CN110146694A - A kind of chloramphenicol direct competitive chemiluminescence high specific immunoassay method - Google Patents
A kind of chloramphenicol direct competitive chemiluminescence high specific immunoassay method Download PDFInfo
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- CN110146694A CN110146694A CN201910347296.7A CN201910347296A CN110146694A CN 110146694 A CN110146694 A CN 110146694A CN 201910347296 A CN201910347296 A CN 201910347296A CN 110146694 A CN110146694 A CN 110146694A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
A kind of method of direct competitive chemiluminescence immunoassay qualitative and quantitative detection chloramphenicol of the present invention.Detection method of the invention includes preparing enzyme mark chloramphenicol resistance antibody, the detection of direct competitive chemiluminescence immunoassay, the chloramphenicol quantitative detection for establishing working curve equation and sample to be tested.The sodium periodate method improved in the present invention is using saturation NaBH4Methanol solution carries out reduction reaction, overcomes NaBH4Aqueous solution is easily oxidized, is unable to the shortcomings that long-term preservation, so as to avoid the limitation of the solution matching while using；Direct competitive chemiluminescence immunoassay detection technique improves the sensitivity of detection efficiency and detection；Traditional tmb substrate, the sensitivity for improving reaction of high degree are substituted using highly sensitive luminol chemiluminescence substrate；The concentration of residual chloromycetin can be fast and accurately obtained by establishing working curve, reduces the complexity of detection and calculating, the application suitable for the detection work of extensive base.
Description
Technical field
The present invention relates to medicament residue detection technique fields, and in particular to a kind of direct competitive chemiluminescence immunoassay is qualitative fixed
The method of amount detection chloramphenicol.
Background technique
With the increasingly raising of living standard, health food is increasingly pursued by consumer, and wherein antibiotic is residual
Health food safety problem caused by staying has become whole world focus of attention.Health food ingredients are complicated, testing concentration compared with
Low, usual sampling quantity is larger, and just to the sensitivity of analysis method and rapidity, more stringent requirements are proposed for this.Direct competitive chemistry
Shine enzyme-linked immune analysis (Direct chemiluminescent enzyme-linked immunosorbent
Assay, D-CLIA) technology combines the immune response of highly sensitive chemiluminescence with high specific, this method gram
The disadvantages of having taken traditional enzyme labelling technique stability and complicated indirect competition operation has high sensitivity, specificity
By force, the range of linearity it is wide, it is easy to operate, do not need the features such as sufficiently expensive instrument and equipment.
Chloramphenicol (chloroamphenicol) is a kind of broad-spectrum antibacterial antibiotic, is had to Grain-positive, negative bacteria
Inhibiting effect.Its antifungal mechanism is by reversibly in conjunction with 50S subunit, blocking the effect for turning peptide acyl enzyme, and interference has amino
Amido acyl-tRNA the terminal of acid is in conjunction with 50S subunit, so that the formation of new peptide chain be made to be obstructed, inhibits protein synthesis.But due to
Chloramphenicol can also be in conjunction with the 70S of human body mitochondria, thus also can inhibit the albumen synthesis of human body mitochondria, generates poison to human body
Property, serious person can inhibit human bone marrow's hematopoiesis function and cause the diseases such as aplastic anemia and granulocytic leukemia,
Therefore the residual chloromycetin in animal-derived food will constitute huge potential hazard to the health of the mankind.It the World Food Programme and defends
Raw tissue is reduced to 0.1 μ g/kg by 10 original μ g/kg to the regulation of the residual quantity of chloramphenicol in food.The Ministry of Agriculture, China in
Also formally the preparations such as chloramphenicol and its salt ester are arranged in " food animal disables veterinary drug and its compound inventory " of publication in 2002
For forbidden drugs, to ensure animal food safety.
Mainly there are instrument analytical method, Indirect cELISA and enzyme-labelled antigen straight for the detection of chloramphenicol at present
Connect competitive enzyme-linked immune method.Instrument analysis technology is mainly based upon high-efficient liquid phase chromatogram technology and liquid chromatography mass combination skill
Art, this method high sensitivity, stability are good.But instrument analysis technology needs complicated and expensive equipment and does some training very often
Experimenter, this method needs complicated pretreatment technology, therefore the application aspect of this method Fast Detection Technique at the scene
There is significant limitation.Furthermore instrument analysis needs to use in pretreatment process and in chromatographic separation process and largely have
Volatile poisonous and harmful reagent, such as methanol, acetonitrile, ethyl acetate, chloroform, are easy to cause damages to experimenter.
Indirect cELISA is a kind of immunological technique combined based on antigen and antibody specific, is analyzed compared to instrument
Technology, this method can be used for the field quick detection of chloramphenicol, but this method is mainly based upon horseradish peroxidase
(HRP) label secondary antibody technology carries out indirect competition reaction, using label enzymatic tetramethyl benzidine (TMB) substrate colour developing to chlorine
Mycin is detected.The complex combination process of more step ELIAS secondary antibodies is reacted in indirect competition reaction compared to direct competitive, when reaction
Between it is longer, cause detection efficiency limited；On the other hand by substrate TMB, itself sensitivity is limited, and the sensitivity of this method is one
There is significant limitation in fixed degree.
Competitive ELISA technology needs to prepare enzyme-specific labeling antibody, traditional enzyme-labelled antibody technique master at present
It to be prepared using sodium periodate method.Horseradish peroxidase HRP is activated using sodium metaperiodate first, after activation
Coupling reaction is carried out with antibody, uses NaBH later4Aqueous solution excessive activating enzymes are restored, terminate reaction, preparation
Horseradish peroxidase-antibody marker.Sodium periodate method labeling effciency is high, but to the NaBH in reaction process4Storage
Condition is more demanding, and current method mainly uses water to configure NaBH4Carry out reduction reaction, and NaBH4It is a kind of strong reduction
Agent easily aoxidizes in water, is difficult to save, therefore experiment requires to reconfigure NaBH every time4Solution, execute-in-place is complicated,
Increase cost consumption and person works' amount.
Summary of the invention
Enzyme mark chloramphenicol antibody is prepared the present invention provides a kind of sodium periodate method of improvement and detects the direct of chloramphenicol
Compete chemiluminescence immunoassay rapid detection method.
The chloramphenicol resistance antibody is mouse antichloromycetin IgG.
The enzyme is horseradish peroxidase.
The coating antigen is the conjugate of chloramphenicol and carrier protein OVA.
The chemiluminescent substrate is made of A liquid and B liquid, and luminescent solution A liquid is sodium peroxydisulfate solution；Luminescent solution B liquid is Shandong
Minot solution.
The present invention is directed to reducing agent NaBH in traditional sodium periodate method4Deficiency not easy to maintain is improved, using first
Alcohol replaces water to dissolve NaBH4, because the solubility of oxygen in methanol is very low, NaBH4Dissolution wherein can effective isolating oxygen
Gas delays oxidation process.Prepare NaBH4Supersaturated methanol solution can be further ensured that NaBH4Reproducibility, therefore can protect for a long time
It deposits, avoids being oxidized in air, so as to efficiently against NaBH4Solution needs the defect of matching while using.
Direct competitive chemiluminescence immunoassay detection method is developed based on prepared enzyme mark chloramphenicol antibody, this method is not required to
The instrument and equipment for wanting complicated pre-treatment program and valuableness, is avoided and is not easy to lacking for on-site test as brought by equipment
It falls into, is in addition immunoreacted and only needs micro organic reagent, so that these chemical reagent be avoided to be made to experimenter and environment
At harm.Direct competitive reaction can reduce by a step antigen-antibody reaction, and reagent needed for reducing experiment improves detection
Efficiency, while using highly sensitive chemiluminescent substrate reagent, it can be improved with the sensitivity for improving reaction of high degree
Detectability.Compared to traditional detection technique, this method can be used for the rapid sensitive detection at chloramphenicol scene.
The present invention provides a kind of method of direct competitive chemiluminescence immunoassay detection chloramphenicol, and the method includes following steps
It is rapid:
(1) antibody marker is prepared
Use sodium metaperiodate NaIO4Horseradish peroxidase HRP is activated.
Further, the horseradish peroxidase after activation and chloramphenicol antibody IgG carry out coupling reaction.
Further, NaBH is added4Reduction reaction is carried out in saturation methanol solution, it is mould to obtain horseradish peroxidase-chlorine
Plain antibody marker.
(2) it is coated with chloramphenicol ELISA Plate
Coating antigen is diluted using carbonate buffer solution, the coating antigen after dilution is subjected to ELISA Plate coating.
Further, ELISA Plate washing is carried out using cleaning solution.
Further, ELISA Plate closing is carried out using confining liquid, obtains the chloramphenicol detection ELISA Plate being coated with.It is placed in 20
It degree Celsius saves backup.
(3) direct competitive chemiluminescence immunoassay detects
Sample is placed in each hole of chloramphenicol coated elisa plate, the antibody marker of step (1) preparation is added, is carried out straight
Connect competitive reaction；Chemiluminescent substrate is added in each hole, measures luminous intensity.
(4) working curve equation is established
Above-mentioned working solution is added preparation in several pieces sample and added by the chloramphenicol standard working solution for preparing gradient concentration
Standard specimen product obtain test solution after processing.
Further, PBS solution is added in mark-on sample, after concussion, extraction of ocean eddies, centrifugation, discards upper-layer fat layer, takes
Interbed solution obtains test solution after water phase filter membrane.
It according to the method for step (3), takes logarithm as abscissa using concentration, using sample percentage luminous value as ordinate, draws
Standard curve.
(5) the chloramphenicol quantitative detection of sample to be tested
Several pieces sample to be tested is weighed, sample to be tested is added PBS solution and discards upper layer after concussion, extraction of ocean eddies, centrifugation
Fat deposit takes intermediate layer solution, solution to be measured is obtained after water phase filter membrane.
Further, luminous intensity is measured using the method for step (3), it is bent that numerical value is substituted into the work that step (4) are established
Line equation calculation goes out the content of chloramphenicol in sample to be tested.
Beneficial effect
Compared with prior art, the beneficial effects of the present invention are:
Labelling technique and detection method of the invention has substantive distinguishing features outstanding and significant progress, changes in the present invention
Good sodium periodate method overcomes NaBH4The shortcomings that aqueous solution is easily oxidized, and is unable to long-term preservation, so as to avoid the solution
The limitation of matching while using.
Direct competitive chemiluminescence immunoassay detection technique in the present invention improves the sensitivity of detection efficiency and detection,
After preliminary preparation (enzyme labelled antibody preparation and antigen coat closing) is completed, live detection time reduce 30min with
On, the detection time of entire detection process greatly shortens, and about 30min can be completed.
The present invention substitutes traditional tmb substrate, the raising of high degree using highly sensitive luminol chemiluminescence substrate
The sensitivity of reaction, detection of the invention are limited to 0.9ng/kg (L).
The present invention can fast and accurately obtain the concentration of residual chloromycetin by establishing working curve, reduce detection
With the complexity of calculating, the application of work is detected suitable for extensive base.
Other features and advantages of the present invention will be illustrated in the following description, also, partly becomes from specification
It obtains it is clear that understand through the implementation of the invention.The objectives and other advantages of the invention can be by specification, right
Specifically noted structure is achieved and obtained in claim and attached drawing.
Detailed description of the invention
Attached drawing is used to provide to further understand technical solution of the present invention, and constitutes part of specification, with this
The specific embodiment of application technical solution for explaining the present invention together, does not constitute the limit to technical solution of the present invention
System.
Fig. 1 is 1 chloramphenicol direct competitive chemiluminescence immunoassay examination criteria curve of the embodiment of the present invention.
Fig. 2 is the structural formula of chloramphenicol in the embodiment of the present invention 2.
Specific embodiment
The present invention is described in more detail below with reference to accompanying drawings, which show the preferred embodiment of the present invention,
It should be understood that those skilled in the art can modify invention described herein and still realize beneficial effects of the present invention.Cause
This, following description should be understood as the widely known of those skilled in the art, and be not intended as limitation of the present invention.
For clarity, not describing whole features of practical embodiments.In the following description, it is not described in detail well known function
And structure, because they can make the present invention chaotic due to unnecessary details.It will be understood that opening in any practical embodiments
In hair, it is necessary to make a large amount of implementation details to realize the specific objective of developer.
To be clearer and more comprehensible the purpose of the present invention, feature, a specific embodiment of the invention is made with reference to the accompanying drawing
Further instruction.It should be noted that attached drawing is all made of very simplified form and using non-accurate ratio, it is only used for
Purpose that is convenient, clearly aiding in illustrating the embodiment of the present invention.
The present embodiment provides a kind of methods of direct competitive chemiluminescence immunoassay detection chloramphenicol, and the method includes following
Step:
Step A: horseradish peroxidase-chloramphenicol antibody marker is prepared
(1) 5mg horseradish peroxidase HRP is weighed to be dissolved in 1mL distilled water；
(2) NaIO of 0.1M is added in (1)4Solution 0.2mL is protected from light stirring 20 minutes at room temperature, activates HRP；
(3) (2) are transferred in pretreated bag filter, are placed in 1mM sodium-acetate buffer (pH4.4) dialysis, 4 DEG C of mistakes
Night；
(4) 10mg chloramphenicol antibody IgG is weighed, (pH8.0) 1mL is dissolved in 0.01M carbonate buffer solution；
(5) solution (3) are taken out, 0.2M carbonate buffer solution (PH9.5) 20 μ L is added, makes the PH of the HRP of the above aldehyde radical
9.0~9.5 are increased to, is rapidly added solution (4), room temperature, which is protected from light, is gently mixed 2 hours progress coupling reactions；
(6) plus 0.1mL is saturated NaBH4Solution (methanol dissolution) mixes, and 4 DEG C are stirred 2 hours, restores excessive aldehyde radical；
(7) above-mentioned liquid is fitted into bag filter, is dialysed at PBS (0.15M, pH 7.4), 4 DEG C overnight, i.e. acquisition horseradish mistake
Oxide enzyme-chloramphenicol antibody marker.Enzyme labelled antibody obtained, which is placed in -20 DEG C and saves, can be used for subsequent direct competitive and exempts from
Epidemic disease reaction.
Step B: based on the enzyme mark chloramphenicol antibody prepared in step A, the quick, sensitive direct of detection chloramphenicol is established
Compete chemical luminous immune detection method.
Step B1: direct competitive chemical luminous immune detection method
(1) it is coated with: coating antigen being diluted to a series of concentration with coating buffer and adds to ELISA Plate, 100 holes μ L/, 37 DEG C of incubations
2h；
(2) wash: extraction raffinate in hole of inclining is washed 3 times with cleaning solution, and 300 holes μ L/ pat dry on blotting paper；
(3) it closes: 150 hole μ L/ of confining liquid is added, 37 DEG C of incubation 1h are directly patted dry after taking-up；
(4) it is loaded: each hole addition sample (standard items) 50 hole μ L/, the enzyme labelled antibody of addition step A preparation, 50 holes μ L/,
37 DEG C of incubation 15min carry out direct competitive reaction；
(5) it washs: same to step (2)；
(6) measure: 50 hole μ L/ of luminol chemiluminescence substrate is added in each hole, measures luminous intensity immediately.
Step B2: working curve equation is established
(1) chloramphenicol drug standards stock solution (methanol dissolution) is chosen, the chlorine for being then diluted to gradient concentration with PBS is mould
Plain standard working solution；
(2) free of contamination blank sample is chosen, several pieces 1.0g (or 1.0mL) sample is then accurately weighed, is respectively placed in
In 50mL polyfluortetraethylene pipe, it is then respectively adding the chloramphenicol standard solution of the various concentration of above-mentioned configuration, is stored at room temperature
The mark-on sample of gradient concentration is made in 30min.Then 10mL PBS solution, concussion, extraction of ocean eddies will be added into every part of sample
5min, 4 DEG C, 9000g centrifugation 10min, discards upper-layer fat layer, intermediate layer solution is taken to cross 0.45 μm of water phase filter membrane, obtain for examination
Solution.
(3) 50 μ L test solutions are respectively taken, are measured using step B1 method, take logarithm as abscissa using concentration, with hair
Luminous intensity is ordinate, carries out four parameter fittings using Origin8.0 software, draws standard curve.
Step B3, the chloramphenicol qualitative and quantitative detection of product to be tested
(1) sample to be tested is taken, several pieces 1.0g (or 1.0mL) sample is then accurately weighed, is respectively placed in 50mL polytetrafluoro
In ethylene tube.Then 10mL PBS solution, concussion, extraction of ocean eddies 10min, 4 DEG C, 9000g centrifugation will be added into every part of sample
5min discards upper-layer fat layer, takes intermediate layer solution to cross 0.45 μm of water phase filter membrane, obtains solution to be measured.
(2) 50 μ L solution to be measured are respectively taken, are measured using step B1 method, luminous intensity is measured.Then sample will be measured
The luminous intensity of product substitutes into the content that the working curve equation calculation that step B2 is established goes out the chloramphenicol in product to be tested.
Each solution used in above-mentioned detection method belongs to the conventional soln of this field, to further clarify, now to molten
Formula of liquid is described in detail:
(1) PBS solution: 0.01mol/L, pH7.4 phosphate buffer
(2) coating buffer: the carbonate buffer solution (CB) of 0.05mol/L, pH9.6
Na2CO3 1.59g
NaHCO3 2.93g
Add distilled water to 1000mL
(3) confining liquid
(4) cleaning solution
(5) luminol chemiluminescence substrate
A liquid: sodium peroxydisulfate stock solution 0.1mol/L
B liquid: Luminol stock solution 0.01mol/L:1.772g luminol 0.1mol/L sodium hydroxide solution dissolves,
It is settled to 1L；
The AB liquid of equivalent is taken to mix before use.
Embodiment 1
The embodiment measures chloramphenicol drug working curve and detection limit, while calculating working curve by the embodiment
Equation.
Working curve equation is established as described below:
It produces chloramphenicol standard solution: choosing chloramphenicol drug standards, then shaken up with acetonitrile dissolution and constant volume is produced
Then chloramphenicol standard solution accurately draws the chloramphenicol that different amounts of chloramphenicol standard solution produces gradient concentration with pipette
Standard solution；
The preparation of test solution: using blank health care product as blank sample, the processing of sample homogeneity accurately weighs respectively
Five parts of 2.0g samples are separately added into the chloramphenicol standard solution of various concentration, are configured to mark-on in 50mL polyfluortetraethylene pipe
It measures at 8 parts of sample of 0~1000 μ g/kg, 8 parts of samples is mixed and stand 30min, then 10mLPBS solution respectively, shake,
Extraction of ocean eddies 5min, 4 DEG C, 9000g be centrifuged 10min, discard upper-layer fat layer, intermediate layer solution taken to cross 0.45 μm of water phase filter membrane,
Obtain test solution.
Measuring method: ready ELISA Plate is added in the 50 μ L of test solution for extracting each concentration respectively with micropipettor
In (enzyme labelled antibody, coating, closing are completed), be added the enzyme labelled antibody of preparation, 50 holes μ L/, 37 DEG C of incubations 15min are carried out directly
Competitive reaction, each hole are added 50 hole μ L/ of luminol chemiluminescence substrate, measure luminous intensity immediately.
Four parameter fittings are carried out using origin8.0 software, are drawn standard working curve (Fig. 1), IC10 is detection limit, line
Property range be IC20-IC80, the results are shown in Table 1.
The standard curve of chloramphenicol, detection limit and the range of linearity in 1 health food of table
Embodiment 2
Sample accuracy test
Before test, chloramphenicol drug standards are chosen first, are then shaken up with acetonitrile dissolution and constant volume produces chloramphenicol mark
Quasi- solution.
The preparation of test solution: selecting free of contamination health food for blank sample, then handles sample homogeneity,
3 parts of 1.0g samples are accurately weighed respectively in 50mL polyfluortetraethylene pipe, are separately added into the chloramphenicol standard solution of various concentration,
It is configured to the sample that scalar quantity is 0.01,0.05,0.2 μ g/kg respectively, mixes and stand 30min, then 10mLPBS solution respectively,
Concussion, extraction of ocean eddies 5min, 4 DEG C, 9000g centrifugation 10min discard upper-layer fat layer, intermediate layer solution are taken to cross 0.45 μm of water phase
Filter membrane obtains test solution.
Measuring method: the coated enzyme of chloramphenicol is added in the 50 μ L of test solution for extracting each concentration respectively with micropipettor
In target, enzyme labelled antibody well prepared in advance, 50 holes μ L/ is added, 37 DEG C of incubation 15min carry out direct competitive reaction, and each hole adds
Enter 50 hole μ L/ of luminol chemiluminescence substrate, measures luminous intensity immediately.
Then the working curve equation calculation chloramphenicol concentration established by embodiment 1, and calculate the rate of recovery.Health food
Middle chloramphenicol TIANZHU XINGNAO Capsul and the coefficient of variation are shown in Table 2.
Chloramphenicol TIANZHU XINGNAO Capsul and the coefficient of variation in 2 health food of table
According to calculated result show this method be applied to health food in chloramphenicol the rate of recovery 69.5-96.4% it
Between, RSD is between 9.6-13.3%.
The foregoing describe the preferred embodiment for the present invention, and however, it is not to limit the invention.Those skilled in the art couple
Embodiment disclosed herein can carry out the improvements and changes without departing from scope and spirit.
Embodiment 3
Cross reacting rate test
Selection has 2 kinds of drug monitoring cross reacting rates of similar structures and similar functions with chloramphenicol.Pass through various drugs
Standard curve respectively obtain its 50% inhibition concentration.This method is calculated to the cross reacting rate of other medicines with following formula.Intersect
React smaller, then the method is better to chloramphenicol detection specificity.
Cross reacting rate (%)=(the chloramphenicol analog concentration for inhibiting concentration/inhibition 50% of 50% chloramphenicol) ×
100%
Experiment sets 3 repetitions, is as a result averaged.
The specificity of 3 chloramphenicol of table detection
The experimental results showed that the developed chemical luminescence detection method of the present invention is only capable of identification chloramphenicol, and to other similar
Object does not have cross reaction, and does not have cross reaction to other similar object.And method reported in the literature is to Thiamphenicol and fluorine at present
Benzene Buddhist nun, which examines, different degrees of cross reaction, which can be used for chloramphenicol high specific and quickly detect.
Claims (8)
1. a kind of method of direct competitive chemiluminescence immunoassay detection chloramphenicol, which is characterized in that the method includes following steps
It is rapid:
(1) antibody marker is prepared
Horseradish peroxidase HRP carries out coupling reaction with chloramphenicol resistance antibody through sodium metaperiodate activation and NaBH is further added4It is full
And methanol solution, horseradish peroxidase-chloramphenicol antibody marker is obtained after sufficiently carrying out reduction reaction；
(2) direct competitive chemiluminescence immunoassay detects
Pretreated sample is placed in each hole of ELISA Plate that chloramphenicol coating is completed, the antibody of step (1) preparation is added
Marker carries out direct competitive reaction；Chemiluminescent substrate is added in each hole after board-washing, measures luminous intensity.
2. the method for detection chloramphenicol according to claim 1, which is characterized in that step (1) uses sodium metaperiodate NaIO4
Horseradish peroxidase HRP is activated.
3. the method for detection chloramphenicol according to claim 1, which is characterized in that the horseradish peroxide after step (1) activation
Compound enzyme HRP is coupled with chloramphenicol antibody IgG.
4. the method for detection chloramphenicol according to claim 1, which is characterized in that step (1) uses NaBH4It is saturated methanol
Solution carries out reduction reaction to the enzyme mark chloramphenicol antibody after coupling.
5. the method for detection chloramphenicol according to claim 1, which is characterized in that step (2) pre-processes before being loaded includes
Be coated with, wash, sealing and etc.；Sample-adding post-processing includes washing step, is not necessarily to indirect competition reaction step, can carry out luminous
Strength detection.
6. the method for detection chloramphenicol according to claim 1, which is characterized in that step (2) described chemiluminescent substrate
For highly sensitive luminol chemiluminescence substrate.
7. a kind of chloramphenicol quantitative analysis method based on claim 1 the method, which is characterized in that the method also includes
Following steps:
A, working curve equation is established
Above-mentioned working solution is added in several pieces sample and prepares mark-on sample by the chloramphenicol standard working solution for preparing gradient concentration
Product obtain test solution after processing；According to the method for claim 1 step (2), the luminous intensity values of mark-on sample are measured, are used
The luminous intensity average value (B) of the standard solution of each concentration obtained divided by dilute solution (0 standard) luminous intensity
It is worth (B0) multiplied by 100%, i.e. percentage luminous value,
Percentage luminous value (%)=(B/B0) × 100%
Using the semilog value of the concentration (ng/mL) of standard solution as X-axis, percentage luminous value is Y-axis, draws standard curve；
B, the chloramphenicol quantitative detection of sample to be tested
Several pieces sample to be tested is weighed, obtains solution to be measured after processing, hair is measured using the method for claim 1 step (2)
Numerical value is substituted into the content that the working curve equation calculation that step a is established goes out chloramphenicol in sample to be tested by light intensity value.
8. a kind of chloramphenicol quantitative analysis method based on claim 1 the method, it is characterised in that the method includes with
Lower sample pre-treatments step:
1.0g (or 1.0mL) health food sample is accurately weighed, is respectively placed in 50mL polyfluortetraethylene pipe.It then will be to every part
10mL PBS solution is added in sample, concussion, extraction of ocean eddies 10min, 4 DEG C, 9000g centrifugation 5min discard upper-layer fat layer, take
Intermediate layer solution crosses 0.45 μm of water phase filter membrane, obtains solution to be measured.
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| US4277437A (en) * | 1978-04-05 | 1981-07-07 | Syva Company | Kit for carrying out chemically induced fluorescence immunoassay |
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