and the like.

An example of an embodiment of the present invention is a polypeptide encoded by the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 (these polypeptides have the amino acid sequences described in SEQ ID NO: 3 and SEQ ID NO: 4, respectively). An antibody molecule comprising these polypeptides, or an antibody fragment comprising these polypeptides. These substances have a binding property to a DNA polymerase derived from a hyperthermophilic archaeon.

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

Antibody molecules comprising a polypeptide encoded by the base sequence described in SEQ ID NO: 1 or 2 is that subclass is not particularly limited, it is preferably an antibody classified into IgG _1.
Specific examples of the antibody fragment containing the polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 1 or 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, for example, an antibody or antibody fragment recombinantly produced by genetic engineering, and is an antibody against KOD DNA polymerase. It also includes the case where an antibody is obtained by linking a gene to an expression vector and transfecting a mammalian cell. The polypeptide, antibody, or antibody fragment of the present invention can also be obtained from a cell deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (Accession Number: FERM P-21784). Further, antibody genes antibody fragment (Fab fragment, F (ab _{') 2} fragments, Fc fragments) may be and each single chain antibody or 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: Amercan Type Culture Collection (ATCC) CCL-61, physics and chemistry) Laboratory BioResource Center RCB0285, RIKEN BioResource Center RCB0403, etc.), human fetal 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, source: ATCC CRL-1651, RIKEN BioResource Center, for example) Ter RCB0539, etc.), human cervical cancer cells (eg, HeLa cells, sources: ATCC CCL-2, ATCC CCL-2.2, RIKEN BioResource Center RCB0007, RIKEN BioResource Center RCB0191), mouse Various tumor cells such as myeloma cells (for example, NS0 cells, available from: RIKEN BioResource Center RCB0213), and the like can be mentioned.

Another embodiment of the present invention is a nonspecific reaction suppression method for removing the influence of nonspecific interference in a method for quantifying an analyte using an immunoassay method, which is described in SEQ ID NO: 1 or 2 A non-specific reaction suppression method comprising a step of allowing a polypeptide encoded by a base sequence or a substance such as an antibody molecule or antibody fragment containing them to coexist 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 inhibitor is not particularly limited, but the nonspecific reaction inhibitor is previously contained in an appropriate buffer 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 non-specific reaction (non-specific reaction substance) in the sample reacts with the non-specific reaction inhibitor, and the non-specific reaction activity in the specific immune reaction step is lost. In the specific immune reaction step, a mixed solution of the sample that has been incubated and the nonspecific reaction inhibitor may be used as it is.

Furthermore, a simple method of using the present invention as a non-specific reaction inhibitor of the present invention is a method of performing a specific immune reaction in the presence of a non-specific reaction inhibitor.
For example, in the case of the most commonly used positive sandwich method, a nonspecific reaction inhibitor 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, the non-specificity that was not completely suppressed in the first immune reaction step Since the reactants are also suppressed, the measurement reliability can be further improved.
Usually, a sufficient effect can be obtained if it is added to the buffer solution of the first immune reaction step.

Also in the one-step sandwich method, non-specific reaction is suppressed by adding a non-specific reaction inhibitor to the buffer of the immune reaction process for reaction.
The nonspecific reaction inhibitor 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 non-specific reaction preferentially reacts with the non-specific reaction inhibitor, so that the non-specific reaction is suppressed.

Another embodiment of the present invention is a nonspecific reaction inhibitor comprising a polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 1 or 2, or a substance such as an antibody molecule or antibody fragment containing the polypeptide.
Moreover, it is an immunoassay kit containing the said nonspecific reaction inhibitor.

The nonspecific reaction inhibitor of the present invention is used by adding it to an appropriate buffer in advance before the immune reaction step, or by adding it to a buffer solution for the immune reaction step.
The concentration of the non-specific reaction inhibitor 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 inhibitor.

The non-specific reaction inhibitor 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 inhibitor of the present invention is preferably added to a buffer solution or labeled antibody solution for reacting a specimen with a solid-phased antibody to form a reagent for a kit. As the medium for suspending the nonspecific reaction inhibitor, 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 inhibitor of the present invention include body fluids such as serum, plasma, spinal fluid, saliva, urine, fecal extract, and the like.
Although not particularly limited as an analyte that can be measured, substances utilized in clinical tests, an in vivo substances and disease markers include, for example, in body fluids, human immunoglobulin, human albumin, human fibrinogen, beta 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. More preferably, it is a sandwich method employed 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. All the PCR described in this example was performed using KOD polymerase (manufactured by Toyobo).

Mouse hybridoma cell line 3G8 that produces an antibody specific to DNA polymerase derived from Thermococcus kodakaraensis KOD1 strain (obtained from: National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center, deposit number: FERM BP-6056) ) Was cultured in CD Hybridoma Medium (GIBCO, product number: 11279-023), total RNA was extracted from 1 × 10 ⁶ cells using RNeasy Plus Mini Kit (product of QIAGEN, product number: 74134), and RiverTra Ace-α Single-stranded cDNA was synthesized using-(registered trademark) (Toyobo Co., Ltd., product number: FSK-101).
After PCR amplification of the heavy and light chains using Mouse Ig-Primer Set (Novagen, product number: 69831-3), the pCI-neo plasmid (Promega, product number: product number: CMV promoter changed to EF-1α promoter) was used. The sequence was confirmed by cloning into the restriction enzyme XbaI-NotI site of E1841). 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.

Comparing the analyzed sequence with the base sequence published in Genbank nucleotide sequence databanks (accession number AB017091, AB017092), 98.9% matches for the heavy chain and 92.4% matches for the light chain and both chains are known The arrangement was different. In addition, in the CDR region (complementary determining region) that contributes to the reactivity with the antigen, it was confirmed that the heavy chain was identical, but the CDR1, CDR2, and CDR3 were different from the sequences in the database in the light chain. It was. FIG. 1 shows a comparison between the light chain base sequence obtained above and the sequence on the database, and FIG. 2 shows a comparison between the heavy chain base sequence and the sequence on the database. The comparison was performed using Genetyx (Geneticx), which is sequence analysis software.

The expression plasmid 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 KOD DNA polymerase was confirmed by ELISA measurement.
Specifically, 50 μl of culture supernatant was added to an ELISA plate on which a DNA polymerase antigen derived from KOD1 at a concentration of 30 μg / ml was immobilized, 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 10,000 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).

The antibody concentration was calculated based on the calibration curve shown in FIG. As a standard, an antibody obtained from the mouse hybridoma cell line 3G8 that produces an antibody specific for KOD DNA polymerase was used. The results are shown in Table 1.
As a result, it was confirmed that the antibody was reactive with the antigen in the same manner as the antibody obtained from the hybridoma cell line 3G8, and the obtained antibody gene was proved to be the target antibody gene.

Whether the anti-KOD DNA polymerase antibody can suppress the influence of HAMA, which is a kind of heterophilic antibody, was confirmed by a positive sandwich ELISA method.

Using three types of antibodies obtained from MAK33-IgG1 (Roche, product number: 1200941), mouse IgG (manufactured by CHEMICON, product number: PP54), mouse hybridoma cell line 3G8 that produces an antibody specific for KOD DNA polymerase. HAMA interference removal effect measurement was performed. As HAMA serum, HAMA serum type 1 (Roche, product number: 1767275) was used.
More specifically, 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) in an amount of 50 μl / well. Phased. In addition, antibody solutions for HAMA suppression diluted with a 0.1% BSA + PBS solution were prepared so that the respective antibodies would be 1 μg / ml and 5 μg / ml. Next, 50 μl of the antibody solution was mixed with 50 μl of HAMA serum, 50 μl / well each was dispensed, and incubated at 25 ° C. for 1 hour. Then, after washing 3 times with PBS-T and sufficiently draining water, Anti-hCEA antibody labeled with peroxidase was diluted 2000 times with 0.1% BSA + PBS solution, added 50 μl / well, and then at 25 ° C. for 1 hour. Incubated. 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 inhibition rate was calculated by measurement with a plate reader (main wavelength: 450 nm, subwavelength: 620 nm).
The results are shown in FIG.

A mouse IgG antibody (manufactured by CHEMICON, PP54), which is a normal anti-mouse antibody, has a HAMA suppression effect of 39% at 1 μg / ml, whereas the anti-KOD DNA polymerase antibody has a high HAMA suppression rate of 48%, which is commercially available. Even when compared with 51% of the HAMA inhibitor MAK33-IgG1, an excellent result was obtained that there was an inhibitory effect equal to or higher than that.
Since the three types of antibodies used in the evaluation are the same mouse antibody and the constant region is the same, the difference in the variable region including the CDR region (complementary determining region) contributing to the reactivity with the antigen is suppressed by HAMA. It is suggested that it affects the effect.

The expression plasmid 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), cultured for 1 day, and then 400 μg. Obtained by performing drug-selective culture for 2 weeks with Ham's F-12 medium (manufactured by Nacalai Tesque, product number: 17458-65) containing / ml G418 disulfate solution (manufactured by Nacalai Tesque, product number: 16513-84) Is cloned by limiting dilution to produce recombinant CHO-K1 cells that produce antibodies specific for KOD DNA polymerase (obtained from: National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center, accession number: FERM P-21784). It was constructed.

Effect of HAMA using the above-mentioned recombinant CHO-K1 cell producing an antibody specific to KOD DNA polymerase and purified with a protein A column (manufactured by GE Healthcare, product number: 17-5079-01) It was confirmed by a positive sandwich ELISA method whether or not it can be suppressed.

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: 3KC534OD75 polymerase) The HAMA interference removal effect was measured using 5 types of antibodies obtained from the mouse hybridoma cell line 3G8 that produces a specific antibody and antibodies obtained from recombinant CHO-K1 cells. As HAMA serum, HAMA serum type 1 (Roche, product number: 1767275) was used.
More specifically, 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) in an amount of 50 μl / well. Phased. In addition, antibody solutions for HAMA suppression diluted with 0.1% BSA + PBS solution were prepared so that the respective antibodies would be 0.5 μg / ml and 1 μg / ml. Subsequently, 50 μl of the HAMA serum was mixed with 50 μl of the antibody solution, each 50 μl / well was dispensed, and incubated at room temperature for 1 hour. Thereafter, the plate was washed 3 times with PBS-T, and after sufficiently removing water, Anti-hCEA antibody labeled with peroxidase was diluted 4000 times with 0.1% BSA + PBS solution and added at 50 μl / well. Incubated for hours. 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 inhibition rate was calculated by measurement with a plate reader (main wavelength: 450 nm, subwavelength: 620 nm).
The results are shown in FIG.

The anti-KOD DNA polymerase antibody derived from the hybridoma and the anti-KOD DNA polymerase antibody derived from the recombinant CHO-K1 cell were confirmed to have almost the same HAMA suppression effect. Mouse IgG antibody (manufactured by CHEMICON, PP54), which is a normal anti-mouse antibody, has a HAMA suppression effect of 61% at 1 μg / ml, whereas HAMA suppression of anti-KOD DNA polymerase antibody derived from recombinant CHO-K1 cells. The rate is as high as 72%, compared with 69% of the commercially available HAMA inhibitor MAK33-IgG1 (Roche, product number: 1200941) and 68% of HBR (SCANTIBODIES LABORATORY, INC., Product number: 3KC534-075). An excellent result was obtained that there was an inhibitory effect equivalent to or better. It was confirmed that the anti-KOD DNA polymerase antibody HAMA suppression effect was extremely effective.

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 (9)

A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 1. A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 2. An antibody molecule comprising the polypeptide according to claim 1 or 2. An antibody fragment comprising the polypeptide according to claim 1 or 2. The antibody fragment according to claim 4, wherein the antibody fragment is Fab, F (ab ') ₂ or scFv. A method for quantifying an analyte using an immunoassay method, which is a nonspecific reaction suppression method for removing the influence of nonspecific interference, wherein the substance according to any one of claims 1 to 5 is contained in a reaction system. A non-specific reaction suppression method including a step of coexistence. The nonspecific reaction suppression method according to claim 6, wherein the immunoassay is a sandwich method. The nonspecific reaction inhibitor containing the substance in any one of Claims 1-5. An immunoassay kit comprising the nonspecific reaction inhibitor according to claim 8.