Saliva pretreatment reagent box and saliva pretreatment process

A pretreatment and kit technology, which is applied in the field of saliva pretreatment kits, can solve the problems such as the decrease of measurement accuracy

Inactive Publication Date: 2004-04-28
GC CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the immunochromatography technique is applied to saliva as a sample, the complex of the labeled antibody and Streptococcus mutans is captured in the membrane holding the labeled antibody, but cannot flow efficiently to the porous membrane immobilized with the captured antibody through capillary phenomenon. The problem that causes the accuracy of the measurement to drop

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0046] Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. If not specified otherwise, operations were carried out at room temperature and the pH was that at 20-25°C.

[0047] (1) Preparation of reagents and test equipment

[0048] 1. Antibody Production

[0049]Streptococcus mutans (ATCC 25175 strain) and Streptococci sobrinus (ATCC 33478 strain) were respectively cultured by using BHI medium (Brain Heart Infusion medium, manufactured by DIFCO Laboratories) overnight at 37°C. After the bacterial cells were collected from the culture solution by centrifugation, they were washed twice with PBS (10 mmol phosphate-buffered saline), and growth was terminated with aqueous formaldehyde. Mice were immunized directly with bacterial lysates, and then hybridomas were established by cell fusion using Kohler and Milstein to obtain two types of antibodies shown below.

[0050] Antibody to ...

Embodiment 1

[0065] An antibody-immobilized porous membrane strip is used in which the SM1 antibody is immobilized at the center and the SM2-labeled antibody is contained in the labeled antibody holder at the other end. Check it in the following way.

[0066] The subjects were asked to chew the gum used for saliva collection for 5 minutes, and the saliva was collected in test tubes. A part of the saliva thus collected was diluted with PBS and spread on MSB medium. After 2 or 3 days of anaerobic culture at 37°C, the number of colonies was determined. After the measurement, some colonies were collected. After the bacterial cells are disrupted, PCR (polymerase chain reaction) is carried out with synthetic DNA primers having specific sequences for Streptococcus mutans or Streptococcus distalis, and the amplified gene fragments are subjected to electrophoresis on agarose gel to identify strain. The number of bacterial cells (CFU / mL) was calculated from the number of colonies confirmed to be...

Embodiment 2

[0072] Using the same antibody-immobilized porous membrane strip as in Example 1 except that the SS1 capture antibody was used instead of the SM1 capture antibody, and the SS2-labeled antibody was used instead of the SM2-labeled antibody, the test was carried out in the following manner.

[0073] A solution (A solution) was prepared by adding 25.0% by weight of sodium chloride to a solution containing 1.0 mol / L sodium hydroxide (component (A)). A solution (BD solution) was prepared by adding 0.5 mol / L citric acid (component (B)) to a solution containing 1.0 mol / L tris(hydroxymethyl)aminomethane (component (D)). An aqueous solution (C solution) containing 5% by weight of polyethylene glycol monooctylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) as a nonionic surfactant (component (C)) was prepared.

[0074] Add 80 µL of BD solution to 250 µL of saliva and stir. Then add 50 μL of C solution to it, and then stir. Finally, 40 μL of A solution was added there...

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Abstract

A pretreatment kit and a pretreatment kit for saliva in identification and quantitative determination of mutans streptococci by immunochromatography utilizing an antigen-antibody reaction, which can remove aggregation caused by mucin and chain formation of mutans streptococci in saliva in a simple operation and can efficiently flow out a complex of a labeled antibody and mutans streptococci from a porous membrane retaining the labeled antibody, contains (A) a 0.01 to 10 mol / L aqueous solution of sodium hydroxide, (B) a 0.01 to 3 mol / L aqueous solution of tartaric acid and / or citric acid, and (C) a nonionic surface active agent and / or an amphoteric surface active agent, in which the component (C) is mixed with the components (A) and / or (B), or is provided separately, and at least one substance selected from the particular metallic salts is contained in at least one of the components (A), (B) and (C) in an amount of 5 to 25% by weight.

Description

technical field [0001] The present invention relates to a saliva pretreatment kit for identifying and quantitatively measuring Streptococcimutans, one of the cariogenic bacteria present in human saliva, by immunochromatography using antigen-antibody reactions, and using the saliva pretreatment kit. Saliva pretreatment method for the processing kit. Background technique [0002] It is known that the presence of Streptococcus mutans in the human oral cavity is closely related to the occurrence of dental caries. Therefore, if the presence and amount of Streptococcus mutans in the human oral cavity can be easily checked, the risk of disease and the current prevalence of disease can be understood. situation, to the benefit of a considerable number of people. [0003] The above is an inspection technique that utilizes the antigen-antibody reaction in the inspection. For example, the immunoenzyme technique, which is a method of identification and quantitative measurement using en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/48G01N1/10G01N33/50G01N33/543G01N33/569
CPCY10T436/10G01N33/56944Y10T436/25Y10T436/107497
Inventor 立野敦史
Owner GC CORP
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