KR20030055187A - The mass preparation method of monoclonal antibodies to hippuric acid - Google Patents

Abstract

The present invention is a mass production method of unicellular antibody against hippuric acid, a metabolite of toluene, which is one of the representative harmful hallucinogens.

Hyplic acid and the transport proteins BSA, KLH, and OVA were prepared by using a coupling reagent and a cross-linker to prepare a protein conjugate for hyplic acid-carrying and then used for the immune and detection reactions. After the immunization by injecting a conjugate prepared with an immunogen into a mouse, spleen cells were harvested for cell fusion with myeloma cells, followed by culturing in HAT medium to detect a cell line producing a monocellular antibody against hypric acid. After screening, the antibody-producing cell line was injected into the abdominal cavity of the mouse to obtain ascites generated, and purified to obtain a large amount of unicellular antibody against hypric acid.

Mass-produced single-cell antibodies have high titers of antibodies, do not cross react with carrier proteins, competitive inhibition occurs with higher concentrations of hypoic acid, and do not cross react with other proteins contained in urine. It is used in diagnostic kit for detecting hypoic acid.

Description

The mass production method of the monoclonal antibody to hypofuric acid {THE MASS PREPARATION METHOD OF MONOCLONAL ANTIBODIES TO HIPPURIC ACID}

The present invention relates to a mass production method of unicellular antibody against hippuric acid (HA), a metabolite of toluene, which is a kind of harmful hallucinogen.

Inhalation of bonds containing large amounts of toluene is a pathological sociological phenomenon peculiar to our country, and the report is rarely reported in developed countries such as the United States and Japan. Therefore, diagnostic kits that can quickly and accurately diagnose inhalation of bonds have not yet been developed worldwide.

Bond inhalation may be measured by measuring the amount of toluene itself in the blood or urine, or by measuring the amount of the toluene metabolites Hippuric Acid, o- cresol, or Mercapturic Acids. Such measurements require expensive equipment such as gas chromatography / mass spectroscopy (GC / MS) or high performance liquid chromatography (HPLC) and well-trained technical personnel. In addition, in order to apply this method, a number of cumbersome and time-consuming processes such as preparation and concentration of a sample through solvent extraction must be involved. Therefore, routine and rapid on-site inspection of bond inhalation is not possible with existing analytical chemical techniques.

The use of immunoassays using antigen-antibody responses is widely recommended to overcome these shortcomings and to obtain objective test results in a quick and convenient manner.

Detection of hypric acid by immunoassay requires specific antibodies that react specifically with hypoic acid present in body fluids, especially urine, but do not react with similar structures. However, since hyplic acid has a low molecular weight and low immunogenicity, it is difficult to obtain a desired antibody by a widely used immunization method.

It is an object of the present invention to efficiently produce a large number of unicellular antibody against hypric acid required for detection of hypric acid by immunoassay. It is also an object of the present invention to provide a kit for diagnosing a bond inhaler with a single cell antibody against the obtained hypoic acid.

1 is a mass production process diagram of a hypoic acid monocell group antibody of the present invention.

Figure 2 is a flow chart of the preparation of the BSA conjugate, which is a hypoacid and a carrier protein.

3 is a process diagram for preparing an OVA conjugate, which is a hypofuric acid and a carrier protein.

4 is a result of confirming the production of the conjugate prepared by using a coupling reagent and a cross-linker of BSA and OVA, which are hyplic acid and a transport protein.

5 is a result of confirming the production of a conjugate prepared by using a coupling reagent and a cross-linker of hypric acid and BSA.

The present invention relates to a mass production method of unicellular antibody against the metabolite hypric acid (HA) of toluene, which is a kind of hallucinogen.

The method of the present invention, using a coupling reagent and a cross-linker of the hydronic acid and the transport proteins BSA, KLH, and OVA to prepare a protein conjugate for hyplic acid-carrying the conjugate, After immunization by injection into mice, splenocytes of mice were harvested and fused with myeloma cells, and then cultured in HAT (hypoxanthine aminopterin thymidine) medium to detect a cell line for detecting monoclonal antibody against hypric acid. After screening, the antibody-producing cell line was injected into the abdominal cavity of a mouse to obtain ascites, which were produced, and purified to obtain a large amount of unicellular antibody against hypric acid.

Mass-produced single-cell antibodies have high titers of antibodies, do not cross react with carrier proteins, competitive inhibition occurs with higher concentrations of hypoic acid, and do not cross react with other proteins contained in urine. It is used for diagnostic kit for detecting hypoic acid.

Referring to the mass production method of a single-cell antibody against hypoic acid of the present invention in more detail.

Hyplic acid and the transport proteins BSA, KLH, and OVA are prepared using a coupling reagent and a cross-linker to prepare a protein conjugate for hypoic acid-carrying.

Immunogen conjugates are immunized by intraperitoneal and subcutaneous injection of mice (Balb / c species).

Splenocytes are harvested from mice and fused with myeloma cells. The myeloma cells do not need to be specific cell lines, they do not produce immunoglobulins and may be myeloma cells lacking HGPRT (hypoxanthine guanine phosphoribosyl transferase).

The cell-fused cells are cultured in HAT medium, and the hypoic acid mononuclear cell secretion cell line is selected using a detection conjugate.

Mice (Balb / c) intended to produce unicellular antibody were pretreated with Pristane (2, 6, 10, 14-tetramethyldecanoic acid) To induce cancer easily.

Ascites is obtained by injecting selected antibody secreting cell lines into the abdominal cavity of pretreated mice.

From the obtained ascites, hypoic acid unicellular antibody is purified.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

Example 1 Preparation of HA-BSA Conjugate as an Immunogen and HA-OVA Conjugate for Detection

HA-BSA conjugate immunogens were prepared using hypoic acid and carrier protein BSA (bovine serum albumin).

Hyplic acid 18.0 mg, DCC 20 mg, and NHS (N-Hydroxy succinic anhydride) 10 mg were mixed in 0.2 mL of DMF (N, N-Dimethylformamide) and stirred at room temperature for 2 hours.

10.0 mg of BSA was dissolved in 10 ml of 10 mM PBS buffer (pH 7.4), and the reacted product was dropped dropwise, followed by stirring at room temperature for 3 hours.

The reaction was dialyzed three times with 10 mM PBS buffer (pH 7.4).

The manufacture of the detection HA-OVA conjugate produced the HA-OVA conjugate for detection by the method similar to the manufacturing method of said HA-BSA conjugate.

Example 2 Preparation of HA-KLH Conjugate as an Immunogen and HA-OVA Conjugate for Detection

HA-KLH conjugate immunogens were prepared using hypoic acid and a carrier protein, KLH (keyhole limpets hemocyanin).

Hyplic acid 18.0 mg, DCC 20 mg, and NHS (N-Hydroxy succinic anhydride) 10 mg were mixed in 0.2 mL of DMF (N, N-Dimethylformamide) and stirred at room temperature for 2 hours.

KLH 10.0 mg was dissolved in 10 ml of 10 mM PBS buffer (pH 7.4), and then the product reacted therein was dropped dropwise, followed by stirring at room temperature for 3 hours.

The reaction was dialyzed three times with 10 mM PBS buffer (pH 7.4).

The manufacture of the detection HA-OVA conjugate produced the HA-OVA conjugate for detection by the method similar to the manufacturing method of said HA-KLH conjugate.

Example 3 Preparation of HA-Lys-KLH Conjugate as an Immunogen and HA-Lys-BSA Conjugate for Detection

Dissolve 10.0 mg of KLH in 10 ml of 10 mM PBS buffer (pH 7.4), add 20 mg of EDC (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) and 10 mg NHS, and react at room temperature. .

Hyplic acid-lysine 30 mg was mixed and reacted by stirring at room temperature for 6 hours.

The reaction was dialyzed three times with 10 mM PBS buffer (pH 7.4).

The manufacture of the detection HA-Lys-BSA conjugate produced the detection HA-Lys-BSA conjugate in the same way as the manufacturing method of said HA-Lys-KLH conjugate.

Example 4 Preparation of HA-KLH Conjugate and HA-BSA Conjugate for Detection

2 mg of Activated SuperCarrier ™ KLH (manufactured by PIERCE) was dissolved in 5 ml of PBS (pH 7.4), and then 10 mg of N-benzoyl-cystine was mixed and reacted at room temperature for 6 hours.

The reaction was dialyzed three times with 10 mM PBS buffer (pH 7.4).

The manufacture of the detection HA-BSA conjugate produced the HA-BSA conjugate for detection by the method similar to the manufacturing method of said HA-KLH conjugate.

Example 5 Preparation of HA-KLH Conjugate and HA-BSA Conjugate for Detection

10 mg of Activated KLH was dissolved in 5 ml of PBS (pH 7.4), and then 19 mg of EDC and 12 mg of NHS were added and stirred at room temperature for 2 hours.

10 mg of N- α -benzoyl-lysine was mixed and reacted by stirring at room temperature for 4 hours.

The reaction was dialyzed three times with 10 mM PBS buffer (pH 7.0).

The manufacture of the detection HA-BSA conjugate produced the HA-BSA conjugate for detection by the method similar to the manufacturing method of said HA-KLH conjugate.

Example 6 Preparation of HA-BSA Conjugate as an Immunogen and HA-OVA Conjugate for Detection

10 mg of Activated BSA is dissolved in 5 ml of PBS (pH 7.4), and then 19 mg of EDC and 12 mg of NHS are added and stirred at room temperature for 2 hours.

10 mg of N- α -benzoyl-lysine was mixed and reacted by stirring at room temperature for 4 hours.

The reaction was dialyzed three times with 10 mM PBS buffer (pH 7.0).

The manufacture of the detection HA-OVA conjugate produced the HA-OVA conjugate for detection by the method similar to the manufacturing method of said HA-BSA conjugate.

Example 7 Preparation of HA-KLH Conjugate and HA-OVA Conjugate for Detection

Dissolve 18 mg of HA, 20 mg of DCC and 10 mg of NHS in 0.2 mL of DMF and stir at room temperature for 1 hour.

To this, add 0.2 mL of PBS solution containing 13 mg of ACA and stir.

After adding 30 mg of DCC and 20 mg of NHS, 10 mg of BSA is added and stirred for 6 hours.

The reaction was dialyzed three times with 10 mM PBS buffer (pH 7.0).

The manufacture of the detection HA-OVA conjugate produced the HA-OVA conjugate for detection by the method similar to the manufacturing method of said HA-KLH conjugate.

Example 8 Immunization of Mice

Mice (Balb / c) were stabilized for 1 week.

70 μl of the HA-BSA conjugate, the immunogen obtained in Example 1, was mixed 1: 1 with complete Freund's adjuvant and injected intraperitoneally and subcutaneously of mice.

At two week intervals after the first immunization, the same amount of HA-BSA conjugate immunogen was immunized three times by mixing with an Incomplete Freund's adjuvant.

Experimental Example 1 Confirmation of Immunization of Mice

Using anti-serum of mice not immunized and mice immunized by the method of Example 1, antibody titers were measured using HA-OVA conjugates by the indirect ELISA method and the results are shown in Table 1.

TABLE 1 Antibody titers measured in antisera of mice

Dilution 1/100 1 / 1,000 1 / 10,000 1 / 100,000 Antiserum 1st - 1.757 0.928 0.372 (1/100) 2nd 2.015 1.831 1.329 0.436 Normal serum (1/100) 0.176 0.069 - - ※ PBS

As shown in Table 1, the antibody titer of the mice not immunized was 0.069 when diluted 1,000-fold, and in the mice immunized by the method of Example 1, the average antibody titer was immunized to 1.794 when diluted 1,000-fold.

Example 9 Cell Fusion

Antibody titers were measured using HA-BSA conjugates by the indirect ELISA method using antiserum of mice immunized in Example 1.

Mice showing the highest antibody titers were selected and injected 100 μg of HA-BSA conjugates dissolved in PBS through the tail vein 3 days prior to cell fusion.

On the day of cell fusion, the spleens of the mice were extracted, washed lightly with 10 ml of RPMI 1640 medium, and lymphocyte cells were extracted using a cell dissociation sieve tissue grinder kit in 30 ml of RPMI medium.

After centrifugation at 400 g , the leukocyte components were separated by hemolysis with a buffer solution. After washing three times with 30 ml of RPMI 1640 medium, the cells precipitated in 10 ml of fresh RPMI 1640 medium were freed and stored in an incubator at 37 ° C.

Myeloma cells used FO cells purchased from Korea Cell Line Bank.

This cell line does not produce immunoglobulins and lacks HGPRT (Hypoxanthin guanine phosphoribosyl transferase) activity.

FO plasmacytoma cells were cultured using RPMI 1640 medium containing 10% FBS, and the cells were dispensed in the same amount of fresh medium the day before cell fusion to increase the health of the cells.

Spleen lymphocyte cells and plasmacytoma cells of the prepared mice were stained with trypan-blue solution (0.04%, Sigma, St. Louis, USA, T-8154) and viable cell numbers were measured using a hemocytometer.

108 spleen lymphocyte cells and 107 plasmacytoma cells were mixed and the mixed cells were washed with RPMI 1640 medium.

After the last wash, the supernatant was completely removed, and the tube containing the precipitated cells was lightly struck with a finger, mixed well, and then maintained at 37 ° C., while 1 ml of 50% (w / v) PEG / DMSO solution was added at a rate of 1 ml / min Fusing while dropping addition.

RPMI 1640 medium was added dropwise in 1 ml increments for 1 minute, and then 30 ml was added at a rate of 3 ml / min.

After fusion, the cells were collected by centrifugation at 400 g for 10 minutes, suspended in a HAT RPMI medium (20% FBS added) to select only the fused cells, and then 50 μl / Each well was dispensed.

In addition, 50 μl / well of the HAT RPMI medium added with 20% of FBS was incubated for 24 hours at 37 ° C. in the presence of 5% CO 2, and then 50 μl / well of the HAT medium was added again.

Thereafter, 100 µl / well of the same medium was exchanged every 3-4 days, and the proliferation of fused cells was observed with an inverted microscope (Olmympus, Japan, IMT2-21-W-PM20-35DX2).

<Example 10> Screening of group cell group antibody secreting cell lines against HA

After cell fusion, the cells were continuously grown under an inverted microscope, and when grown to the extent of 10-20% at the bottom of the well, the medium stock of the upper layer of the well was taken and the antibody titer was measured using the HA-BSA conjugate by the indirect ELISA method.

Antibody titers were negative control and O.D. When wells with a difference of more than 0.5 were selected, when the cells grew about 50% in the wells, they were transferred to a 24 well culture plate and cultured.

Cells grown 10-20% in the culture plate was again measured in antibody titers by indirect ELISA method is shown in Table 2 below for the four wells of high titers. Normal serum and PBS were used as negative control.

TABLE 2 Selected Antibody Producing Cell Lines

Control Conjugate (BSA-OVA) OVA Antiserum (1/100) 1.725 1.499 Normal media 0.048 0.042 Supernatant Conjugate (BSA-OVA) OVA 2A11 1.529 0.073 3E6 1.544 0.076 4F5 1.533 0.072 5B7 1.607 0.073

Of these, 5B7 was selected and cultured by limiting dilution to 1 cell / well in a 96 well culture plate, and the antibody titers were measured by indirect ELISA to select four cell lines that secrete monoclonal antibodies as shown in Table 3 below.

At this time, anti-HA-BSA serum was used as a positive control and normal media was used as a negative control.

<Table 3> Result of 1st Limited Dilution Dispensing Culture

Antiserum (1/100) 1.118 5B71E3 1.086 Normal media 0.541 5B1G6 1.109 Antiserum (1/100) 1.212 5B71G9 1.084 Normal media 0.512 5B71D11 1.055

5B1G6, 5B71G9, and 5B71D11 cell lines were cultured by diluting the cells with secondary restriction dilution to 0.5 cell / well, and measuring the antibody titers by indirect ELISA method. It was.

At this time, anti-HA-BSA serum was used as a positive control and normal medium as a negative control.

<Table 4> Result of second limit dilution aliquot culture

Antiserum (1/100) 0.858 5B71G61D2 1.141 5B71G61D5 0.975 Normal media 0.052 5B71G91G6 0.944 5B71G91C10 1.011 Antiserum (1/100) 0.853 5B71D111B11 1.015 5B71D111F5 1.024 Normal media 0.050 5B71D111F9 0.989

Example 11 Preparation of Ascite Fluid

To facilitate cancer induction, 0.5 ml of Pristen was injected into the abdominal cavity of three mice (Balb / c) older than 8 weeks.

One week after the injection of cycithene, at least 5 × 10 7 cells of 5B71G61D2, 5B71G91C10, and 5B71D111F5 monoclonal antibody-secreting cell lines selected in Example 10 were suspended in 400 μl of RPMI 1640 medium, and then inoculated into the abdominal cavity of mice. .

Inoculated mice were observed for abdominal swelling at intervals of 2 to 3 days.

A week after the inoculation, the abdomen was swollen to a nearly pregnant size, and the ascites was obtained by inserting it into the abdomen of the mouse using a sterile 16G needle.

Each obtained ascites was centrifuged at 600 g at 4 ° C. for 10 minutes, the supernatant was removed and the fat layer was removed.

Example 12 Purification of Monoclonal Antibodies Against Hypric Acid

To each of the plurality obtained in Example 11 was added 1M Tris-HCl buffer (pH 8.0) to 1/10 of the plurality of volumes to prevent the pH change, and then saturated with a saturated ammonium sulfate solution 1: It was mixed with 1 and left at 4 degreeC for about 1 hour.

The supernatant was completely removed by centrifugation at 10,000 g for 20 minutes, and then dissolved in 20 mM sodium phosphate buffer (pH 7.0) in the same volume as a plurality of initial volumes.

Ammonium sulfate was completely removed by dialysis four times at 4 ° C. for three hours using a 100-fold volume of PBS (pH 7.0) buffer.

Protein G column (volume: 1 ml, Supelco, Bellefonte, USA, 54840-U) was washed with 20 mM sodium phosphate buffer (pH 7.0) to equilibrate the column, and then 1 ml of the dialysis sample was added. .

After washing with the same buffer to wash the proteins unbound with protein G, bound IgG was eluted with 0.1MGlycine-HCl buffer (pH 2.7).

The pH was restored to the original state by adding 1/10 of 1M Tris-HCl buffer solution (pH 9.0) of the eluate volume to the eluted sample.

The eluted sample was concentrated (200-500 μg / ml) using a concentrator (2 ml, Vivascience, USA, 99 VSO248) to prepare a single cell antibody against HA of the present invention.

Experimental Example 1 Isotype Determination of Monoclonal Antibody

The subclass of unicellular antibody produced by the seven cell lines established in Example 10 was identified using Immunopure® Monoclonal Antibody Isotyping Kit I. As a result, as shown in Table 5, all cell lines secrete IgG2b with κ light chain.

<Table 5> Subclass analysis of unicellular antibody

IgG1 IgG2a IgG2b IgG3 IgA IgM Kappa Lamda Normal 5B71G61D2 0.122 0.472 1.214 0.096 0.125 0.097 1.095 0.174 0.133 5B71G61D5 0.097 0.541 1.123 0.086 0.110 0.095 1.077 0.168 0.120 5B71G91G6 0.099 0.254 1.105 0.074 0.134 0.074 0.854 0.175 0.115 5B71G91C10 0.111 0.234 1.004 0.092 0.127 0.101 0.150 0.159 0.099 5B71D111B11 0.115 0.412 1.018 0.097 0.133 0.095 1.763 0.161 0.097 5B71D111F5 0.114 0.354 1.188 1.002 0.130 0.093 1.987 0.179 0.104 5B71D111F9 0.105 0.501 0.035 1.017 0.121 0.091 0.963 0.170 0.107

Experimental Example 2 Measurement of Titer of Monoclonal Antibody to Prepared HA

The titer of unicellular antibody against HA obtained in Example 12 was measured by ELISA method, and the results are shown in Table 6.

TABLE 6 Titers of unicellular antibody against HA

(Antibody concentration: ng / ml)

One 10 100 1,000 10,000 5B71G61D2 0.207 0.742 1.452 1.657 1.954 5B71G91C10 0.244 0.975 1.643 1.685 1.986 5B71D111F5 0.317 1.125 1.766 1.988 2.071 PBS 0.179

The titers of these antibodies showed high titers of 0.74-1.13 ng / ml at concentrations of 10 ng / ml.

Experimental Example 3 Cross-Reactivity Measurement of Monoclonal Antibody and Carrier Protein

The cross-reactivity of the unicellular antibody prepared in Example 12 of the present invention with the carrier protein was measured by an Indirect ELISA method, and the results are shown in Table 7.

<Table 7> Cross-Reactivity Results of Hyproic Acid Monoclonal Antibody and Carrier Protein

Cell line HA-BSA Conjugate BSA KLH 5B71G61D2 1.022 0.052 0.069 5B71G61D5 1.097 0.041 0.053 5B71G91G6 1.099 0.054 0.065 5B71G91C10 1.111 0.034 0.064 5B71D111B11 1.115 0.042 0.048 5B71D111F5 1.114 0.054 0.068 5B71D111F9 1.105 0.061 0.065 Antiserum (1/100) 1.334 0.461 0.133 Normal media 0.071 0.062 0.089

As shown in Table 7, all seven unicellular antibody antibodies did not cross react at all with the immunogen or the proteins used for analysis.

Experimental Example 4 Competitive Inhibition of Antibody Using ELISA

Competitive ELISA was performed to determine whether established monocellular antibodies reacted with pure hypoic acid, and the results are shown in Table 8.

<Table 8> Competitive inhibition rate (%) of mononuclear antibody secreting cell lines against hypoic acid

HA conc. (Μg / ml) 10 100 1,000 5B71G61D2 - 3.0% 42.0% 5B71G91C10 8.4% 1.9% 43.7% 5B71D111F5 9.0% 21.0% 76.0%

As a result, it was observed that competitive inhibition occurred more severely as the concentration of HA increased for all the antibodies purified from three cell lines (5B71G61D2, 5B71G91C10, 5B71D111F5).

Under the same conditions of HA concentration of 1,000 µg / ml and antibody concentration of 50 ng / ml, 76.0% of 5B71D111F5 antibodies, 43.7% of 5B71G91C10, and 42.0% of 5B71G61D2 showed inhibition.

As a result, the affinity of the antibody isolated from the cell line 5B71D111F5 with the highest inhibition rate showed the highest affinity.

Competitive ELISA of antibodies isolated from the 5B71D111F5 cell line again with the same concentration of antibodies in order to examine the broader range of competitive inhibition was performed. It was observed that 91.2% of inhibition was observed at μg / ml (see Table 9).

<Table 9> Competitive Inhibition Ratio of Hyperic Acid Monoclonal Antibody-secreting Cell Lines According to Hypric Acid Concentration (%)

HA conc. (Μg / ml) 0 3.2 16 80 150 400 2,000 10,000 PBS OD 1.742 1.169 1.424 1.401 1.389 1.157 0.784 0.650 0.522 % Suppression 0 27.5 29.9 30.4 30.2 48.9 85.1 91.2 100

Provided is a method of producing a large number of unicellular antibody against hypoic acid according to the present invention.

Monoclonal antibodies produced by the present invention have high titers of antibodies, do not cross react with carrier proteins, and competitive inhibition occurs with increasing concentrations of hypoic acid.

Monoclonal antibodies produced in large quantities by the present invention are used in diagnostic kits for detecting hypoic acid.

Claims (4)

In the method for producing a single cell antibody against hypoic acid (HA), Hyplic acid and the transport proteins BSA, KLH, and OVA were prepared using a coupling reagent and a cross-linker to prepare a protein conjugate for hyplic acid-carrying. The conjugate was injected into the abdominal cavity and subcutaneously of mice (Balb / c) for immunization. Splenocytes from immunized mice were harvested and fused with myeloma cells. While fused cells were cultured in HAT (hypoxanthine aminopterin thymidine) medium, Cell lines producing unicellular antibody were selected using the HA-OVA conjugate for detection. Selected unicellular antibody-producing cell lines were injected intraperitoneally of mice (Balb / c), To obtain the resulting ascite, Consisting of a method of purifying the ascites obtained to obtain unicellular antibody against hypoic acid, Method for mass production of unicellular antibody against hypoic acid. The method of claim 1, Bacterial harvesting mice (Balb / c) were treated with 2,6,10,14-tetramethyldecanoic acid in advance to create conditions suitable for inducing celiac cancer, Characterized in that ascites is generated by injecting the selected unicellular antibody-producing cell line into the abdominal cavity of a mouse (Balb / c), Mass production method of unicellular antibody against hypoic acid. The method according to claim 1 or 2, Myeloma cells do not produce immunoglobulins and are characterized by a lack of HGPRT (hypoxanthine guanine phosphoribosy transferase) activity. Mass production method of unicellular antibody against hypoic acid. Prepared by the method of claim 1, The titer of the antibody is high, does not cross react with the carrier protein, The higher the concentration of hypoic acid, the more competitive inhibition occurs. A single cell group antibody against hypoic acid used in a diagnostic kit for detecting hypoic acid.