CN108037277A - It is a kind of to distinguish Brucella abortus vaccine immunity and the method for natural infection state - Google Patents
It is a kind of to distinguish Brucella abortus vaccine immunity and the method for natural infection state Download PDFInfo
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- CN108037277A CN108037277A CN201711281982.6A CN201711281982A CN108037277A CN 108037277 A CN108037277 A CN 108037277A CN 201711281982 A CN201711281982 A CN 201711281982A CN 108037277 A CN108037277 A CN 108037277A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/23—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Brucella (G)
Abstract
Brucella abortus vaccine immunity and the method for natural infection state are distinguished the present invention relates to a kind of.This method is established on the basis of to brucella IgG and IgM antibody hypotype detection method, susceptible animal model is built, China is obtained and is often used as the antibody dynamic regularity of natural infection by the use of brucella attenuated vaccine (A19 plants of S2 plants of pig kind and ox kind) vaccine immunity and standard velogen strain (2308 plants of ox kind).And according to the characteristics of natural infection group and vaccine immunity group, determine 5 kinds of states, establish the method for distinguishing natural infection and vaccine immunity；And this method is verified, confirm its reliability.This law only need to detect its IgG and IgM at the same time to a sample and natural infection and vaccine immunity can be distinguished, method is simple and convenient, accuracy is high, the operating time is short and cheap, suitable for all kinds of laboratory operations, meet the requirement of scale detection, this method has been filled up both at home and abroad in the technological gap in this field, and technical support is provided for cloth disease prevention and control.
Description
Technical field
Brucella abortus vaccine immunity and the method for natural infection state are distinguished the present invention relates to a kind of --- belong to biology system
Product detection technique field.
Technical background
Brucellosis develops in a healthy way as one of important zoonosis, serious threat public safety and aquaculture.It is right
For domestic animal, the cardinal symptom of cloth disease has undulant fever, weight loss, becomes thin, Milk Production decline, then draws for pregnant female
Play miscarriage, stillborn foetus, long with the symptom such as infertile, bring serious threat to international trade, also influence the outlet of relevant animal product, quilt
World Organization for Animal Health (OIE) is classified as B class infectious diseases, and China is also classified as one of two class infectious diseases.Except a small number of flourishing states
Family is outer, and cloth disease has generation in country of more than 170, the world, wherein the most serious with popularities such as the Central Asia, the Middle East.
China's cloth disease was once effectively controlled in 60~eighties of last century, but in recent years, the morbidity quantity of animal cloth disease
Elevated trend year by year is but presented with population risk.Public health security is seriously affected as one kind and aquaculture develops in a healthy way
Zoonosis, prevention and control cloth disease seem extremely urgent.Cloth disease has been included into《Long-term animal epidemic prevention rule in country
Draw (2012~the year two thousand twenty)》.The country of cloth disease is being eliminated, vaccine immunity is usually taken and quarantines and slaughters arranging of being combined
Impose and achieve the purpose that to purify colony.
Vaccine immunity is one of measure of maximally efficient control and prevention cloth disease on animal.S19 plants of B. abortus
Important function, especially S2 plant are played with the S2 plants of prevention and control for China's cloth disease of pig kind brucella, it has China in previous generation
Record and develop the sixties, good protecting effect is respectively provided with to various cloth disease susceptible animals, and virulence is relatively low, the pair produced after immune
React and relatively low to sanitarian security threat.But above two vaccine is smooth type bacterial strain, its produce antibody with
Natural infection is difficult to differentiate between, and the use to vaccine brings very big puzzlement.
The content of the invention
It is an object of the invention to establish a kind of to distinguish animal Brucella abortus vaccine immunity and natural infection
Technical method, the indirect ELISA method for Brucella abortus IgG and IgM subclass antibodies that the present invention is established by early period
(CN05067812A and CN06771182A), respectively to subcutaneous vaccination immune cattle Brucella live vaccine (A19 plants), oral immunity
Brucella abortus live vaccine (S2 plants), and the 2308 plants strong malicious viable bacteria adult ox blood that manually () infects of injection Brucella abortus
IgG and IgM hypotypes in liquid carry out keeping track its Fluctuation, different conditions (immune/infection) critical value are determined, to establish
Brucella abortus is immunized/the antibody subtype growth and decline model of natural infection, and by antibody subtype brucella vaccine is immunized or
Natural infection carries out antidiastole and provides technical method, meets the needs of China's cloth disease epidemiology survey.
Technical scheme
1. a kind of method for distinguishing Brucella abortus vaccine immunity and natural infection state of the present invention, is by dividing
Horizontal for brucella specific IgG and IgM subclass antibodies in ox body, infection/immune state of definite detected ox is analysed, it is real
Now to the differentiation of Brucella abortus vaccine immunity or natural infection, meet brucellosis (cloth disease) epidemiology survey, we
Method is diagnostic for non-disease.
2. a kind of method for distinguishing Brucella abortus vaccine immunity and natural infection state of the present invention, also resides in Bu Lu
The detection of Salmonella specific IgG and IgM subclass antibodies level, is to be based on specific Brucella abortus IgG antibody detection kit
Carried out with Brucella abortus IgM antibody detection kit.
3. a kind of method for distinguishing Brucella abortus vaccine immunity and natural infection state of the present invention, also resides in really
Surely infection/the immune state for being detected ox is to be immunized and simulate the ox of brucella natural infection by building brucella vaccine
Internal IgG and IgM Fluctuations, and divide and judge scope to realize, that is, judge the finger of infection/immune state of tested animal
It is demarcated as：
It is that Brucella abortus IgG subclass antibodies are positive, as the P% of serum sample when the P% values >=20% of serum sample
It is that Brucella abortus IgG subclass antibodies are negative during value ＜ 20%；
It is that Brucella abortus IgM subclass antibodies are positive, as the P% of serum sample when the P% values >=85% of serum sample
It is that Brucella abortus IgM subclass antibodies are negative during value ＜ 85%.
4. a kind of method for distinguishing Brucella abortus vaccine immunity and natural infection state of the present invention, also resides in it
The index of the middle infection/immune state for judging tested animal is that foundation is from following result of the test：
When inspection result is：IgG, S/P >=60%；When IgM, S/P≤85%, state 1 is identified as, is natural infection；Inspection
It is IgG to test result, S/P >=60%；IgM, S/P >=85%, is identified as state 2, is that initial stage is immunized in natural infection or A19；Examine
As a result it is IgG, S/P is between 20%~60%；IgM, S/P >=85%, is identified as state 3, is immunized for A19；Inspection result is
IgG, S/P are between 20%~60%；IgM, S/P≤85%, is identified as state 4, and later stage or infection later stage is immunized for A19；Examine
As a result it is：IgG, S/P ＜ 20%；IgM, S/P >=85%, is identified as state 5, is immunized or acute infection for S2.
5. a kind of method for distinguishing Brucella abortus vaccine immunity and natural infection state of the present invention, data therein
Processing is adapted to complete using computer disposal.
The specific embodiment of the invention
The present invention is respectively with the Brucella abortus indirect ELISA antibody assay kit for IgG and IgM
(CN05067812A and CN06771182A) measures antibody titer, its method is shown in note 1.
(claim 1. the application establishes brucella disease live-vaccine (A19 plants) between following：A19 vaccines) it is anti-in immune cattle body
Body changing rule,
The negative ox of cloth disease more than 50 is selected, injecting immune is carried out by the operation instruction of A19 strain vaccines, and when different
Between point collection blood sample, separate serum.Respectively with the Brucella abortus indirect ELISA antibody assay kit for IgG and IgM
(CN05067812A and CN06771182A) measures antibody titer, records the P values of each stage measure：
The Adult Bovine of 92 Brucella antibody feminine genders is immunized in the present invention with A19 vaccine injections respectively, and 7 after immune
My god, 15 days, 30 days, 60 days, 90 days, 120 days and 150 days gather serum sample respectively and carry out antibody test, to describe A19 vaccines
After immune, the changing rule of IgG and IgM in immune cattle body.Result of study is found (see Fig. 1), the 7th day after A19 vaccine immunities, its
Internal IgG and IgM antibody begin to occur, and peak at the same time at the 15th day, start progressively to decline afterwards.For IgG,
After immune 15 days, A19 vaccine immunity group antibody levels are decreased obviously, to IgG at 90 days average close to critical value, its group
Internal different time sections positive rate is respectively 62.64% (the 7th day), 84.78% (the 15th day), 78.26% (the 30th day), 75%
(the 60th day), 36.95% (the 90th day), 27.17% (the 120th day) and 14.22% (the 150th day).Different time sections antibody water
Put down there is significant difference, wherein significant difference (p between the 60th day and the 90th day<0.01), illustrate animal 60 days immune
Afterwards, the content of its internal IgG is decreased obviously.Compared with IgG, the change of its IgM of A19 vaccine immunity groups becomes apparent, and is immunized
IgM antibody level peaks after 15 days, afterwards rapid decrease, and at the 60th day, its average value was close to critical value, to 120
Then it is wholly absent after it.In different time sections, the changes of contents of IgM is fairly obvious, the 15th day and the 30th day, the 30th day and the 60th
My god, between the 90th day and the 120th day, antibody titer has significant difference (p<0.01).According to colony in difference after immune
The arithmetic average of phase antibody level draws antibody trend rule figure (see Fig. 2), in figure it can be found that A19 be immunized after 15 days, its
IgG levels are higher than critical value, progressively decline afterwards, to substantially below critical value at 90 days；IgM peaks on the 15th day, thereafter
Continue to decline, after 60 days, less than critical value.
(claim 2. the application establishes brucella disease live-vaccine (S2 plants) between following：S2 vaccines) antibody in immune cattle body
Changing rule
S2 vaccines are the most widely used Brucella live vaccines in China, and the change for studying its different subtype antibody becomes
Gesture, the important logo for finding the different immune states of differentiation are more meaningful.
The negative ox of cloth disease more than 50 is selected, oral immunity is carried out by the operation instruction of S2 vaccines, and in the different time
Point collection blood sample, separates serum.Respectively with the Brucella abortus indirect ELISA antibody assay kit for IgG and IgM
(CN05067812A and CN06771182A) measures antibody titer, records the P values of each stage measure：
Compared with A19, recommendation immunization route of the S2 vaccines to ox is oral, its immune group animal internal antibody potency is overall
The characteristics of potency is low, decline is fast is presented (see Fig. 3).Specifically, IgM subclass antibodies are being immunized rear 7 in most of test ox body
It starts to produce, and by 15 days when peaks.In peak period, its antibody level (P% values) is 3 times or so of critical value, big portion
Its P% value of sample is divided to be between 100~200.Its antibody level declines rapidly after 15 days, during by the 60th day, community average
Below critical value, during by the 90th day, most of sample (55/56) is less than critical value in colony.It is significantly different with A19
, its IgG subclass antibodies content of S2 vaccine immunities colony is very low, except 30 days indivedual its antibody water of sample (4/56) after immune
Outside flat slightly higher and critical value, most of to be below critical value in whole monitoring cycle, this is the most important spy of S2 immune cattles
One of point.Relative to A19 vaccine immunity groups, the horizontal change of S2 vaccine group different time sections IgM subclass antibodies is more violent.S2 epidemic diseases
Its IgM subclass antibodies is the 7th day and the 15th day after seedling is immunized, the 15th day with the 30th day, the 30th day with the 60th day, the 60th day and the
There were significant differences (p between 90 days, and 90 and the 120th days<0.05)；For IgG, although most of sample is entirely monitoring
The interior antibody of phase is below critical value, but the 30th day with the 60th day and the 60th day with there is also significant difference (p within the 90th day<
0.05).Antibody trend rule figure (see Fig. 4) is drawn according to the arithmetic average of colony's different times antibody level after immune,
By Fig. 2 compare with Fig. 4 it can be seen from the ox that is immunized of S2 vaccine groups, its IgG mean antibody levels will be significantly lower than A19 vaccines
Immune group, and IgM, although its peak is less than A19 vaccine groups, after the 15th day, its horizontal and subordinate's amplitude and A19 vaccine groups
Relatively.
3. the application establishes brucella with reference to IgG and IgM Fluctuations measure in strong malicious (2308 plants) infected cattle body
Antibody change
The antibody of 2308 infected cattles.The negative ox of cloth disease 4 is selected to carry out artificial challenge in P3 laboratories, and when different
Between point collection blood sample, separate serum.Respectively with the Brucella abortus indirect ELISA antibody assay kit for IgG and IgM
(CN05067812A and CN06771182A) measures antibody titer, records the P values of each stage measure：
In the application, we to test ox simulate certainly using brucella with reference to strong malicious 2308 plants of B. abortus
The experiment so infected, and tracked the trend rule of its antibody variation (see Fig. 5).It turns out that with vaccine immunity group (including
A19 vaccine groups and S2 vaccine groups) to compare, infected group has the characteristics that antibody generation evening, duration are long.IgM subclass antibodies exist
Just starting to detect within two weeks after infection, by the 30th day when peaks, afterwards rapid decrease, and by the 90th day when is less than critical value,
It is also only 2 times of critical value during IgM antibody potency highest, the antibody level difference between different infected individuals is smaller.Relative to
IgM, although IgG antibody generation time is later, persistently rises thereafter, and is always maintained at stablizing in whole supervised period.
4. the application establishes the antibody rule tendency chart of different subtype and the division scope of different Infection Status is determined
In order to more intuitively grasp the difference between different groups, we are according to infected group (2308 plants) and immune group
The changing rule of (including A19 and S2) antibody subtype, incorporates the trend of IgG and IgM antibody change, has obtained being used for cloth Shandong
The analysis curve that Salmonella vaccine immunity is distinguished with natural infection (see Fig. 6).In figure, No. 1 and No. 2 lines represent artificial challenge
Antibody trend after 2308 plants of bacterium, 3, No. 4 lines represent the antibody trend after immune A19 vaccines, and 4, No. 5 lines represent immune S2
Variation tendency after vaccine；There are arrow 1, the representative IgM of 3, No. 5,2,4,6 solid lines of no arrow represent IgG.According under each model
Antibody level, by the antibody law curve of merging in the longitudinal axis (Y-axis, S/P values, represent antibody level) and transverse axis (X-axis, day, table
Show the time after infection/immune) on be respectively divided into three regions.On the longitudinal axis, S/P is IgG antibody negative areas less than 20%, high
It is IgG antibody positive region in more than 20%；S/P is IgM antibody negative areas less than 85%, higher than 85% is IgM
Antibody positive region.Tendency chart shows that S2 vaccine groups IgG is constantly in negative areas, and its IgG of A19 is since the 15th day,
Its average value maintains 35% or so always, declines after 60 days, and by the 90th day when begins lower than critical value.2308 plants of bacterium senses
Its average value of dye group rises and keeps higher level always after being higher than critical value after 30 days.It is poor according to different group antibody rules
It is different, the time of X-axis artificially can be divided into three phases, the first stage, the stage was properly termed as to infect/be immunized in 15 days latter
Antibody ascent stage, wherein A19 vaccine immunities group IgG and IgM, S2 vaccine group IgM subclass antibodies increase in this stage, and
Peak within 15 days；And 2308 plants of bacterium infected group its IgM then lag the next stage and just reach peak, in this stage, its antibody
Though rising, critical value is still below.Second stage i.e. infection/immune 15~90 days latter, is referred to as the antibody decline stage, at this
Downward trend is presented in stage, A19 vaccine immunities group and S2 vaccine immunities group its antibody, both IgM subclass antibodies average values
Critical value was below after 90 days at the 60th day near critical value.Its IgG antibody of A19 vaccine immunities group declines with IgM
Gesture is essentially identical, more slow；Entirely different trend is then presented in this stage for infected group, and crest was presented in its IgM, with 30 days
For boundary, rapid decrease, its average value are above critical value to antibody at this stage again after 15 days rapid increases；And IgG then exists
Start occur and be always maintained at higher level after 30 days.Phase III, i.e., be referred to as antibody disappearance rank after immune/infection 90 days
Section, the stage remove.Still on critical value, each subclass antibodies of remaining group have detected 2308 plants of bacterium infected group IgG antibodies
Less than.Analysis chart 6, it can be found that at second stage, especially 60~90 day.2308 plants of bacterium infected groups and A19 vaccine immunities
There are certain intersection, such case can have the real conditions of the strong viral infections of accurate judgement or vaccine immunity certain group
Interference, runs into such case, can be detected by gathering (with first time sample collection interval more than 2 weeks) sample again,
To evade the erroneous judgement for being in moment.
According to infection/immune model of foundation, and the testing result of a large amount of clinical samples is combined, by IgG and IgM antibody water
The flat state for being divided into 5 infection/be immunized (result sees attached list 1)：1. natural infection (IgG, S/P >=60%；IgM, S/P≤
85%.Be identified as state 1), 2. natural infection or A19 vaccine immunity initial stage (IgG, S/P >=60%；IgM, S/P >=85%.Mark
Know for state 2), 3. (IgG, S/P are between 20%~60% for A19 vaccine immunities；IgM, S/P >=85%.Be identified as state 3), 4.
A19 vaccine immunity later stages or 2308 plants of bacterium infection later stages, (IgG, S/P were between 20%~60%；IgM, S/P≤85%.It is identified as
State 4), 5. S2 vaccine immunities or acute infection (IgG, S/P<20%；IgM, S/P >=85%.It is identified as state 5).For shape
State 2 and state 4, in order to ensure real result is reliable, it is necessary to collecting sample is detected again after 15 days, the knot detected again
Fruit can relatively accurately judge infection/immune state of tested animal, it judges the index of infection/immune state of tested animal
It is set to：
It is that Brucella abortus IgG subclass antibodies are positive, as the P% of serum sample when the P% values >=20% of serum sample
Value<It is that Brucella abortus IgG subclass antibodies are negative when 20%；
It is that Brucella abortus IgM subclass antibodies are positive, as the P% of serum sample when the P% values >=85% of serum sample
Value<It is that Brucella abortus IgM subclass antibodies are negative when 85%.
1 different conditions of table draw the line
5. the clinical sample of pair clear background is detected, confirm the reliability of the present invention.
The method established according to the present invention, we have carried out condition adjudgement by 50 parts of clinical samples to clear background, as a result
The judging result of 92% (46/50) is consistent with actual conditions (detailed results are shown in 1 table 2 of embodiment).There are two for state 2 and state 4
Kind is possible, and verification result is found, is shared 6 samples and is judged as being in state 2, wherein 5 samples are natural infection, 1 sample
For A19 vaccine immunities, illustrate this stage be in natural infection state situation it is in the majority.For state 4,6 samples are have detected altogether,
Wherein 2 are A19 vaccine immunities, account for the 33% of sum, under the conditions of defining herein, compare and be difficult to differentiate between natural infection or A19
Vaccine, which is immunized, it is necessary to combine second of result using detection, to be judged.The above results prove, what the present invention was established
Method, reliablely can be immunized brucella vaccine and be identified with natural infection state.
Brief description of the drawings
Antibody titer and immunization time after Fig. 1 A19 vaccine immunity oxen (my god) graph of a relation is wherein：GG schemes A and is dripped for IgG antibody
Spend (P%) and immunization time (my god) relation；Scheme B for IgM antibody titre (P%) and immunization time (my god) (* * * represent difference to relation
Extremely notable p<0.001, * * represents p<0.01, * represents p<0.05).
The Fluctuation of IgG antibody and IgM antibody after Fig. 2 A19 vaccine immunity oxen.
Antibody titer and immunization time after Fig. 3 S2 vaccine immunity oxen (my god) graph of a relation is wherein：Figure A is IgG antibody titre
(P%) with immunization time (my god) relation；Scheme B for IgM antibody titre (P%) and immunization time (my god) (* * * represent difference pole to relation
Notable p<0.001, * * represents p<0.01, * represents p<0.05).
The Fluctuation of IgG antibody and IgM antibody after Fig. 4 S2 vaccine immunity oxen.
Fig. 5 with antibody titer after 5 oxen of the last 2308 toadstool artificial challenge and infection time (my god) graph of a relation figure A for infection after
IgG antibody titre (P%) in ox body and infection time (my god) relation；B is schemed for IgM titres (P%) and infection time after infection
(my god) relation.
Under Fig. 6 vaccine immunities and natural infection state, 1 and 2 be respectively 2308 bacterium in IgG and IgM antibody Fluctuation figure
The antibody dynamic regularity of the metainfective IgG and IgM of strain；3 and 4 be respectively A19 vaccine immunities after IgG and IgM antibody variation
Rule；5 and 6 be respectively S2 vaccine immunities after IgG IgM antibody dynamic regularity.
Microbial resources information of the present invention
Microorganism according to the present invention is：Pig kind brucella (Brucella suis) S2 plants (CVCC70502), is
Pig kind brucella 2 types of biology, are brucellosis attenuated live vaccines Reference Strains；B. abortus (Brucella
Abortus) A19 plants (or S19 plants, CVCC 70202), are biological 1 type of B. abortus, are that the sick and weak poison of brucella is living
Vaccine Reference Strains；B. abortus 2308 (CVCC 788) is velogen strain, and 3 plants of the above is by Zhongguancun South St., Haidian District, Beijing City
Veterinary microorganism culture presevation administrative center of China of China Veterinery Drug Inspection Office identification of street 8, keeping and supply are (see in
Veterinary medicament supervision institute of state, Chinese veterinary microorganism culture presevation administrative center write, Chinese animal doctor's strain catalogue (second edition),
Scientia Agricultura Sinica technology publishing house, 2002, p35, p25, p24).
Advantages of the present invention
Brucella abortus vaccine immunity and the method for natural infection state are distinguished the present invention relates to a kind of.This method is established
On the basis of brucella IgG and IgM antibody hypotype detection method, susceptible animal model is built, obtains China Chang Yongbu Shandongs
The antibody rule of Salmonella attenuated vaccine (A19 plants of S2 plants of pig kind and ox kind) and standard velogen strain (2308 plants of ox kind).Then root
According to natural infection group and the characteristics of vaccine immunity group, it is determined that 5 kinds of states, establish and distinguish natural infection and vaccine immunity
Method；And the method for foundation is verified, confirm its reliability.This law only need to detect a sample at the same time its IgG and
IgM can distinguish natural infection and vaccine immunity, and method is simple and convenient, accuracy is high, the operating time is short low with price
It is honest and clean, suitable for all kinds of laboratory operations, meet the requirement detected on a large scale, this method compensate for both at home and abroad in the technology in this field
Blank, technical support is provided for cloth disease prevention and control.
Embodiment
Following embodiments do not limit the invention to further illustrate technical scheme.
Embodiment 1
--- the clinical sample of clear background is detected, confirms the reliability of the present invention.
The method established according to the present invention, we have carried out condition adjudgement by 50 parts of clinical samples to clear background, as a result
The judging result of 92% (46/50) is consistent with actual conditions (the results are shown in Table 2).There are two kinds of possibility, verification for state 2 and state 4
It is judged as being in state 2 it turns out that sharing 6 samples, wherein 5 samples are natural infection, 1 sample is immunized for A19, says
The situation that this bright stage is in natural infection state is in the majority.For state 4,6 samples are have detected altogether, wherein 2 are exempted from for A19
Epidemic disease, accounts for the 33% of sum, under the conditions of defining herein, compares and is difficult to differentiate between natural infection or A19 vaccine immunities, it is necessary to reference to the
The secondary result using detection is judged.The above results prove that the method that the present invention is established can be reliablely to cloth Shandong
Salmonella vaccine immunity is identified with natural infection state.
2 clinical sample result verification of table
Note 1
IELISA method operation instructions
1. usage
(1) sample treatment：Animal's whole blood is taken, after blood clotting, 10min is centrifuged with 4000r/min, collects supernatant, blood
Clearly should be limpid, no haemolysis.
The preparation of (2) 1 × cleaning solutions：Before use, recovering 20 × cleaning solution to room temperature (20~25 DEG C), and shake, make
Crystallization dissolving (5~10min can be heated in 37 DEG C of water-baths), then makees 1 with deionized water:20 dilutions, fully mix.
(3) serum that is diluted in of serum and control serum to be checked is diluted serum to be checked, negative control sera and sun in plate
Property control serum with sample diluting liquid make 1:50 dilutions.
(4) operating procedure
1) it is loaded：Take antigen coated microplate (according to sample how much, removable gradation use), with 1 × cleaning solution, 300 μ l/ holes
Board-washing 1 time, discards cleaning solution.The serum to be checked, positive control serum and the negative control sera that have diluted are added separately to
In elisa plate, 100 μ l/ holes, wherein positive control serum and negative control sera respectively add 2 holes (hole 1, hole 2).After sample-adding,
37 DEG C of effect 30min.Reaction plate is taken out, discards reaction solution, 300 μ 1 × cleaning solutions of l are added per hole, washs 3 times, gets rid of for last 1 time
Do or pat dry.
2) enzyme labeling antibody：HRP- rabbit-antis ox IgG enzyme labelled antibodies or HRP- goat-anti ox IgM enzyme labelled antibody dilutions are made
1:After 200 dilutions, 100 μ l, 37 DEG C of effect 30min are added per hole.Reaction plate is taken out, discards reaction solution, 300 μ l1 are added per hole
× cleaning solution, wash 3 times, it is last 1 time drying or pat dry..
3) colour developing and termination：Substrate solution A and B are pressed 1:After 1 (V/V) mixing, it is added to immediately in ELISA reaction plates,
100 μ l/ holes, room temperature lucifuge develop the color after 15min, add 50 μ l terminate liquids to terminate reaction per hole.
2. result judgement：After reaction terminating, OD is measured with microplate reader in 15min450nmValue.
(1) computational methods：
(2) establishment condition is tested：
When positive control is averaged OD450nm>=0.8, negative control is averaged OD450nm<0.2 and P%<20% (IgG) or 85%
(IgM) when, experiment is set up.
(3) result judgement：
It is that Brucella abortus IgG subclass antibodies are positive when the P% values >=20% of serum sample；As the P% of serum sample
Value<It is that Brucella abortus IgG subclass antibodies are negative when 20%.
It is that Brucella abortus IgM subclass antibodies are positive when the P% values >=85% of serum sample；As the P% of serum sample
Value<It is that Brucella abortus IgM subclass antibodies are negative when 85%.
Claims (5)
1. a kind of distinguish Brucella abortus vaccine immunity and the method for natural infection state, it is characterised in that by analyzing in ox body
It is horizontal for brucella specific IgG and IgM subclass antibodies, determine infection/immune state of detected ox, realize to ox cloth
Shandong Salmonella vaccine immunity or the differentiation of natural infection, meet brucellosis epidemiology survey, and this method diagnoses for non-disease
Property.
2. a kind of differentiation Brucella abortus vaccine immunity and the method for natural infection state, its feature described in claim 1 exist
In the detection of brucella specific IgG and IgM subclass antibodies level, detected based on specific Brucella abortus IgG antibody
What kit and Brucella abortus IgM antibody detection kit carried out.
3. a kind of differentiation Brucella abortus vaccine immunity and the method for natural infection state, its feature described in claim 1 exist
In determining that infection/immune state of detected ox is to be immunized and simulate brucella natural infection by building brucella vaccine
Ox body in IgG and IgM Fluctuations, and divide and judge scope to realize, that is, judge infection/immune state of tested animal
Index be set to：
It is that Brucella abortus IgG subclass antibodies are positive, as the P% values ＜ of serum sample when the P% values >=20% of serum sample
It is that Brucella abortus IgG subclass antibodies are negative when 20%；
It is that Brucella abortus IgM subclass antibodies are positive, as the P% values ＜ of serum sample when the P% values >=85% of serum sample
It is that Brucella abortus IgM subclass antibodies are negative when 85%.
4. a kind of differentiation Brucella abortus vaccine immunity and the method for natural infection state described in claim 1,3, its feature
The index for being to judge infection/immune state of tested animal is that foundation is from following result of the test：
When inspection result is：IgG, S/P >=60%；When IgM, S/P≤85%, state 1 is identified as, is natural infection；Examine knot
Fruit is IgG, S/P >=60%；IgM, S/P >=85%, is identified as state 2, is that initial stage is immunized in natural infection or A19；Inspection result
For IgG, S/P is between 20%~60%；IgM, S/P >=85%, is identified as state 3, is immunized for A19；Inspection result is IgG, S/P
Between 20%~60%；IgM, S/P≤85%, is identified as state 4, and later stage or infection later stage is immunized for A19；Inspection result is：
IgG, S/P ＜ 20%；IgM, S/P >=85%, is identified as state 5, is immunized or acute infection for S2.
5. a kind of described in claim 1 distinguish Brucella abortus vaccine immunity and the method for natural infection state, it is characterised in that
Data processing therein can be completed using computer disposal.
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| CN201711281982.6A CN108037277A (en) | 2017-12-07 | 2017-12-07 | It is a kind of to distinguish Brucella abortus vaccine immunity and the method for natural infection state |
| FR1870903A FR3074912B1 (en) | 2017-12-07 | 2018-08-03 | METHOD FOR DISTINGUISHING A STATE OF IMMUNIZATION BY THE VACCINE AGAINST BRUCELLA ABORTUS FROM A STATE OF NATURAL INFECTION |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101092605A (en) * | 2007-05-25 | 2007-12-26 | 中国人民解放军军事医学科学院微生物流行病研究所 | Mutant strain of Brucella bacterin with weak poison, constructing method, and application |
| CN105067812A (en) * | 2015-08-06 | 2015-11-18 | 中国兽医药品监察所 | Bovine brucella indirect ELISA antibody detection kit |
| CN106771182A (en) * | 2016-11-29 | 2017-05-31 | 中国兽医药品监察所 | Brucella abortus IgM subclass antibodies indirect ELISA testing kits |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1336576C (en) * | 1986-09-26 | 1995-08-08 | Malcolm B. Perry | Immunoassays for discriminating between brucellosis infections and vaccinations |
| AU2006326283B2 (en) * | 2005-12-12 | 2012-01-19 | Ac Immune S.A. | Therapeutic vaccine |
| MY150105A (en) * | 2006-01-17 | 2013-11-29 | Forsgren Arne | A novel surface exposed haemophilus influenzae protein (protein e; pe) |
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2017
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| CN101092605A (en) * | 2007-05-25 | 2007-12-26 | 中国人民解放军军事医学科学院微生物流行病研究所 | Mutant strain of Brucella bacterin with weak poison, constructing method, and application |
| CN105067812A (en) * | 2015-08-06 | 2015-11-18 | 中国兽医药品监察所 | Bovine brucella indirect ELISA antibody detection kit |
| CN106771182A (en) * | 2016-11-29 | 2017-05-31 | 中国兽医药品监察所 | Brucella abortus IgM subclass antibodies indirect ELISA testing kits |
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| FR3074912B1 (en) | 2022-07-29 |
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