JP4578401B2 - Method for producing immobilized antibody - Google Patents

Description

  The present invention relates to a method for producing an immobilized antibody for use in an immunological assay.

  Microbiological testing is an important test item in medical testing, food testing, and environmental measurement.

  A wide variety of microorganisms exist in the oral cavity, and it is known that microorganisms that cause infections may exist among these microorganisms. For example, it is known that caries-related bacteria and periodontal disease-related bacteria are strongly involved in the occurrence of caries and periodontal disease. In recent years, it has been clarified that microorganisms present in the oral cavity may be involved in the development of diseases that have not been related to conventional infectious diseases, such as heart disease, gastric ulcer, cancer, etc. The microbiological examination is important not only in the dental field but also in the medical field.

  The measurement of microorganisms in the sample liquid has been performed by the conventional culture method, and there was a problem that it took a long time until the result became clear, but the immunological measurement method was developed, and the test time was greatly shortened. . The immunological measurement method is a method of detecting a microorganism with high sensitivity using an antibody against a specific antigen possessed by the microorganism (Non-patent Document 1).

  Detection sensitivity can be further enhanced by measuring an antigen extract prepared by extracting an antigen from a microorganism by an immunological method. The reasons for this are considered to be 1) the antigen is solubilized and becomes a low molecule, so that the antigen-antibody reaction is likely to proceed, and 2) the epitope is exposed. As a method for extracting a microbial antigen, various methods such as a surfactant treatment, an enzyme treatment, and a heat treatment have been put into practical use, and a nitrite extraction method (Non-patent Document 2) is suitable as a method for extracting a sugar chain antigen. )It has been known. This method is particularly effective for extracting sugar chain antigens of microorganisms belonging to the genus Streptococcus such as group A streptococci and oral streptococci. The method is a method in which an aqueous nitrite solution is reacted with a sugar chain, the sugar chain is cleaved and released from the cells, and a general nitrite extraction method includes an aqueous nitrite solution (in many cases sodium nitrite) and Nitrous acid is generated by mixing an acid solution (acetic acid, hydrochloric acid, nitric acid, etc.), and the microorganism is reacted with nitrous acid for a sufficient time.

  The immunological method used to measure the amount of glycan antigen in the glycan antigen extract prepared as described above is generally an immobilized antibody obtained by immobilizing an antibody on an insoluble immobilization carrier. Used to detect antigens. In general, an immobilized antibody is produced by bringing an antibody solution in which an antibody is dissolved in an aqueous solution such as a buffer solution into contact with an immobilization carrier for a certain period of time, thereby allowing the antibody to bind to the insoluble carrier. When nonspecific adsorption of substances other than antigen to the carrier for immobilization occurs during measurement of the amount of antigen in the sample liquid, the background at the time of measurement increases, making accurate measurement impossible. Is generally bound to a carrier for immobilization and then blocked with a protein such as bovine serum albumin (hereinafter sometimes abbreviated as BSA), casein, gelatin, skim milk or the like.

  However, even if the antibody-immobilized carrier is blocked with a protein as described above, the nonspecific reaction cannot be completely removed or the nonspecific reaction can be removed, but the sensitivity of the measurement is reduced. However, there are cases where satisfactory results cannot be obtained by blocking with conventional proteins.

  A method of blocking agglutination reaction particles (immobilized antibody) with nonpolar amino acids such as alanine and valine instead of protein has been proposed (Patent Document 1). By adopting this method, a protein blocking method is used to measure the amount of anti-treponema / paridum antibody in serum, the amount of hemoglobin in stool suspension, and the amount of anti-adult T cell leukemia virus antibody in serum. It is disclosed that non-specific reactions can be suppressed without lowering the sensitivity compared to the above, but as will be described later, glycan antigens extracted from microorganisms by the nitrite extraction method are measured by the present inventors. It became clear that the effect was insufficient when targeted.

Edited by The Biochemical Society of Japan, Laboratory for Neonatal Chemistry 12 Molecular Immunology III Antigens / Antibodies / Captures, Tokyo Chemical Dojin, 1992 Takei Tsutomu, Osaka University Dental Journal, 35, 93-109, 1990 JP-A-5-288750

  The present invention has been made in view of the above circumstances, and is a method for producing an immobilized antibody used for immunological measurement of a sugar chain antigen extracted by a nitrous acid extraction method, and the sensitivity of the immunological measurement method is improved. It is an object of the present invention to provide a method for producing an immobilized antibody that can sufficiently suppress a non-specific reaction without lowering.

  The present inventors have intensively studied to solve the above problems. As a result, the sugar chain extracted by the nitrite extraction method using the immobilized antibody prepared by coexisting tris (hydroxymethyl) aminomethane and a nonpolar amino acid when contacting the antibody with the immobilization carrier. When antigen was measured, it was found that satisfactory results were obtained in both sensitivity and specificity. Further studies have been made and the present invention has been completed.

  That is, the present invention is a method for producing an immobilized antibody for use in measuring the amount of a sugar chain antigen in a sugar chain antigen extract obtained by extracting a sugar chain antigen from a microorganism by a nitrite extraction method by an immunological method, A method for producing an immobilized antibody, comprising contacting an antibody corresponding to the sugar chain antigen with an immobilization carrier in the presence of tris (hydroxymethyl) aminomethane and a nonpolar amino acid.

  By using the immobilized antibody prepared by the method for producing an immobilized antibody of the present invention, the sugar chain antigen extracted by the nitrite extraction method can be measured with high sensitivity and accuracy.

  In the method for producing an immobilized antibody of the present invention, an antibody corresponding to a sugar chain antigen to be measured, which will be described later, is immobilized in the presence of tris (hydroxymethyl) aminomethane (hereinafter sometimes abbreviated as tris) and a nonpolar amino acid. Contact with the carrier.

  Examples of the antibody that can be used in the present invention include a polyclonal antibody, a monoclonal antibody, a partial degradation product (Fab, Fab ′, Fab′2, etc.) of the antibody, and an active fragment (antigen recognition site of the antibody) of the antibody. Existing partial structures and the like are also included. The globulin class of the antibody is not limited and includes any globulin class currently known.

  As the carrier for immobilization, conventionally known ones such as membranes, particles, plates, test tubes and the like can be used without particular limitation. For example, conventionally known materials such as nitrocellulose, PVDF, latex, gelatin, metal colloid, polystyrene, polypropylene, and polycarbonate can be used without any limitation.

  In the present invention, the reaction in which the antibody and the immobilization carrier are brought into contact with each other and the antibody is bound to the immobilization carrier is performed in the presence of Tris and a nonpolar amino acid. As a result, the obtained immobilized antibody can accurately and accurately measure the sugar chain antigen extracted by the nitrite extraction method. The reason for this is not clear, but it is presumed that the above-described Tris and the nonpolar amino acid interact with each other to greatly enhance the blocking effect.

  When the suitable density | concentration of the tris used by this invention is illustrated, it is 5-30 mM.

  In the present invention, as the nonpolar amino acid, a known amino acid having a nonpolar side chain can be used without limitation, but in general, those belonging to α-amino acid are preferable, and glycine, alanine, valine, leucine, isoleucine, proline are particularly preferable. Nonpolar amino acids constituting a protein selected from phenylalanine, tryptophan, and methionine are preferred. A suitable concentration of these nonpolar amino acids is 5 to 50 mM, more preferably 5 to 30 mM. These nonpolar amino acids may be used alone or in combination. When combining a plurality of nonpolar amino acids, the total amino acid concentration is set to 5 to 30 mM.

  By allowing Tris and a nonpolar amino acid to coexist, an inhibitory effect on a nonspecific reaction (reduction in background) can be obtained at a lower concentration than when Tris or a nonpolar amino acid alone is used. Tris or a non-polar amino acid alone can provide a non-specific reaction inhibitory effect, but it must be at a higher concentration than when both components coexist. In this case, a decrease in sensitivity is observed simultaneously with the suppression of the non-specific reaction. There is a case.

  The method of bringing the antibody into contact with the immobilization carrier is, for example, adding Tris and a non-polar amino acid to the above-mentioned suitable concentration in a liquid (antibody solution) in which the antibody is dissolved, and fixing the immobilization carrier to a certain amount. It can be carried out by contact for a period of time. The pH of the solution during the production of the immobilized antibody is preferably kept within a certain range (for example, pH 4 to 9). For this purpose, it is preferable to contact the antibody and the solidifying carrier in a buffer solution. . As the buffer solution, for example, a phosphate buffer solution, a GOOD buffer solution, a carbonate buffer solution or the like can be used. As a specific method for bringing an antibody into contact with an immobilization carrier, for example, an antibody solution (antibody concentration 0.001 to 5 mg / ml) containing an antibody, Tris and a non-polar amino acid is prepared, and the antibody solution and immobilization carrier are used. The method of contacting a support | carrier at 4-56 degreeC for 2 minutes or more can be shown. As another method, a buffer solution (for example, Tris hydrochloride buffer) is prepared with Tris or nonpolar amino acid, and an antibody solution containing no buffer components other than Tris and nonpolar amino acid is prepared to produce an immobilized antibody. it can.

  When the antibody is brought into contact with the immobilization carrier, surfactants, saccharides, and salts can be mixed in addition to the above buffer components, Tris, and nonpolar amino acids from the viewpoint of storage stability of the antibody. . As the surfactant, for example, Triton X-100, Tween 20 and the like can be suitably used, and as the saccharide, for example, glucose and sucrose can be suitably used, and as the salt, for example, sodium chloride and potassium chloride can be suitably used. In addition, proteins can be mixed at a dilute concentration (e.g., 0.5% or less) that is usually less than the concentration effective as a blocking agent. Depending on the antibody, the storage stability may be greatly improved by mixing a specific protein. In such a case, addition of a protein at a low concentration is effective.

  The immobilized antibody prepared as described above is preferably washed once or more with a phosphate buffer or the like.

  The amount of the glycan antigen in the glycan antigen extract extracted from the microorganism by the nitrite extraction method is measured by an immunoassay using the immobilized antibody thus prepared.

  In the present invention, microorganisms refer to bacteria, Rickettsiae, Chlamydia, Mycoplasma, and unicellular eukaryotes. Examples of specimen fluids containing these microorganisms include microorganism culture solutions, microorganism suspensions, foods and suspensions thereof, throat swabs, body fluids such as saliva, plasma, serum, urine, and plaque suspensions. Liquid and the like. In order to measure microorganisms present in the oral cavity, it is particularly preferable to use a sample fluid containing saliva, plaque, or a mixture thereof.

  Examples of microorganisms that cause infections contained in a sample fluid containing saliva, dental plaque, or a mixture thereof include caries-related bacteria, periodontal disease-related bacteria, upper respiratory tract infection-causing bacteria, and opportunistic infections. . When specific examples are displayed for each, the caries-related bacteria include Streptococcus mutans, Streptococcus sobrinus, and other bacteria belonging to mutans streptococci, Lactobacillus, Bacteria belonging to the genus Actinomyces include porphyromonas gingivalis, actinomycetemcomitomitus Bacterium, and actinomycetem bacterium bacterium. ), Treponema denticola, Prevotella intermedia, Prevotella nigrescens, etc., and the upper respiratory tract-causing bacteria, A group of Streptococcus (Gr. (Micoplasma pneumoniae), Chlamydia pneumoniae, etc., and Candida sp., Staphylococcus aureus, and Pseudomonas (Pseudomonas) are examples of opportunistic infections.

  The nitrite extraction method may be carried out according to a known method, for example, by generating nitrous acid by mixing an aqueous nitrite solution and an aqueous acid solution, and extracting a sugar chain antigen in the aqueous nitrite solution. . As the nitrite aqueous solution, for example, 0.2 to 8M sodium nitrite and potassium nitrite can be used, and as the acid aqueous solution, 0.5 to 4M acetic acid, nitric acid, hydrochloric acid, citric acid and the like can be used. As a specific method of the nitrite extraction method, the suspension containing the microorganisms is centrifuged and the supernatant is discarded, and 0.005 to 0.5 ml of the nitrite aqueous solution and the acid aqueous solution as described above are respectively added to the precipitate. The method of making it react by adding and leaving to stand at 15-50 degreeC for 1 to 10 minutes can be illustrated.

  Alternatively, the nitrite aqueous solution and the acid aqueous solution are mixed in advance to prepare a nitrite aqueous solution containing 0.2 to 4 M nitrite and 0.5 to 4 M acid, and the nitrite aqueous solution contains microorganisms. Nitrous acid extraction can also be carried out by adding 0.01 to 1 ml to the precipitate and leaving it at 15 to 50 ° C. for 1 to 10 minutes.

  Since the sugar chain antigen extract after the reaction is strongly acidic and cannot be used as it is in an immunological assay, for example, 1 to 3 M sodium hydroxide, 0.5 to 2 M Tris, 0.5 to 1 M sodium bicarbonate It is preferable to adjust the pH of the extract to 7.0 to 8.5 by adding a basic aqueous solution such as

  Specific methods of immunological measurement methods for measuring sugar chain antigens in the sugar chain antigen extract thus produced include immunoagglutination, optical immunoassay, labeled immunoassay, and combinations thereof. Any conventionally known method can be used without limitation.

  Hereinafter, these immunological measurement methods will be described.

[Immunoagglutination]
This method is a method for detecting and quantifying an antigen in a sugar chain antigen extract using an agglutination reaction of an insoluble carrier based on an antigen-antibody reaction. Semi-quantitative methods include latex agglutination and microtiter methods, and quantitative measurement methods include latex quantification and the like.

  For example, when the amount of antigen in a sugar chain antigen extract is immunologically measured using the latex agglutination method, an antibody that binds to a microorganism sugar chain antigen (hereinafter also simply referred to as an antibody) is immobilized on latex beads. After preparing a measurement reagent composed of sensitized antibody-sensitized particles, the measurement reagent and a sugar chain antigen extract are mixed, and the degree of aggregation of the sensitized particles after the antigen-antibody reaction is visually or optically measured. It can be measured by detecting.

[Optical immunoassay method]
This method is a method for detecting a change in turbidity of an aggregate resulting from an antigen-antibody reaction when an antigen-antibody reaction is carried out by contacting an antibody and a sugar chain antigen extract, or a thin antibody on which an antibody is immobilized. A method in which a sugar chain antigen extract is brought into contact with a layer (hereinafter also referred to as an antibody layer), and a change in the refractive index of the antibody layer resulting from an antigen-antibody reaction is detected as a change in transmitted light, surface plasmon wave, etc. It is a method for optically detecting the presence or absence of an antibody reaction.

[Labeled immunoassay]
The method comprises contacting an antigen with a measurement reagent containing a labeled antibody obtained by binding various labeled substances such as radioactive substances, enzymes, various dyes, colloids, and various particles to an antibody, and a sugar chain antigen extract. After performing the antibody reaction, the amount of the labeling substance bound to the antigen in the sugar chain antigen extract, ie, the radioactivity, enzyme activity, fluorescence intensity, coloring, etc. derived from the labeling substance is measured to extract the sugar chain antigen. This is a method for detecting and quantifying an antigen in a liquid.

  In this method, for example, an antigen-antibody reaction is performed by contacting a measurement reagent comprising an insoluble carrier (particle, membrane, immunoplate, etc.) on which an antibody is immobilized with a sugar chain antigen extract, and then the antibody is labeled with a labeling substance. After further antigen-antibody reaction by contacting with another measurement reagent containing the labeled antibody, the amount of the labeled substance is measured, or the sugar chain antigen extract and the sugar chain antigen labeled with the labeled substance are mixed After the antigen-antibody reaction by contacting with a measurement reagent consisting of an insoluble carrier with immobilized antibody, the antigen in the sugar chain antigen extract is detected and quantified by measuring the amount of the labeled substance bound to the antibody. can do.

  Labeling substances include radioactive iodine, radioactive carbon, etc. as radioactive substances, peroxidase, alkaline phosphatase, galactosidase, etc. as enzymes, various dyes, fluorescent dyes such as fluorescein isothiocyanate, tetramethylrhodamine, etc., colloidal gold colloid Carbon colloids and the like, and various latex particles can be colored latex particles. When enzyme labeling is performed, a method such as direct labeling by a covalent bond such as a thiol group and a maleimide group, an amino group and an aldehyde group, or a labeling via a biotin-avidin complex can be used. In addition, alkaline phosphatase and peroxidase are used as labeling enzymes, and in the case of the former enzyme, a chemiluminescent substance such as a dioxetane derivative, and in the case of the latter enzyme, a chemiluminescent substance such as a luminol derivative is used as the enzyme substrate. When used as, the luminescence of the substrate can also be detected.

  The operations, procedures and the like in these various labeled immunoassay methods are not particularly different from those generally employed, and can be applied to known non-competitive methods, competitive methods, sandwich methods, and the like. In addition to the antibody, a secondary antibody labeled with each of the labeling substances described above, a substance capable of binding to an antibody such as protein A, and the like can also be used for detection and quantification of a sugar chain antigen.

  In the labeled immunoassay method, a conventionally used method can be used without particular limitation depending on the label to be used. Among them, a radioimmunoassay method using a radioactive substance as a label, an enzyme immunoassay method using an enzyme as a label, Fluorescent immunoassay methods that use dyes, especially fluorescent dyes as labels, and chemiluminescent immunoassay methods that use chemiluminescent substances as labels for enzymes as labels are highly quantitative, so when performing highly accurate quantitative measurements It is preferable to adopt these measurement methods. In addition, the flow-through immunoassay method, immunochromatography method, and latex agglutination method using colloids or various particles as labels are characterized by simple operation.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by a following example.

Production Example 1 [Preparation of purified polyclonal antibody against Streptococcus mutans]
(1) [Preparation of cell sample suspension]
Brain Heart Infusion (hereinafter sometimes abbreviated as “BHI”) (DIFCO) (3.7 g) was dissolved in 100 ml of pure water and then autoclaved to prepare a BHI liquid medium. After culturing Ingbritt (Streptococcus mutans, serotype c) in 2 ml of BHI liquid medium under anaerobic conditions (N ₂ : H ₂ : CO ₂ = 80: 10: 10) at 37 ° C. for 5 hours, the culture solution Was centrifuged at 4000 g for 5 minutes, the medium components in the supernatant were removed, and the cell pellets were collected.

Subsequently, the precipitate was suspended in 5 ml of phosphate physiological saline buffer (pH 7.4) (hereinafter sometimes abbreviated as PBS), and the same centrifugation operation was performed three times to wash the precipitate. Thereafter, the obtained bacterial cell precipitate was suspended in PBS, adjusted to A ₆₀₀ = 1.0, and used as an Ingbritt cell sample suspension. In addition, after ultrasonically treating the bacterial cell sample suspension, it is appropriately diluted and then added to a BHI medium plate, and the number of colonies produced is counted and multiplied by the dilution factor of the bacterial cell sample suspension. The bacterial cell concentration of the sample suspension was determined to be about 1 × 10 ⁹ cells / ml.

(2) [Preparation of antiserum against Streptococcus mutans]
Immunization was performed as follows. That is, in the first week, 0.5 ml of Ingbritt cell sample suspension was injected into the auricular vein to the rabbit 5 times for 5 consecutive days. In the second week, 1.0 ml of the bacterial cell sample suspension was injected into the auricular vein into the rabbit 5 times for 5 consecutive days. In the third week, 2.0 ml of the cell sample suspension was injected into the ear vein of the rabbit 5 times for 5 consecutive days. The fourth week was immunized as in the third week. After confirming the increase in titer by the degree of agglutination reaction of the bacterial cells using a slide glass, blood was collected according to a standard method one week after the final immunization to obtain an antiserum against Streptococcus mutans.

(3) [Purification of polyclonal antibody against Streptococcus mutans]
Ingbritt was cultured at 37 ° C. for 12 hours under anaerobic conditions in 1 L of autoclaved BHI liquid medium. The culture solution was centrifuged at 4000 g for 5 minutes to remove the culture medium component of the supernatant and collect the bacterial cell precipitate. Next, the precipitate was suspended in 100 ml of PBS and subjected to the same centrifugation three times to wash the precipitate.

The Ingbritt cells were washed, suspended in 0.1 M Tris-HCl buffer (pH 8.0), and adjusted to A ₆₀₀ = 15. Pronase (Wako Pure Chemical Industries, Ltd.) was added to this so that it might become 5 mg / ml, and it heat-retained at 37 degreeC for 1 hour. After completion of the reaction, the mixture was centrifuged to collect the cell precipitate. Subsequently, the precipitate was suspended in 20 ml of PBS and subjected to the same centrifugation three times to wash the precipitate. Subsequently, it was washed 3 times with 20 ml of 0.1 M glycine hydrochloride buffer (pH 2.0), and further washed 3 times with 20 ml of PBS to prepare a protease-treated cell suspension (A ₆₀₀ = 12.5).

  Next, the protease-treated cell suspension and 0.5 ml of the antiserum prepared in (2) were mixed and reacted at 4 ° C. for 60 minutes. The mixed solution was centrifuged at 4000 g for 5 minutes to recover the cells. The cells were suspended in 10 ml of PBS and washed by performing the same centrifugation operation three times.

  Next, the cells are suspended in 0.5 ml of 0.1 M glycine hydrochloride buffer (pH 2.0), the adsorbed antibody is eluted, the supernatant is recovered by centrifugation, and 1 M Tris-HCl (pH 9.0) is collected. Was added to adjust the pH to 7.4. The same elution operation was performed 4 times, and the protein amount of each fraction was measured by absorbance at 280 nm.

Next, the eluate was added to a column packed with 1 ml of Protein A-Sepharose (Amersham Pharmacia Biotech) previously equilibrated with PBS, washed 5 ml, and then 5 ml of 0.1 M glycine-hydrochloric acid buffer (pH 3.0). 1M Tris-hydrochloric acid (pH 9.0) was immediately added to adjust to pH 7.4. The eluted fraction of IgG was confirmed by measuring _A280 . The IgG fraction was collected and dialyzed against 0.01 M phosphate buffer (4 ° C., 3 days).

  As described above, about 1 mg of a polyclonal antibody obtained by purifying antiserum (0.5 ml) with protease-treated cells was obtained.

Example 1 [Measurement of Streptococcus mutans by antibody-immobilized latex particles produced by coexistence of Tris and glycine]
Using the anti-Streptococcus mutans antibody prepared in Production Example 1, an antibody solution (antibody concentration 0.1 mg / ml) consisting of 10 mM phosphate buffer containing Tris and glycine was prepared. Table 1 shows the concentrations of Tris and glycine in the antibody solution. Next, 1 ml of 0.5% polystyrene latex particles (JSR) was added to this antibody solution (1 ml) and left at room temperature for 1 hour. The antibody-bound latex particles are then isolated by centrifugation and washed once with 0.01 M phosphate buffer (hereinafter sometimes referred to as buffer A) containing 0.05 M sodium chloride to immobilize the antibody. It was suspended in buffer A so that the latex particles were 0.5%.

Ingbritt cultured in Production Example 1 (1) was suspended in buffer A to prepare a 10 ⁷ to 10 ⁵ CFU / ml bacterial solution, which was used as a sample solution. The sample liquid (0.5 ml) was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was removed. To the precipitate, 0.02 ml of 2M acetic acid solution and 0.02 ml of 1M sodium nitrite aqueous solution were added and mixed well, and then allowed to stand at room temperature for 10 minutes. 0.09 ml of 1M Tris (pH not prepared) was added to prepare a sugar chain antigen extract. An equal amount of the antibody-immobilized latex suspension was added to the sugar chain antigen solution, mixed, and allowed to stand at room temperature for 30 minutes, and then the aggregation of latex particles was visually observed. The results are shown in Table 1.

It was found that when Tris and glycine were allowed to coexist when the antibody was brought into contact with the immobilization carrier, nonspecific reaction could be suppressed without reducing sensitivity. A tris concentration of 5-30 mM was suitable. 10 ⁵ CFU / ml of mutans bacteria could be detected in the glycine concentration range of 5-50 mM, but the degree of aggregation of 10 ⁶ CFU / ml was stronger at 5-30 mM. In the case of Tris alone, a nonspecific reaction inhibitory effect was observed at 100 mM and in the case of glycine alone at 500 mM, but at the same time, the measurement sensitivity was also lowered.

Comparative Example 1 [Measurement of Streptococcus mutans with antibody-immobilized latex particles subjected to blocking treatment with protein]
The anti-Streptococcus mutans antibody prepared in Production Example 1 was suspended in 0.01 M phosphate buffer so as to be 0.1 mg / ml. Next, 1 ml of 0.5% polystyrene latex particles (JSR) was added to this antibody solution (1 ml) and left at room temperature for 1 hour. Next, 2 ml of 2% BSA solution was added and allowed to stand at room temperature for 1 hour to prepare antibody-immobilized latex particles. The washing of the antibody-immobilized latex particles, the extraction of sugar chain antigens by the nitrous acid extraction method from Ingbritt cells, and the measurement by the latex agglutination method were carried out in the same manner as in Example 1. The results are shown in Table 1. When blocking with BSA, the sensitivity decreased.

Example 2 [Measurement of Streptococcus mutans by antibody-immobilized latex particles produced by coexisting non-polar amino acids other than glycine and Tris]
Streptococcus mutans was measured in the same manner as in Example 1 except that the nonpolar amino acids shown in Table 2 were used instead of glycine in the presence of 10 mM Tris. The results are shown in Table 2.

Nonpolar amino acids other than glycine showed the same effect as glycine. However, although the lower limit of detection was 10 ⁵ CFU / ml for all amino acids, the strength of the agglutination reaction was slightly weaker than that of glycine.

Claims (2)

  A method for producing an immobilized antibody for use in measuring the amount of a sugar chain antigen in a sugar chain antigen extract obtained by extracting a sugar chain antigen from a microorganism by a nitrous acid extraction method by an immunological method, comprising tris (hydroxymethyl) A method for producing an immobilized antibody, comprising contacting an antibody corresponding to the sugar chain antigen with an immobilization carrier in the presence of aminomethane and a nonpolar amino acid. The method for producing an immobilized antibody according to claim 1, wherein the nonpolar amino acid is glycine.