CN104101596A - Detection method for complement-mediated serum antibody-dependent bactericidal activity - Google Patents
Detection method for complement-mediated serum antibody-dependent bactericidal activity Download PDFInfo
- Publication number
- CN104101596A CN104101596A CN201410362470.2A CN201410362470A CN104101596A CN 104101596 A CN104101596 A CN 104101596A CN 201410362470 A CN201410362470 A CN 201410362470A CN 104101596 A CN104101596 A CN 104101596A
- Authority
- CN
- China
- Prior art keywords
- serum
- hole
- volume
- checked
- doubly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention relates to a detection method for complement-mediated serum antibody-dependent bactericidal activity. The detection method comprises the following steps: bacterium suspension preparing; sample processing; bactericidal detecting; coloration judging. When the positive serum control group turns out the positive result, and the negative serum control group, the complement blank control group and the bacterial activity control group all turn out the negative results, the color development or tip-shaped precipitation of the serum to be detected turns out the negative result, that is, the serum to be detected fails to have the bactericidal activity; the color development failure or lamellar precipitation of the serum to be detected turns out the positive result, that is, the serum to be detected has the bactericidal activity. The detection method dispenses with naked eyes for each viable counting, and whether the serum to be detected has the bactericidal activity can be intuitively and rapidly judged through the color development condition; the control groups are simultaneously provided for each micro-porous plate to be adopted as the condition that the detection result is true; the detection steps are convenient, fast, reliable and accurate.
Description
Technical field
The present invention relates to after bacterial vaccine immune animal or healthy population, the serum antibody of generation has functional sterilizing or bacteriostatic activity assessment technique field.
Technical background
Serum antibody sterilizing power detects, and can weigh the functional characteristic of the immunoglobulin (Ig) of specific species.Serum antibody sterilizing power detects and relies on antibody recognition condition, by classical pathway, activates, and the antigen that surface is exposed is combined with complement, antibody, causes antibacterial or bacteriolyze.Existing serum antibody sterilizing power detects, and its step and result decision method are complicated, particularly need people's naked eyes directly to carry out count plate, and error is larger, tests length consuming time, and restricted application.The epidemic meningitis serum antibody sterilizing power of take below test illustrates its existing serum antibody sterilizing power detection method concrete operations as example:
1, collecting cells
Breakdown bacterial classification, after recovery is cultivated on solid medium, is inoculated in amplification cultivation in fluid nutrient medium.Uniform bacteria suspension is made in the dilution of bacterium liquid.On the spectrophotometer that to get bacteria suspension be 540nm in wavelength, survey its optical density value, then (for example: 4000 colony-forming units/milliliters) be diluted to required ultimate density.
2, bactericidin detects
2.1 serum dilutions to be checked
First in the every hole of microwell plate, add 25 μ l dilutions.With pipettor, from often ranked first, add 25 μ l through 56 ℃, the serum to be checked of 30min deactivation in hole, after pressure-vaccum 5～10 times, sucking-off 25 μ l, to next hole, so make continuous doubling dilution to last hole.
2.2 bactericidal reaction
In every hole, add 12.5 μ l complements, then add the bacteria suspension 12.5 μ l that diluted, on oscillator, middling speed vibration, after 5 minutes, in 37 ℃ of cultivations 25 minutes, then is vibrated 5 minutes.
After 2.3 sterilizations, colour developing is cultivated
Every hole adds 50 μ l has melted after the TTC agar that is chilled to 45 ℃ of left and right, and microwell plate is put 37 ℃ of conditions and cultivated observations after 15～20 hours.
3, result is judged
3.1 contrasts arrange
Positive reference serum contrast; 4 hole complement controls (respectively one of dilution, complement, bacterium liquid check that complement natural sterilization is active); 4 hole bacterial growth contrasts (each 12.5 μ l of dilution, deactivation complement and target bacterium bacterium liquid).During each detection, at least every 5 microwell plates, should there is one group of above-mentioned three kinds of control test.
3.2 count plate result of determination
3.2.1 bacteria suspension blank check
After each hole drips off bacteria suspension, by two one end that are added drop-wise to respectively an agar plate of this bacteria suspension, along two lines that pull, tilt dirty, in 37 ℃, cultivate clump count and the purity thereof of every bacteria suspension of inferior daily inspection with microwell plate simultaneously.
3.2.2 check the result that three kinds of contrasts arrange:
Should there is numerous red point-like petites in each hole with bacterial growth contrast of complement; Positive reference serum should reach original definite sterilization titre.If when red point-like bacterium colony number is less than significantly complement control hole or does not occur red point-like bacterium colony in trial each hole, be judged as the sterilization positive; If when the bacterium colony number in institute's prospect hole approaches complement control hole half or more, be judged as sterilization feminine gender.
3.2.3 sterilization result criterion
Visual inspection by people is also calculated bacterium colony number (count plate) one by one seriatim, how many according to red point-like clump count object, with symbol "+" log.When if complement and target bacteria growing contrast the bacterium normal growth in each hole, should there are numerous red point-like bacterium colonies, can be designated as " ++++".When trial each Kong Yuqi comparison, clump count reduces below 30%, is designated as " +++ "; Reduce by 50% left and right and be designated as " ++ "; Reduce by 70% left and right and be designated as "+"; Every hole is less than 10 bacterium colonies and is designated as " ± ".With bacterial growth, become to be "+" and following person is judged as the sterilization positive; With " ++ " and above person, be judged to sterilization feminine gender.Take and occur the highest titre that the high dilution of serum of the sterilization positive is bactericidin.
Therefore, existing serum antibody sterilizing power testing result is judged need count by visual inspection the mode of the count plate of bacterium colony, obtain the viable count of serum group to be checked and growth control group and compare, if serum group viable count to be checked reduces 70% with respect to growth control group, being judged to the positive.Because the visual field on microwell plate is poor, visual inspection counting bacterium colony error rate is high, often occurs leaking the situation of meter or repeat count, and resultant error is larger, and workload is large, very complicated.If bacteria suspension excessive concentration, bacterium colony is intensive growth lawn in the form of sheets in small hole, does not observe single bacterium colony, causes being judged sterilization testing result.
Summary of the invention
The technical problem to be solved in the present invention is to overcome existing serum antibody sterilizing power detection method to exist and need count bacterium colony by visual inspection, often there is leaking the situation of meter or repeat count, miscount rate is high, and resultant error is larger, and workload is large, the defect of very complicated.Its objective is that a kind of step of exploitation is simple, visual result is easy to differentiate, the detection method of the serum antibody dependence bactericidal activity of the reliable complement-mediated of decision method (Complement-Mediated Antibody-Dependent Bactericidal Activity Test, CMAB).
Method of the present invention is with antigen (vaccine or thalline) immunity test animal (comprising Strains of Mouse, rabbit, rhesus macaque) or healthy population, specific serum after adaptive immune, again specific serum antibody, complement (non-homogeneous with immune animal) and detection bacterium are mixed to rear reaction in order, select suitable viable bacteria developer to distinguish the activated thalline of tool and dead bacterium, what make that experimenter can quicklook judges test findings.
Technical scheme provided by the present invention is as follows:
1. a detection method for the serum antibody dependence bactericidal activity of complement-mediated, comprises the following steps:
(1) preparation of bacteria suspension and sample preparation
1. collecting cells
Recovery bacterial classification on Solid agar culture inclined-plane, is seeded to amplification cultivation in fluid nutrient medium and obtains bacterium liquid, determines the concentration of bacterium liquid according to turbidimetry, with physiological saline, bacterium liquid is diluted to working concentration and obtains bacteria suspension;
2. reagent is prepared
It is 0.1%～3% chromogenic reagent solution that developer is mixed with quality volume fraction, by chromogenic reagent solution filtration sterilization, in 2～8 ℃, keeps in Dark Place;
3. sample preparation
Complement is to be the serum of immune animal injecting normal saline with the nonhomologous animal blood serum of immune animal, negative serum, positive serum is the diagnostic serum of commercially available object bacteria, complement is diluted to 5～10 times, positive serum and negative serum are diluted to respectively former times～10 times, the complement of 5～10 times of dilutions is mixed to rear ice bath for the ratio of 1:1～1:2 by volume with bacteria suspension and within standing 2～30 minutes, obtain activating bacteria suspension;
(2) sterilization detects
1. application of sample dilution
Get in the sample well that serum to be checked adds to aseptic microwell plate, with physiological saline, carry out doubly dilution; In positive control sample hole, negative control sample well, add respectively the negative serum that is diluted to the positive serum of former times～10 times and is diluted to former times～10 times; Complement blank hole and bacterial activity control wells add respectively fluid nutrient medium, and each contrast repeats three holes;
2. bactericidal reaction
Except bacterial activity control wells, each Kong Jun of all the other in microwell plate adds 3. described activation bacteria suspension of step (1), in bacterial activity control wells, add 1. described bacteria suspension of step (1), then microwell plate is hatched 1～8 hour in 37 ℃ of conditions, in each hole of microwell plate, add the nutrient culture media of 37 ℃ of preheatings again, then hatch 1～12 hour;
(3) colour developing is judged
1. colour developing, adds after the chromogenic reagent solution that step (1) 2. prepared at each Kong Zhongjun of microwell plate, in 37 ℃, hatches colour developing in 0.5～6 hour; Or by microwell plate in 2～8 ℃ of standing over night, the chromogenic reagent solution that adds step (1) 2. to prepare at each Kong Zhongjun of microwell plate, hatches colour developing in 0.5～6 hour in 37 ℃;
2. judge
Colour developing or occur that tip-like is precipitated as negative findings, does not have bactericidal activity, does not develop the color or occurs the positive result of flaky precipitate having bactericidal activity; When the positive result of positive serum control group, when negative serum control group, complement blank group and bacterial activity control group are negative findings, serum to be checked develops the color or occurs that tip-like is precipitated as negative findings, does not have bactericidal activity; Serum to be checked does not develop the color or occurs the positive result of flaky precipitate, has bactericidal activity.
2. according to the detection method of the serum antibody dependence bactericidal activity of the complement-mediated described in technical scheme 1, it is characterized in that: step (1) bacterial classification that 1. collecting cells is recovered is Salmonella typhi, bacillus paratyphosus genus, Escherichia coli genus, hemolytic vibrio or shigella dysenteriae.
3. according to the detection method of the serum antibody dependence bactericidal activity of the complement-mediated described in technical scheme 2, it is characterized in that:
Step (1) is when 1. bacterial classification is Salmonella typhi in collecting cells, and described Solid agar culture and fluid nutrient medium are the buffered peptone water nutrient culture media in CNS GB4789.4-2010, and described working concentration is 10 * 10
5～10 * 10
8/ ml;
Step (1) the 2. developer described in reagent preparation is tetrazole purple or new tetrazole;
Step (2) 1. in application of sample dilution in every hole of sample well doubly the serum volume to be checked after dilution and step (2) 2. the volume ratio of the activation bacteria suspension volume described in bactericidal reaction be 2:1～4:1, and the serum volume to be checked after doubly diluting in every hole of sample well all equates, the amount of the positive serum that every hole in positive control hole adds, the amount of the negative serum that every hole of negative control hole adds, the amount of the fluid nutrient medium that the amount of the fluid nutrient medium that every hole in complement blank hole adds and every hole of bacterial activity control wells add all with every hole of sample well in the consumption equal-volume of the serum to be checked after dilution doubly, the fluid nutrient medium that complement blank hole and bacterial activity control wells add is respectively the buffered peptone water nutrient culture media in CNS GB4789.4-2010,
Step (2) 2. activates bacteria suspension volume described in bactericidal reaction be 20 μ l～40 μ l, the amount of the bacteria suspension adding in bacterial activity control wells and activation bacteria suspension equal-volume, the consumption of the nutrient culture media of 37 ℃ of preheatings that add in each hole of microwell plate is in sample well doubly doubly 2.5～3 times of the serum volume to be checked after dilution, and the nutrient culture media of 37 ℃ of described preheatings is the buffered peptone water nutrient culture media in CNS GB4789.4-2010;
Serum equal-volume to be checked after doubly diluting in the amount of the chromogenic reagent solution that 2. step (1) that step (3) adds in 1. developing the color in each hole of microwell plate prepared and sample well.
4. according to the detection method of the serum antibody dependence bactericidal activity of the complement-mediated described in technical scheme 2, it is characterized in that:
Step (1) 1. in collecting cells bacterial classification be bacillus paratyphosus while belonging to, described Solid agar culture and fluid nutrient medium are the buffered peptone water nutrient culture media in CNS GB4789.4-2010, described working concentration is 10 * 10
5～10 * 10
8/ ml;
Step (1) the 2. developer described in reagent preparation is tetrazole purple or new tetrazole;
Step (2) 1. in application of sample dilution in every hole of sample well doubly the serum volume to be checked after dilution and step (2) 2. the volume ratio of the activation bacteria suspension volume described in bactericidal reaction be 2:1～4:1, and the serum volume to be checked after doubly diluting in every hole of sample well all equates, the amount of the positive serum that every hole in positive control hole adds, the amount of the negative serum that every hole of negative control hole adds, the amount of the fluid nutrient medium that the amount of the fluid nutrient medium that every hole in complement blank hole adds and every hole of bacterial activity control wells add all with every hole of sample well in the consumption equal-volume of the serum to be checked after dilution doubly, the fluid nutrient medium that complement blank hole and bacterial activity control wells add is respectively the buffered peptone water nutrient culture media in CNS GB4789.4-2010,
Step (2) 2. activates bacteria suspension volume described in bactericidal reaction be 20 μ l～40 μ l, the amount of the bacteria suspension adding in bacterial activity control wells and activation bacteria suspension equal-volume, in every hole that the consumption of the nutrient culture media of 37 ℃ of preheatings that add in each hole of microwell plate is sample well doubly doubly 2.5～3 times of the serum volume to be checked after dilution, the nutrient culture media of 37 ℃ of described preheatings is the buffered peptone water nutrient culture media in CNS GB4789.4-2010;
Serum equal-volume to be checked after doubly diluting in the amount of the chromogenic reagent solution that 2. step (1) that step (3) adds in 1. developing the color in each hole of microwell plate prepared and sample well.
5. according to the detection method of the serum antibody dependence bactericidal activity of the complement-mediated described in technical scheme 2, it is characterized in that:
Step (1) 1. in collecting cells bacterial classification be Escherichia coli while belonging to, described Solid agar culture and fluid nutrient medium are the EC broth bouillon in CNS GB 4789.38-2012, described working concentration is 8 * 10
5/ ml～8 * 10
8/ ml;
Step (1) the 2. developer described in reagent preparation is tetrazole purple or new tetrazole;
Step (2) 1. in application of sample dilution in every hole of sample well doubly the serum volume to be checked after dilution and step (2) 2. the volume ratio of the activation bacteria suspension volume described in bactericidal reaction be 2:1～4:1, and the serum volume to be checked after doubly diluting in every hole of sample well all equates, the amount of the positive serum that every hole in positive control hole adds, the amount of the negative serum that every hole of negative control hole adds, the amount of the fluid nutrient medium that the amount of the fluid nutrient medium that every hole in complement blank hole adds and every hole of bacterial activity control wells add all with every hole of sample well in the consumption equal-volume of the serum to be checked after dilution doubly, the fluid nutrient medium that complement blank hole and bacterial activity control wells add is respectively the EC broth bouillon in CNS GB 4789.38-2012,
Step (2) 2. activates bacteria suspension volume described in bactericidal reaction be 20 μ l～40 μ l, the amount of the bacteria suspension adding in bacterial activity control wells and activation bacteria suspension equal-volume, in every hole that the consumption of the nutrient culture media of 37 ℃ of preheatings that add in each hole of microwell plate is sample well doubly doubly 2.5～3 times of the serum volume to be checked after dilution, the nutrient culture media of 37 ℃ of described preheatings is the EC broth bouillon in CNS GB 4789.38-2012;
Serum equal-volume to be checked after doubly diluting in the amount of the chromogenic reagent solution that 2. step (1) that step (3) adds in 1. developing the color in each hole of microwell plate prepared and sample well.
6. according to the detection method of the serum antibody dependence bactericidal activity of the complement-mediated described in technical scheme 2, it is characterized in that:
Step (1) is when 1. bacterial classification is hemolytic vibrio in collecting cells, described Solid agar culture and fluid nutrient medium are 3% sodium chloride tryptone soya broth in CNS GB 4789.7-2013, and described working concentration is 18 * 10
5/ ml～18 * 10
8/ ml;
Step (1) the 2. developer described in reagent preparation is that triphenyl tetrazolium chloride or tetrazole are purple;
Step (2) 1. in application of sample dilution in every hole of sample well doubly the serum volume to be checked after dilution and step (2) 2. the volume ratio of the activation bacteria suspension volume described in bactericidal reaction be 2:1～4:1, and the serum volume to be checked after doubly diluting in every hole of sample well all equates, the amount of the positive serum that every hole in positive control hole adds, the amount of the negative serum that every hole of negative control hole adds, the amount of the fluid nutrient medium that the amount of the fluid nutrient medium that every hole in complement blank hole adds and every hole of bacterial activity control wells add all with every hole of sample well in the consumption equal-volume of the serum to be checked after dilution doubly, the fluid nutrient medium that complement blank hole and bacterial activity control wells add is respectively 3% sodium chloride tryptone soya broth in CNS GB 4789.7-2013,
Step (2) 2. activates bacteria suspension volume described in bactericidal reaction be 20 μ l～40 μ l, the amount of the bacteria suspension adding in bacterial activity control wells and activation bacteria suspension equal-volume, in every hole that the consumption of the nutrient culture media of 37 ℃ of preheatings that add in each hole of microwell plate is sample well doubly doubly 2.5～3 times of the serum volume to be checked after dilution, the nutrient culture media of 37 ℃ of described preheatings is 3% sodium chloride tryptone soya broth in CNS GB 4789.7-2013;
Serum equal-volume to be checked after doubly diluting in the amount of the chromogenic reagent solution that 2. step (1) that step (3) adds in 1. developing the color in each hole of microwell plate prepared and sample well.
7. according to the detection method of the serum antibody dependence bactericidal activity of the complement-mediated described in technical scheme 2, it is characterized in that:
Step (1) is when 1. bacterial classification is shigella dysenteriae in collecting cells, and the Shigella that described Solid agar culture and fluid nutrient medium are in CNS GB4789.5-2012 increases bacterium broth bouillon, and described working concentration is 8 * 10
5/ ml～8 * 10
8/ ml;
Step (1) the 2. developer described in reagent preparation is tetrazole purple or new tetrazole;
Step (2) 1. in application of sample dilution in every hole of sample well doubly the serum volume to be checked after dilution and step (2) 2. the volume ratio of the activation bacteria suspension volume described in bactericidal reaction be 2:1～4:1, and the serum volume to be checked after doubly diluting in every hole of sample well all equates, the amount of the positive serum that every hole in positive control hole adds, the amount of the negative serum that every hole of negative control hole adds, the amount of the fluid nutrient medium that the amount of the fluid nutrient medium that every hole in complement blank hole adds and every hole of bacterial activity control wells add all with every hole of sample well in the consumption equal-volume of the serum to be checked after dilution doubly, the Shigella that the fluid nutrient medium that complement blank hole and bacterial activity control wells add is respectively in CNS GB4789.5-2012 increases bacterium broth bouillon,
Step (2) 2. activates bacteria suspension volume described in bactericidal reaction be 20 μ l～40 μ l, the amount of the bacteria suspension adding in bacterial activity control wells and activation bacteria suspension equal-volume, in every hole that the consumption of the nutrient culture media of 37 ℃ of preheatings that add in each hole of microwell plate is sample well doubly doubly 2.5～3 times of the serum volume to be checked after dilution, the nutrient culture media of 37 ℃ of described preheatings is that the Shigella in CNS GB4789.5-2012 increases bacterium broth bouillon;
Serum equal-volume to be checked after doubly diluting in the amount of the chromogenic reagent solution that 2. step (1) that step (3) adds in 1. developing the color in each hole of microwell plate prepared and sample well.
Compared with prior art, the beneficial effect of the inventive method is:
1, the inventive method has proposed a kind of new technical scheme, without prior art, adopt naked eyes to carry out one by one the method for count plate, only by developing the color or not developing the color and just can judge intuitively, fast whether serum to be checked has bactericidal activity, its detecting step is easy, quick, reliably accurate, with the naked eye count plate is often prone to Lou meter or repeat count, miscount rate is high to have overcome prior art, resultant error is larger, and workload is large, the defect of very complicated.
2, the inventive method has been set up positive control, negative control, complement blank hole, bacterial activity contrast at every microwell plate simultaneously, and take each contrast as due result as testing result establishment condition, testing result be take each contrast as checking, makes the testing result of the inventive method reliably accurate.
Accompanying drawing explanation
Fig. 1 is that the detection method of the serum antibody dependence bactericidal activity of complement-mediated of the present invention detects the rear result example of colour developing.
Embodiment
By following instance, further illustrate the present invention.The present invention is not limited to embodiment, and example is only for the present invention solves one of case.
Following embodiment is common method without specified otherwise.Vaccine used provides by Yunnan Watson Biotechnology Co., Ltd, and Ye Kexiang the said firm buys, the said firm address: university science and technology park, Ke Falu Yunnan Province, Kunming, Yunnan Province new and high-tech development zone North, postcode 650106.
Embodiment 1 PA combined vaccine immune serum bactericidin detects
The preparation of 1 bacteria suspension and sample preparation
1.1 collecting cells
Recovery PA bacterial strain on agar medium inclined-plane, cultivates 22 hours for 37 ℃, with the appropriate lawn of oese picking, is inoculated in fluid nutrient medium, and 37 ℃ are shaken and educate 18 hours, and bacterium liquid is diluted to 10 * 10 with physiological saline than turbid
7/ ml obtains bacteria suspension, and described Solid agar culture and fluid nutrient medium are the buffered peptone water nutrient culture media in CNS GB4789.4-2010.
1.2 reagent are prepared
Developer is mixed with the chromogenic reagent solution of 0.1% (W/V) with physiological saline, aseptic filtration, and 2～8 ℃ keep in Dark Place, and described developer is that tetrazole is purple.The sterilizing of 96 orifice plates: 96 orifice plates are put into 75% ethanol and soaked 15 minutes, irradiates 30 minutes (or 96 orifice plates of sterilizing of commodity in use) under uviol lamp.
1.3 sample preparation
Complement is healthy experiment guinea pig serum, with physiological saline, complement is done to 5 times of dilutions; Negative serum is the serum obtaining after healthy mice injecting normal saline, and positive serum is commercially available Salmonella O:2 factor diagnostic serum, and negative serum and positive serum are done 10 times of dilutions with physiological saline respectively.By the complement and 10 * 10 of 5 times of dilutions
7the bacteria suspension equal-volume of/ml mixes, and within ice bath standing 20 minutes, obtains activating bacteria suspension.
2 sterilizations detect
2.1 application of sample dilutions
1. get each blood serum sample 20 μ l to be checked and add to respectively 1
#～14
#in the 1st hole of group sample well, with 60 μ l physiological saline, do 4 times of dilutions, then from 1
#～14
#in the 1st hole of group sample well, get in the 40 rear serum to the to be checked of μ l dilution 2 holes, with 40 μ l physiological saline, do doubly dilution, so do and be doubly doubly diluted to the 6th hole (extension rate is in Table 1), serum volume to be checked after doubly diluting in the every hole of sample well is 40 μ l, and in the serum volume to be checked in the every hole of sample well after times dilution and step 2.2 bactericidal reaction, 1. the volume ratio of described activation bacteria suspension volume is 2:1.
2. positive control sample hole adds 10 times of dilution positive serum 40 μ l, repeats three holes; Negative control sample well adds 10 times of dilution negative serum 40 μ l, repeats three holes; Complement blank hole adds fluid nutrient medium 40 μ l, repeats three holes; Bacterial activity control wells adds 40 μ l fluid nutrient mediums, repeats three holes.The fluid nutrient medium that complement blank hole and bacterial activity control wells add is the buffered peptone water nutrient culture media in standard GB/T 4789.4-2010.
2.2 bactericidal reaction
1. except bacterial activity control wells, each hole of all the other of 96 orifice plates add respectively each 20 μ l of activation bacteria suspension described in step 1.3 (with the serum volume ratio to be checked after dilution be doubly 1:2).Every hole of bacterial activity control wells adds 5 * 10
7the bacteria suspension of/ml (not containing complement) 20 μ l.Application of sample and extension rate are in Table 1.
2. 96 orifice plates are hatched 2 hours in 37 ℃ of conditions, then in each hole of 96 orifice plates, add the nutrient culture media 100 μ l (be in the every hole of sample well doubly doubly 2.5 times of the serum volume 40 μ l to be checked after dilution) of 37 ℃ of preheatings, then hatch 3 hours.The nutrient culture media of 37 ℃ of described preheatings is the buffered peptone water nutrient culture media in CNS GB4789.4-2010.
Table 1. serum samples diluted table
3 colour developing result of determination
In every hole of 96 orifice plates, add the chromogenic reagent solution 40 μ l of 0.1% (W/V) described in the present embodiment 1.2, hatch colour developing in 1 hour for 37 ℃.
Colour developing is judged and interpretation of result: the PA combined vaccine of three batches is immune 3 groups of healthy mices respectively, and 40/group, after collection immunity, the 35th day mice serum carries out CMAB detection.Control group result is set up, i.e. the positive result of positive serum control group, and negative serum control group, complement blank group and bacterial activity control group are negative findings, and testing result is reliable, and serum antibody sterilization titre to be checked is up to 1:128.The positive rate of rotation of serum to be checked under different extension rates is in Table 2.
Three batches of PA combined vaccine CMAB testing results of table 2.
A. positive rate of rotation (%)=[positive findings number/gross sample number] * 100%, gross sample number is 40.
Embodiment 2 paratyphoid B combined vaccine immune serum bactericidins detect
The preparation of 1 bacteria suspension and sample preparation
1.1 collecting cells
Recovery paratyphoid B bacterial strain on agar medium inclined-plane, cultivates 22 hours for 37 ℃.With the appropriate lawn of oese picking, be inoculated in fluid nutrient medium, 37 ℃ are shaken and educate 18 hours.Bacterium liquid is diluted to 10 * 10 with physiological saline than turbid
7/ ml obtains bacteria suspension, and described Solid agar culture and fluid nutrient medium are the buffered peptone water nutrient culture media in CNS GB4789.4-2010.
1.2 reagent are prepared
Developer is mixed with the chromogenic reagent solution of 0.1% (W/V), aseptic filtration with physiological saline.2～8 ℃ keep in Dark Place.The sterilizing of 96 orifice plates: 96 orifice plates are put into 75% ethanol and soaked 15 minutes, irradiates 30 minutes (or 96 orifice plates of sterilizing of commodity in use) under uviol lamp.Described developer is new tetrazole.
1.3 sample preparation
Complement is healthy experiment guinea pig serum, with physiological saline, complement is done to 5 times of dilutions; Negative serum is the serum obtaining after healthy mice injecting normal saline, and positive serum is commercially available Salmonella O:4 diagnostic serum, and negative serum and positive serum are done 10 times of dilutions with physiological saline respectively.By the complement of 5 times of dilutions and 10 * 10
7the bacteria suspension equal-volume of/ml mixes, and within ice bath standing 20 minutes, obtains activation bacteria suspension.
2 sterilizations detect
2.1 application of sample dilutions
1. get each blood serum sample 20 μ l to be checked and add to respectively 1
#～14
#in the 1st hole of group sample well, with 60 μ l physiological saline, do 4 times of dilutions, then from 1
#～14
#in the 1st hole of group sample well, get in the 40 rear serum to the to be checked of μ l dilution 2 holes, with 40 μ l physiological saline, do doubly dilution, so do and be doubly doubly diluted to the 6th hole (extension rate is in Table 1), serum volume to be checked after doubly diluting in the every hole of sample well is 40 μ l, and in the serum volume to be checked in the every hole of sample well after times dilution and step 2.2 bactericidal reaction, 1. the volume ratio of described activation bacteria suspension volume is 2:1.
2. positive control sample hole adds 10 times of dilution positive serum 40 μ l, repeats three holes; Negative control sample well adds 10 times of dilution negative serum 40 μ l, repeats three holes; Complement blank hole adds fluid nutrient medium 40 μ l, repeats three holes; Bacterial activity control wells adds 40 μ l fluid nutrient mediums, repeats three holes.The fluid nutrient medium that complement blank hole and bacterial activity control wells add is respectively the buffered peptone water nutrient culture media in CNS GB4789.4-2010.
2.2 bactericidal reaction
1. except bacterial activity control wells, each hole of all the other of 96 orifice plates add respectively each 20 μ l of activation bacteria suspension (with the serum volume ratio to be checked after dilution be doubly 1:2).Every hole of bacterial activity control wells adds 5 * 10
7each 20 μ l of the bacterium liquid of/ml (not containing complement).Application of sample and extension rate are in Table 1.
2. 96 orifice plates are hatched 2 hours in 37 ℃ of conditions.In each hole of 96 orifice plates, add again the nutrient culture media 100 μ l of 37 ℃ of preheatings, then hatch 3 hours.The nutrient culture media of 37 ℃ of described preheatings is the buffered peptone water nutrient culture media in CNS GB4789.4-2010.
3 colour developing result of determination
In every hole of 96 orifice plates, add the chromogenic reagent solution 40 μ l of 0.1% (W/V) described in the present embodiment 1.2, hatch colour developing in 1 hour for 37 ℃.
Colour developing is judged and interpretation of result: we have prepared 40 healthy mices of paratyphoid B combined vaccine immunity of batch, and after collection immunity, the 35th day mice serum carries out CMAB detection.Control group result is set up, i.e. the positive result of positive serum control group, and negative serum control group, complement blank group and bacterial activity control group are negative findings, and testing result is reliable, and serum antibody sterilization titre to be checked is up to 1:128.The positive rate of rotation of serum to be checked under different extension rates is in Table 3.
Table 3. paratyphoid B combined vaccine CMAB testing result
A. positive rate of rotation (%)=[positive findings number/gross sample number] * 100%, gross sample number is 40.
Embodiment 3 typhoid Vi polysaccharide combined vaccine immune serum bactericidins detect
The preparation of 1 bacteria suspension and sample preparation
1.1 collecting cells
Recovery Salmonella typhi bacterial strain on agar medium inclined-plane, cultivates 22 hours for 37 ℃, with the appropriate lawn of oese picking, is inoculated in fluid nutrient medium, and 37 ℃ are shaken and educate 18 hours, and bacterium liquid is diluted to 10 * 10 with physiological saline than turbid
5/ ml obtains bacteria suspension, and described Solid agar culture and fluid nutrient medium are the buffered peptone water nutrient culture media in standard GB/T 4789.4-2010.
1.2 reagent are prepared
Developer is mixed with the chromogenic reagent solution of 0.1% (W/V) with physiological saline, aseptic filtration, and 2～8 ℃ keep in Dark Place.The sterilizing of 96 orifice plates: 96 orifice plates are put into 75% ethanol and soaked 15 minutes, irradiates 30 minutes (or 96 orifice plates of sterilizing of commodity in use) under uviol lamp.Described developer is that tetrazole is purple.
1.3 sample preparation
Complement is healthy experiment guinea pig serum, with physiological saline, complement is done to 5 times of dilutions; Negative serum is the serum obtaining after healthy mice injecting normal saline, and positive serum is commercially available Salmonella Vi diagnostic serum, and negative serum and positive serum are done 10 times of dilutions with physiological saline respectively.By the complement of 5 times of dilutions and 10 * 10
5the bacteria suspension equal-volume of/ml mixes, and within ice bath standing 20 minutes, obtains activating bacteria suspension.
2 sterilizations detect
2.1 application of sample dilutions
1. get each blood serum sample 40 μ l to be checked and add to respectively 1
#～14
#in the 1st hole of group sample well, with 40 μ l physiological saline, do 2 times of dilutions, then from 1
#～14
#in the 1st hole of group sample well, get in the 40 rear serum to the to be checked of μ l dilution 2 holes, with 40 μ l physiological saline, do doubly dilution, so do and be doubly doubly diluted to the 6th hole (extension rate is in Table 4), the serum volume to be checked after doubly diluting in the every hole of sample well is 40 μ l.In serum volume to be checked after doubly diluting in the every hole of sample well and step 2.2 bactericidal reaction, 1. the volume ratio of described activation bacteria suspension volume is 2:1.
2. positive control sample hole adds 10 times of dilution positive serum 40 μ l, repeats three holes; Negative control sample well adds 10 times of dilution negative serum 40 μ l, repeats three holes; Complement blank hole adds fluid nutrient medium 40 μ l, repeats three holes; Bacterial activity control wells adds 40 μ l fluid nutrient mediums, repeats three holes.The fluid nutrient medium that complement blank hole and bacterial activity control wells add is the buffered peptone water nutrient culture media in CNS GB4789.4-2010.
2.2 bactericidal reaction
1. except bacterial activity control wells, all the other 93 holes of 96 orifice plates add respectively each 20 μ l of activation bacteria suspension (with the serum volume ratio to be checked after dilution be doubly 1:2).Every hole of bacterial activity control wells adds 5 * 10
5each 20 μ l of the bacteria suspension of/ml (not containing complement).Application of sample and extension rate are in Table 4.
2. 96 orifice plates are hatched 2 hours in 37 ℃ of conditions.In each hole of 96 orifice plates, add again the nutrient culture media 100 μ l of 37 ℃ of preheatings, then hatch 3 hours.2～8 ℃ of standing over night.The nutrient culture media of 37 ℃ of described preheatings is the buffered peptone water nutrient culture media in Chinese Home standard GB4789.4-2010.
Table 4. serum samples diluted table
3 colour developing result of determination
Take out 96 orifice plates, in every hole of 96 orifice plates, add the chromogenic reagent solution 40 μ l of 0.1% (W/V) described in the present embodiment 1.2, hatch 1 hour observations for 37 ℃.
Colour developing is judged and interpretation of result: we have prepared the immune 3 groups of healthy mices of typhoid Vi polysaccharide combined vaccine difference of three batches, and 40/group, after collection immunity, the 35th day mice serum carries out CMAB detection.Control group result is set up, i.e. the positive result of positive serum control group, and negative serum control group, complement blank group and bacterial activity control group are negative findings, and testing result is reliable, and serum antibody sterilization titre to be checked is up to 1:64.The positive rate of rotation of serum to be checked under different extension rates is in Table 5.
Three batches of typhoid Vi polysaccharide combined vaccine CMAB testing results of table 5.
A. positive rate of rotation (%)=[positive findings number/gross sample number] * 100%, gross sample number is 40.
Embodiment 4 Escherichia coli immunity Chinchilla rabbit whole cells surpass exempts from serum sterilizing antibody test
The preparation of 1 bacteria suspension and sample preparation
1.1 collecting cells
Recovery Escherichia coli bacterial strain on agar medium inclined-plane, cultivates 18 hours for 37 ℃, with the appropriate lawn of oese picking, is inoculated in fluid nutrient medium, and 37 ℃ are shaken and educate 15 hours.Bacterium liquid is diluted to 8 * 10 with physiological saline than turbid
5/ ml obtains bacteria suspension, and described Solid agar culture and fluid nutrient medium are the EC broth bouillon in CNS GB 4789.38-2012.
1.2 reagent are prepared
Developer is mixed with the chromogenic reagent solution of 1% (W/V) with physiological saline, aseptic filtration, and 2～8 ℃ keep in Dark Place.The sterilizing of 96 orifice plates: 96 orifice plates are put into 75% ethanol and soaked 15 minutes, irradiates 30 minutes (or 96 orifice plates of sterilizing of commodity in use) under uviol lamp.Described developer is for being tetrazole purple.
1.3 sample preparation
Complement is healthy experiment guinea pig serum, with physiological saline, complement is done to 5 times of dilutions; Negative serum is the serum obtaining after healthy Chinchilla rabbit injecting normal saline, and positive serum is commercially available Escherichia coli diagnostic serum, and negative serum and positive serum are done 10 times of dilutions with physiological saline respectively.By the complement of 5 times of dilutions and 8 * 10
5the bacteria suspension equal-volume of/ml mixes, and within ice bath standing 20 minutes, obtains activating bacteria suspension.
2 sterilizations detect
2.1 application of sample dilutions
1. get each blood serum sample 20 μ l to be checked and add to respectively 1
#～7
#in the 1st hole of group sample well, with 60 μ l physiological saline, do 4 times of dilutions, then from 1
#～7
#in the 1st hole of group sample well, get in the 40 rear serum to the to be checked of μ l dilution 2 holes, with 40 μ l physiological saline, do doubly dilution, so do and be doubly doubly diluted to the 12nd hole (extension rate is in Table 6).In serum volume to be checked after doubly diluting in the every hole of sample well and step 2.2 bactericidal reaction, 1. the volume ratio of described activation bacteria suspension volume is 2:1.
2. positive control hole adds 10 times of dilution positive serum 40 μ l, repeats three holes; Negative control hole adds 10 times of dilution negative serum 40 μ l, repeats three holes; Complement blank hole adds fluid nutrient medium 40 μ l, repeats three holes; Bacterial activity control wells adds 40 μ l fluid nutrient mediums, repeats three holes.The fluid nutrient medium that complement blank hole and bacterial activity control wells add is the EC broth bouillon in CNS GB 4789.38-2012.
2.2 bactericidal reaction
1. except bacterial activity control wells, each hole of all the other of 96 orifice plates add respectively activate described in step 1.3 each 20 μ l of bacteria suspension (with the serum volume ratio to be checked after dilution be doubly 1:2).Every hole of bacterial activity control wells adds 4 * 10
5each 20 μ l of the bacteria suspension of/ml (not containing complement).Application of sample and extension rate are in Table 6.
2. 96 orifice plates are hatched 4 hours in 37 ℃ of conditions, then in each hole of 96 orifice plates, add the nutrient culture media 100 μ l of 37 ℃ of preheatings, then hatch 1.5 hours.The nutrient culture media of 37 ℃ of described preheatings is the EC broth bouillon in standard GB/T 4789.38-2012.
Table 6. serum samples diluted table
3 colour developing result of determination
Take out 96 orifice plates, in every hole of 96 orifice plates, add the chromogenic reagent solution 40 μ l of 1% (W/V) described in the present embodiment 1.2, hatch 0.5 hour observations for 37 ℃.
Colour developing is judged and interpretation of result: we by the Escherichia coli after deactivation with physiological saline than the turbid suitable concentration that is diluted to, 7 healthy Chinchilla rabbits of injecting immune, obtain the super serum of exempting from of Escherichia coli.To Chinchilla rabbit is super, exempts from serum and carry out CMAB detection.Control group result is set up, it is the positive result of positive serum control group, negative serum control group, complement blank group and bacterial activity control group are negative findings, testing result is reliable, serum antibody sterilization titre to be checked is up to 1:8192, and in 1:8 extension rate Xiayang, rate of rotation reaches 100%, illustrates that super the exempting from of Escherichia coli Chinchilla rabbit that we prepare has the uncommon bacteria antibody of Chinese People's Anti-Japanese Military and Political College's intestines dust in serum, antibody sterilization titre is high, can be used as self-control detection and uses with the super serum of exempting from.
Embodiment 5 shigella dysenteriae immunity Chinchilla rabbit whole cells surpass exempts from serum sterilizing antibody test
The preparation of 1 bacteria suspension and sample preparation
1.1 collecting cells
Recovery shigella dysenteriae bacterial strain on agar medium inclined-plane, cultivates 18～22 hours for 37 ℃, with the appropriate lawn of oese picking, is inoculated in fluid nutrient medium, and 37 ℃ are shaken and educate 8～10 hours, and bacterium liquid is diluted to 8 * 10 with physiological saline than turbid
5/ ml obtains bacteria suspension, and the Shigella that described Solid agar culture and fluid nutrient medium are in CNS GB4789.5-2012 increases bacterium broth bouillon.
1.2 reagent are prepared
Developer is mixed with the chromogenic reagent solution of 2% (W/V) with physiological saline, aseptic filtration, and 2～8 ℃ keep in Dark Place.Commercial 96 orifice plates of sterilizing are put in superclean bench external packing is carried out to ultraviolet sterilization.Described developer is new tetrazole.
1.3 sample preparation
Complement is healthy experiment guinea pig serum, with physiological saline, complement is done to 5 times of dilutions; Negative serum is the serum obtaining after healthy Chinchilla rabbit injecting normal saline, and positive serum is commercially available Shigella diagnostic serum, and negative serum and positive serum are done 10 times of dilutions with physiological saline respectively.By the complement of 5 times of dilutions and 8 * 10
5the bacteria suspension equal-volume of/ml mixes, and within ice bath standing 30 minutes, obtains activating bacteria suspension.
2 sterilizations detect
2.1 application of sample dilutions
1. get each blood serum sample 80 μ l to be checked and add to respectively 1
#～7
#in the 1st hole of group sample well, with 80 μ l physiological saline, do 2 times of dilutions, then from 1
#～7
#in the 1st hole of group sample well, get in the 80 rear serum to the to be checked of μ l dilution 2 holes, with 80 μ l physiological saline, do doubly dilution, so do and be doubly doubly diluted to the 12nd hole (extension rate is in Table 7), the serum volume to be checked after doubly diluting in the every hole of sample well is 80 μ l.In serum volume to be checked after doubly diluting in the every hole of sample well and step 2.2 bactericidal reaction, 1. the volume ratio of described activation bacteria suspension volume is 4:1.
2. positive control hole adds 10 times of dilution positive serum 80 μ l, repeats three holes; Negative control hole adds 10 times of dilution negative serum 80 μ l, repeats three holes; Complement blank hole adds fluid nutrient medium 80 μ l, repeats three holes; Bacterial activity control wells adds 80 μ l fluid nutrient mediums, repeats three holes.The Shigella that the fluid nutrient medium that complement blank hole and bacterial activity control wells add is in CNS GB4789.5-2012 increases bacterium broth bouillon.
2.2 bactericidal reaction
1. except bacterial activity control wells, each hole of all the other of 96 orifice plates add respectively each 20 μ l of activation bacteria suspension described in step 1.3 (with the serum volume ratio to be checked after dilution be doubly 1:4).Every hole of bacterial activity control wells adds 4 * 10
5each 20 μ l of the bacteria suspension of/ml (not containing complement).Application of sample and extension rate are in Table 7.
2. 96 orifice plates are hatched 6 hours in 37 ℃ of conditions.In each hole of 96 orifice plates, add again the nutrient culture media 60 μ l of 37 ℃ of preheatings, hatch 3 hours.The nutrient culture media of 37 ℃ of described preheatings is that the Shigella in CNS GB4789.5-2012 increases bacterium broth bouillon.
Table 7. application of sample and extension rate
3 colour developing result of determination
Take out 96 orifice plates, in every hole of 96 orifice plates, add the chromogenic reagent solution 40 μ l of 2% (W/V) described in the present embodiment 1.2, hatch 2 hours observations for 37 ℃.
Colour developing is judged and interpretation of result: we by the Shigella dysenteriae after deactivation with physiological saline than the turbid suitable concentration that is diluted to, 7 healthy Chinchilla rabbits of injecting immune, obtain the super serum of exempting from of Shigella dysenteriae.To Chinchilla rabbit is super, exempts from serum and carry out CMAB detection, control group result is set up, it is the positive result of positive serum control group, negative serum control group, complement blank group and bacterial activity control group are negative findings, testing result is reliable, serum antibody sterilization titre to be checked is up to 1:4096, in 1:8 extension rate Xiayang, rate of rotation reaches 100%, illustrate that super the exempting from of Shigella dysenteriae Chinchilla rabbit that we prepare has anti-Shigella dysenteriae antibody in serum, antibody sterilization titre is high, can be used as self-control detection and uses with the super serum of exempting from.
Embodiment 6 hemolytic vibrios immunity Chinchilla rabbit whole cells surpass exempts from serum sterilizing antibody test
The preparation of 1 bacteria suspension and sample preparation
1.1 collecting cells
Recovery hemolytic vibrios on agar medium inclined-plane, cultivates 18～24 hours for 37 ℃, with the appropriate lawn of oese picking, is inoculated in fluid nutrient medium, and 37 ℃ are shaken and educate 8～10 hours, and bacterium liquid is diluted to 18 * 10 with physiological saline than turbid
5/ ml obtains bacteria suspension, and described Solid agar culture and fluid nutrient medium are 3% sodium chloride tryptone soya broth in standard GB/T 4789.7-2013,
1.2 reagent are prepared
Developer is mixed with the chromogenic reagent solution of 2% (W/V), aseptic filtration with physiological saline.2～8 ℃ keep in Dark Place.Commercial 96 orifice plates of sterilizing are put in superclean bench external packing is carried out to ultraviolet sterilization.Described developer is triphenyl tetrazolium chloride.
1.3 sample preparation
Complement is healthy experiment guinea pig serum, with physiological saline, complement is done to 5 times of dilutions; Negative serum is the serum obtaining after healthy Chinchilla rabbit injecting normal saline, and positive serum is commercially available vibrio parahaemolytious diagnostic serum, and negative serum and positive serum are done 10 times of dilutions with physiological saline respectively.By the complement of 5 times of dilutions and 18 * 10
5the bacteria suspension equal-volume of/ml mixes, and within ice bath standing 30 minutes, obtains activating bacteria suspension.
2 sterilizations detect
2.1 application of sample dilutions
1. get each blood serum sample 160 μ l to be checked and add to respectively 1
#～7
#in the 1st hole of group sample well, then from 1
#～7
#in the 1st hole of group sample well, get in 80 μ l serum to the to be checked, 2 holes, with 80 μ l physiological saline, do doubly dilution, so do and be doubly doubly diluted to the 12nd hole (extension rate is in Table 8), the serum volume to be checked after doubly diluting in every hole, test specimen hole is 80 μ l.In serum volume to be checked after doubly diluting in the every hole of sample well and step 2.2 bactericidal reaction, 1. the volume ratio of described activation bacteria suspension volume is 4:1.
2. positive control hole adds 10 times of dilution positive serum 40 μ l, repeats three holes; Negative control hole adds 10 times of dilution negative serum 80 μ l, repeats three holes; Complement blank hole adds fluid nutrient medium 80 μ l, repeats three holes; Bacterial activity control wells adds 80 μ l fluid nutrient mediums, repeats three holes.The fluid nutrient medium that complement blank hole and bacterial activity control wells add is 3% sodium chloride tryptone soya broth in CNS GB 4789.7-2013.
2.2 bactericidal reaction
1. except bacterial activity control wells, each hole of all the other of 96 orifice plates add respectively each 20 μ l of activation bacteria suspension (with the serum volume ratio to be checked after dilution be doubly 1:4).Every hole of bacterial activity control wells adds 9 * 10
5each 20 μ l of the bacteria suspension of/ml (not containing complement).Application of sample and extension rate are in Table 8.
2. 96 orifice plates are hatched 6 hours in 37 ℃ of conditions, then in each hole of 96 orifice plates, add the nutrient culture media 60 μ l of 37 ℃ of preheatings, then hatch 3 hours.The nutrient culture media of 37 ℃ of described preheatings is 3% sodium chloride tryptone soya broth in CNS GB4789.7-2013.
Table 8. application of sample and extension rate
3 colour developing result of determination
Take out 96 orifice plates, in every hole of 96 orifice plates, add the chromogenic reagent solution 40 μ l of 2% (W/V) described in the present embodiment 1.2, hatch 2 hours observations for 37 ℃.
Colour developing is judged and interpretation of result: we by the hemolytic vibrios after deactivation with physiological saline than the turbid suitable concentration that is diluted to, 7 healthy Chinchilla rabbits of injecting immune, obtain the super serum of exempting from of hemolytic vibrios.To Chinchilla rabbit is super, exempts from serum and carry out CMAB detection, control group result is set up, it is the positive result of positive serum control group, negative serum control group, complement blank group and bacterial activity control group are negative findings, testing result is reliable, serum antibody sterilization titre to be checked is up to 1:2048, in 1:4 extension rate Xiayang, rate of rotation reaches 85.7%, illustrate that super the exempting from of hemolytic vibrios Chinchilla rabbit that we prepare has hemolytic vibrios antibody in serum, antibody sterilization titre is high, can be used as self-control detection and uses with the super serum of exempting from.
Claims (7)
1. a detection method for the serum antibody dependence bactericidal activity of complement-mediated, comprises the following steps:
(1) preparation of bacteria suspension and sample preparation
1. collecting cells
Recovery bacterial classification on Solid agar culture inclined-plane, is seeded to amplification cultivation in fluid nutrient medium and obtains bacterium liquid, determines the working concentration of bacterium liquid according to turbidimetry, with physiological saline, bacterium liquid is diluted to working concentration and obtains bacteria suspension;
2. reagent is prepared
It is 0.1%～3% chromogenic reagent solution that developer is mixed with quality volume fraction, by chromogenic reagent solution filtration sterilization, in 2～8 ℃, keeps in Dark Place;
3. sample preparation
Complement is to be the serum of immune animal injecting normal saline with the nonhomologous animal blood serum of immune animal, negative serum, positive serum is the diagnostic serum of commercially available object bacteria, complement is diluted to 5～10 times, positive serum and negative serum are diluted to respectively former times～10 times, the complement of 5～10 times of dilutions is mixed to rear ice bath for the ratio of 1:1～1:2 by volume with bacteria suspension and within standing 2～30 minutes, obtain activating bacteria suspension;
(2) sterilization detects
1. application of sample dilution
Get in the sample well that serum to be checked adds to aseptic microwell plate, with physiological saline, carry out doubly dilution; In positive control sample hole, negative control sample well, add respectively the negative serum that is diluted to the positive serum of former times～10 times and is diluted to former times～10 times; Complement blank hole and bacterial activity control wells add respectively fluid nutrient medium, and each contrast repeats three holes;
2. bactericidal reaction
Except bacterial activity control wells, each Kong Jun of all the other in microwell plate adds 3. described activation bacteria suspension of step (1), in bacterial activity control wells, add 1. described bacteria suspension of step (1), then microwell plate is hatched 1～8 hour in 37 ℃ of conditions, in each hole of microwell plate, add the nutrient culture media of 37 ℃ of preheatings again, then hatch 1～12 hour;
(3) colour developing is judged
1. colour developing, adds after the chromogenic reagent solution that step (1) 2. prepared at each Kong Zhongjun of microwell plate, in 37 ℃, hatches colour developing in 0.5～6 hour; Or by microwell plate in 2～8 ℃ of standing over night, the chromogenic reagent solution that adds step (1) 2. to prepare at each Kong Zhongjun of microwell plate, hatches colour developing in 0.5～6 hour in 37 ℃;
2. judge
Colour developing or occur that tip-like is precipitated as negative findings, does not have bactericidal activity, does not develop the color or occurs the positive result of flaky precipitate having bactericidal activity; When the positive result of positive serum control group, when negative serum control group, complement blank group and bacterial activity control group are negative findings, serum to be checked develops the color or occurs that tip-like is precipitated as negative findings, does not have bactericidal activity; Serum to be checked does not develop the color or occurs the positive result of flaky precipitate, has bactericidal activity.
2. the detection method of the serum antibody dependence bactericidal activity of complement-mediated according to claim 1, is characterized in that: step (1) bacterial classification that 1. collecting cells is recovered is Salmonella typhi, bacillus paratyphosus genus, Escherichia coli genus, hemolytic vibrio or shigella dysenteriae.
3. the detection method of the serum antibody dependence bactericidal activity of complement-mediated according to claim 2, is characterized in that:
Step (1) is when 1. bacterial classification is Salmonella typhi in collecting cells, and described Solid agar culture and fluid nutrient medium are the buffered peptone water nutrient culture media in CNS GB4789.4-2010, and described working concentration is 10 * 10
5～10 * 10
8/ ml;
Step (1) the 2. developer described in reagent preparation is tetrazole purple or new tetrazole;
Step (2) 1. in application of sample dilution in every hole of sample well doubly the serum volume to be checked after dilution and step (2) 2. the volume ratio of the activation bacteria suspension volume described in bactericidal reaction be 2:1～4:1, and the serum volume to be checked after doubly diluting in every hole of sample well all equates, the amount of the positive serum that every hole in positive control hole adds, the amount of the negative serum that every hole of negative control hole adds, the amount of the fluid nutrient medium that the amount of the fluid nutrient medium that every hole in complement blank hole adds and every hole of bacterial activity control wells add all with every hole of sample well in the consumption equal-volume of the serum to be checked after dilution doubly, the fluid nutrient medium that complement blank hole and bacterial activity control wells add is respectively the buffered peptone water nutrient culture media in CNS GB4789.4-2010,
Step (2) 2. activates bacteria suspension volume described in bactericidal reaction be 20 μ l～40 μ l, the amount of the bacteria suspension adding in bacterial activity control wells and activation bacteria suspension equal-volume, the consumption of the nutrient culture media of 37 ℃ of preheatings that add in each hole of microwell plate is in sample well doubly doubly 2.5～3 times of the serum volume to be checked after dilution, and the nutrient culture media of 37 ℃ of described preheatings is the buffered peptone water nutrient culture media in CNS GB4789.4-2010;
Serum equal-volume to be checked after doubly diluting in the amount of the chromogenic reagent solution that 2. step (1) that step (3) adds in 1. developing the color in each hole of microwell plate prepared and sample well.
4. the detection method of the serum antibody dependence bactericidal activity of complement-mediated according to claim 2, is characterized in that:
Step (1) 1. in collecting cells bacterial classification be bacillus paratyphosus while belonging to, described Solid agar culture and fluid nutrient medium are the buffered peptone water nutrient culture media in CNS GB4789.4-2010, described working concentration is 10 * 10
5～10 * 10
8/ ml;
Step (1) the 2. developer described in reagent preparation is tetrazole purple or new tetrazole;
Step (2) 1. in application of sample dilution in every hole of sample well doubly the serum volume to be checked after dilution and step (2) 2. the volume ratio of the activation bacteria suspension volume described in bactericidal reaction be 2:1～4:1, and the serum volume to be checked after doubly diluting in every hole of sample well all equates, the amount of the positive serum that every hole in positive control hole adds, the amount of the negative serum that every hole of negative control hole adds, the amount of the fluid nutrient medium that the amount of the fluid nutrient medium that every hole in complement blank hole adds and every hole of bacterial activity control wells add all with every hole of sample well in the consumption equal-volume of the serum to be checked after dilution doubly, the fluid nutrient medium that complement blank hole and bacterial activity control wells add is respectively the buffered peptone water nutrient culture media in CNS GB4789.4-2010,
Step (2) 2. activates bacteria suspension volume described in bactericidal reaction be 20 μ l～40 μ l, the amount of the bacteria suspension adding in bacterial activity control wells and activation bacteria suspension equal-volume, in every hole that the consumption of the nutrient culture media of 37 ℃ of preheatings that add in each hole of microwell plate is sample well doubly doubly 2.5～3 times of the serum volume to be checked after dilution, the nutrient culture media of 37 ℃ of described preheatings is the buffered peptone water nutrient culture media in CNS GB4789.4-2010;
Serum equal-volume to be checked after doubly diluting in the amount of the chromogenic reagent solution that 2. step (1) that step (3) adds in 1. developing the color in each hole of microwell plate prepared and sample well.
5. the detection method of the serum antibody dependence bactericidal activity of complement-mediated according to claim 2, is characterized in that:
Step (1) 1. in collecting cells bacterial classification be Escherichia coli while belonging to, described Solid agar culture and fluid nutrient medium are the EC broth bouillon in CNS GB 4789.38-2012, described working concentration is 8 * 10
5/ ml～8 * 10
8/ ml;
Step (1) the 2. developer described in reagent preparation is tetrazole purple or new tetrazole;
Step (2) 1. in application of sample dilution in every hole of sample well doubly the serum volume to be checked after dilution and step (2) 2. the volume ratio of the activation bacteria suspension volume described in bactericidal reaction be 2:1～4:1, and the serum volume to be checked after doubly diluting in every hole of sample well all equates, the amount of the positive serum that every hole in positive control hole adds, the amount of the negative serum that every hole of negative control hole adds, the amount of the fluid nutrient medium that the amount of the fluid nutrient medium that every hole in complement blank hole adds and every hole of bacterial activity control wells add all with every hole of sample well in the consumption equal-volume of the serum to be checked after dilution doubly, the fluid nutrient medium that complement blank hole and bacterial activity control wells add is respectively the EC broth bouillon in CNS GB 4789.38-2012,
Step (2) 2. activates bacteria suspension volume described in bactericidal reaction be 20 μ l～40 μ l, the amount of the bacteria suspension adding in bacterial activity control wells and activation bacteria suspension equal-volume, in every hole that the consumption of the nutrient culture media of 37 ℃ of preheatings that add in each hole of microwell plate is sample well doubly doubly 2.5～3 times of the serum volume to be checked after dilution, the nutrient culture media of 37 ℃ of described preheatings is the EC broth bouillon in CNS GB 4789.38-2012;
Serum equal-volume to be checked after doubly diluting in the amount of the chromogenic reagent solution that 2. step (1) that step (3) adds in 1. developing the color in each hole of microwell plate prepared and sample well.
6. the detection method of the serum antibody dependence bactericidal activity of complement-mediated according to claim 2, is characterized in that:
Step (1) is when 1. bacterial classification is hemolytic vibrio in collecting cells, described Solid agar culture and fluid nutrient medium are 3% sodium chloride tryptone soya broth in CNS GB 4789.7-2013, and described working concentration is 18 * 10
5/ ml～18 * 10
8/ ml;
Step (1) the 2. developer described in reagent preparation is that triphenyl tetrazolium chloride or tetrazole are purple;
Step (2) 1. in application of sample dilution in every hole of sample well doubly the serum volume to be checked after dilution and step (2) 2. the volume ratio of the activation bacteria suspension volume described in bactericidal reaction be 2:1～4:1, and the serum volume to be checked after doubly diluting in every hole of sample well all equates, the amount of the positive serum that every hole in positive control hole adds, the amount of the negative serum that every hole of negative control hole adds, the amount of the fluid nutrient medium that the amount of the fluid nutrient medium that every hole in complement blank hole adds and every hole of bacterial activity control wells add all with every hole of sample well in the consumption equal-volume of the serum to be checked after dilution doubly, the fluid nutrient medium that complement blank hole and bacterial activity control wells add is respectively 3% sodium chloride tryptone soya broth in CNS GB 4789.7-2013,
Step (2) 2. activates bacteria suspension volume described in bactericidal reaction be 20 μ l～40 μ l, the amount of the bacteria suspension adding in bacterial activity control wells and activation bacteria suspension equal-volume, in every hole that the consumption of the nutrient culture media of 37 ℃ of preheatings that add in each hole of microwell plate is sample well doubly doubly 2.5～3 times of the serum volume to be checked after dilution, the nutrient culture media of 37 ℃ of described preheatings is 3% sodium chloride tryptone soya broth in CNS GB 4789.7-2013;
Serum equal-volume to be checked after doubly diluting in the amount of the chromogenic reagent solution that 2. step (1) that step (3) adds in 1. developing the color in each hole of microwell plate prepared and sample well.
7. the detection method of the serum antibody dependence bactericidal activity of complement-mediated according to claim 2, is characterized in that:
Step (1) is when 1. bacterial classification is shigella dysenteriae in collecting cells, and the Shigella that described Solid agar culture and fluid nutrient medium are in CNS GB4789.5-2012 increases bacterium broth bouillon, and described working concentration is 8 * 10
5/ ml～8 * 10
8/ ml;
Step (1) the 2. developer described in reagent preparation is tetrazole purple or new tetrazole;
Step (2) 1. in application of sample dilution in every hole of sample well doubly the serum volume to be checked after dilution and step (2) 2. the volume ratio of the activation bacteria suspension volume described in bactericidal reaction be 2:1～4:1, and the serum volume to be checked after doubly diluting in every hole of sample well all equates, the amount of the positive serum that every hole in positive control hole adds, the amount of the negative serum that every hole of negative control hole adds, the amount of the fluid nutrient medium that the amount of the fluid nutrient medium that every hole in complement blank hole adds and every hole of bacterial activity control wells add all with every hole of sample well in the consumption equal-volume of the serum to be checked after dilution doubly, the Shigella that the fluid nutrient medium that complement blank hole and bacterial activity control wells add is respectively in CNS GB4789.5-2012 increases bacterium broth bouillon,
Step (2) 2. activates bacteria suspension volume described in bactericidal reaction be 20 μ l～40 μ l, the amount of the bacteria suspension adding in bacterial activity control wells and activation bacteria suspension equal-volume, in every hole that the consumption of the nutrient culture media of 37 ℃ of preheatings that add in each hole of microwell plate is sample well doubly doubly 2.5～3 times of the serum volume to be checked after dilution, the nutrient culture media of 37 ℃ of described preheatings is that the Shigella in CNS GB4789.5-2012 increases bacterium broth bouillon;
Serum equal-volume to be checked after doubly diluting in the amount of the chromogenic reagent solution that 2. step (1) that step (3) adds in 1. developing the color in each hole of microwell plate prepared and sample well.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410362470.2A CN104101596A (en) | 2014-07-28 | 2014-07-28 | Detection method for complement-mediated serum antibody-dependent bactericidal activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410362470.2A CN104101596A (en) | 2014-07-28 | 2014-07-28 | Detection method for complement-mediated serum antibody-dependent bactericidal activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104101596A true CN104101596A (en) | 2014-10-15 |
Family
ID=51669925
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410362470.2A Pending CN104101596A (en) | 2014-07-28 | 2014-07-28 | Detection method for complement-mediated serum antibody-dependent bactericidal activity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104101596A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104587461A (en) * | 2015-01-05 | 2015-05-06 | 云南沃森生物技术股份有限公司 | Typhoid and paratyphoid A and B polysaccharide-protein conjugated polyvalent combined vaccine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011008400A2 (en) * | 2009-06-16 | 2011-01-20 | Novartis Ag | High-throughput complement-mediated antibody-dependent and opsonic bactericidal assays |
| CN102203276A (en) * | 2008-08-27 | 2011-09-28 | 奥克兰儿童医院及研究中心 | Complement factor h-based assays for serum bactericidal activity against neisseria meningitidis |
-
2014
- 2014-07-28 CN CN201410362470.2A patent/CN104101596A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102203276A (en) * | 2008-08-27 | 2011-09-28 | 奥克兰儿童医院及研究中心 | Complement factor h-based assays for serum bactericidal activity against neisseria meningitidis |
| WO2011008400A2 (en) * | 2009-06-16 | 2011-01-20 | Novartis Ag | High-throughput complement-mediated antibody-dependent and opsonic bactericidal assays |
Non-Patent Citations (3)
| Title |
|---|
| 夏丹等: "2012 年广州市黄埔区健康人群C 群脑膜炎奈瑟菌血清杀菌力抗体水平分析", 《华南预防医学》, vol. 40, no. 2, 30 April 2014 (2014-04-30), pages 171 - 173 * |
| 梁昊宇: "甲型副伤寒沙门氏菌杀菌抗体测定法的建立", 《第五次全国免疫诊断暨疫苗学术研讨会》, 31 May 2012 (2012-05-31), pages 244 - 247 * |
| 邓俊兴: "韶关市 2009－2011 年健康人群 C 群脑膜炎奈瑟菌血清杀菌力抗体检测结果分析", 《热带医学杂志》, vol. 14, no. 2, 28 February 2014 (2014-02-28), pages 238 - 240 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104587461A (en) * | 2015-01-05 | 2015-05-06 | 云南沃森生物技术股份有限公司 | Typhoid and paratyphoid A and B polysaccharide-protein conjugated polyvalent combined vaccine |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Robbins et al. | Detecting the enterotoxigenicity of Staphylococcus aureus strains | |
| WO2012000309A1 (en) | Sterility test method and totally enclosed bacterial ampoule incubator used by it | |
| Bugno et al. | Performance survey and comparison between rapid sterility testing method and pharmacopoeia sterility test | |
| López-Amorós et al. | Use of two oxonols and a fluorescent tetrazolium dye to monitor starvation of Escherichia coli in seawater by flow cytometry | |
| CN107192656A (en) | A kind of coelomocyte of Apostichopus japonicus phagocytic rate detection method based on flow cytometer | |
| CN105132519B (en) | A kind of selective medium and Escherichia coli quantitative detecting method for Escherichia coli quantitative detection | |
| CN105713859B (en) | Method for detecting multiple antibiotic residues in bifidobacterium breve and milk and application | |
| CN102643919B (en) | Detection kit and detection method for viable bacteria in vibrio parahaemolyticus in food | |
| CN102288586A (en) | Method for determining minimal inhibitory concentration of drug | |
| CN102071244B (en) | Method for accurately detecting toxicity of water quality by using photobacterium toxicity test | |
| CN104101596A (en) | Detection method for complement-mediated serum antibody-dependent bactericidal activity | |
| CN103045524A (en) | Vibrio qinghaiensis Q67B and separation, screening and application thereof | |
| Turner et al. | Changes in viability and macromolecular content of long‐term batch cultures of Salmonella typhimurium measured by flow cytometry | |
| CN101943660B (en) | Method for detecting psychrophilic bacteria in crude milk by utilizing aminopeptidase method | |
| CN101307291B (en) | Microbe auto culturing system using nano-sensor | |
| CN102304585A (en) | Immunocapture PCR (polymerase chain reaction) detection kit of staphylococcus aureus and using method of kit | |
| CN102967545A (en) | Method for detecting content of active bacteria | |
| Noble et al. | Heterotrophic plate counts of surface water samples by using impedance methods | |
| CN103923827B (en) | Full-automatic quantitative microorganism preparing instrument | |
| CN104267067A (en) | Method based on smell sensor and used for predicting growth stage of pseudomonas aeruginosa as meat typical putrefying bacterium | |
| CN103614453A (en) | Gastrointestinal bacteria detection method for medical microbiology experiment | |
| CN1837357A (en) | A culture medium and method for culturing micro vibrio cholerae therewith | |
| CN103499600B (en) | A kind of method differentiating bacterial infection kind in ascites due to cirrhosis | |
| CN203117162U (en) | Integrated membrane biosensor for quickly detecting escherichia coli | |
| CN208187983U (en) | A kind of blood culture report sun detection device |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20141015 |