JP2002308900A - Anti-human hepatic triglyceride lipase antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily measure blood HL with a high sensitivity. SOLUTION: The anti-human hepatic triglyceride lipase antibody or its fragment recognizes an amino acid sequence represented by sequence number 1 or 2 or an amino acid sequence wherein one or few amino acids of these amino acids are deleted, replaced or added. The anti-human HL antibody has a high specificity against human HL, does not respond to LPL and is therefore useful as an antibody for human HL measurement. By using this antibody, human HL can be easily measured with a high sensitivity, which is advantageous for arteriosclerosis and liver function diagnoses.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an anti-human hepatic triglyceride having a high antigen recognition specificity useful as a diagnostic agent for familial human hepatic triglyceride lipase deficiency, liver disease / malnutrition, arteriosclerosis, etc. The present invention relates to a lipase antibody and a reagent using the same.

[0002]

BACKGROUND OF THE INVENTION Human hepatic triglyceride lipase (Hepat
ic triglyceride lipase (HL) is a 65 kDa glycoprotein synthesized by Hepatocyte. Lipoprotein lipase (Lipop
HL catalyzes the hydrolysis of triglycerides, causing the conversion of triglyceride-rich chylomicrons and very low density lipoproteins (VLDL) into cholesterol-rich remnant particles. Each lipase is a heparin-binding protein and is found in post-heparin plasma (PHP) of humans and experimental animals. Early studies established a role for HL in remnant clearance. Inhibition of HL activity by injection of anti-HL IgG into rats or monkeys impairs liver uptake of radioactively labeled remnant particles, resulting in accumulation of plasma VLDL and LDL fractions. In humans, the presence of familial HL deficiency results in elevated plasma levels of remnant-like particles, including cholesterol and triglycerides, leading to premature atherosclerotic disease. Expression of human HL in transgenic mice and rabbits exhibits a reduction in total plasma cholesterol. H of high fat diet load
In L knockout mice, expression of HL results in correction of the abnormal lipid profile and a clear decrease in aortic cholesterol. These results support the anti-atherogenic role of HL. Since HL is a glycoprotein synthesized in the liver and having a short half-life, it is attracting attention as an extremely highly organ-specific diagnostic marker that reflects liver function and nutritional status.

[0003] As a method for quantifying HL in body fluids of healthy persons and patients suffering from the above-mentioned various diseases, two kinds of methods (active amount and protein amount) having different principles are mainly used. Since HL is an enzyme anchored on the surface of hepatic endothelial cells, heparin is injected intravenously and taken out of the blood in a free state. The plasma after heparin administration is called post-heparin plasma (PHP). Currently PH
It is known that two types of lipases that degrade triglyceride are present in P, and lipoprotein lipase (Lipoprotein lipase, lipase) anchored via glycans to the surface of capillary endothelial cells of HL and extrahepatic tissue. LPL). In order to accurately measure HL, it is necessary to separate and quantify HL and LPL. As a method of quantifying HL by an activity amount,
In general, there is a method in which one of two enzymes (HL and LPL) is inactivated by some method, and the remaining enzyme is measured under optimal conditions. Representative methods include (1) a method of inactivating LPL with 1 M NaCl and then measuring the remaining HL activity in PHP, and (2) separating HL and LPL by NaCl concentration using a heparin sepharose column. After elution, there is a method of measuring the activity of each enzyme. However, after the selective neutralization of NaCl, the method (1)
It is known that NaCl adversely affects the remaining active enzyme and partially inactivates the activity, and the column method of (2) is known to be time-consuming and has a problem in the recovery rate.

[0004] Regarding the method of measuring HL by the amount of protein,
Yasuyuki Ikeda et al. (Medical Immunology
, Vol. 18, No. 4, page 643, 1989) reports a quantification method using a monoclonal antibody.
However, the operation is complicated, such as using an insoluble carrier derived from the cell wall, for which a difference between raw material lots is known, and requiring centrifugation. Further, the antigen recognition site of this monoclonal antibody is not known, and its use is limited to antigen measurement and the like.

[0005]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an anti-human HL antibody having binding specificity to human HL, and a method for measuring blood HL simply and with high sensitivity using the antibody. Is to do.

[0006]

Under such circumstances, the present inventors have conducted various studies to obtain an anti-HL antibody having high binding specificity (antigen recognition specificity). The two sequence portions are the predominant sites of the immune response of human HL, and we succeeded in obtaining an anti-human HL antibody having extremely high binding specificity (antigen recognition specificity) for these sequences. According to the method, human HL can be measured simply and with high sensitivity, and the antibody can be used not only for HL measurement, but also for separation / purification of human HL, and gene abnormalities having abnormalities in the present sequence portion or sequences other than the present sequence. The present inventors have found that the present invention is extremely useful for the detection of infectious diseases and completed the present invention.

That is, the present invention relates to an amino acid sequence represented by SEQ ID NO: 1 or 2;
It is intended to provide an anti-human HL antibody or a fragment thereof that recognizes an amino acid sequence in which several amino acids have been deleted, substituted or added. The present invention also provides a hybridoma that produces the above-mentioned anti-human HL monoclonal antibody. The present invention also provides a human HL measurement reagent containing the above-mentioned anti-human HL antibody, a fragment thereof, or a labeled product thereof. The present invention also provides a method for measuring human HL in a human blood-derived sample, which comprises reacting the above-mentioned anti-human HL antibody, a fragment thereof, or a labeled product thereof with the sample. Further, the present invention provides a method for separating or purifying human HL by affinity chromatography using an immobilized anti-human HL antibody or an immobilized fragment obtained by immobilizing the above anti-human HL antibody or a fragment thereof on an insoluble carrier. Things.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The anti-human HL antibody of the present invention
L-reactive and recognizes an amino acid sequence whose antigen recognition site is represented by SEQ ID NO: 1 or 2 or an amino acid sequence in which one to several amino acids have been deleted, substituted or added. It is. The number and location of amino acid mutations here are not particularly limited. "One to several" means a number that can be generated by a commonly used technique, for example, site-directed mutagenesis, and specifically means about 1 to 10, preferably 1 to 6
And more preferably 1 to 3.

The anti-human HL antibody of the present invention preferably has reactivity with human HL and does not react with human LPL.

The anti-human HL antibody of the present invention includes both a monoclonal antibody and a polyclonal antibody, but a monoclonal antibody is preferred. A particularly preferred example is the monoclonal antibody 3A2 produced by hybridoma 3A2 deposited as FERM P-18181 and recognizing SEQ ID NO: 1.

SEQ ID NO: 1 or 2 recognized by the antibody of the present invention
The amino acid sequence shown by is the site where the immune response is predominant in human HL. Such an antibody against the site where human HL has an immune response can be produced, for example, by the following steps (a), (b) and (c).

(A) Preparation and selection of immunizing antigen The step (a-1) of expressing a recombinant protein and preparing it as an antigen using the primary structure of the amino acid sequence and base sequence of the protein, and the basic steps of protein This is a step (a-2) of selecting and synthesizing a sequence of a synthetic peptide in which the animal species is clear in the hydrophilic region and the primary structure by utilizing the properties. As a method for selecting a peptide, the Hop and Wood method (Hopp & W
oods method, Proceedings National Academy of Sciences (Proc. Natl. Acad. Sci. USA), Vol. 78, pp. 3824-3828, 1981), Perker antigen
icity method, Protrusion index antigenicity method and Wellin
The gantigenicity method can be used. The type of the peptide to be synthesized is preferably 5 to 10 or more, more preferably 5 to 10.

(B) Identification of the predominant site of the immune response of the protein (a) a peptide obtained by immunizing a mammal with the protein antigen expressed in (a-1) and synthesizing the obtained antiserum in (a-2) Reacting each with a solid-phased carrier, and evaluating the distribution of an antibody group against a specific peptide in the antiserum, thereby identifying an immunologically superior site. (A) A mammal is immunized with the protein antigen expressed in (a-1), the spleen cells of the immunized animal are fused with myeloma cells, and the resulting hybridoma cell culture supernatant is subjected to (a-
A step of allowing the peptides synthesized in 2) to react with the solid-phased carriers, and evaluating the distribution of the antibody group against the specific peptide in the hybridoma cell group, thereby specifying the site where the immune response is superior. (C) an antigen obtained by binding the peptide synthesized in (a-2) to a carrier protein and mixing an equal amount of each peptide and the conjugate of the carrier protein, or mixing an equal amount of each peptide to obtain a carrier protein Immunizing a mammal with an antigen conjugated with
The obtained antiserum was reacted with a carrier on which the peptide synthesized in (a-2) was immobilized, and the distribution of the antibody group against the specific peptide in the antiserum was evaluated to identify the site where the immune response was superior. Process. (D) an antigen obtained by binding the peptide synthesized in (a-2) to a carrier protein and mixing the respective peptides and the conjugate of the carrier protein in equal amounts, or mixing the respective peptides in equal amounts and mixing the carrier protein Immunizing a mammal with an antigen conjugated with
The spleen cells of the immunized animal are fused with myeloma cells, and the resulting hybridoma cell culture supernatant is reacted with a carrier on which the peptide synthesized in (a-2) has been immobilized, and the hybridoma cell group is identified. A step of identifying an immune response dominant site by evaluating the distribution of an antibody group against the peptide of (1).

The above steps (a) and (a) facilitate the production of an antibody having a selectively high binding specificity (antigen recognition specificity), and further evaluate the specificity of the antibody after the establishment of hybridoma cells. Processes (Western blotting, enzyme immunoassay with plateau immobilized, immunoprecipitation, expensive pepset (PepSet ^TM , Kurashiki Boseki Co., Ltd.)
Company)) can be largely omitted. 5-10 each
The complexity of immunizing mammals with individual peptides can be eliminated, and labor and cost can be reduced. The above process (c)
According to (d), the complexity of immunizing a mammal with 5 to 10 peptides each can be eliminated, and labor and cost can be reduced.

(C) Production of Antibody Against Immune Response Predominant Site in Protein From the hybridoma cells obtained in the above steps (a) and (c), a hybridoma producing an antibody recognizing the immune response superior site in the protein And then reacting the recombinant protein and two antigens of a peptide corresponding to the immune response dominant site on the solid-phased carrier, recognizing the immune response dominant site, and having high binding specificity to the protein ( Although a hybridoma cell having antigen recognition specificity can be produced, it is preferable to produce a monoclonal antibody by the following steps (e), (f) and (g).

(E) immunizing a mammal with a protein antigen isolated from a biological sample or the protein antigen expressed in (a-1), and fusing spleen cells of the immunized animal with myeloma cells; From the obtained hybridoma cells, the immune antigen and the peptide group corresponding to the immune response dominant site are allowed to react with a carrier on which each is immobilized, and the immunological dominant site is recognized, and high binding specificity to the immune antigen (antigen recognition specificity) Producing monoclonal antibodies by screening hybridoma cells having

(F) A mammal is immunized with an antigen obtained by binding a peptide corresponding to a site superior to the immune response in the protein identified in (b) to a carrier protein, and spleen cells and myeloma cells of the immunized animal are immunized. And reacting the resulting hybridoma cells with a carrier on which two types of antigens, a recombinant protein and a peptide corresponding to the immune response dominant site, are respectively immobilized, and recognizing the immune response dominant site, A step of producing a monoclonal antibody by screening hybridoma cells having high binding specificity (antigen recognition specificity).

(G) A mammal is immunized with an antigen in which a peptide corresponding to the immune response dominant site in the protein identified in (b) is bound to a carrier protein, and the immune response dominant site is recognized, and A step of producing an antiserum and a polyclonal antibody having a high binding specificity (antigen recognition specificity), and reacting the antiserum with a carrier on which a peptide corresponding to the immune response dominant site has been immobilized, followed by a conventional method. Isolating the carrier from the carrier, and enriching the polyclonal antibody group having high binding specificity (antigen recognition specificity).

Human HL used as an antigen includes:
An amino acid sequence having the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence may be mentioned. Examples of the peptide having a specific amino acid sequence in human HL protein include, for example, the amino acid sequence of human HL represented by SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1.
In a peptide having an amino acid sequence in which one or more amino acids have been deleted, substituted or added, or in the amino acid sequence of human HL represented by SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2, one or more amino acids are Peptides having a deleted, substituted or added amino acid sequence are included.
The above-mentioned recombinant HL (SEQ ID NO: 3), peptides comprising the sequences of SEQ ID NO: 1 and SEQ ID NO: 2,
It can be obtained by chemical synthesis or gene expression by a conventional method. The method of peptide synthesis is based on a general method as described in “Experimental Medicine (separate volume) Cell Engineering Handbook” (edited by Toshio Kuroki et al., Published by Yodosha, pp. 66-74, 1992). Can be synthesized. For example, t
-BOC (tert-butyloxylcarbony 1) method, Fmoc (9-fluor
enylmethyloxycarbony 1) method or MAP (Multiple A
ntigen Peptide) method can be used. As a method based on gene expression, a gene prepared by inserting a gene having an amino acid sequence capable of being expressed into an expression vector, transforming a host cell with the expression vector, and culturing the transformed cell can be used. it can. As host cells, any of prokaryotic cells (eg, E. coli) and eukaryotic cells (eg, CHO cells) can be used. In order to make the bond with the carrier effective, an amino acid such as cysteine, lysine, arginine or the like can be added to the N- or C-terminal of the peptide represented by SEQ ID NO: 1 or 2. When cysteine is added to the N- or C-terminal, the amino group or carboxy group of cysteine is made free, and the other end is blocked. Alternatively, the synthesis may be performed with the amino terminal free, the carboxy terminal converted to an amide, and a bisimide ester may be used for bonding to the carrier. As the peptide carrier, proteins derived from mammals such as albumin and globulin, proteins such as keyhole limpet hemocyanin, microorganisms such as inactivated tuberculosis bacterium, polyamino acids such as polylysine and polyasparagine, and polysaccharides can be used. .

The DNA encoding human HL includes, for example, human HL having the amino acid sequence of SEQ ID NO: 3.
Can be used (SEQ ID NO: 4). The DNA consisting of the nucleotide sequence of SEQ ID NO: 4 can be chemically synthesized according to a conventional method, and a primer designed according to the nucleotide sequence of SEQ ID NO: 4 using mRNA extracted from human liver according to a conventional method as a template. RT-P
It can also be amplified by the CR method. Furthermore, DNA encoding human HL can also be obtained from a human liver-derived cDNA library prepared according to a conventional method using a probe designed according to the nucleotide sequence of SEQ ID NO: 4. For preparation of human HL from DNA encoding human HL, for example, a recombinant vector containing DNA encoding human HL is prepared, a suitable host cell is transformed from the vector, and the transformant is transformed into an appropriate host cell. It can be carried out by purifying a culture obtained by culturing in a medium.

The monoclonal antibody of the present invention is described in Experimental Medicine (separate volume) Cell Engineering Handbook (edited by Toshio Kuroki et al., Published by Yodosha, pp. 66-74, 1992) and an introduction to experimental procedures for monoclonal antibodies (Tamie Ando) Author, published by Kodansha,
1991) and the like, and can be produced from a hybridoma (fused cell) produced by so-called cell fusion according to a general method for producing a monoclonal antibody. Such a hybridoma is obtained by immunizing a mammal with an antigen to obtain an antibody-producing cell, preparing a hybridoma from the antibody-producing cell and a myeloma cell line (myeloma cell) having no autoantibody-producing ability, and cloning the hybridoma. And a clone that produces a monoclonal antibody showing specific affinity for the antigen used for immunization of the mammal.

Specifically, using purified human HL as an antigen,
Alternatively, a peptide consisting of the sequence of SEQ ID NO: 1 or 2, which is a part of the amino acid sequence of the human HL, or a conjugate thereof with a carrier is used as an antigen, and the antigen is used as a mammal (for producing human antibodies). Genetically engineered transgenic animals such as human antibody-producing mice are also included), specifically, mice, rats, hamsters, guinea pigs or rabbits, and more specifically, mice, rats or hamsters, subcutaneously, Immunization is performed by one or several injections intramuscularly, intravenously, in a footpad or intraperitoneally. Usually, immunization is performed 1 to 4 times about every 1 to 14 days after the initial immunization, and antibody producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization.

Hybridomas can be created according to, for example, the method of Koehler and Milstein et al. (Nature, Vol. 256, 495-497, 1975) and modification methods analogous thereto. That is, the antibody-producing cells contained in the spleen, lymph node, bone marrow, tonsil, etc., preferably in the spleen obtained from the mammal immunized as described above, and preferably mouse, rat, guinea pig, hamster, rabbit or human Etc., and more preferably, by cell fusion with myeloma cells having no autoantibody-producing ability derived from mammals, more preferably mice, rats or humans. For example, mouse-derived myeloma cells include, for example, mouse-derived myeloma P3 / X
63-AG8.653 (653), P3 / NS1 / 1-
Ag4-1 (NS-1), P3 / X63-Ag8. U1
(P3U1), SP2 / O-Ag14 (Sp2 / O, S
p2) PA1, F0 or BW5147, rat-derived myeloma 210RCY3-Ag. 2.3. , Human-derived myeloma U-266AR1, GM1500-6TG-
A1-2, UC729-6, CEM-AGR, D1R1
1 or CEM-T15 can be used.

The screening of hybridomas producing monoclonal antibodies is carried out by culturing the hybridomas, for example, in a microtiter plate, and examining the reactivity of the culture supernatant in the wells in which proliferation has been observed with the antigen used for immunization of the mammal. For example, it can be measured by an enzyme immunoassay such as RIA and ELISA. Here, a hybridoma that efficiently produces an antibody against human HL can be selected by the steps (e) and (f) of (c) described above.

The method for cloning the hybridoma producing the desired monoclonal antibody may be a conventional one, and is not particularly limited. The cloning of the hybridoma can be performed by, for example, a limiting dilution method, a soft agar method, a fibrin gel method, a fluorescence excitation cell sorter method, or the like.

The production of the monoclonal antibody from the hybridoma can be performed by culturing the hybridoma in vitro or in vivo and isolating it from the obtained culture supernatant or ascites of a mammal. In vitro culture can be performed using a known nutrient medium used to produce monoclonal antibodies, or any nutrient medium derived and adjusted from a known basal medium. As the basal medium, for example, Ham'F12 medium,
Low calcium medium such as MCDB1553 medium or low calcium MEM medium and MCDB104 medium, MEM
Medium, D-MEM medium, RPMI-1640 medium, AS
A high calcium medium such as an F104 medium or an RD medium can be used. The basic medium can contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances depending on the purpose. In vivo culture can be carried out using mouse, rat, guinea pig, hamster, rabbit, or the like, preferably mouse, or rat, more preferably mouse ascites. For isolation and purification of the monoclonal antibody, the above-mentioned culture supernatant or ascites can be obtained by using a saturated ammonium sulfate, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE, DE52, etc.), an anti-immunoglobulin column or It can be performed by subjecting it to affinity column chromatography such as a protein A column.

The polyclonal antibody of the present invention can be prepared according to a general method described in the same literature as that described for the preparation of the monoclonal antibody. That is, mammals such as mice, rats, guinea pigs, rabbits, goats, pigs, horses or cows, and birds such as chickens, preferably rats, guinea pigs, rabbits or goats, or any peptide of SEQ ID NO: 1 or 2, or A mixture thereof or a conjugate thereof with a carrier is immunized by a method as described in the method for producing a monoclonal antibody, and a serum, milk or egg containing an antibody reactive with human HL produced thereby is purified. It can be prepared by isolation and purification in the same manner as in the case of the above monoclonal antibody.

The method for producing a polyclonal antibody using a purified protein is a well-known method. However, if there is a very small amount of antigen-containing contaminants in an antigen, antibodies against them may be produced together. It is a problem. Also, when trying to obtain purified proteins from human biological samples, ethical issues, problems with worker health due to infectious substances in raw materials, problems with differences between product lots, etc.
There are many problems. On the other hand, the method of the present invention using the peptide having the sequence of SEQ ID NO: 1 or 2 has no problem derived from the raw material, and the difference between production lots can be almost ignored. The sequence has many advantages and is extremely useful as compared with a method using a purified protein derived from a biological sample, for example, it can be easily obtained if the facility has facilities for protein expression and peptide synthesis. In addition, SEQ ID NO: 1 or 2 is designed so as to selectively produce a high binding specificity (antigen recognition specificity) antibody to a site where an immune response of a protein is superior, and the advantage as an immunizing antigen is extremely high. The anti-human HL antibody of the present invention obtained as described above is converted into an antibody fragment, ie, Fab ′ or F (ab ′) ₂ by a conventional method such as treatment with a protease such as pepsin or papain. In the measurement method and the purification method of the present invention, not only the anti-human HL antibody of the present invention but also such an antibody fragment can be used.

The use of the anti-human HL antibody of the present invention, a fragment thereof, or a labeled product thereof makes it possible to easily and sensitively detect or quantify HL in a human blood-derived specimen. The sample derived from human blood is not particularly limited, but examples include plasma, serum and the like.

In order to measure a sample, an immunoassay may be carried out using the antibody obtained above. As a measuring method, an immunoturbidimetric assay, an enzyme immunoassay, a latex immunoassay, a radioimmunoassay, a phloromunoassay, an immunochromatographic assay, an optical immunoassay, or the like can be used. As the measurement system of the present invention, a radioimmunoassay (R
IA), a known immunoassay such as an enzyme immunoassay (EIA), a fluorescent immunoassay (FIA), an immunoturbidimetric assay, an immunoprecipitation assay, or an immunochromatographic assay can be used to measure human HL. it can. It is advantageous to use enzymes as labels, such as peroxidase,
Alkaline phosphatase, β-D-galactosidase and the like are preferred. It is also advantageous to use a label that can be visually identified, such as colloidal gold, latex, or carbon particles.

One example of the immunoassay is a so-called competitive immunoassay. For example, a certain amount of human HL labeled with a radioisotope or the like is mixed with a sample, and an anti-human HL antibody is further mixed to react the human HL and the labeled human HL in the sample. Since the human HL in the sample competes with the labeled human HL and reacts with the anti-human HL antibody, the presence of human HL in the sample reduces the reaction with the labeled human HL. After the reaction, whether the anti-human HL antibody is previously bound to the solid phase carrier,
The bound, unlabeled human HL is separated by reacting the anti-immunoglobulin antibody, protein A, with the anti-human HL antibody. The unbound fraction is washed by a commonly used method, and human HL can be measured by detecting labeled radioisotopes and the like.

As another example of the immunoassay, there is a so-called sandwich system. For example, an anti-human HL antibody is bound to a carrier commonly used in immunoassays such as microtiter plates, beads, nitrocellulose membranes, nylon membranes, etc., and this is brought into contact with the specimen, whereby human HL in the specimen is transferred onto the carrier. With the anti-human HL antibody at The unbound fraction is washed by a generally used method, and is contacted with an anti-human HL antibody labeled with a radioisotope, an enzyme, a fluorescent substance, biotin, etc., and the human bound with the anti-human HL antibody on the carrier is washed. React with HL. The unbound fraction is washed by commonly used methods, and labeled radioisotopes, enzymes, fluorescent substances,
By detecting biotin and the like, human HL can be measured. In addition, it is also possible to label one of the above-mentioned antibodies with a visually determinable substance such as colloidal gold, latex or carbon particles, and apply it to the above-mentioned sandwich system or competition method to make the determination. As the anti-human HL antibody and the labeled anti-human HL antibody used in this measurement system, any of a monoclonal antibody, a polyclonal antibody, and a combination thereof can be used.

What is essential here is to combine the antibodies so that the complex of the antibody bound to the carrier and the human HL can bind to the labeled antibody. It can be selected by assembling and applying it to the system.

[0034] An agglutination assay is also an advantageous embodiment, producing particles coated with one of the aforementioned antibodies and particles coated with another antibody. The binding of the particles to the substance to be detected causes agglomeration, which can determine or detect a change in turbidity. In addition, sandwich ELISA, which is an immunoassay method capable of measuring a typical human HL protein concentration, is described.
In the case of Method A, it is preferable to immobilize the monoclonal antibody 3A2 of the present invention on a plastic plate and use the polyclonal antibody of the present invention as a secondary antibody and a peroxidase-labeled anti-rabbit antibody as a tertiary antibody.

Human HL contained in a sample is separated or purified by affinity chromatography using an immobilized anti-human HL antibody or a fragment obtained by immobilizing the anti-human HL antibody or fragment of the present invention on an insoluble carrier. be able to.

The insoluble carrier used for separating or purifying human HL includes (1) polystyrene resin,
In addition to plates, test tubes, tubes, or other materials with an internal volume such as plates, test tubes, or tubes made of water-insoluble substances such as polycarbonate resin, silicone resin, and nylon resin, as well as beads, balls, filters, and membranes, etc. (2) Cellulose carrier, agarose carrier, polyacrylamide carrier, dextran carrier, polystyrene carrier, polyvinyl alcohol carrier, polyamino acid carrier, porous silica carrier and the like can be used.

As such affinity chromatography, for example, (a) using the filter, membrane or the like of the above (1) as an insoluble carrier, immobilizing the anti-human HL antibody of the present invention or an antibody fragment thereof, A method of separating human HL contained in a sample by bringing the sample into contact with the insoluble carrier, and (b) using the cellulosic carrier as described in (2) above as the insoluble carrier and using the anti-human HL of the present invention. The antibody or its antibody fragment is immobilized by a conventional method (physical adsorption, polymerization by cross-linking, sealing in a matrix or immobilization by non-covalent bonding, etc.), and this insoluble carrier is made of glass, plastic or stainless steel. Packed into columns such as
Samples (eg, body fluid samples such as plasma and serum, culture supernatants, centrifugal supernatants, etc.) are placed on columns (eg, columnar columns).
And a method for separating or purifying human HL contained in a sample by elution.

Specific examples of the insoluble carrier (2) used in the affinity chromatography of the above (b) are not particularly limited as long as the anti-human HL antibody of the present invention or an antibody fragment thereof can be immobilized thereon. Amersham Pharmacia Biotech, a commercial product
(amersham pharmacia biotech) Sepharose 2B, Sep
harose 4B, Sepharose 6B, CNBr-Activated 4B, AH-Sepha
rose 4B, CH-Sepharose4B, Activated CH-Sepharose 4B, E
poxy-activated Sepharose 6B, Activated thiol-Sephar
ose 4B, Sephadex, CM-Sephadex, ECH-Sepharose 4B, EAH-S
epharose 4B, NHS-activated Sepharose or Thiopro
Bio-Rad (Bio-Rad) such as pyl Sepharose 6B
-Gel A, Cellex, Cellex AE, Cellex-CM, Cellex PAB, Bio-G
el P, Hydrozide Bio-Gel P, Aminoethyl Bio-Gel P, Bio-
Gel CM, Affi-Gel 10, Affi-Gel 15, Affi-Prep 10, Affi-G
el Hz, Affi-Prep Hz, Affi-Gel 102, CMBio-Gel A, Affi-G
el heparin, Affi-Gel 501 or Affi-Gel 601 etc., Chromagel A, Chromagel P, Enzafix P-HZ, Enzafix P-SH or Enzafix P-AB, etc., manufactured by Wako Pure Chemical Industries, Ltd., Serva (Serva) AE-Cellurose, C
M-Cellurose or PAB Cellurose can be mentioned.

[0039]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (Production of Recombinant Human HL Protein) (1) Preparation of cDNA Encoding Human HL Using a commercially available human liver-derived cDNA library (Takara Shuzo Co., Ltd.) as a template, specific primers [ sense primer: 5 ^{'-GGAATTCGGACAAAGCCTGAAACCAG-3'} (SEQ ID NO: 5), antisense primer: 5'-GTTTCGCTTTC
TAGTCTACTCCTAGGC-3 ′ (SEQ ID NO: 6)], and PCR was carried out under the following conditions to clone human HL cDNA. That is, using specific primers, PC
The DNA fragment amplified by R is cleaved with two kinds of restriction enzymes EcoRI and BamHI under the following conditions, and separated by agarose electrophoresis according to a conventional method to obtain about 1.4 Kb.
After cutting out the DNA fragment of p together with the gel, Geneclean II
(Manufactured by BIO 101) to obtain a desired cDNA fragment.

<PCR reagent conditions> Takara Ex Taq ^™ (5 units / μl) 0.25 to 0.5 μl 10X Ex Taq ^™ Buffer 10 μl dNTP Mixture (2.5 mM each) 8 μl Template (cDNA library) 1 μl Primer (SEQ ID NO: 5) 0.2 to 0.5 μl 1.0 μl Primer (SEQ ID NO: 6) 0.2-1.0 μl Sterile distilled water To final volume of 100 μl

<PCR Temperature Reaction Conditions> The three steps of 95 ° C. and 5.0 minutes or less were repeated 37 times. 95 ℃, 0.5min 65 ℃, 1.0min 72 ℃, 2.0min

<Restriction enzyme reaction conditions> A restriction enzyme buffer (50 mM NaCl, 100 mM) was used per 10 µl of each obtained PCR product.
MTirs-HCl, 10 mM MgCl ₂ , 0.025% Triton X-100, pH 7.
5) with 1 μl and restriction enzymes EcoRI and BamHI (Bio
Labs) (2 μl), and allowed to react at 37 ° C. overnight for digestion.

About 1.4 Kbp obtained in this way.
Was inserted into a pMAL-p2 vector (manufactured by BioLabs) cut with two kinds of restriction enzymes EcoRI and BamHI under the above conditions using DNA Ligation Kit (manufactured by Takara Shuzo Co., Ltd.). Then, this pMA
The L-p2_HL vector was introduced into E. coli JM109 competent cells (Invitrogen). Further, plasmid DNA was extracted from the Escherichia coli, and plasmid DN
The sequence of A is performed, and the target c of about 1.4 Kbp is obtained.
The nucleotide sequence of the DNA fragment was confirmed. It was confirmed that the thus obtained PCR product of about 1.4 Kbp was a cDNA encoding the target human HL.

(2) Preparation of Recombinant Human HL Protein A plasmid constructed so that the human HL cDNA obtained above is incorporated downstream of maltose binding protein (MBP) and expressed as a binding protein between MBP and HL ( p
Escherichia coli transformed with MAL-p2_HL vector) was inoculated into 5 mL of LB medium containing 100 μg / mL Ampicillin and cultured with shaking at 37 ° C. overnight. 100 μl of this culture
The cells were inoculated into 100 mL of LB medium containing g / mL of ampicillin and 0.3 mM of isopropylthiogalactoside (IPTG), and cultured with shaking at 37 ° C. for 2 hours. The culture is collected by centrifugation (4,000 × g, 10 min), and the cells are centrifuged at 200 m.
The cells were suspended in 20 mM Tris buffer (pH 7.4) containing M NaCl and 1 mM EDTA (hereinafter referred to as buffer (A)), centrifuged (4,000 × g, 10 min), and the cells were lysed. Washed. The cells were suspended in 100 mL of the buffer solution (A) and then sonicated to disrupt the cells. Centrifuge (9,000 ×
g, 10 min), and the supernatant was recovered as a crude enzyme solution. Amylose resin (BioLabs) equilibrated with buffer (A) by filling the obtained crude purified enzyme in a glass column tube
Was added. After adsorbing and thoroughly washing the enzyme with buffer (A),
Elution was carried out with a buffer (A) containing 10 mM maltose. The elution pattern was observed by absorbance at a wavelength of 280 nm to obtain a purified recombinant human HL / MBP binding protein fraction.

Example 2 (Anti-human HL monoclonal antibody
Preparation of (a) Selection and preparation of immunizing antigen In general, a highly hydrophilic site in an amino acid sequence, which is considered to be a site exposed to the outside as a protein structure, is used for screening an immunizing antigen or a hybridoma cell. HL protein structure analysis was performed. The analysis of the base sequence and the amino acid sequence used the dedicated software DNASIS-Macv.3.6 (Hitachi Software). The Hopp & Woods method, the Proceedings National Academy of Sciences (Proc. Natl. Acad. Sci. US)
A), volume 78, pages 3824 to 3828, 1981
Year). In addition, considering that it is important to immunize a region where the species difference of the animal is clearly recognized in the amino acid of the antigen as an amino acid sequence presented as the antigen when immunizing a certain animal species with a heterologous antigen, A search was performed and sequences with more species differences were selected. Human HL
Hydrophobicity (Hydrophobicit)
y) was calculated. Although it is strongly hydrophobic as a whole,
There are hydrophilic regions at the center and at the C-terminus. Homology search results (Multiple alignment) between the HL amino acid sequences of the animals for which the gene sequences have been determined. Overall, the HL amino acid sequences show almost no difference in animal species, with only slight differences between the N- and C-termini. Was. Estimation of the hydrophobic site and homology evaluation of amino acid sequences of different HLs of different origin animals, and further comprehensive evaluation of the structural characteristics of individual amino acids and binding to carriers, and use as antigens or producing antibodies The sequence used for hybridoma selection was determined.

From the above point of view, the peptide synthesized and used in this example is a peptide consisting of the amino acid sequence of positions 24 to 43 in the amino acid sequence of human HL represented by SEQ ID NO: 3 (SEQ ID NO: 1; 45-6
A peptide having the third amino acid sequence (hereinafter referred to as “peptide 2”), a peptide having the 255-275th amino acid sequence (hereinafter referred to as “peptide 3”), 330
Peptides consisting of the amino acid sequence of positions 338 to 338 (hereinafter referred to as “peptide 4”); peptides consisting of the amino acid sequences of positions 367 to 385 (hereinafter referred to as “peptide 5”); Peptide 6 "), a peptide having the amino acid sequence at positions 467 to 476 (hereinafter referred to as" peptide 7 "), and a peptide having the amino acid sequence at positions 490 to 499 (SEQ ID NO: 2; hereinafter referred to as" peptide 8 "). The synthesis of these peptides is based on tBOC (tert-buty
loxylcarbonyl) method, Fmoc (9-fluorenylmethyloxycarbony
l) The method was performed.

In the method of binding a synthetic peptide to a carrier protein, keyhole limpet hemocyanin (KL
H) was used. KLH for 10mg carrier (PIERCE)
Was dissolved in 1 mL of distilled water, and Sulfo-SMCC (PI
2 mg of ERCE) was dissolved in 1 mL of distilled water.
0 μl and 200 μl of the KLH solution were mixed and stirred at room temperature for 1 hour. It was added to PD-10 (Pharmacia Biotech) equilibrated with 0.1 M phosphate buffer (pH 7.4). Elution was performed with a 0.1 M phosphate buffer (pH 7.4), and the elution pattern was measured by absorbance at a wavelength of 280 nm. After recovering maleimide-activated KLH according to the elution pattern, 0.5 mL of a synthetic peptide (4 mg / mL distilled water) was added, the mixture was stirred at room temperature for 2 hours, dialyzed against 100-fold volume of PBS, and subjected to immunological antigen or enzyme immunoassay Subject to law.

(B) Identification of the Predominant Site of the Immune Response of the Protein (b-1) The mammal was immunized with the protein antigen expressed in (a), and the obtained antiserum was synthesized by the peptide synthesized in (a). By reacting with the immobilized carrier and evaluating the distribution of the antibody group against the specific peptide in the antiserum,
The step of specifying the immune response superior site was performed as follows.

When recombinant human HL is used, the immunization method is to subcutaneously or intravenously or intraperitoneally in a BALB / c mouse of 8 to 10 weeks, and purify the purified recombinant human HL with an appropriate adjuvant such as Freund. Adjuvant or Ribi Ajuvant System,
The first immunization (day 0) was carried out by injection together with RIBI IMMUNOCHEM RESEARCH). 14 days after the first immunization, 2
On days 8 and 42, booster immunization was performed by injecting subcutaneously or intraperitoneally with purified recombinant human HL. Two weeks after the final administration, whole blood was collected, and the serum was obtained. When a conjugate of a synthetic peptide and a carrier protein was used, 0.3 mg of the conjugate was combined with Freund's Complete adjuvant (Freud's Complete).
adjuvant, manufactured by Sigma) were mixed in equal amounts, and injected into several tens of places on the skin of the rabbit limbs and back for immunization. The same administration was performed 5 to 6 times during 2 to 3 months. Two weeks after the final administration, whole blood was collected, and the serum was obtained. The antibody group in the mouse serum was assayed using an enzyme immunoassay in which the synthetic peptide synthesized after the logical and strategic selection shown below was immobilized.

(B-2) The peptide synthesized in (a) is bound to a carrier protein, and an antigen in which each peptide and the conjugate of the carrier protein are mixed in equal amounts, or each peptide is mixed in equal amounts. A mammal is immunized with an antigen conjugated to a carrier protein, and the resulting antiserum is reacted with a carrier on which each of the peptides synthesized in (a) is immobilized to form a group of antibodies against a specific peptide in the antiserum. The step of identifying the site where the immune response is dominant by evaluating the distribution was performed as follows.

Regarding the conjugate of the synthetic peptide and the carrier,
After each synthetic peptide was bound to a carrier to form a conjugate with the carrier, (1) an immunizing antigen obtained by mixing equal amounts of the conjugates of peptides 2, 4 to 8 with the carrier, and (2) peptide 1 and 2 of the immunizing antigen obtained by mixing equal amounts of the conjugate with the carrier 2
Using different types, the complexity of immunizing rabbits with each peptide was eliminated, and labor saving was achieved. With respect to the obtained antiserum, an antibody group in mouse serum was assayed using an enzyme immunoassay in which the following synthetic peptide was immobilized.

Peptides 1 to 8 were dissolved in high-purity distilled water at a concentration of 1 mg / mL. After preparing each dissolved peptide at a concentration of 10 μg / mL in a 20 mM phosphate buffer (pH 7.4: hereinafter referred to as PBS) containing 150 mM NaCl, a 96-well EIA
50 μl / well was dispensed into the plate and left at room temperature for 2 hours to immobilize the antigen on the plate. 0.05% Tween 2
After washing twice with PBS containing 0 (hereinafter referred to as T-PBS), a blocking reagent (Block Ace, manufactured by Dainippon Pharmaceutical Co., Ltd.) was dispensed at 350 μl / well and left at room temperature for 1 hour to allow protein binding on the bottom surface. Residues were blocked. After washing twice with T-PBS, each of the above-prepared mouse serum and rabbit serum is diluted 100-fold in T-PBS as a primary antibody, dispensed at 50 μl / well, and left at room temperature for 2 hours. . After washing twice with T-PBS, a 50,000-fold diluted solution of a goat anti-mouse immunoglobulin-peroxidase conjugate and an anti-rabbit immunoglobulin-peroxidase conjugate (manufactured by TAGO) was dispensed as a second antibody in an amount of 50 μl / well. And left at room temperature for 1 hour. After washing twice with T-PBS, an OPD substrate solution (60 mg of o-phenylenediamine @ amine dihydrochloride was added to a citrate / phosphate buffer (pH
A solution prepared by adding 20 μl of 30% hydrogen peroxide to a solution of 5.2) dissolved in 20 mL) was dispensed in 50 μl / hole portions, and after coloring, 1.5 N sulfuric acid solution was dispensed in 50 μl / hole portions. Stop the reaction. The absorbance is measured with a plate reader at a main wavelength of 492 nm and a sub wavelength of 620 nm. Recombinant human HL
Sera obtained by immunizing mice with antisera (a), and sera obtained by immunizing rabbits with an immunizing antigen obtained by mixing equal amounts of conjugates of peptides 2 and 4 to 8 with a carrier were used as antisera. (B) A serum obtained by immunizing a rabbit with an immunizing antigen obtained by mixing equal amounts of the conjugates of peptides 1 and 2 with a carrier was used as an antiserum (c). The results are shown in Table 1 and FIG.

[0054]

[Table 1]

In the serum (a) obtained by immunizing mice with recombinant human HL, the group of antibodies against peptide 1 is predominant, and peptide 1 has a superior immune response in the amino acid sequence of human HL. Site. Peptide 2, 4-8
In the antiserum (b), a serum obtained by immunizing an immunizing antigen rabbit obtained by mixing an equal amount of the conjugate with the carrier with the carrier of the present invention, the antibody group against peptide 8 is predominant, and peptide 8 is
Being a site of superior immune response in the amino acid sequence of L,
It became clear. A serum obtained by immunizing a rabbit with an immunizing antigen obtained by mixing equal amounts of the conjugates of the peptides 1 and 2 with the carrier was used in the antiserum (c).
Antibody group is dominant, and peptide 1 is human HL
Was the site where the immune response was dominant in the amino acid sequence.

From the above results, when producing an anti-human HL monoclonal antibody, an antibody-producing cell was obtained by immunizing a mammal with peptide 1 or 8, and the myeloma having no autoantibody-producing ability with the antibody-producing cell was obtained. It is apparent that the hybridoma can be produced by preparing a hybridoma from a lineage cell (myeloma cell), cloning the hybridoma, and selecting a clone that produces a monoclonal antibody having a specific affinity for the antigen used for immunizing the mammal. It became. Similarly, when producing an anti-human HL polyclonal antibody, it was revealed that an antiserum can be obtained and produced by immunizing a mammal with peptide 1 or 8. In addition, when obtaining an anti-HL antibody against peptide 1, a mouse or rabbit is preferable as a mammal, and when obtaining an anti-HL antibody against peptide 8, a rabbit is preferable as a mammal.

(C) To the site where the immune response predominates in the protein
Production of Antibodies (c-1) (b) A mammal corresponding to an antigen obtained by binding a peptide corresponding to an immunological response dominant site to a carrier protein in a protein identified in (b) was immunized with spleen cells of the immunized animal. The hybridoma cells were fused with myeloma cells, and the resulting hybridoma cells were combined with the recombinant protein and the peptide corresponding to the site superior to the immune response.
Monoclonal antibodies are produced by reacting each type of antigen with a solid-phased carrier, recognizing the predominant site of the immune response, and screening for hybridoma cells with high binding specificity (antigen recognition specificity) for proteins. The steps performed were as follows.

The immunization method used was BALB / c of 8 to 10 weeks old.
Subcutaneously, intravenously, or intraperitoneally in a mouse, a conjugate of peptide 1 with a carrier is added to an appropriate adjuvant (for example, Freund's adjuvant or lividjuvant system (RIBI Ajuvant System, RIBI IMMUNOCHEM REM).
The first immunization (day 0) was carried out by injection together with SEARCH (manufactured by SEARCH). On days 14, 28 and 42 after the initial immunization, booster immunization was carried out by injecting the conjugate of peptide 1 with the carrier subcutaneously or intraperitoneally, and two days before the preparation of the monoclonal antibody-producing hybridoma described below. In the same manner, on the previous day, immunization was performed by collecting and immunizing, and spleen cells were prepared from mice and used for cell fusion.

8-azaguanine-resistant mouse myeloma cell P
3-U1 was cultured (37 ° C., CO ₂ , 5% aeration) in a normal medium (medium containing 1.5 mM glutamine and 13% fetal bovine serum in RPMI-1640), and after 4 days, 2 × 10 ^7.
The above cells were obtained.

The immunized mouse spleen cells (1 × 10 ⁸ ) and mouse myeloma cells (2 × 10 ⁷ ) thoroughly washed with RPMI-1640 (manufactured by Nissui Pharmaceutical Co., Ltd.) were mixed and centrifuged at 1500 rpm for 5 minutes. Splenocytes obtained as a precipitate and P3-U1
After dissolving the mixed cell group, 1 mL of a mixed solution of 50% polyethylene glycol and 10% DMSO was added with stirring, and 2 minutes later, RPMI-1640 was gradually added so that the total volume became 50 mL. did. 1000rpm
After centrifugation for 5 minutes at, the supernatant was discarded, the cells were loosened loosely, and the HAT medium ( ^10-4 M hypoxanthine, 1.5 × 10 ⁻⁵ M thymidine, and 4 × 10 ⁵ aminopterin in the normal medium) was added. 30 mL of medium containing ^-7 M) and 5 mL
Gently suspend cells with a volumetric pipette and add 5% CO ₂
The cells were cultured at 37 ° C. for 2 hours in an incubator. 1500
After centrifugation at rpm for 5 minutes, the supernatant was discarded, the cells were loosened loosely, suspended in HAT medium, dispensed into a 96-well culture plate at 200 μl / well, and incubated at 37 ° C. in a 5% CO ₂ incubator at 37 ° C. Cultured for ~ 14 days.

The screening of the hybridoma producing the anti-human HL monoclonal antibody was carried out using an enzyme immunoassay in which the antigen was immobilized. After the recombinant human HL and peptide 1 prepared in Example 1 were prepared at a concentration of 1 μg / mL in PBS, 50 μl / well was dispensed into a 96-well EIA plate, and allowed to stand at room temperature for 2 hours to immobilize the antigen on the plate. It has become. 350 μl / well of T-PBS was dispensed and left at room temperature for 1 hour to block protein-binding residues on the bottom surface.
After washing twice with T-PBS, 50 μL /
The hybridoma culture supernatant was placed in a 96-well EIA plate as a primary antibody in a clean bench in a 96-well EIA plate.
It was transferred at 0 μl / well and left at 4 ° C. overnight or at room temperature for 2 hours. After washing twice with T-PBS, as a second antibody,
A 5000-fold dilution of a goat anti-mouse immunoglobulin-peroxidase conjugate (manufactured by TAGO) was dispensed at 50 μl / well and left at room temperature for 1 hour. 2 with T-PBS
After washing twice, an OPD substrate solution (a solution obtained by dissolving 60 mg of o-phenylenediamine dihydrochloride in 20 mL of citrate / phosphate buffer (pH 5.2)) was added with 20 μL of 30% hydrogen peroxide.
l) was dispensed at 50 μl / well, and after coloring, 1.
The reaction is stopped by dispensing 50 μl / well of 5N sulfuric acid solution. Measure the absorbance with a plate reader at the main wavelength of 492 nm,
The measurement was performed at a sub wavelength of 620 nm.

Cloning was repeated 2 to 4 times by the limiting dilution method in the wells in which the hybridoma producing the anti-HL monoclonal antibody was observed, and those in which stable antibody production was recognized were regarded as the anti-HL monoclonal antibody-producing hybridoma strain. Selected. The established hybridoma 3A2 was deposited on January 24, 2001 with the Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Biotechnology and Industrial Technology, under the deposit number FERM P-
No. 18181 was deposited.

(C-2) Mass Preparation of Monoclonal Antibody 10 weeks after pristane treatment (0.5 mL / animal of 2,6,10,14-tetramethylpentadecane was intraperitoneally administered and bred for 1 to 2 weeks) Aged mice (BALB / c) were injected intraperitoneally with 1 × 10 ⁶ cells / animal of each of the above hybridoma strains. After 10 to 21 days, the hybridoma line became ascites cancer, and ascites (4 to 10) from a mouse having ascites in the abdominal cavity.
(ml / animal) and centrifuged to remove solids. After the supernatant was salted out with 50% ammonium sulfate, it was dialyzed overnight with PBS to obtain a crude purified monoclonal antibody.

(C-3) A mammal is immunized with an antigen obtained by binding a peptide corresponding to an immunological response dominant site in the protein identified in (b) to a carrier protein, and the immune response dominant site is recognized. A step of producing an antiserum and a polyclonal antibody having high binding specificity (antigen recognition specificity) for the protein, and reacting the antiserum with a carrier having a peptide corresponding to an immune response dominant site immobilized thereon, After isolating the carrier according to a conventional method, the step of fractionating the antibody fraction from the carrier and concentrating the polyclonal antibody group having high binding specificity (antigen recognition specificity) was carried out as follows.

The peptide 1 or 8 prepared in (a) above and 0.1 to 0.3 mg of the conjugate of the carrier (KLH) and Freund's Complete adjuvant (S
igma), and the mixture was injected into dozens of rabbit limbs and skin on the back to immunize. 5 to 2 months to 3 months
The same administration was performed six times. Two weeks after the final administration, whole blood was collected, and the serum was obtained. After 50% ammonium sulfate salting out of serum, P
It was dialyzed overnight against BS to obtain a crudely purified polyclonal antibody.

(C-4) (Anti-human HL antibody, monoclonal
Purification of Null Antibody and Polyclonal Antibody) The monoclonal antibody 3A2 obtained above and the partially purified monoclonal antibody and the roughly purified polyclonal antibody
An equal amount of APS II binding buffer (manufactured by Bio-Rad) was added, and the mixture was filtered through a 0.45 μm filter (manufactured by Millipore) to remove white precipitate. The resulting filtrate is HiTrap Protein A
It was added with an affinity column (Pharmacia Biotech). After washing with MAPS II binding buffer, MAPS II elution buffer (Bio-Rad)
(3 mL / tube, about 10 tubes). The elution pattern was measured by the absorbance at a wavelength of 280 nm, and the fraction containing the antibody was collected according to the elution pattern. (C-5) (Determination of isotype) Mouse monoclonal antibody isotype determination kit (Z
Using YMED), the procedure was performed according to the experimental operation protocol attached to the kit, and the isotype of monoclonal antibody 3A2 was determined. 3A2 was confirmed to be IgG _2a , κ.

Example 3 ( Affinity Chromatograph)
Purification of Human HL by A ) High-purity human HL was purified from human PHP by affinity chromatography using the purified monoclonal antibody 3A2 prepared in Example 2. (1) Preparation of immobilized monoclonal antibody (adsorbent for column packing) CNBr-activated Sepharose 4B (CNBr-activated Sep.)
harose 4B, manufactured by Pharmacia Biotech)
The operation was performed according to the attached protocol. CNBr
The activated Sepharose 4B is swollen with 1 mM hydrochloric acid for 20 minutes, and washed with 1 mM hydrochloric acid on a Buchner funnel with a glass filter having G3 grade pores. 0.5M gel
Suspended in a 0.1 M sodium bicarbonate (pH 8.3) buffer containing sodium chloride (pH 8.3), dialyzed overnight against the binding buffer, and mixed at room temperature for 2 hours. Then, let the antibody and the gel bind. Centrifuge at 11000 rpm for 5 minutes to remove the supernatant,
After suspending in 0.1 M Tris buffer (pH 8.0), the suspension is left at room temperature for 2 hours. After filtration through a glass filter, the plate is washed alternately with a binding buffer and 0.1 M acetate buffer (pH 4.0) containing 0.5 M sodium chloride on the glass filter. Finally, it is suspended in an appropriate amount of PBS and packed in a column.

(2) Purification by Heparin Sepharose 4B Chromatography Peripheral blood after heparin loading, collected from a plurality of healthy persons,
Blood cells were removed by centrifugation according to a conventional method to obtain human plasma PHP, which was stored at -80 ° C until use. -80
200mL of human plasma stored at ℃ is dissolved in a thermostat at 37 ℃ and centrifuged to remove insolubles (5000 × g, 30min, 4 ℃)
did. This PHP was filled into a glass column tube (2 × 40 cm, manufactured by Bio-Rad), and 0.3M NaCl and 10%
Glycerol was added to Heparin-Sepharose 4B equilibrated with 5 mM phosphate buffer (pH 7.4). 0.3M Na
5 mM phosphate buffer containing Cl and 10% Glycerol (pH
After washing in 7.4), 0.3-1.5M NaCl, 10%
The mixture was eluted with a 5 mM phosphate buffer (pH 7.4) containing Glycerol in a concentration gradient (8 mL / tube, 65 tubes). The elution pattern was determined by HT using an enzyme immunoassay in which the fractions indicated below were immobilized on the basis of the absorbance at a wavelength of 280 nm and the fractions shown below.
The discrimination state of GL and LPL was tested.

Each fraction was collected in 96-well E
The mixture was dispensed at 50 μl / well into an IA plate, left at room temperature for 2 hours, and immobilized on an antigen plate. 350 T-PBS
The mixture was dispensed in μl / well and left at room temperature for 1 hour to block protein-binding residues on the bottom surface. After washing twice with T-PBS, 50 μl / well of T-PBS was dispensed, and the purified anti-human HL monoclonal antibody 3A2 prepared in Example 3 and the purified anti-LPL monoclonal antibody (particularly, The monoclonal antibody described in Japanese Unexamined Patent Application Publication No. 05-223822 is suitable.
The solution was dispensed at 50 μl / well and left at room temperature for 2 hours. T-
After washing twice with PBS, goat anti-mouse immunoglobulin-peroxidase conjugate (TAGO
(Manufactured by K.K.) in 50 μl / well.
It was left at room temperature for 1 hour. After washing twice with T-PBS,
OPD substrate solution (60% o-phenylenediamine dihydrochloride)
mg solution in 20 mL of citrate / phosphate buffer solution (pH 5.2) and 20 μl of 30% hydrogen peroxide) were dispensed in 50 μl / well portions, and after coloring, 1.5 N The reaction is stopped by dispensing 50 μl / well of sulfuric acid solution. Measure absorbance with a plate reader at main wavelength 492 nm and sub wavelength 6
Measured at 20 nm.

FIG. 2 shows the results. The HL protein detected by the purified anti-human HL monoclonal antibody 3A2 prepared in Example 2 was eluted at a NaCl concentration of about 0.8 M, and was purified by Chao-Fu Cheng et al. (The Journal of Biological Chemistry). The Journal of B
iological Chemistry), Vol. 260, No. 19, No. 1
0720-10727, 1985), which is consistent with the results of the anti-human HL monoclonal antibody 3 of the present invention.
This indicates that A2 specifically recognizes human HL. In addition, the anti-human HL monoclonal antibody 3 of the present invention
A2 shows no cross-reactivity with LPL eluted at about 1.5 M NaCl concentration detected by the anti-LPL monoclonal antibody, and an antigen-antibody reaction against human HL is observed. At least for human lipoprotein lipase
It was proved that no antigen-antibody reaction was observed.

(3) Purification by Affinity Chromatography The solution containing HL prepared in (2) above was dialyzed against PBS overnight, and then added to the affinity column prepared in (1). The column was washed with PBS, and eluted with PBS containing 3 M NaSCN.
Measured by absorbance at nm. The results are shown in FIG. According to the elution pattern, fractions containing human HL were collected and
Dialyzed to prepare purified human HL of extremely high purity.

Analysis was performed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) according to a conventional method. FIG. 4 shows the results. Electrophoresis / spot image analysis software Densitograph Ver.4.03C (manufactured by ATTO Corporation)
Was used to evaluate the molecular weight and purity.
It was confirmed to be of extremely high purity, consisting of one kind of C.00.

Example 4 Establishment of Quantitative Method for Human HL by Sandwich ELISA (1) Preparation of Immobilized Antibody Diluted purified monoclonal antibody 3A2 or purified polyclonal antibody to 10 μg / mL in PBS, and 96-well EIA
50 μl / well was dispensed into the plate and left at room temperature for 2 hours to immobilize the antibody on the plate. After washing twice with T-PBS, 350 μl / well of T-PBS containing 1% BSA was dispensed and allowed to stand at room temperature for 1 hour to block protein-binding residues on the bottom surface. After removing the contents, 1% BSA and 1%
350 μl / T-PBS containing 0% sucrose
The mixture was dispensed into holes, left at room temperature for 1 hour, and the contents were removed and air-dried.

(2) Peroxidase Labeling of Monoclonal and Polyclonal Antibodies A solution prepared by dissolving peroxidase (hereinafter referred to as HRP) at a concentration of 10 mg / mL in 5 mM sodium acetate buffer (pH 4.5) was added to sodium metaperiodate. With distilled water
A solution dissolved at a concentration of 00 mg / mL was added at a ratio of HRP: sodium metaperiodate = 1 mg: 0.43 mg, and the solution was added in a dark place.
The reaction was performed at room temperature for 30 minutes. Next, the reaction solution was added to a column of Sephadex G-25 (1.5 × 30 cm, manufactured by Pharmacia Biotech) equilibrated with 5 mM sodium acetate buffer (pH 4.5), and the activated HRP fraction was removed. Collected. Next, the purified monoclonal antibody and polyclonal antibody were added to a 0.1 M sodium bicarbonate buffer solution (pH 9.
The solution dialyzed overnight in 3) was mixed with an activated HRP fraction at a weight ratio of 1: 1 and reacted at room temperature in the dark for 1 hour. After the reaction, sodium tetrahydroborate is added with distilled water at 2 mg / mL.
Solution dissolved at a concentration of 1
The mixture was mixed with 0 μl, and reacted at room temperature in the dark for 15 minutes. After the reaction, an equal amount of saturated ammonium sulfate was added and left on ice for 1 hour.
After centrifugation at 12,000 rpm for 10 minutes, the supernatant was removed to recover the HRP-labeled antibody, which was suspended in PBS. Wavelength 2
The absorbance at 80 nm was measured, and the following test was performed using the absorbance.

(3) Quantitative system by sandwich ELISA (2-antibody method) The immobilized antibody, monoclonal antibody or polyclonal antibody prepared in (1) (hereinafter referred to as immobilized microplate) is used. A measurement sample (plasma before heparin loading, PHP, purification, diluted with PBS containing 0.2% BSA and 0.05% Triton X-100 (hereinafter referred to as BT-PBS) in each well of the solid-phased microplate. HL) 50μ
1 / well was added and incubated for 2 hours at room temperature. After washing the solid-phased microplate twice with T-PBS, BT
-50 μl of the HRP-labeled monoclonal or polyclonal antibody solution prepared in (2) diluted with PBSB
Per well, and incubated at room temperature for 1 hour. After washing the immobilized microplate twice with T-PBS, OP
D substrate solution (60 mg of o-phenylenediamine dihydrochloride dissolved in 20 mL of citrate / phosphate buffer (pH 5.2), and a solution obtained by adding 20 μl of 30% hydrogen peroxide to 50 μl / well) After color development, the reaction was stopped by dispensing 50 μl / well of 1.5 N sulfuric acid solution, and the absorbance was measured with a plate reader at a main wavelength of 492 nm and a subwavelength of 620 nm.
Was measured.

(4) Quantitative system by sandwich ELISA (3 antibody method) Immobilized antibody prepared in (1), monoclonal antibody 3A
2 (hereinafter, immobilized microplate). A measurement sample (plasma before heparin loading, PHP, purified HL) diluted with BT-PBS was added to each well of the solid-phased microplate at 50 μl / well and incubated at room temperature for 2 hours. After the immobilized microplate was washed twice with T-PBS, a solution obtained by diluting a polyclonal antibody to 10 μg / mL with BT-PBS as a second antibody was dispensed in 50 μl / well portions and incubated at room temperature for 1 hour. After washing the immobilized microplate twice with T-PBS, as a third antibody,
A 5000-fold dilution of a goat anti-rabbit immunoglobulin-peroxidase conjugate (manufactured by TAGO) was dispensed at 50 μl / well and left at room temperature for 1 hour. After washing the solid-phased microplate twice with T-PBS, an OPD substrate solution (60 mg of o-phenylenediamine dihydrochloride) was dissolved in 20 mL of a citrate / phosphate buffer (pH 5.2). %
A solution obtained by adding 20 μl of hydrogen peroxide was dispensed at 50 μl / hole at a time, and after coloring, 1.5 N sulfuric acid solution was dispensed at 50 μl / well to stop the reaction. The absorbance was measured with a plate reader at a main wavelength of 492 nm and a sub wavelength of 620 nm. Or
A TMB substrate (manufactured by PIERCE) was dispensed at 50 μl / well and color development was performed, and then a 2.0N sulfuric acid solution was dispensed at 50 μl / well to stop the reaction. Absorbance of 450 with plate reader
Measured in nm. The HL concentration in the subject was determined from a calibration curve prepared using purified HL and standard PHP as standard substances, respectively.

(5) Preparation of calibration curve (purified HL) Using the purified HL prepared in Example 3 as a standard substance, (4)
A calibration curve was prepared using the sandwich ELISA established in the above. FIG. 5 shows the results. A good calibration curve was obtained in the concentration range of 10 to 1000 ng / mL (y = 0.315 Ln
(X) -0.5275, correlation coefficient: R ² = 0.996
3). The HL concentration in the subject was calculated from the calibration curve.

(6) Confirmation of the accuracy of the measurement system From the calibration curve prepared in (5), the PHP measured simultaneously was increased by 40 times, 80 times and 160 times, and the HL concentration at each point was calculated. The accuracy of the measurement system was confirmed from the dilution linearity. FIG. 6 shows the results. Good dilution linearity was shown up to 40-160 times (y = 894.41x-0.9684,
Correlation coefficient: R ² = 0.9857).

(7) Examination of Specificity of Sandwich ELISA System In order to examine the specificity, the plasma before heparin loading and the plasma after heparin loading (PHP) were measured at 40-fold, 80-fold and 160-fold, and the HL at each point was measured. The concentration was calculated to confirm specificity. FIG. 7 shows the results. Heparin-loaded plasma (PH
P) shows good dilution linearity (y = 5) from 40 to 160 times.
51.94x + 2.2692, correlation coefficient: R ² = 0.9
816), which revealed that heparin was injected intravenously and HL anchored on the surface of liver endothelial cells was taken out in the blood in a free state and specifically measured. On the other hand, the plasma before heparin loading has HL anchored on the surface of hepatic endothelial cells, and there is almost no HL free in the blood,
A small amount of HL (y = 24.878x + 5.3756, correlation function: R ² = 0.9322) could be measured.

[0080]

EFFECT OF THE INVENTION The anti-human HL antibody of the present invention has high specificity for human HL and does not react to LPL.
It is useful as an antibody for measuring L. Therefore, using the antibody of the present invention, human HL can be measured simply and with high sensitivity, which is advantageous for diagnosing arteriosclerosis and liver function.

[0081]

[Sequence List] SEQUENCE LISTING <110> Daiichi Pure Chemicals Co., Ltd. <120> Anti-HL Antibody <130> P01421304 <140> <141> <160> 6 <170> PatentIn Ver. 2.1 <210> 1 < 211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide based on hydrophilicity of human HL <400> 1 Gly Gln Ser Leu Lys Pro Glu Pro Phe Gly Arg Arg Ala Gln Ala Val 1 5 10 15 Glu Thr Asn Lys 20 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide based on hydrophilicity of human HL <400> 2 Lys Ser Lys Thr Ser Lys Arg Lys Ile Arg 1 5 10 <210> 3 <211> 499 <212> PRT <213> Homo sapiens <400> 3 Met Asp Thr Ser Pro Leu Cys Phe Ser Ile Leu Leu Val Leu Cys Ile 1 5 10 15 Phe Ile Gln Ser Ser Ala Leu Gly Gln Ser Leu Lys Pro Glu Pro Phe 20 25 30 Gly Arg Arg Ala Gln Ala Val Glu Thr Asn Lys Thr Leu His Glu Met 35 40 45 Lys Thr Arg Phe Leu Leu Phe Gly Glu Thr Asn Gln Gly Cys Gln Ile 50 55 60 Arg Ile Asn His Pro Asp Thr Leu Gln Glu Cys Gly Phe Asn Ser Ser 65 70 75 80 Leu Pro Leu Val Met Ile Ile His Gly Trp Ser Val Asp Gly Val Leu 85 90 95 Glu Asn Trp Ile Trp Gln Met Val Ala Ala Leu Lys Ser Gln Pro Ala 100 105 110 Gln Pro Val Asn Val Gly Leu Val Asp Trp Ile Thr Leu Ala His Asp 115 120 125 His Tyr Thr Ile Ala Val Arg Asn Thr Arg Leu Val Gly Lys Glu Val 130 135 140 Ala Ala Leu Leu Arg Trp Leu Glu Glu Ser Val Gln Leu Ser Arg Ser 145 150 155 160 His Val His Leu Ile Gly Tyr Ser Leu Gly Ala His Val Ser Gly Phe 165 170 175 Ala Gly Ser Ser Ile Gly Gly Thr His Lys Ile Gly Arg Ile Thr Gly 180 185 190 Leu Asp Ala Ala Gly Pro Leu Phe Glu Gly Ser Ala Pro Ser Asn Arg 195 200 205 Leu Ser Pro Asp Asp Ala Asn Phe Val Asp Ala Ile His Thr Phe Thr 210 215 220 Arg Glu His Met Gly Leu Ser Val Gly Ile Lys Gln Pro Ile Gly His 225 230 235 240 Tyr Asp Phe Tyr Pro Asn Gly Gly Ser Phe Gln Pro Gly Cys His Phe 245 250 255 Leu Glu Leu Tyr Arg His Ile Ala Gln His Gly Phe Asn Ala Ile Thr 260 265 270 Gln Thr Ile Lys Cys Ser His Glu Arg Ser Va l His Leu Phe Ile Asp 275 280 285 Ser Leu Leu His Ala Gly Thr Gln Ser Met Ala Tyr Pro Cys Gly Asp 290 295 300 Met Asn Ser Phe Ser Gln Gly Leu Cys Leu Ser Cys Lys Lys Gly Arg 305 310 315 320 Cys Asn Thr Leu Gly Tyr His Val Arg Gln Glu Pro Arg Ser Lys Ser 325 330 335 Lys Arg Leu Phe Leu Val Thr Arg Ala Gln Ser Pro Phe Lys Val Tyr 340 345 350 His Tyr Gln Leu Lys Ile Gln Phe Ile Asn Gln Thr Glu Thr Pro Ile 355 360 365 Gln Thr Thr Phe Thr Met Ser Leu Leu Gly Thr Lys Glu Lys Met Gln 370 375 380 Lys Ile Pro Ile Thr Leu Gly Lys Gly Ile Ala Ser Asn Lys Thr Tyr 385 390 395 400 400 Ser Phe Leu Ile Thr Leu Asp Val Asp Ile Gly Glu Leu Ile Met Ile 405 410 415 Lys Phe Lys Trp Glu Asn Ser Ala Val Trp Ala Asn Val Trp Asp Thr 420 425 430 Val Gln Thr Ile Ile Pro Trp Ser Thr Gly Pro Arg His Ser Gly Leu 435 440 445 Val Leu Lys Thr Ile Arg Val Lys Ala Gly Glu Thr Gln Gln Arg Met 450 455 460 Thr Phe Cys Ser Glu Asn Thr Asp Asp Leu Leu Leu Arg Pro Thr Gln 465 470 475 480 Glu Lys Ile Phe Val Lys Cys Glu Ile Lys S er Lys Thr Ser Lys Arg 485 490 495 Lys Ile Arg <210> 4 <211> 1553 <212> DNA <213> Homo sapiens <400> 4 ccgagaaatg gacacaagtc ccctgtgttt ctccattctg ttggttttat gcattag ttggttttat gcatctgacctagt gccctgag ttggttgcctagt gccctgag gaagaccaga ttcctgctct ttggagaaac 180 caatcagggc tgtcagattc gaatcaatca tccggacacg ttacaggagt gcggcttcaa 240 ctcctccctg cctctggtga tgataatcca cgggtggtcg gtggacggcg tgctagaaaa 300 ctggatctgg cagatggtgg ccgcgctgaa gtctcagccg gcccagccag tgaacgtggg 360 gctggtggac tggatcaccc tggcccacga ccactacacc atcgccgtcc gcaacacccg 420 ccttgtgggc aaggaggtcg cggctcttct ccggtggctg gaggaatctg tgcaactctc 480 tcgaagccat gttcacctaa ttgggtacag cctgggtgca cacgtgtcag gatttgccgg 540 cagttccatc ggtggaacgc acaagattgg gagaatcaca gggctggatg ccgcgggacc 600 tttgtttgag ggaagtgccc ccagcaatcg tctttctcca gatgatgcca attttgtgga 660 tgccattcat acctttacgc gggagcacat gggcctgagc gtgggcatca aacagcccat gagcat ctgact gagcat ctgact gagcactcgccact ccctaga 780 gctctacaga catattgccc agcacggctt caatgccatc acccagacca taaaatgctc 840 ccacgagcga tcggtgcacc ttttcatcga ctccttgctg cacgccggca cgcagagcat 900 ggcctacccg tgtggtgaca tgaacagctt cagccagggc ctgtgcctga gctgcaagaa 960 gggccgctgc aacacgctgg gctaccacgt ccgccaggag ccgcggagca agagcaagag 1020 gctcttcctc gtaacgcgag cccagtcccc cttcaaagtt tatcattacc agttaaagat 1080 ccagttcatc aaccaaactg agacgccaat acaaacaact tttaccatgt cactactcgg 1140 aacaaaagag aaaatgcaga aaattcccat cactctgggc aaaggaattg ctagtaataa 1200 aacgtattcc tttcttatca cgctggatgt ggatatcggc gagctgatca tgatcaagtt 1260 caagtgggaa aacagtgcag tgtgggccaa tgtctgggac acggtccaga ccatcatccc 1320 atggagcaca gggccgcgcc actcaggcct cgttctgaag acgatcagag tcaaagcagg 1380 agaaacccag caaagaatga cattttgttc agaaaacaca gatgacctac tacttcgccc 1440 aacccaggaa aaaatcttcg tgaaatgtga aataaagtct aaaacatcaa agcgaaagat 1500 cagatgagat ttaatgaaga cccagtgtaa agaataaatg aatcttactc cct 1553 <210> 5 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Descripti on of Artificial Sequence: Designed peptide based on hydrophilicity of human HL <400> 5 Gly Gly Ala Ala Thr Thr Cys Gly Gly Ala Cys Ala Ala Ala Gly Cys 1 5 10 15 Cys Thr Gly Ala Ala Ala Cys Cys Ala Gly 20 25 < 210> 6 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide based on hydrophilicity of human HL <400> 6 Gly Thr Thr Thr Cys Gly Cys Thr Thr Thr Cys Thr Ala Gly Thr Cys 1 5 10 15 Thr Ala Cys Thr Cys Cys Thr Ala Gly Gly Cys 20 25

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a view showing a result of specifying a site where an immune response is predominant in human HL.

FIG. 2 is a diagram showing the results of assaying the fractionation state of HTGL and LPL using an enzyme immunoassay in which the fraction fractionated by heparin Sepharose 4B chromatography was immobilized.

FIG. 3 is a diagram showing a chromatogram of human HL by affinity chromatography using monoclonal antibody 3A2.

FIG. 4 is an electrophoretogram showing the degree of purification of human HL purified by affinity chromatography using monoclonal antibody 3A2.

FIG. 5: Sandwich ELI established according to the invention
It is a figure which shows the calibration curve of the quantification method of human HL by SA.

FIG. 6 shows a sandwich ELI established according to the invention.
It is a figure which shows the accuracy of the method of quantifying human HL by SA.

FIG. 7: Sandwich ELI established according to the invention
It is a figure which shows the specificity of the human HL quantification method by SA.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/573 C12P 21/08 // C12P 21/08 C12N 5/00 B (72) Inventor Kazunori Saito Ibaraki 3-3-1 Koyodai, Ryugasaki, Japan F-term (reference) at Daiichi Kagaku Pharmaceutical Co., Ltd. Diagnostics Research Laboratories CA25 CA46 4H045 AA11 AA30 BA10 CA40 DA76 EA50 FA72 FA74

Claims (6)

[Claims] 1. An anti-human hepatic triglyceride lipase antibody or a fragment thereof which recognizes the amino acid sequence represented by SEQ ID NO: 1 or 2 or an amino acid sequence in which one to several amino acids have been deleted, substituted or added. 2. The anti-human hepatic triglyceride lipase antibody or a fragment thereof according to claim 1, which is a monoclonal antibody. 3. A hybridoma that produces the monoclonal antibody according to claim 2. 4. A reagent for measuring human hepatic triglyceride lipase, comprising the anti-human hepatic triglyceride lipase antibody according to claim 1 or 2, a fragment thereof, or a labeled product thereof. 5. A method for measuring human hepatic triglyceride lipase in a human blood-derived sample, which comprises reacting the anti-human hepatic triglyceride lipase antibody according to claim 1 or 2, a fragment thereof, or a labeled product thereof. Law. 6. An affinity chromatography using an immobilized anti-human hepatic triglyceride lipase antibody or a fragment thereof obtained by immobilizing the anti-human hepatic triglyceride lipase antibody or a fragment thereof according to claim 1 or 2 on an insoluble carrier. A method for separating or purifying human hepatic triglyceride lipase.