CN1309420C - Anticarious preparation of antibody - Google Patents

Abstract

The present invention relates to a biological product used for oral cavity decayed tooth resistance, which is prepared from one or more than two kinds of antiserum, salicylic acid, menthol and gallic acid prepared by immunizing several kinds of streptococcus mutans strains resulting in oral cavity decayed teeth by rabbits after the streptococcus mutans strains are cultivated in a mixed way, and the concentration diluting range combined after mother liquor of all components is diluted comprises: 20 to 80 X of antiserum, 10 to 80 X of salicylic acid, 4 to 40 X of menthol and 1 to 8 X of gallic acid. All combined bacterium restraining effects are higher than that of monospecific antiserum, and a decayed tooth resisting preparation of an antibody has obvious antibacterial activity to usual and main decayed tooth resulting bacteria in oral cavities and is nontoxic and nonirritant through detection. Therefore, the biological product can be used for producing oral cavity medicaments to prevent and treat the decayed teeth, and oral cavity vaccines can be added in daily living articles, containing toothpaste, collutory and chewing gum to prevent the decayed teeth and other oral diseases.

Description

The anti-dental caries preparation of antibody

Technical field: the present invention relates to a kind of goods of dental, be specifically related to the biological product of the anti-dental caries in oral cavity.

Background technology: for a long time, both at home and abroad with fluoride as main caries preventive agent, but, also expose some problems of fluoride along with using widely.Show according to relevant department's investigation: form haloform even fluoride trace in water also may combine with Organic substance.Haloform is a kind of carcinogen with genotoxicity; The prolonged application of fluoride can produce the bacterial strain of anti-the fluorine, the not only anti-fluorine of this bacterial strain, and still can so that dental caries, especially when it is present in mixing during bacterial plaque, cariogenic potential is stronger.

In view of the problem of above existence, that the various countries scholars are devoted to seek is new, better, fundamentally eliminates the research of the preventing decayed tooth preparation that causes the dental caries substance.More external scientists successively find and have confirmed that Streptococcus mutans is the main dental caries substance that causes.Thus, many scholars are from immunologic angle, and how fundamentally at preventing decayed tooth has been done a large amount of research, and has obtained great achievement.Studies show that active immunity and passive immunity preventing decayed tooth all are effective technologies.But because the active immunity vaccine may produce the reaction that other is unfavorable for human body in tissue, so active immunity is not carried out application as yet in the mankind.And passive immunity is directly to import resistance streptococcus antibody (immunoglobulin) in the oral cavity, thereby reaches the purpose of preventing decayed tooth, has avoided the existing problem of active immunity, therefore, becomes the focus of current preventing decayed tooth research.At present, in being applied to put into practice mainly be anti-dental caries tooth egg yolk antibody (anti-DC IgY).Anti-dental caries tooth IgY can suppress the activity of mutans streptococcus bacterial enzyme significantly, and the anti-dental caries tooth IgY for preparing tires and can reach 320, and is proved to be safety non-toxic; Also make the cariogenic bacteria inactivation, thereby can reach the purpose for the treatment of both the principal and secondary aspects of a disease owing to just combining behind the anti-dental caries tooth IgY contact cariogenic bacteria with thalline.

Yet, although anti-dental caries tooth IgY can suppress the activity of mutans streptococcus bacterial enzyme significantly, but still the difficult direct effect that reaches chemical preventing decayed tooth preparations such as present widely used fluoride.Therefore, how strengthening and use the effect that antibody suppresses cariogenic bacteria, then is the problem that important needs are researched and solved.

Summary of the invention: technical problem to be solved by this invention is the defective that overcomes above-mentioned prior art, a kind of growth with the mutans streptococcus in the unusual obvious suppression dental plaque is provided, the activity that can also obviously suppress some important periodontal pathogen, not only have good anticaries action, and can prevent the anti-dental caries preparation of antibody of other oral disease.

The present invention solves the problems of the technologies described above the technical scheme that is adopted: the anti-dental caries preparation of antibody by several Streptococcus mutans bacterial strain Mixed culture that cause dental caries with the oral cavity after the synergist of one or more in the antiserum of rabbit immunity preparation and salicylic acid, menthol, the gallic acid form; Solution with antibody titer 640, salicylic acid 0.138g/ml, menthol 0.01g/ml, gallic acid 0.047g/ml concentration during four kinds of combination of components is mother solution, mixes after diluting X times, and each component concentrations dilution range is: antiserum 20～80X; Salicylic acid 10～80X; Menthol 4～40X; Gallic acid 1～8X; During four kinds of combination of components of anti-dental caries preparation, antibody titer improves, and sero-fast extension rate increases, the component concentrations expanded range.

Be described in further detail the present invention below: by cultivating indicator bacteria, observe indicator bacteria bacterium colony state and growth change, preparation indicator bacteria antiserum is measured indicator bacteria antiserum, salicylic acid, menthol and the gallic acid inhibitory action to the indicator bacteria growth, measures inhibition zone size and minimum inhibitory concentration.By Orthogonal Experiment and Design, indicator bacteria antiserum, salicylic acid, menthol and gallic acid are made up the back respectively by the finite concentration certain proportion find to have obvious synergistic effect suppressing the indicator bacteria growth.

One, the preparation of antiserum (antibody)

1, preparation material

1.1, strain

Strain source: buy from key lab of the oral cavity biomedical engineering Ministry of Education of West China College of Stomatology Sichuan University.

Strain name and numbering: Streptococcus mutans (Streptococcus Porteus) S.mulans AHT, S.mulansBHT, S.mulans MT8148.Strain is preserved by the test chamber lyophilizing.(hereinafter to be referred as indicator bacteria).

1.2, culture medium

Solid medium: glucose 2%, yeast extract 1%, peptone 1%, agar 1.6%, 7.0,121 ℃ of sterilizations of pH 20min.

Fluid medium: glucose 2%, yeast extract 1%, peptone 1%, 7.0,121 ℃ of sterilizations of pH 20min.

The performance measurement culture medium: glucose 2%, yeast extract 1%, peptone 1%, pH 7.0.In culture fluid, add several 1.6% bromocresol purple aqueous solutions and be purple.The about 10ml of packing test tube, and Durham's fermentation tube is inverted in the culture fluid 121 ℃ of sterilization 20min.

2, the enrichment culture of strain and determination of activity

2.1, the cultivation of indicator bacteria

2.1.1, enrichment culture

The a small amount of freeze dried mixed indicators of picking inserts in the fluid medium, and 37 ℃ leave standstill cultivation 72 hours.When the culture fluid muddiness, liquid level does not have film, and after the vibration when fluctuation thing of spun silk sample is arranged, inserts in another fresh liquid culture medium, and inoculum concentration 20% one 30% continues to cultivate 72h.

2.1.2, dilution cultivates

Draw proliferating liquid 1ml and add in the 9ml sterilized water, be diluted to 10 successively ^-6, get different dilution factor bacterium liquid 1ml respectively in culture dish, add to melt then and be cooled to solid medium 10-15ml about 45 ℃, shake up, add the 10ml culture medium again after waiting to solidify, shake up, make the double-layer plate that contains bacterium, cultivated 2-5 days in 37 ℃.

2.1.3, determination of activity

Selection has the white small-sized bacterium colony of transparent circle, and access performance is measured in the culture medium, and supplementing culture medium is apart from mouth of pipe 2cm place then, and sealing orifice was cultivated 2-3 days for 37 ℃, observed the culture fluid change in color.When choosing indicator bacteria and homotype lactate fermentation, owing to produce lactic acid, the culture medium color becomes yellow by purple; If when institute's bacterial strain of choosing and heterofermentation, not only the culture medium color changes, and since aerogenesis, gassy in the Du Shi pipe.

2.1.4, the preservation of thalline

Bacterium liquid behind the enrichment culture through the centrifugal 4min of 12000r/min, is removed supernatant, and 0.9% normal saline flushing, thalline are put into 4 ℃ of refrigerators and are preserved.

3, preparation antiserum

3.1, antigenic preparation

Cultured indicator bacteria of picking and PBS phosphate buffer mixing are mixed with the bacteria suspension that concentration is 1mg/ml, get bacteria suspension 2ml and complete Freund's adjuvant mixing and emulsifying (bacteria suspension: complete Freund's adjuvant=1: 1), standby after fully mixing.

3.2, the thalline immunity

Immunity is preceding from the about 3ul blood of tame rabbit ear vein collection, serum in contrast.

The injecting program of making the rabbit immunity of indicator bacteria antigen is as follows:

Table 1

Time	Dosage	Approach
			The 1st day the 7th day the 13rd day the 19th day	0.5ml 1.0ml 2.0ml 2.5ml	Ear vein ear vein ear vein ear vein

3.3, the blood sampling

Adopt the about 3ml of examination blood after injection is last, tiring when examination blood reaches more than 1: 500, but then heart is adopted whole blood, otherwise wants booster injection 1-2 time.

3.4, sero-fast separation preserves

Leave standstill through room temperature or 37 ℃ from the blood of immunizing rabbit heart collection and to be placed on refrigerator overnight, allow serum fully separate out, in case of necessity through the centrifugal 10min of 3000r/min, PBS washing 2 times, add 1% thimerosal in 1: 100 ratio, divide the penicillin bottle of packing into, it is standby to put-20 ℃ of preservations.

3.5, sero-fast titration

3.5.1, antiserum dilution

Press table 2 double dilution method dilution antiserum

Table 2 dilution process

Serum dilution	1∶ 10	1∶ 20	1∶ 40	1∶ 80	1∶ 160	1∶ 320	1∶ 640	1∶ 1280	1∶ 2560	1∶ 5120	CK
												Serum (ml) normal saline	0.1 0.9	0.5 0.5	0.5 0.5	0.5 0.5	0.5 0.5	0.5 0.5	0.5 0.5	0.5 0.5	0.5 0.5	0.5 0.5	0 0.5

(ml)

3.5.2, antigen diluent

The indicator bacteria antigen of preserving is diluted with normal saline, make it be equivalent to the concentration of Maxwell opacity tube the 7th pipe.

3.5.3, titration

Add 0.5ml indicator bacteria antigenic dilution respectively in 11 serum tubes, the antiserum uniform mixing of antigen and equivalent is put in 37 ℃ of water-baths and was hatched 2～4 hours, measures by following criterion and tires:

++ ++ 100% coagulation (coagulation fully, upper strata become clear)

+++about 75% coagulation (most coagulations, upper strata are slightly muddy)

++ about 50% coagulation (partial coagulation, the upper strata is more muddy)

+ about 25% coagulation (seldom partial coagulation, upper strata muddiness)

-no coagulation (liquid muddiness)

Maximum dilution multiple with appearance " ++ " reaction is sero-fast tiring.After measured, sero-fast the tiring of indicator bacteria is 640.

3.6, antiserum suppresses the indicator bacteria growth measurement

3.6.1, the screening of best bacteria concentration

Picking indicator bacteria thalline 0.5000g is diluted in the 5ml normal saline, and concentration is 0.1g/ml, and is standby.Get 10 clean sterilized test tubes, be diluted to 10,20,40,80,160,320,640,1280,2560,5120 times of totally 11 Concentraton gradient successively.Medium sterilization, dull and stereotyped, cooling, every ware is inoculated the indicator bacteria diluent 50ul of variable concentrations respectively, each 3 repetition, 37 ℃ of incubators are cultivated 18h, observation experiment result.

By relatively, be evenly distributed with the 10th indicator bacteria bacterium colony of managing concentration bacterium liquid, consistent in density, upgrowth situation is good, the most suitable bacteriostatic experiment of doing.Therefore,, standby with this concentration as bacteriostatic experiment concentration.The bacterium turbidimetric analysis turbidimetry, preparation fungus suspension is a blank with the normal saline, 721 type spectrophotometric determination bacteria concentrations, wavelength 530nm measures the 10th pipe bacterium liquid absorbance, OD ₄₂₀Value is 0.33.

3.6.2, bacteriostatic test

Adopt the Oxford agar diffusion method to measure the size of inhibition zone.

Antiserum is diluted to 10,20,40,80,100,200,400,800,1000,1200 times of totally 10 Concentraton gradient successively.Medium sterilization, dull and stereotyped, cooling, every ware coating indicator bacteria liquid 50ul, 4 repetitions are drawn the 200ul antiserum and are added in the culture dish that is coated with indicator bacteria liquid, and 37 ℃ of incubators are cultivated 18h, the results are shown in Table 4.

Table 4 antiserum fungistatic effect:

The antiserum extension rate	Fungistatic effect (mm)
		Mother liquor 10 20 40* 80 100 200 400 800 1,000 1200	There is not antibacterial phenomenon, a large amount of coagulations.There is not antibacterial phenomenon, micro-coagulation.10.07 10.18 9.34 10.20 11.49 10.91 11.30 11.76 10.24 9.93 10.25 10.16 no obvious antibacterial phenomenons do not have obvious antibacterial phenomenon and do not have antibacterial phenomenon and do not have antibacterial phenomenon and do not have antibacterial phenomenon and do not have antibacterial phenomenon

Tiring is that sero-fast 20～80X diluent of 640 has inhibitory action to indicator bacteria, wherein with fungistatic effect the best of the normal saline diluent of 40X.

Two, the preparation of synergist and fungistatic effect are measured

1, salicylic acid

Salicylic acid (salicylic acid), it is oxybenzoic acid, be a kind of secondary metabolite of plant, it extensively is present in plant kingdom, has outer can also being discharged in the air with the form of methyl salicylate divided by free form and glucoside form in vivo.Salicylic acid has multiple physiological regulatory action, and most typical physiological action is to induce the biological systemic acquired resistance that produces, and is the biological essential factor that produces systemic acquired resistance.Many SA of experimental results show that are important systemic acquired resistance (Systemic acquired resistance, endogenous signal molecules SAR).

1.1, the preparation

Salicylic acid, Huamei Bio-Engrg Co., buys by Beijing.Take by weighing salicylic acid (analytical pure) 6.9000g, fully grind to form smalls, precentagewise concentration is dissolved in 50% alcoholic solution, is settled to 50ml, and concentration is 1M, and is standby as mother solution.Dilute 10,20,40,80,100,200,400,600,800,1000,1200 totally 11 Concentraton gradient successively.

1.2, fungistatic effect measures

Medium sterilization, dull and stereotyped, cooling, every ware coating indicator bacteria liquid 50ul, 4 repetitions are drawn the 200ul salicylic acid in the culture dish that is coated with indicator bacteria liquid.Make blank with 50% ethanol, 4 repetitions, 37 ℃ of incubators were cultivated 18 hours.Measurement result sees Table 5.

Table 5 fungistatic effect

The salicylic acid extension rate	Inhibition zone (mm)
		10* 20 40 80 100 200 400 800 1000 1200	19.48 19.63 19.76 19.54 15.40 15.30 15.15 15.67 12.25 11.51 10.10 11.17 11.11 11.02 10.00 9.95 outer shrouds are darker; Not obvious outer shroud is dark, and not obvious no antibacterial phenomenon does not have antibacterial phenomenon and do not have antibacterial phenomenon and do not have antibacterial phenomenon

50% ethanol blank, 4 repetitions all do not produce inhibition zone, illustrate that 50% ethanol does not suppress the growth of indicator bacteria, do not have bacteriostasis, and the interference measurement salicylic acid is not to the action effect of indicator bacteria; Salicylic acid has fungistatic effect in the 50% ethanol dilution degree scope of 10～80X as can be seen from Table 5, with fungistatic effect the best of 10X.

2, menthol

Menthol also is a kind of secondary metabolite of plant, artificial is the processed goods of Dipterocarpaceae aiphyllium Borneolum Syntheticum (Dryobalanops Arornatica Gaertnerf) resin, or the extract of feverfew (Blumea Balsamifera), or be the be processed into product of raw material through chemosynthesis with Camphora, Oleum Terebinthinae.Menthol belongs to volatile medicine, is called baras camphor in organic chemistry, is insoluble in water, dissolves in the aquiferous ethanol, is made up of the chemical compound of two isoprene units, belongs to the polyenoid compounds of group.Menthol is to extract from contain the volatile oil plant, also belongs to alcohols.The in-vitro antibacterial test of Mu Shi shows: menthol can suppress or kill 5 kinds of common bacterias such as staphylococcus aureus, B-mode bacteriolyze streptococcus, viridans streptococci, escherichia coli, its minimum inhibitory concentration (MIC) is 1.0%-2.0%, minimum bactericidal concentration (MFC) is 1.5%-2.0%, and is antibacterial for contacting.Its main component Borneolum Syntheticum is consistent with the antibacterial effect of isoborneol, has not both had antagonism between two components and has not also had synergism.Discover that its minimum bactericidal concentration (MFC) is 10%.Menthol application clinically a few days ago is very extensive, and particularly the application in cardiovascular and cerebrovascular disease is noticeable especially.

2.1, the preparation

Menthol, (Borneolum Syntheticum 59.9%, isoborneol 37.5%), Huamei Bio-Engrg Co., buys by Beijing.Take by weighing menthol (analytical pure) 1.0000g, fully grind to form smalls, precentagewise concentration is dissolved in 45% alcoholic solution, is settled to 100ml, and is standby as mother solution.Be diluted to 0,2,4,8,10,20,40,80,160,320,640 totally 11 Concentraton gradient successively with 45% ethanol.

2.2, fungistatic effect measures

Medium sterilization, dull and stereotyped, cooling, every ware coating indicator bacteria liquid 50ul, 4 repetitions pipette 200ul menthol liquid and go in the culture dish.45% ethanol is made blank, 4 repetitions, and 37 ℃ of incubators were cultivated 18 hours.Measurement result sees Table 6.

Table 6 fungistatic effect

The menthol extension rate	Inhibition zone (mm)
		Mother liquor 24 8* 10 20 40 80 160 320 640	Not having antibacterial phenomenon does not have antibacterial phenomenon 10.25 10.51 10.51 10.36 13.86 13.47 14.77 14.12 11.03 11.45 11.38 11.16 10.08 9.65 9.85 10.12 9.50 9.41 9.71 9.69 no antibacterial phenomenons and does not have antibacterial phenomenon and do not have antibacterial phenomenon and do not have antibacterial phenomenon

45% ethanol blank, 4 are repeated all not produce inhibition zone, illustrate that 45% ethanol does not suppress the growth of indicator bacteria, do not have bacteriostasis, and the interference measurement menthol is not to the action effect of indicator bacteria.The measurement result explanation menthol of table 6 has fungistatic effect in the 45% ethanol dilution degree scope of 4～40X, with fungistatic effect the best of 8X.

3, gallic acid

Gallic acid (Gallic acid) can extract from plants such as Galla Chinensis, Caesalpinia spinosaKuntze soybean pod and get. and gallic acid has precipitation to protein, after the ulcer surface of skin, mucosa contacts, its tissue protein promptly is solidified, and causes one deck tunicle and is astriction; The protein of glandular cell is solidified and causes secretion, produces the mucosa drying, and the proteinic precipitation of teleneuron can be faint local anesthesia phenomenon.Gallic acid can form not dissolved compound with number of metal, glycoalkaloids, thereby as antidote.Galla Turcica (Galla Helepensis) has astriction to small intestinal, can alleviate enteritis, prevents diarrhoea.In addition, staphylococcus aureus, streptococcus, streptococcus pneumoniae, typhoid fever, paratyphoid fever, dysentery, anthrax, diphtheria, bacillus pyocyaneus all there is inhibitory action.

3.1, the preparation

Gallic acid, Huamei Bio-Engrg Co., buys by Beijing.Take by weighing gallic acid 4.7035g, precentagewise concentration is dissolved in the distilled water, is settled to 100ml, and concentration is 0.25M, and is standby as mother solution.Dilute 0,2,4,8,10,20,40,60,80,100 totally 10 Concentraton gradient successively.

3.2, fungistatic effect measures

Medium sterilization, fall dull and stereotyped, cooling, every ware coating indicator bacteria liquid 50ul, 3 repetitions are drawn 200ul Galla Turcica (Galla Helepensis) acid solution to the culture dish that is coated with indicator bacteria, 37 ℃ of incubators were cultivated 18 hours.Measurement result sees Table 7.

Table 7, fungistatic effect

Extension rate	Inhibition zone (mm)
				Mother liquor 2* 48 10 20 40 80 100	25.81 21.52 17.61 11.94 no phenomenons do not have phenomenon and do not have phenomenon and do not have phenomenon and do not have phenomenon	25.40 21.77 17.54 11.88 no phenomenons do not have phenomenon and do not have phenomenon and do not have phenomenon and do not have phenomenon	25.51 21.40 17.67 11.72 no phenomenons do not have phenomenon and do not have phenomenon and do not have phenomenon and do not have phenomenon

Find out that from the measurement result of table 7 diluent of mother solution and 2～8X all has fungistatic effect.

Three, the potentiation of antiserum and synergist various combination is measured:

1, the factor and the level of orthogonal test investigation

Whether having potentiation for inquiring into antiserum and salicylic acid, menthol, gallic acid various combination, is evaluation index with the size of inhibition zone, carries out orthogonal test.Investigate the influence to the inhibition zone size of inhibition indicator bacteria growth of salicylic acid, menthol, gallic acid and 4 kinds of principal elements of antiserum of variable concentrations, the concentration combination of inhibition indicator bacteria growth is selected in test by four factors, four horizontal combination orthogonal experiments.

Factor and level that table 8 orthogonal test is investigated

Level	Factor
					Salicylic acid concentration	Menthol concentration	Antiserum concentration	Gallic acid concentration
	1	10X	4X	20X					1X
2					20X	8X	40X	2X
	3	40X	20X	60X					4X
4					80X	40X	80X	8X

The concentration of above mother solution is: serum titer 640, salicylic acid 0.138g/ml, menthol 0.01g/ml, gallic acid 0.047g/ml." X " be extension rate.

2, orthogonal test method and result

With serum 20-80X, menthol 4-40X, salicylic acid 10-80X, gallic acid 1-8X is the interval, each material is selected for use four above concentration to make up respectively to carry out orthogonal experiment.

According to above-listed combination preparation successively one by one, draw every kind of material 1ml respectively and be mixed in the Boiling tube, fully mixing pipettes mixed liquor 1ml in the eppendorf pipe, to measure the fungistatic effect of each combination, each concentration.

Medium sterilization, dull and stereotyped, cooling, every ware is coated with newborn cloth indicator bacteria liquid 50ul, draws the mixed liquor 200ul that has prepared successively and be placed in the cup of Oxford three repetitions of every processing from the eppendorf pipe, behind 37 ℃ of cultivation 18h, observe the fungistatic effect (seeing Table 9) of different factor levels combinations.

Table 9.1. synergist and antiserum orthogonal experiments (three repeat meansigma methods)

Numbering	Antiserum	Salicylic acid	Inhibition zone (mm)	Numbering	Antiserum	Salicylic acid	Inhibition zone (mm)
								1	20X	10X	17.21	9	60X	10X	18.87
2	20X	20X	15.24	10	60X	20X	15.77
								3	20X	40X	10.71	11	60X	40X	10.34
4	20X	80X	9.99	12	60X	80X	10.66
								5	40X	10X	19.12	13	80X	10X	19.79
6	40X	20X	15.55	14	80X	20X	12.96
								7	40X	40X	10.47	15	80X	40X	11.10
8	40X	80X	10.03	16	80X	80X	9.98
														Overall average	12.98

Table 9.2. synergist and antiserum orthogonal experiments (three repeat meansigma methods)

Numbering	Antiserum	Gallic acid	Inhibition zone (mm)	Numbering	Antiserum	Gallic acid	Inhibition zone (mm)
								1	20X	2X	21.34	7	60X	2X	22.69
2	20X	4X	18.19	8	60X	4X	14.10
								3	20X	8X	12.10	9	60X	8X	13.04
4	40X	2X	20.46	10	80X	2X	23.43
								5	40X	4X	16.33	11	80X	4X	17.44
6	40X	8X	11.87	12	80X	8X	11.67
														Overall average	16.88

Table 9.3. synergist and antiserum orthogonal experiments (three repeat meansigma methods)

Numbering	Antiserum	Menthol	Inhibition zone (mm)	Numbering	Antiserum	Menthol	Inhibition zone (mm)
								1	20X	4X	10.28	6	40X	40X	10.17
2	20X	8X	12.49	7	80X	4X	10.35
								3	20X	40X	9.95	8	80X	8X	12.51
4	40X	4X	11.13	9	80X	40X	9.96
								5	40X	8X	13.27
						Overall average	11.12

Table two kinds of synergists of 9.4. and antiserum are handed over result of the test (three repeat meansigma methods)

Numbering	Antiserum	Salicylic acid	Menthol	Inhibition zone (mm)	Numbering	Antiserum	Salicylic acid	Menthol	Inhibition zone (mm)
										1	20X	10X	4X	17.70	33	60X	10X	4X	18.45
2	20X	10X	8X	17.38	34	60X	10X	8X	17.69
										3	20X	10X	20X	19.96	35	60X	10X	20X	17.51
4	20X	10X	40X	19.30	36	60X	10X	40X	15.33
										5	20X	20X	4X	15.26	37	60X	20X	4X	12.43
6	20X	20X	8X	12.05	38	60X	20X	8X	12.21
										7	20X	20X	20X	12.89	39	60X	20X	20X	11.38
8	20X	20X	40X	12.61	40	60X	20X	40X	12.00
										9	20X	40X	4X	10.12	41	60X	40X	4X	11.44
10	20X	40X	8X	10.27	42	60X	40X	8X	10.91
										11	20X	40X	20X	10.15	43	60X	40X	20X	11.77
12	20X	40X	40X	9.87	44	60X	40X	40X	9.99
										13	20X	80X	4X	9.67	45	60X	80X	4X	9.79
14	20X	80X	8X	10.11	46	60X	80X	8X	9.66
										15	20X	80X	20X	9.38	47	60X	80X	20X	9.83
16	20X	80X	40X	9.61	48	60X	80X	40X	9.35
										17	40X	10X	4X	19.92	49	80X	10X	4X	19.87

18	40X	10X	8X	18.99	50	80X	10X	8X	20.01
										19	40X	10X	20X	17.68	51	80X	10X	20X	17.77
20	40X	10X	40X	13.32	52	80X	10X	40X	16.67
										21	40X	20X	4X	14.45	53	80X	20X	4X	12.70
22	40X	20X	8X	11.06	54	80X	20X	8X	13.13
										23	40X	20X	20X	10.81	55	80X	20X	20X	13.44
24	40X	20X	40X	10.09	56	80X	20X	40X	12.10
										25	40X	40X	4X	10.11	57	80X	40X	4X	11.01
26	40X	40X	8X	10.30	58	80X	40X	8X	10.20
										27	40X	40X	20X	10.22	59	80X	40X	20X	9.45
28	40X	40X	40X	9.60	60	80X	40X	40X	10.19
										29	40X	80X	4X	9.39	61	80X	80X	4X	10.00
30	40X	80X	8X	9.98	62	80X	80X	8X	9.98
										31	40X	80X	20X	9.45	63	80X	80X	20X	9.74
32	40X	80X	40X	9.46	64	80X	80X	40X	9.65
																		Overall average	12.29

Table two kinds of synergists of 9.5. and antiserum orthogonal experiments (three repeat meansigma methods)

Numbering	Antiserum	Menthol	Gallic acid	Inhibition zone (mm)	Numbering	Antiserum	Thin what alcohol	Gallic acid	Inhibition zone (mm)
										1	20X	4X	2X	14.76	10	40X	8X	4X	16.91
2	20X	4X	4X	12.81	11	40X	10X	2X	13.28
										3	20X	8X	2X	15.49	12	40X	10X	4X	13.26
4	20X	8X	4X	14.21	13	80X	4X	2X	16.17
										5	20X	10X	2X	15.38	14	80X	4X	4X	10.11
6	20X	10X	4X	12.25	15	80X	8X	2X	19.89
										7	40X	4X	2X	13.99	16	80X	8X	4X	11.90
8	40X	4X	4X	11.02	17	80X	10X	2X	13.56
										9	40X	8X	2X	18.74	18	80X	10X	4X	10.99
								Overall average	13.63

Table two kinds of synergists of 9.6. and antiserum orthogonal experiments (three repeat meansigma methods)

Numbering	Antiserum	Gallic acid	Salicylic acid	Inhibition zone (mm)	Numbering	Antiserum	Gallic acid	Salicylic acid	Inhibition zone (mm)
										1	20X	2X	10X	17.20	4	40X	2X	10X	18.88
2	20X	4X	10X	15.94	5	80X	4X	10X	14.32
										3	40X	2X	10X	16.31	6	80X	2X	10X	11.67
								Overall average	15.72

Table three kinds of synergists of 9.7. and antiserum orthogonal experiments (three repeat meansigma methods)

Numbering	Antiserum	Salicylic acid	Menthol	Gallic acid	Inhibition zone (mm)	Numbering	Antiserum	Salicylic acid	Menthol	Gallic acid	Inhibition zone (mm)
												1	20X	10X	8X	2X	21.04	17	60X	10X	8X	2X	19.97
2	20X	10X	8X	4X	16.79	18	60X	10X	8X	4X	17.03
												3	20X	10X	20X	2X	15.55	19	60X	10X	20X	2X	22.33
4	20X	10X	20X	4X	14.91	20	60X	10X	20X	4X	16.19
												5	20X	40X	8X	2X	14.61	21	60X	40X	8X	2X	14.66
6	20X	40X	8X	4X	13.14	22	60X	40X	8X	4X	14.65
												7	20X	40X	20X	2X	14.99	23	60X	40X	20X	2X	15.21
8	20X	40X	20X	4X	12.05	24	60X	40X	20X	4X	13.30
												9	40X	10X	8X	2X	22.55	25	80X	10X	8X	2X	23.54
10	40X	10X	8X	4X	17.87	26	80X	10X	8X	4X	22.93
												11	40X	10X	20X	2X	17.99	27	80X	10X	20X	2X	16.70
12	40X	10X	20X	4X	14.06	28	80X	10X	20X	4X	14.05
												13	40X	40X	8X	2X	13.00	29	80X	40X	8X	2X	15.36
14	40X	40X	8X	4X	12.10	30	80X	40X	8X	4X	12.03
												15	40X	40X	20X	2X	12.44	31	80X	40X	20X	2X	12.53
16	40X	40X	20X	4X	10.78	32	80X	40X	20X	4X	11.10
																						Overall average	15.79

More than " X " be the mother solution extension rate of every kind of material.

3, potentiation interpretation of result

Can find out from table 9.1～9.7:

3.1 its inhibition zone of antiserum that the uses a kind of synergist meansigma methods of high minimum sees Table 9.1,9.2,9.3 than the peak 11.76 high 3.12 and the 5.79[of monospecific antiserum respectively, serum adds salicylic acid: (9.98+19.79)/and 2=14.88; Serum adds gallic acid: (11.67+23.43)/and 2=17.55]; (serum adds salicylic acid to overall average: 12.98, serum adds gallic acid: 16.88, serum adds menthol: 11.12) and antiserum add the highest minimum meansigma methods of menthol: (9.95+12.51)/2=11.23 all is higher than sero-fast 20X, 40X, the meansigma methods 9.74 of three kinds of concentration of 80X (seeing Table 6); Use a kind of sero-fast minimum 9.95 of synergist to be higher than the sero-fast minimum 9.34 of independent use.

3.2, its inhibition zone of antiserum of using two kinds of synergists the meansigma methods of high minimum than using sero-fast inhibition zone maximum separately: 11.76 is high by 2.30 respectively, 3.24, add menthol and salicylic acid 3.51[see Table 9.4,9.5,9.6 serum: (8.11+20.01)/2=14.06, serum adds gallic acid and menthol: (10.11+19.89)/2=15.00, serum adds gallic acid and salicylic acid: (11.67+18.88)/2=15.27]; Overall average is higher by 0.53,1.87 respectively than the peak 11.76 that uses serum separately, 3.96 (serum adds salicylic acid and menthol: 12.29, and serum adds gallic acid and menthol: 13.63, serum adds gallic acid and salicylic acid: 15.72).

3.3, its inhibition zone of antiserum of using three kinds of synergists the meansigma methods of high minimum see Table 9.7 serum than the inhibition zone maximum 11.76 high 5.40[that use serum separately and add salicylic acid and add gallic acid and add menthol: (10.78+23.54)/2=17.16]: 11.76 high by 4.03 (serum adds salicylic acid and adds gallic acid and add menthol overall average: 15.79) than using sero-fast peak separately; The minimum 10.78 of four kinds of materials is higher than the sero-fast minimum 9.34 of independent use.

The above results illustrates that the potentiation that one or more synergist adds in the antiserum is obvious.When the antibody titer raising, sero-fast dilution range enlarges.

Stomatology engineering education key lab of West China College of Stomatology Sichuan University anti-dental caries preparation of antagonist carries out antibacterial activity and detects, detect the anaerobe drug sensitivity test meat Soup dilution method that adopts NCCLs to recommend, measure the anti-dental caries preparation of antibody to the most common and topmost cariogenic bacteria in oral cavity: Streptococcus mutans and actinomyces viscosus, and important periodontal pathogen: the antibacterial activity of the unicellular bacterium of porphyromonas, the result shows that the inspection product all have tangible antibacterial activity to three kinds of experimental strains.

Table 10 inspection product are to the MIC and the MBC (g/L) of three strain experimental bacteria

	Porphyromonas gingivalis	Streptococcus mutans	Viscosity lonizing radiation bacterium
				MIC	1.56	3.12	3.12
MBC	3.12	3.12	3.12

Above result shows, the anti-dental caries preparation of antibody has the growth of the mutans streptococcus in the unusual obvious suppression dental plaque, can also obviously suppress the activity of some important periodontal pathogen, not only has good anticaries action, and can prevent other oral disease.

According to Hunan Province Center for Disease Control (CDC) " the anti-dental caries preparation of antibody " product is carried out the acute toxicity test result and shows, to the acute oral LD of Kunming kind male and female mice ₅₀Greater than 20000mg/Kgbw, true border non-poisonous material; 24 hours, 48 hours, the 72 hours acute eye irritation reaction results to white rabbit are that cornea, red film, conjunctival congestion and chemosis integral mean value are 0, belong to nonirritant.

Therefore the anti-dental caries preparation of antibody can be used for producing the mouth cavity medicine of preventing and treating dental caries, the oral cavity vaccine of child's preventing decayed tooth, also can be used for oral health products and be added into people's articles for daily use such as toothpaste, rinse liquid, chewing gum etc. in be used for preventing decayed tooth and anti-oral disease.

The specific embodiment:

Join in the 80X serum with the 4X gallic acid, doubly add in the toothpaste and make toothpaste in the back, or make collutory etc. and can prevent dental caries and periodontitis effectively with distilled water diluting 600-800.

Be mixed and made into synergist with 10X salicylic acid and 20X menthol and join in the 80X serum,, can be specifically designed to the anti-dental caries tooth health care of child with can be made into special-purpose anti-dental caries spray health product behind 500 times of the distilled water dilutings.

Be mixed and made into synergist with 2X gallic acid, 10X salicylic acid and 8X menthol and join in the 80X serum, can be made into preventing decayed tooth oral cavity medicine spray with 200 times of distilled water dilutings.The medicine of making is sprayed on the affected part, oral cavity can not only prevent dental caries effectively, but also periodontitis is had therapeutical effect preferably.

Claims (2)

1, the anti-dental caries preparation of a kind of antibody, it is characterized in that anti-dental caries preparation by several Streptococcus mutans bacterial strain Mixed culture that cause dental caries with the oral cavity after the synergist of one or more in the antiserum of rabbit immunity preparation and salicylic acid, menthol, the gallic acid form.

2, by the anti-dental caries preparation of the described antibody of claim 1, solution with antibody titer 640, salicylic acid 0.138g/ml, menthol 0.01g/ml, gallic acid 0.047g/ml concentration when it is characterized in that four kinds of combination of components of anti-dental caries preparation is mother solution, mix after diluting, the blended concentration dilution scope of each component is:

20～80 times of antiserum dilutions

10～80 times of salicylic acid dilutions

4～40 times of menthol dilutions

1～8 times of gallic acid dilution.