JPH0483171A - Method for immunologically measuring coliform group - Google Patents

Abstract

PURPOSE:To singularly measure only coliform group at high sensitivity by making an animal immune to coliform group so as to obtain an antibody, and absorbing the antibody into a microorganism other than coliform group, and then subjecting the antibody to affinity refining, and using the antibody as a labeled or solid antibody. CONSTITUTION:An antibody obtained by making an animal immune to coliform group serving as a material to be measured, preferably its ultrasonically crushed antigen, or an antibody intersecting a microorganism other than coliform group, preferably its ultrasonically crushed antigen is absorbed by a microorganism other than coliform group and removed. Next the antibody absorbed is subjected to affinity refining of elusion method whereby the antibody is coupled with the antibody of the ultrasonically crushed antigen of the coliform group and made to pass through a packed column. The antibody obtained is prepared into a labeled or solid antibody for use in a noncompetitive method, especially sandwich method, which is a method of measuring oxygen immunity. The labeled or solid antibody thus obtained is used to singularly measure the coliform group at high sensitivity.

Description

[Detailed Description of the Invention] A. Industrial Application Field The present invention relates to an immunoassay method for coliform bacteria, and in particular, it hardly reacts with microorganisms other than coliform bacteria and can measure coliform bacteria with high sensitivity. Concerning immunoassay methods for coliform bacteria.

B0 Summary of the Invention The present invention is an immunoassay method for coliform bacteria, in which antibodies obtained by immunizing coliform bacteria are absorbed by microorganisms other than coliform bacteria, and then affinity purified and used as a label or a solid-phase antibody. This makes it possible to measure coliform bacteria specifically and with high sensitivity.

C9 Prior Art At present, the following methods are generally known as methods for quantifying microorganisms.

(1) A method of counting the number of bacterial colonies with the naked eye (written by Arai et al.,
“The latest microbial testing methods in practice” P62. Industrial Technology Society).

(2) Method of quantifying from the amount of enzyme produced by microorganisms [(Shigeru Yamanaka et al., “Japan Society of Food Hygiene, 49th Academic Lecture Abstracts” P29, 1985), (Minor Ohashi et al., “Japan Society for Fermentation Engineering Lecture Abstracts”) “, P73゜1989)).

(3) Method using antigen-antibody reaction [5arafian et al.
et al,,I,Med,Microbiol,,
15,541.1982), the quantitative method of Escherichia coli by Dadd et al. (D, C9Dadd et al., Inf.
Ection and Immunity, 38, 76
4.1982).

Quantitative method of rice blast fungus by Kitagawa et al. (Tsunehiro Kitayo.

Special public Sho 6O-46461)E.

However, each of these methods has the following problems.

(1) Problems with the method of counting bacterial colonies with the naked eye Since this method measures after culturing for 24 hours, it is not possible to obtain rapid results. Furthermore, all operations are performed aseptically, so skill is required.

(2) Problems with the method of quantifying the amount of enzyme produced by microorganisms a) Yamanaka et al.'s method requires 10 hours, including culturing, to obtain results, and requires complicated procedures such as pH adjustment and centrifugation. Requires operation.

b) In the method of Ohashi et al., the detection sensitivity is 106 pieces/t.
Therefore, the sensitivity is insufficient compared to the target sensitivity of 3×10' pieces/il.

(3) Problems with the method using antigen-antibody reaction a) S,
The method of 5arafian et al.

In the method of C. Dadd et al., the bacterial strain used to obtain antibodies against bacteria can be measured with high sensitivity, but other bacterial strains can hardly be measured, and it is difficult to comprehensively quantify multiple bacterial strains such as coliform bacteria. Not suitable.

b) Kitagawa et al.'s method requires two days for measurement, and
There is one more step than the Sandermanch method.

As explained above, existing quantitative methods rely on measurement time.

It can be said that there are many problems in terms of specificity, sensitivity, and measurement operation.

Therefore, the present inventors were able to specifically detect coliform bacteria,
Furthermore, a method was proposed in which the measurement could be completed in about 3 hours and the measurement could be performed with even higher sensitivity (Japanese Patent Application No. 109664/1999).

D0 Problems to be Solved by the Invention However, the method according to the above application still has a measurement sensitivity of about 104 particles/ml, which is lower than the target sensitivity of 3×10' particles/wealth liter.
had not been reached. This suggests that specific binding to Escherichia coli at antigen concentration is insufficient, which prevents a reduction in the cross rate with bacteria other than coliform bacteria.

Therefore, the present invention was devised to solve this problem, and the antibody obtained by immunizing coliform bacteria is absorbed by microorganisms other than coliform bacteria, and then affinity purified and used as a labeled or solid-phase antibody. By using this method, the specific binding is increased, that is, the cross rate with bacteria other than coliform bacteria is reduced, and this increases the target sensitivity of 3×10' particles/coliform bacteria when measuring coliform bacteria.
The purpose is to achieve yal.

Means and effects for solving the E1 problem, that is, the immunoassay method for coliform bacteria according to the present invention, involves absorbing antibodies obtained by immunizing coliform bacteria with microorganisms other than the coliform bacteria, and then
The solution is to use it as a label or solid-phase antibody after affinity purification.

The present invention will be explained in more detail below.

The antibodies used in the method of the present invention include coliform bacteria, which is a substance to be measured, and preferably the sonicated antigen of coliform bacteria in animals.
For example, antibodies (IgG) obtained by immunizing rabbits, goats, guinea pigs, etc., or fragments with antigen-binding activity obtained by treating these antibodies with proteases (
Among F a b), microorganisms other than coliform bacteria, which are the substances to be measured, preferably antibodies that cross the sonicated antigen are absorbed and removed by microorganisms other than coliform bacteria, and then the absorbed antibodies are covered with Elution method by affinity purification in which the ultrasonicated antigen of the coliform bacteria, which is the substance to be measured, is bound to a carrier, such as a CNBr-activated carrier, and passed through a packed column 1. For example, a method by pH change, preferably glycine-hydrochloric acid buffer. An affinity purified method such as a method using a pH change using a liquid or a method using chaotropic ions, preferably a method using chaotropic ions using sodium thiocyanate is used.

Next, the obtained antibody was measured using a non-competitive enzyme immunoassay method (1).
In particular, it is prepared as a labeled antibody or solid-phase antibody for use in the Sand-Deutsch method.

Using the labeled antibody or solid-phase antibody thus obtained, coliform bacteria can be measured with high sensitivity and specificity.

F. Examples Hereinafter, a detailed explanation of the immunoassay method for coliform bacteria according to the present invention will be explained based on examples.

Example 2 Coliform bacteria and bacteria other than coliform bacteria were measured using the combinations of solid-phase antibodies and labeled antibodies shown in Table 1, and the sensitivity and crossover rate were determined.

The immunoassays are shown in Procedure 6.7, respectively.

■ Operation procedure A Operation procedure 1 Separation method of coliform bacteria ■ Inflow water and effluent from a sewage treatment plant were appropriately diluted and spread on a dioxycholate medium.

■ Next, after culturing at 37°C overnight, red colonies were harvested.

(Left below) First, the method for separating coliform bacteria and non-coliform bacteria is explained in step 1.
2. Antiserum preparation method and antiserum absorption method.

and procedure 3.4 for the preparation of affinity-purified antibodies.
．． 5, and the method for preparing labeled antibodies and Enzyme B Operation procedure 2 General bacterial isolation method (inject subcutaneously into rabbits from the large intestine at intervals of 1 to 2 weeks) Appropriate dilution of influent and effluent water from a sewage treatment plant Then, it was spread on an ordinary agar medium.

(2) After culturing overnight at 37°C, the colonies were harvested and inoculated into a dioxycholate medium.

(2) After further culturing at 37°C overnight, colonies other than the red colonies were separated.

C Operation Procedure 3 Antiserum Preparation Method ■ The coliform bacteria isolated according to Operation Procedure 1 was cultured in a normal nutrient liquid medium overnight at 37°C with shaking.

■ The bacteria cultured in (■) were collected, washed, and then disrupted by ultrasonication (5 minutes under water cooling).

■　■ was mixed with Freund's adjuvant.

did.

■　■ was repeated 2 to 4 times.

■ Whole blood was collected and antiserum was obtained.

D Operation procedure 4 Antiserum absorption operation ■ General bacteria isolated according to operation procedure 2 were disrupted by ultrasonic waves.

■■woCNBr-activated Sepharose 4B■ (manufactured by Pharmacia) was bonded to obtain an absorption carrier.

② The carrier prepared in ② was packed into a column (IX 3 cm).

② The antiserum prepared in step 3 was passed through the column in ②. 0.1 moA'/A' borate buffer (pH 8.5) was used as the elution buffer.

■ IgG, F (ab) from the antiserum obtained in ■
z was prepared.

E Operation procedure 5 Preparation method of affinity purified antibody ■ The coliform bacteria isolated in operation procedure 1 were disrupted by ultrasonic waves.

(2) was bound to CNBr-activated Sepharose 4B (manufactured by Pharmacia) to prepare a carrier for affinity purification.

② The carrier prepared in ② was packed into a column (IX 3 cm).

■ 30mg/mI anti-coliform IgG2mj! was added to the column (■).

■ 0.01 containing 0.15 mol/l sodium chloride
Sufficiently fill the mol/l phosphate buffer (pH 7,2) column with
Washed.

■ Q, 1mol/j! Glycine-HCl buffer (pH2
, 3), and add 1 mol/l IJ solution to 0.
．． Aliquots of 1 mA' were placed into 1 ml test tubes.

■ The affinity purified anti-coliform group 1gG obtained in ■ is 0.
The buffer was exchanged to 1 mol/A' borate buffer (pH 8,5) by gel filtration.

F Operation procedure 6 Preparation method of labeled antibody ■ 0.1 mol containing 15 mg/mA' gelcol
5.6/l in a test tube containing 1 mA' of phosphate buffer.
Add 60 μl of dimethylformamide containing mg of r-maleimidobutyryloxy-N-succimide and incubate at 30°C.
The mixture was reacted for 1 hour with stirring.

(2) Desalting was performed using a PD-10 column (manufactured by Pharmacia) to obtain maleimidized COD. Elution is 0.1moA'/
I phosphate buffer (pH 6,0).

■ 0.1 m containing 9 mg of F(ab)'z fraction of each antibody.
β-
0.1 mol containing 0.8 mg of mercaptoethylamine
Add 50μ of /V phosphate buffer (pH 6,0) and
After reacting at ℃ for 1.5 hours with stirring, PD-1
Desalting was performed using a 0 column.

(2) 3 mg of the maleimidized COD obtained in (2) and 5.4 mg of the Fab' fraction of each antibody obtained in (2) were mixed and allowed to stand overnight at 4°C.

■　TSKG3000SW (21.5X60cm.

(manufactured by Tosoh) to obtain a labeled antibody.

G Operating procedure 7 Enzyme immunoassay ■ Each antigen solution 0.1mj? and ■ solution (Q, 1 mol/A' borate buffer containing 0.1% BSA (pH 8.5))
0.2 ml was placed in a well, and one polystyrene ball coated with anti-coliform IgG was added.

(2) After reacting at room temperature for 1 hour, the solid phase was washed three times with distilled water.

(2) 0.3 ml of labeled antibody prepared according to procedure 6 was added.

(2) After reacting at room temperature for 1 hour, the solid phase was washed three times with distilled water.

■ Q, 0.01 mol containing 5 mof/l glucose
Add 0.3 ml of /l acetate buffer (pH 5,1) at 37°C.
The mixture was allowed to react for 30 minutes.

■ Take 0.1 ml of the reaction solution and make 2 x 10-7 mol
0.2 mol/II carbonate buffer containing /1 luminol (
pH 9.8) and 6 x 10-"mo//l potassium ferricyanide solution were added at 0.5 mA' each, and 1
The amount of chemiluminescence was measured for 6 to 45 seconds.

Example 1 Measurement method using an antibody that was affinity purified after absorption with bacteria other than coliform bacteria as a labeled antibody ■ 0.10", 5X10", 10', 5X10'.

10', 5X10', 10', 5X10', 10' (I
A calibration curve was prepared according to Operation Procedure 6 using ultrasonically disrupted coliform bacteria and non-coliform bacteria at a concentration of I/ml as antigens. As the labeled antibody, an antibody that was absorbed by bacteria other than coliform bacteria and then affinity purified was used. In addition, the crossover rate (%) of bacteria other than coliform bacteria is 106 cells/ml, and the amount of chemiluminescence when bacteria other than coliform bacteria is used as an antigen is 103 cells/ml.
Dividing the amount of chemiluminescence when 1 coliform bacteria is used as an antigen is 1
The value was multiplied by 00.

■ The results are shown in Figure 1. 10 as shown in Figure 1
It can be seen that coliform bacteria can be measured in the range of 3 to 5 x 10' cells/ml. Also, the crossing rate (crossing rate (%): 10'
chemiluminescence amount of bacteria other than coliform bacteria/ml/106/ml
The chemiluminescence amount of coliform bacteria in ml x100) is 1.9%
and low. These values show that the method of using, as a labeled antibody, an antibody that has been absorbed by bacteria other than coliform bacteria and then affinity purified is effective for specific measurement of coliform bacteria.

Comparative Example 1 Measurement method using an antibody absorbed by bacteria other than coliform bacteria as a labeled antibody ■ Measurement of coliform bacteria by the same method as in Example 1 except that an antibody absorbed by bacteria other than coliform bacteria was used as a labeled antibody. The crossover rate was calculated.

■ The results are shown in Figure 2. 5X as shown in Figure 2
It can be seen that coliform bacteria can be measured in the range of 103 to 5×106 cells/ml. Moreover, the crossing rate is 7.7%. These values indicate that the sensitivity is lower at 115 and the crossover rate is about 4 times higher than in Example 1. Further 10', 10
The S/N of ', 10' pieces/ml is also about 1/1 compared to Example 1.
It was as low as 3-1/2.

Comparative Example 2 Measurement method using an affinity-purified antibody as a labeled antibody ■ Measurement of coliform bacteria and cross-over rate were determined in the same manner as in Example 1 except that an affinity-purified antibody was used as a labeled antibody.

■ The results are shown in Figure 3. 10 as shown in Figure 3
It can be seen that 3 to 106 coliform bacteria/ml can be measured, but the crossover rate is 39.3%, which is about 20 times higher than in Example 1. Moreover, the S/N of 10' and 105106 cells/ml was also smaller than that of Example 1, at 1/2 to 415.

Comparative Example 3 Measurement method using an untreated antibody as a labeled antibody ■ Measurement of coliform bacteria and cross-over rate were determined by the same method as in Example 1 except that an untreated antibody was used as a labeled antibody.

■ The results are shown in Figure 4. 10 as shown in Figure 4
It can be seen that 4 to 5 x 10' coliform bacteria/ml can be measured. Moreover, the crossing rate is 74.1%. Both of these values show that they are considerably inferior to Example 1.

Furthermore, the S/N of 10', 105106 cells/ml was also determined in Example 1.
It was about 1/8 to 1/2 lower than that of .

Table 2 shows the calibration range, crossover rate, and S/N of coliform bacteria 10' and 105.10'pieces/mA' when measuring coliform bacteria in Example 1 and Comparative Example 1□ 2.3 described above. . As shown in Table 2, it can be seen that Example 1 is superior to Comparative Examples 1, 2.3 in terms of calibration range, specific binding, and crossover rate.

(Leaving space below) Table 2 Example of summary of coliform measurement results■ Coliform bacteria and bacteria other than coliform bacteria were measured using the combinations of solid-phase antibodies and labeled antibodies shown in Table 3, and the sensitivity and crossover rate were determined. .

(Leaving space below) Table 3 Combinations of solid-phase antibodies and labeled antibodies. The enzyme immunoassay method was performed using the operating procedure 1. shown in Example I. 2. 3°4.5.6 and 7 were followed.

Example 2 Assay method using affinity-purified antibody after absorption with bacteria other than coliform bacteria as a solid-phase antibody■ Example 1 except that a solid-phase antibody is used instead of a labeled antibody
Measurement of coliform bacteria and cross-over rate were determined using the same method as above.

■ The results are shown in Figure 5. 10 as shown in Figure 5
It can be seen that coliform bacteria can be measured in the range of 3 to 5 x 10' cells/ml. Also, the crossover rate is as low as 2.3%. From these values, it can be seen that the method using, as a solid-phase antibody, an antibody that has been absorbed by bacteria other than coliform bacteria and then affinity purified is effective for specific measurement of coliform bacteria.

Comparative Example 4 Measurement method using an antibody absorbed by bacteria other than coliform bacteria as a solid-phase antibody■ Measurement method using an antibody absorbed by bacteria other than coliform bacteria by the same method as in Example 2 except that an antibody absorbed by bacteria other than coliform bacteria was used as the solid-phase antibody. Measurements and crossover rates were determined.

■ The results are shown in Figure 6. 5X as shown in Figure 6
It can be seen that coliform bacteria can be measured in the range of 10" to 5 x 10' cells/ml. Also, the crossover rate is 9.8%. These values have a lower sensitivity of 115 than in Example 2, and the crossover rate is It shows that it is about 3 times as high.
The S/N at 10' and 10' cells/ml was also lower than that in Example 1, at about 1/7 to 1/3.

Comparative Example 5 Measurement method using affinity-purified antibody as solid-phase antibody ■ Measurement of coliform bacteria and cross-over rate were determined by the same method as in Example 2 except that an affinity-purified antibody was used as the solid-phase antibody.

■ The results are shown in Figure 7. 10 as shown in Figure 7
Although it is possible to measure ~5×10' coliform bacteria/m, the crossover rate is 47.9%, which is approximately 20 times higher than in Example 2. N
It was also as low as 1/2 to 2/3 compared to Example 2.

Comparative Example 6 Measurement method using an untreated antibody as a solid-phase antibody ■ Measurement of coliform bacteria and cross-over rate were determined in the same manner as in Example 2, except that an untreated antibody was used as a solid-phase antibody.

■ The results are shown in Figure 8. 10 as shown in Figure 8.
It can be seen that 4 to 5 x 10' coliform bacteria/ml can be measured. Moreover, the crossing rate is 66.7%. Both of these values show that they are considerably inferior to Example 2.

Furthermore, the S/N of 10' and 10' 106 pieces/ml was also lower than that of Example 2, at about 1/2 to 2/3.

Table 4 shows the calibration range, crossover rate, and S/N of coliform bacteria 10', 10', and 10' pieces/ml when measuring coliform bacteria in Example 2 and Comparative Examples 4 and 5.6 described above. . As shown in Table 4, Example 2 is different from Comparative Examples 4 and 5.
It can be seen that the calibration range, specific binding, and crossover rate are all superior to that of Sample 6.

(Leaving space below) Table 4 Summary of coliform bacteria measurement results G0 Effect of the invention When measuring coliform bacteria, the present invention uses antibodies that have been affinity-purified after absorption with bacteria other than coliform bacteria, either labeled or solid-phase antibodies. Compared to using absorbed antibodies (Comparative Examples 1.4), affinity-purified antibodies (Comparative Examples 2 and 5), and untreated antibodies (Comparative Examples 3 and 6) as solid-phase antibodies, , increases the sensitivity of immunoassays, reduces the cross-over rate with bacteria other than the large intestine, and increases specific binding.

Therefore, according to the coliform immunoassay method of the present invention, even if either the labeled antibody or the solid-phase antibody is an untreated antibody, as long as the other is absorbed and used as an affinity-purified antibody. , it is possible to measure coliform bacteria with a high sensitivity of 3×10” cells/ml, which is the target sensitivity, and with low cross-reactivity.

[Brief explanation of the drawing]

Figures 1 and 5 are graphs showing the measurement results of coliform bacteria using labeled antibodies and solid-phase antibodies, respectively, which were absorbed with bacteria other than coliform bacteria and then affinity purified.
Figures 3 and 6 are graphs showing the measurement results of coliform bacteria using antibodies absorbed by bacteria other than coliform bacteria as labeled antibodies and solid-phase antibodies, respectively. Figures 3 and 7 are labeled antibodies and solid-phase antibodies, respectively. Figures 4 and 8 are graphs showing the measurement results of coliform bacteria using an affinity-purified antibody as a labeled antibody and an untreated antibody as a solid-phase antibody, respectively. 1 other person

Claims (1)

[Claims] (1) An immunoassay method for coliform bacteria, which comprises absorbing an antibody obtained by immunizing coliform bacteria with a microorganism other than the coliform bacteria, and then affinity-purifying the antibody and using it as a label or a solid-phase antibody.