KR100505783B1 - Method of immunoassay for the detection of teicoplanin - Google Patents

Abstract

The present invention relates to a quantitative analysis (direct competitive ELISA) of teicoplanin (TP), a rapid and precise glycopeptide antibiotic using specific antibodies. Immunizing animals to produce anti-TP antibodies. The conditions for direct competitive ELISA (cdELISA) using TP-HRP conjugate and specific antibodies purified using Protein A column were established. The detection limit was 0.3 ng / ml (ppb), and the detection sensitivity was very good. Anti-TP antibody has very specificity because it has no cross-reactivity with other antibiotics (glycopeptide and non-glycopeptide) other than teicoplanin (TP). Analysis of teicoplanin (TP) in the culture samples of teicoplanin (TP) producing strains by cdELISA showed a relatively good correlation with the results obtained by the inhibition hollow test (r = 0.778). . Therefore, the teicoplanin (TP) assay by cdELISA according to the present invention is faster, simpler, easier, more economical and more precise than conventional instrumental or specific methods, and has been shown to be particularly effective in analyzing multiple samples simultaneously. . In addition, the teicoplanin (TP) assay by cdELISA established in the present invention is expected to be useful for the development of detection kits.

Description

Method of immunoassay for the detection of teicoplanin

The present invention is to provide an immunoassay method of antibiotic Teicoplanin (Teicoplanin).

The analysis of antibiotics generally takes advantage of the properties that inhibit the growth of microorganisms. Once the physical and chemical properties of the material have been identified, instrumental analysis by HPLC and the like is possible. Instrument analysis method of Teicoplanin (Aefinity), such as affinity (affinity) method to remove impurities and then by reversed phase HPLC, LC-FAB-MS (liquid chromatography to fast atom bombardment mass spectrometry) method Etc. However, these microbial methods and instrumental analysis methods are disadvantageous in that they are complicated, laborious, and take a long time to detect.

Meanwhile, vancomycin-based glycopeptide antibiotics such as teicoplanin, vancomycin, ristocetin, aristopasin, avoparcin, and actaplanin, A-47934 , A-41030, and A-35512-B are complexes with the D-alanin-D-alanin termini, a pentapeptide precursor of chain growth of peptidoglycan, a component of microbial cell walls. Using the principle of forming a method, a solid phase enzyme receptor assay (SPERA) and a receptor-antibody sandwich assay (RASA) have been developed as a quick and easy assay using a microplate.

  SPERA assay is a method of analyzing various types of glycopeptide antibiotics, and it is possible to detect ε-aminocaproyl-D-alanin-D-alanin conjugated bovine serum albumin (BSA-ε-Aca-D-Ala-D-Ala). After coating the microplate as a receptor and using a teicoplanin-HRP conjugate as a competitor, the HRP after competition with the antibiotic (or sample) The results of substrate reactions to horseradish peroxidase are compared to the calibration curve. The detection range was generally 0.04-4 ug / ml (ppm).

In RASA assays, coating BSA-ε-Aca-D-Ala-D-Ala on the microplate as a receptor is the same as for SPERA assay, but in the next step teicoplanin ( After treatment with teicoplanin or a sample and the anti-teicoplanin antibody-HRP conjugate, it is quantitatively analyzed as a result of the final substrate reaction. Therefore, unlike SPERA, only teicoplanin was specifically analyzed among glycopeptide antibiotics in a non-competitive sandwich method, and the detection limit was 0.03 ng / ml (ppb).

These methods were used to analyze antibiotics remaining in the living body such as blood and ascites fluid after administration of glycopeptide antibiotics.

In addition, the FPIA (competitive homogeneous fluorescence polarization immunoassay), developed as a method for analyzing teicoplanin using the specificity of an antibody, has a detection limit of 1.5 ug / ml (ppm) and vancomycin for vancomycin. Cross reaction was 0.2%.

As the conventional analysis method of teicoplanin (teicoplanin), the above-described microbial method or instrumental analysis has a disadvantage in that it takes a lot of labor and takes a long time to detect or is complicated and difficult.

Meanwhile, solid phase enzyme receptor assay (SPERA) and receptor-antibody sandwich assay (RASA) have been developed as a quick and simple assay that can replace microbial methods as described above, but these methods are known as ε- as receptors. Aminocaproyl-D-alanin-D-alanin conjugated bovine serum albumin (BSA-ε-Aca-D-Ala-D-Ala) is used.

Therefore, the researchers of the present invention produced specific antibodies and established a direct competitive ELISA assay to develop a simpler and more efficient teicoplanin assay than the above methods. The introduction of linked immunosorbent assay (cdELISA) method not only does not use the above BSA-ε-Aca-D-Ala-D-Ala for analysis, but also reduces the analysis process by one step. The present invention was completed.

The immunoassay method of the antibiotic Teicoplanin TP of the present invention is roughly configured as follows.

1) production and purification of specific antibodies against teicoplanin,

2) establishing conditions of cdELISA,

3) cross-reactivity of anti-teicoplanin antibodies by cdELISA,

4) the effect of extraction solvent on the analysis of teicoplanin (TP),

5) Analyze teicoplanin (TP) in culture and extract samples.

Therefore, the present invention forms a conjugate with teicoplanin with bovin serum albumin (BSA), and then forms an adjuvant and an emulsion to an experimental animal rabbit. Providing a method for producing an anti-teicoplanin antibody, characterized by producing an antibody having excellent specificity for teicoplanin in the serum by immunization,

Furthermore, the direct competing enzyme immunoassay characterized by precisely analyzing teicoplanin using the anti-teicoplanin antibody and teicoplanin-horseradish peroxidase (TP-HRP), which are the specific antibodies thus produced. ELISA, cdELISA) provides a method of performing,

In addition, in the enzyme immunoassay method, it provides a sample pretreatment method characterized in that quantification of teicoplanin by dilution without removing impurities in the analysis of teicoplanin in the microbial culture,

Furthermore, the present invention provides a comprehensive analysis system for enzyme immunoassay characterized in that teicoplanin is analyzed quickly, easily, precisely, economically and efficiently by carrying out the enzyme immunoassay and pretreatment of the sample.

Hereinafter, the present invention will be described with specific examples.

I material

Teicoplanin (TP) used in this experiment was TARGOCID ^?? (Gruppo Lepetit SPA, Italy) was used and Horseradish peroxidase (HRP) for conjugation, TRIZMA ^?? as a coating buffer ^. PRE-SET CRYSTALS (tris (hydroxymethyl) aminomethane, 0.05 M, pH 9.0), phosphate buffered saline (PBST; 0.01 M phosphate buffer with 0.138 M NaCl, 0.0027 M KCl, 0.05% Tween 20), Phosphate-citrate buffer tablets (0.05 M phosphate-citrate buffer, pH 5.0, 1 tablet / 100ml), substrate 5'-tetramethyl benzidine dihydrochloride (TMB), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), goat anti-rabbit IgG-HRP, Freund's complete adjuvant and incomplete adjuvant, Phosphate Buffered Saline (PBS; 0.01 M phosphate buffer with 0.138 M NaCl, 0.0027 M KCl, pH 7.2), dialysis tubing (benzoylated cellulose tubing ) Was purchased from Sigma. Ultra Link ^TM Immobilized Protein A column and bovine serum albumin (BSA) for conjugation were purchased from Pierce.

Ⅱ Experimental Method

1. Preparation of teicoplanin bovine plasma albumin conjugate (TP-BSA conjugate)

A TP-BSA conjugate was prepared for use as an antigen for coating in immunogen and ELISA. 20 mg of lyophilized bovine serum albumin (BSA) was dissolved in 2 ml of distilled water to make a BSA solution (in PBS), and 20 mg of teicoplanin (TP) was dissolved in 0.5 ml of PBS to prepare a TP solution. After preparation, these two solutions were mixed. This TP-BSA solution was added to 100 mg of EDC and dissolved with slow stirring. This reaction was performed at room temperature for 24 hours, and then dialyzed with PBS, and absorbance was measured at a wavelength of 280 nm on a Jasco V-550 UV / VIS spectrophotometer to obtain a TP-BSA conjugate. The prepared TP-BSA was confirmed by electrophoresis.

2. Preparation of teicoplanin-horseradish peroxadase conjugate (TP-HRP conjugate).

The TP-HRP conjugate was prepared as follows for direct competition ELISA. 2.5 mg of teicoplanin (TP) is prepared by dissolving in 0.5 ml of phosphate buffer (PBS), 10 mg of hose ready peroxidase (HRP) is dissolved in 0.5 ml of 25% ethanol, and then mixed with EDC 188 Mg was added and reacted at room temperature for 30 minutes. Then, the mixed solution was left at 4 ° C. for 16 hours, dialyzed in PBS, and the recovered solution was precipitated with acetone. The acetone precipitation process is as follows. Acetone made at −20 ° C. was added to the recovered solution in twice the volume of the recovered solution, left at −20 ° C. for 10 minutes and then centrifuged (1,500 g, 10 minutes). Then, the supernatant was discarded, the precipitate was dissolved in PBS, then acetone precipitated again, purified by Sephadex G-25 column, absorbance at 280 nm with a spectrophotometer, and refrigerated for storage. Used.

3. Implementation of SDS-PAGE.

SDS-PAGE was performed by Laemmli method (Laemmli, 1970). Separating gel concentration was 10% acrylamide and 5% stacking gel was used. Protein molecular weight determination by SDS-PAGE was performed using standard molecular weight markers (myosin, 200,000 kDa; phophorylase B, 97.4 kDa; bovine serum albumin, 68 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 29 kDa; β-lactoglobulin, 18.4 kDa; lysozyme, 14.3 kDa; Gibco BRL). The protein content of the egg white treated sample was 10 ug and electrophoresis was performed at 100 V for 1 hour. After electrophoresis, protein staining was performed using coomassie brilliant blue R-250 (CB).

4. Antiserum Production for Teicoplanin (TP)

To produce polyclonal antibodies against teicoplanin (TP), two New Zealand White rabbits weighing 2.5-3 kg were used as experimental animals. Dissolve TP-BSA in sterile saline to make 1 mg / ml, mix 1: 1 with Freund's Complete Adjuvant, and use Micro-mate ^?? Emulsions were made using interchangeable syringes. Next, primary immunization was performed by subcutaneously injecting 1 ml (0.5 mg / head) into the soles of two rabbits, respectively. Additional immunizations from 2nd to 5th were performed at 2 week intervals (3, 5, 7 weeks after the first immunization), and then 1 ml (0.5 mg / head) was prepared using Freund's incomplete adjuvant. ) Was injected subcutaneously. One week after immunization, blood was drawn from the vein of rabbit's ear and the first and second blood volume was 10 ml per horse and the 3, 4 and 5 blood volume was about 100 ml per animal. Each blood was left at room temperature for 1 hour to coagulate the blood, stored in the refrigerator for one day, and the antisera were separated. 1 ml each was dispensed into a microcentrifuge tube, and sodium azide was added to 0.02%, and then used for the next experiment while refrigerated.

5. Purification of Antibodies

Antibodies were purified from polyclonal antisera using 1 ml of Ultra Link ^™ Immobilized Protein A column according to Pierce's product instructions. Equilibrate in advance with a binding buffer, mix 2 ml of antiserum and 2 ml of binding buffer, and load it into a column, and then load Protein A column about 15 ml with binding buffer. After washing with water, 1 mL of 1 M phosphate buffer (pH 7.6) was added to the microcentrifuge, which was previously eluted with elution buffer, to separate IgG polyclonal antibody. This was used for the next experiment with concentration determination and refrigeration.

6. non-competitive indirect ELISA

Indirect non-competitive ELISA was performed for antibody titer as follows. TP-BSA used as an immunogen was dissolved in a coating buffer (TRIZMA ^?? PRE-SET CRYSTALS (tris hydroxymethyl) aminomethane 0.05 M, pH 9.0) to make 2 ug / ml, and the solution was added to each well of a microplate. 100 ul per well) was dispensed and allowed to stand overnight at 4 ° C. for coating. After washing three times with 150 ul of washing buffer PBST, and then using anti-TP serum diluted in multiples of PBST as a primary antibody, 100 ul of each well was added, and then the antigen-antibody at room temperature for 1 hour. The reaction was carried out. After washing again with washing buffer as described above, the goat anti-rabbit IgG-HRP conjugate, a secondary antibody, was diluted several times with PBST and added to 100 ul per well, followed by reaction at room temperature for 1 hour. Then 100 ul of substrate solution per well (0.01% TMB in phosphate-citrate buffer pH 5.O, 0.001% H ₂ O ₂ , prepared immediately before use) for 30 minutes at room temperature, followed by 2 MH ₂ SO ₄ was added to each well by 50 ul to stop the color reaction. Color development was measured by absorbance at a wavelength of 450 nm with a microplate reader (THERMOmax, Molecular Devices, USA). This process was all performed three times.

7. Competitive direct ELISA (cdELISA)

CdELISA conditions were established to determine the concentration of teicoplanin (TP) in the sample. In this case, unlike the indirect non-competitive ELISA, the anti-tecoplanin antibody (anti-TP antibody) purified by the Protein A column is diluted with a coating buffer (coating buffer) to make 2 ug / ml and then per well 100 ul was dispensed and coated overnight at 4 ° C. After washing three times with 150 ul of washing buffer (PBST), the same amount of TP-diluted TP (10 ^-1 to 10 ⁴ ng / ml) and ¹ / 300-diluted TP-HRP was mixed in the PBST. After adding 100 ul per well, the reaction was performed at room temperature for 1 hour. After washing with washing buffer again, the substrate reaction was carried out, and the subsequent process was the same as in the case of indirect non-competitive ELISA.

8. Investigating the Matrix Effect

To see the matrix effect of methanol as an extraction solvent, dilute methanol with PBST to make methanol concentrations of 0, 0.3%, 1%, 3%, 10%, 30% and 100%, and add teicoplanin to each solvent. TP) was dissolved at a concentration of 1 ng / ml. These solutions were diluted step by step and competed with TP-HRP for the anti-teicoplanin antibody. Contention was used above 7. Direct contention ELISA method.

9. Processing of Sample

Teicoplanin (TP) producing strains were cultured and the contents of TP were analyzed among the various samples produced. Samples include (1) supernatant cultured with the Actinoplanes teicomyceticus MSL 2211 strain (patent application 13665/1999), and (2) teicoplanin by blocking feedback inhibition during the culture of these strains. (TP) the supernatant cultured with beads (HP-205) to increase the yield, (3) the culture solution of the MSL 2211 strain containing beads (bead) was centrifuged at 14,000xg for 10 minutes and then the precipitate Extracted with 100% methanol was used. All samples except (3) were centrifuged at 14,000xg for 10 minutes and diluted with PBST at appropriate magnification to use for analysis.

III. Experimental Results and Discussion

1. TP-BSA conjugate preparation

The TP-BSA conjugate prepared for use as an immunogen was identified by SDS-PAGE. Teicoplanin (TP) having a molecular weight of 1.9 kDa did not show a band, and BSA having a molecular weight of 66 kDa conjugated with teicoplanin (TP) to show a good binding band at a position of 68 kDa or more. Could have guessed. Therefore it was used as an immunogen for antibody production (see Figure 1).

In Figure 1, lanes 1 and 5 are molecular weight markers, 2 is BSA, 3 is teicoplanin, 4 is teicoplanin-BSA conjugate.

 2. Production and Purification of Antisera for Teicoplanin (TP)

Antibody values of antisera produced in rabbits against teicoplanin (TP) were compared by indirect non-competitive ELISA method. As shown in Figure 2, the production of specific antibodies began to increase after the second immunization compared to normal rabbit serum (NRS), showing a maximum after the fourth and fifth immunity. The 4th blood collection of rabbit No. 1 showed the highest antibody titer, which was purified by protein A column and desalted for use in the next experiment. (See Fig. 2)

 In Figure 2, each rabbit was immunized with teicoplanin at 0, 2, 4, 6 and 8 weeks, blood collection was taken once every week after immunization, NRS was normal rabbit serum (normal rabbit serum) Indicates.

3. Establishment of Direct Contention ELISA

As stated in the experimental method, conditions for the cdELISA using purified specific antibody and TP-HRP conjugate were set. In other words, when the anti-TP antibody is coated, direct competition ELISA (cdELISA) is performed on TP diluted to each concentration (10 ⁻¹ to 10 ⁴ ng / ml), and the standard curve is shown in FIG. 3. As a result, the standard teicoplanin (TP) gradually decreased at concentrations of about 3 ng / ml (ppb) and up to 300 ng / ml. It was found possible.

In FIG. 3, polyclonal anti-teicoplanin antibodies were absorbed into microtiter plates, and serially diluted teicoplanin (TP 10 ⁻¹ to 10 ⁴ ng / ml) and 1 / Teicoplanin-HRP conjugate diluted to 300 was added to the wells and incubated at room temperature for 1 hour, TMB / H ₂ O ₂ substrate was added for 30 minutes and the reaction was terminated with 2M H ₂ SO ₄ . .

4. Cross-reactivity of Anti-TP Antibodies by Direct Competition ELISA

(1) Reactivity with Glycopeptide Antibiotics

Teicoplanin (TP) is a glycopeptide-based antibiotic known as vancomycin class, and thus the cross-reactivity of the anti-TP antibody against vancomycin and ristocetin, the same class, was examined (Fig. 4). Reference). As a result, it was found that both ristocetin and vancomycin did not compete with the competitor TP-HRP conjugate, indicating that anti-TP antibodies do not react with these glycopeptide antibiotics. Could.

In FIG. 4, teicoplanin-HRP conjugate and antibiotics (TP, teicoplanin; R, ristocetin; V, vancomycin) are added to the wells coated with anti-teicoplanin antibody and competition is achieved. For 1 hour at room temperature.

(2) Reactivity of non-glycopeptide antibiotics

To examine the properties of anti-TP antibodies, the cross-reactivity of the antibiotics lincomycin, erythromycin, amoxicillin, penicillin-G, and spiramycin was measured by cdELISA. Learned by. As a result, it was shown that the anti-TP antibody has little reactivity with these non-glycopeptide antibiotics (see FIG. 5).

In Figure 5, Teicoplanin-HRP conjugate,

Antibiotics (TP, teicoplanin; L, lincomycin; E, erythromycin; A, amoxicillin; P, penicillin-G; S, spiramycin) were added to wells coated with anti-teicoplanin antibody Incubated at room temperature for 1 hour for competition.

5. Effect of Extraction Solvent on Teicoplanin (TP) Analysis

The effect of methanol as an extractant on the competition of cdELISA was examined. Methanol was diluted with PBST to make methanol concentrations of 0, 0.3%, 1%, 3%, 10%, 30% and 100% solutions, and the tecoplanin (TP) was dissolved in each of them to perform their cdELISA reaction. The standard curve was created by examining. As a result, the standard curve of teicoplanin (TP) at methanol concentration of 3% or less was very similar to that of teicoplanin (TP) diluted with PBST only. However, the overall color development was slightly lower at the methanol concentration of 10% or higher, and no contention occurred at 100% methanol. Therefore, if the cdELISA was performed by diluting the methanol ratio to 3% or less, the influence of the extraction solvent was found to be negligible (see FIG. 6).

In FIG. 6, methanol was diluted using wash buffer (PBST) while varying at a concentration of 0-100%.

6. Quantitative Analysis of Teicoplanin (TP) in Culture Samples

Teicoplanin (TP) content in various cultures was analyzed by cdELISA, and the results were compared with the analysis results of teicoplanin (TP) by the microbial method (Inhibition hollow test) (see Table 1). .

   In the cdELISA assay, the cultures were diluted with PBST (1/100, 1/1000, or 1/10000) to rule out matrix effects. The supernatants (Nos. 4 to 6) of the Actinoplanes teicomyceticus MSL 2211 strain with HP-20 bead showed little content or activity of teicoplanin (TP) in both methods. The results were in good agreement. In addition, in the remaining samples, both methods showed more than a certain concentration or activity regardless of methanol extraction.

However, the microorganism-activated samples showed similar values between groups (9.1-12.9 mm in diameter), but the difference in concentration was relatively large (3-13 ug / ml) in the cdELISA results. In the case of microbial method, it is considered that the activity difference of teicoplanin (TP) in the sample is difficult to know due to the problem of inaccurate precision of the test method because of high activity. On the other hand, in the case of cdELISA, it was thought that more accurate measurement of teicoplanin (TP) was possible by diluting the sample and analyzing the sample by diluting the sample to a concentration that can be read from the standard curve. This tendency is well illustrated in the correlation plot between the results by the two methods (see Figure 7).

In Figure 7, teicoplanin concentration was analyzed by cdELISA assay, the antibiotic efficacy in the culture was analyzed by inhibition hollow test (inhibition hollow test).

Nevertheless, the correlation between the two assays is relatively good (r = 0.778), especially for screening teicoplanin (TP) content in many samples, and cdELISA is very effective because it is simple, precise and rapid. It seems to be.

Table 1. Quantitative Analysis of Teicoplanin in Culture Samples by cdELISA

¹⁾ Samples were centrifuged at 14,000 g for 15min.

²⁾ Activity was determined by inhibition hollow test.

³⁾ Concentration was determinde by cdELISA.

⁴⁾ Above strains are known as Actinoplanes teichomyceticus MSL 2211 in Korean Patent Application no 13665/1999 and consumers can always obtain them in commerce.

1. Teicoplanin (TP) -BSA conjugate was prepared and immunized with experimental animals to produce and purify good specific antibodies.

2. The conditions of direct competitive ELISA (cdELISA) using this specific antibody and TP-HRP conjugate were established, and the detection limit was 0.3 ng / ml (ppb), indicating good detection sensitivity.

3. Anti-TP antibodies have very specificity because they have no cross-reactivity to antibiotics other than teicoplanin (TP) (glycopeptide and non-glycopeptide).

4. The analysis of teicoplanin (TP) in culture samples by cdELISA showed a relatively good correlation with the analysis by microbial analysis (r = 0.778).

5. The teicoplanin (TP) assay by cdELISA has been shown to be quick, simple, easy, economical and precise, particularly effective for screening multiple samples simultaneously.

6. The teicoplanin (TP) assay by cdELISA established in the present invention is expected to be useful for the development of detection kits.

Therefore, it can be seen that the present invention is an industrially very useful invention.

Figure 1 is an electrophoresis picture showing the SDS-PAGE pattern of the teicoplanin-BSA conjugate, lanes 1 and 5 are molecular weight markers, number 2 is BSA, number 3 is teicoplanin, number 4 is teicoplanin Nin-BSA conjugate.

Figure 2 is a graph showing the production of anti-teicoplanin antibodies in rabbits, each rabbit was immunized with teicoplanin at 0,2,4,6 and 8 weeks, blood collection It was taken once every week after immunization and NRS represents normal rabbit serum.

FIG. 3 is a graph showing the standard curve of cdELISA for teicoplanin with anti-deicoplanin antibodies, wherein the polyclonal anti-teicoplanin antibody was absorbed into microtiter plates and was continuous. Teicoplanin (TP 10 ^-1 to 10 ⁴ ng / ml) and 1/300 diluted teicoplanin-HRP conjugate were added to the wells and incubated at room temperature for 1 hour, and TMB / H ₂ O ₂ substrate was added for 30 minutes and the reaction ended with 2M H ₂ SO ₄ .

4 is a graph showing the cross-reactivity of the anti-teicoplanin antibody by direct competition ELISA, teicoplanin-HRP conjugate and antibiotics (TP, teicoplanin; R, ristocetin; V, vancomycin) Was added to wells coated with anti-teicoplanin antibody and incubated at room temperature for 1 hour for competition.

5 is a graph showing the reactivity of the anti-teicoplanin antibody of various antibiotics by cdELISA, teicoplanin-HRP conjugate,

Antibiotics (TP, teicoplanin; L, lincomycin; E, erythromycin; A, amoxicillin; P, penicillin-G; S, spiramycin) were added to wells coated with anti-teicoplanin antibody Incubated at room temperature for 1 hour for competition.

6 is a graph showing the effect of methanol concentration on teicoplanin analysis by cdELISA. Methanol was diluted with washing buffer (PBST) at a concentration of 0-100%.

Figure 7 is a graph showing the relationship between teicoplanin concentration and antibiotic efficacy, teicoplanin concentration was analyzed by cdELISA assay, the antibiotic efficacy in the culture was analyzed by inhibition hollow test (inhibition hollow test).

Claims (4)

a) Glycopeptide antibiotic teicoplanin (TP) forms a conjugate with bovine serum albumin (BSA) and forms an conjugate with an adjuvant and an emulsion. ) To produce anti-teicoplanin antibody with high specificity for teicoplanin in serum by immunization with rabbits for experimental animals. b) The precipitate obtained by centrifuging the culture solution of Actinoplanes teicomyceticus MSL 2211 (Accession No .; KCTC 8926P) strain to which HP-20 resin was added was extracted with methanol, and then the extract was PBST (Phosphate buffered saline: Pre-dilution with 0.01 M phosphate buffer with 0.138 M NaCl.0.0027 M KCl, 0.05% Tween 20) c) Analyzing teicoplanin (TP) to a concentration of 3 ng / ml to 300 ng / ml (ppb) using the antibody and teicoplanin (TP) -horseradish peroxidase (HRP) obtained in a). Competing direct immunoassay (competitive direct ELISA, cdELISA). delete delete delete