CN1256589C - Indirect enzyme-linked immunosorbent test method for quantitative detecting red tide hinge algae - Google Patents
Indirect enzyme-linked immunosorbent test method for quantitative detecting red tide hinge algae Download PDFInfo
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- CN1256589C CN1256589C CN 200410052856 CN200410052856A CN1256589C CN 1256589 C CN1256589 C CN 1256589C CN 200410052856 CN200410052856 CN 200410052856 CN 200410052856 A CN200410052856 A CN 200410052856A CN 1256589 C CN1256589 C CN 1256589C
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Abstract
The present invention relates to a method for the quantitative detection of heterosigma akashiwo through indirect enzyme linked immunosorbent assay, which is used for the technical field of marine organisms. Antibodies for identifying heterosigma akashiwo are used as a basis. An enzyme labeled plate is coated with a standard sample of heterosigma akashiwo and a sample to be detected, and after incubation at 37 DEG C, the enzyme labeled plate is sealed by solution of defatted milk powder. After the plate is washed, diluted solution of specific anti-heterosigma akashiwo antiserum is added. After incubation at 37 DEG C, the plate is washed, and enzyme labeled second antibody solution is added. After incubation at 37 DEG C and plate washing, enzymatic reaction substrates are added, and after incubation, reaction terminating solution is added to terminate reaction. Then, an enzyme labeled instrument is utilized to measure the absorbance value of each hole on the plate, and a standard curve of heterosigma akashiwo detection is obtained through data transformation. The curve shape range is calculated to obtain a linear equation, and heterosigma akashiwo in the sample to be detected is quantitated. The method of the present invention can be used for the rapid and convenient quantitative detection of heterosigma akashiwo and can overcome the defects of the conventional detection of heterosigma akashiwo.
Description
Technical field
The present invention relates to a kind of method of detection by quantitative Heterosigma akashiwo, specifically is a kind of indirect enzyme-linked immunosorbent assay method of detection by quantitative Heterosigma akashiwo.Be used for the marine biotechnology field.
Background technology
Heterosigma akashiwo (Heterosigma akashiwo) is a kind of common harmful algae that is distributed widely in world's immediate offshore area, it also is the common red tide plankton in China coastal waters, the report that repeatedly causes red tide was once arranged, bring grave danger for sea fishery and human health.Therefore, detect Heterosigma akashiwo in time, accurately and rapidly, it is significant to understand its dynamic change in the ocean at any time.On taxonomy, Heterosigma akashiwo belongs to the different curved Trentepohlia of Xanthophyta.Traditional discrimination method, promptly distinguish Heterosigma akashiwo from morphology, mainly be to utilize optical microscope Direct observation and counting, there is certain difficulty during operation, and process is loaded down with trivial details, time-consuming, effort, workload is very big for a long time when sample size, so is mainly used in the qualitative of algae and separates, and is not suitable for the quantitative examination of algae.Therefore, develop the new method and the new technology that are used for the fast detecting Heterosigma akashiwo and have realistic meaning.But, at present little about the progress of this respect both at home and abroad, do not form as yet and very effectively carry out qualitative and mature technology quantitative measurement this algae.Indirect enzyme-linked immunologic adsorption test method based on antibody technique has advantages such as special, sensitive, efficient, easy to operate, that expense is cheap, and can analyze a large amount of samples simultaneously, is a kind of effective means of target antigen being carried out qualitative and quantitative analysis.At present, the indirect enzyme-linked immunosorbent assay method mainly detects and microbiology detects application in the research in medical science, and it is then less to be used for report that whole frustule is detected.In addition, though the preparation process of frustule antibody is simple, be difficult for obtaining the antibody of high specific.
Find through retrieval prior art, Xin Zeyu etc. are in " hi-tech communication " (2003.3, the 13rd volume, the 74-79 page or leaf) " foundation and the application of two kinds of Chaetoceros enzyme linked immunosorbent detection methods " delivered on, this article has been introduced and has been prepared mouse-anti and revolve chain Chaetoceros and weak Chaetoceros polyclonal antibody, found that two kinds of antibody and three kinds of Chaetoceros belong to marine alga very strong cross reaction is arranged, and to belong to marine alga cross reactions very little or do not have with other.Use these two kinds of antibody and set up the state of conflict enzyme linked immunosorbent assay detection method of revolving chain and weak Chaetoceros respectively, detection limit reaches 146 and 521 frustules respectively.This competition enzyme-linked immunosorbent adsorption test has higher detection sensitivity, but this detection method need be used more frustule cracked solution coated elisa plate in advance, therefore need carry out a large amount of Continuous Cultivation of frustule, has increased the workload of testing process.In addition, the antibody that does not have acquisition to have high specific between kind makes the actual application value that detects reduce greatly.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art and defective, a kind of indirect enzyme-linked immunosorbent assay method by high specific polyclonal antibody detection by quantitative Heterosigma akashiwo is provided, make it quick, easy quantitatively, and objective, exactly this algae is detected.
The present invention is achieved by the following technical solutions, detection method provided by the invention, it is the indirect enzyme-linked immunosorbent assay method, discern the antibody of Heterosigma akashiwo as the basis with selectivity ground, at first with Heterosigma akashiwo standard model and sample coated elisa plate to be checked, hatching the back for 37 ℃ seals with skimmed milk power solution, add the anti-Heterosigma akashiwo antiserum of specificity dilute solution after washing plate, wash plate after hatching for 37 ℃, add ELIAS secondary antibody solution, hatch for 37 ℃, add enzyme reaction substrate after washing plate, hatch the back and add cessation reaction liquid cessation reaction; Use the light absorption value in each hole on the microplate reader assay plate then, through after the data-switching, draw the typical curve of Heterosigma akashiwo detection by quantitative, its linear range is carried out quantitatively the Heterosigma akashiwo in the testing sample according to the equation that obtains through calculating straight-line equation.
Below by step the present invention is done further concrete the qualification:
(1) with Heterosigma akashiwo standard model and sample coated elisa plate to be checked, every hole 100 microlitres, 37 ℃ were sealed with 10% skimmed milk power solution after 20-40 minute, wash plate after 20-40 minute for 37 ℃, add the anti-Heterosigma akashiwo antiserum of specificity dilute solution, hatch after 10-20 minute for 37 ℃ and wash plate, add ELIAS secondary antibody solution, hatched 10-20 minute for 37 ℃, add enzyme reaction substrate after washing plate, hatch and add cessation reaction liquid cessation reaction after 10-15 minute.
(2) with the light absorption value in each hole on the microplate reader assay plate, calculate corresponding absorptance % then, computing formula is A/A
0* 100%, A
0Be saturated light absorption value, value 3.5.A is the light absorption value in each hole, then calculate the Logit value of each hole absorptance, with be ordinate, common logarithm value with algae cell density in the standard model gradient dilution liquid is a horizontal ordinate, draw out the typical curve of Heterosigma akashiwo detection by quantitative, find out its range of linearity and calculate the Logit value of absorptance and the straight line correlation equation of the common logarithm value of corresponding algae cell density, linearly dependent coefficient must be more than 0.99, according to the linear equation that obtains, the Logit value of substitution testing sample hole absorptance, promptly obtain the common logarithm value of corresponding algae cell density, through exponent arithmetic finish to Heterosigma akashiwo in the testing sample quantitatively.
Principle of the present invention is that specific antibody can be discerned specific antigenic determinant single-mindedly, thereby can carry out qualitative and detection by quantitative to antigen, under 37 ℃ of conditions of hatching, frustule in standard model and the sample to be checked can be adsorbed by the ELISA Plate of high absorption affinity, remove by closed process in unnecessary protein adsorption site, add after the specific antisera dilute solution of high-affinity, frustule meeting on the ELISA Plate and the antibodies in the solution, can calculate frustule content in the corresponding sample to be checked by the antibody amount that detects combination on the ELISA Plate, therefore can carry out quantitative measurement to unknown sample exactly.
Antibody of the present invention, it can obtain via Heterosigma akashiwo immunization experiment animal, and these animals used as test generally include (but being not limited to) rabbit, mouse, pig, dog, ox, sheep, horse etc.Specific antibody can directly use, the specificity of antibody can be via some conventional means on the other hand, antibody sealing technique of knowing as those skilled in that art etc., be improved, in addition, antibody itself also can carry out mark and uses with radioactive isotope, biotin, enzyme, fluorescein or other chemiluminescent substances.
Anti-Heterosigma akashiwo antibody of the present invention has species specificity preferably.Utilize indirect enzyme-linked immunosorbent assay to measure cross reaction between described antibody and other algae, the result shows that it can only discern Heterosigma akashiwo and other algae of nonrecognition, so can be used for the qualitative identification of Heterosigma akashiwo.
Described with Heterosigma akashiwo standard model coated elisa plate, be meant: with the gradient dilution solution coated elisa plate that contains the Heterosigma akashiwo cell through accurate light microscopic counting.Described with Heterosigma akashiwo sample coated elisa plate to be checked, be meant: with natural sea-water sample to be checked or natural sea-water sample concentration solution coated elisa plate.
This method has not only overcome the shortcoming in the Heterosigma akashiwo conventional sense, and it is few to analyze required sample size, and detection limit is low, and is highly sensitive.
Owing to the invention provides the method for quick, easy this algae of detection by quantitative, make the dynamic change of Heterosigma akashiwo in certain sea area of monitoring or the plant's maritime interior waters become possibility.By the dynamic data that monitors, can grasp the quantity of this kind algae in the seawater, and in time adjust to reduce economic loss; Simultaneously, these dynamic datas are combined with other data (such as the quantity of nutritive salt, other species, hydrometeorological data etc.), the influencing each other relation that can be in the scientific research marine ecology between each factor provides some useful helps.
Embodiment
Below in conjunction with embodiment the inventive method is done further to understand:
Embodiment 1. is used to detect the preparation of the antibody of Heterosigma akashiwo
The used Heterosigma akashiwo algae kind of the present invention is provided by the Life Science and Technology department of the Chinese Academy of Sciences of Chinese Marine University.By obtaining pure culture at microscopically picking single algae cell repeatedly.Used nutrient culture media is conventional f/2 nutrient culture media, and condition of culture is: illumination/dark cycle is 14h/10h, and intensity of illumination is 3000Lx, and cultivation temperature is 18 ℃-22 ℃.
Be in the Heterosigma akashiwo nutrient solution of exponential phase, after formaldehyde fixed (final concentration 0.5-1%), the centrifugal 10min of 10000r/min collects frustule, clean one time with distilled water again, phosphate buffered saline(PBS) is cleaned twice, per step, all by centrifugal collection frustule, centrifugal condition was 10000r/min, 10min.Change frustule over to the 1.5ml centrifuge tube, get rid of residual moisture ,-20 ℃ of preservations are standby.Face and use preceding taking-up, the phosphate buffered saline(PBS) resuspension of every effective 0.6ml sterilization, mixing, with isopyknic Fu Shi Freund's complete adjuvant (Sigma company) mixing and emulsifying, fully, emulsion is used for immunizing rabbit to the emulsification degree as far as possible, select hypodermic injection and muscle injection mode, dosage is 10
7Individual cell/rabbit.Interval booster immunizations after two weeks, except that adjuvant was changed to freund 's incomplete adjuvant, other step was constant, afterwards every two all booster immunizations once.In a week behind the 3rd booster immunization, rabbit ear vein is got blood, and separation of serum detects serum titer with conventional enzyme linked immunosorbent assay, and when serum titer arrived higher level, the rabbit whole serum was gathered in no longer immunity, and check back packing is stored in-20 ℃.The anti-Heterosigma akashiwo antiserum titre of Huo Deing is 1: 22000 at last.
The specific evaluation of embodiment 2. anti-Heterosigma akashiwo antiserums
Present embodiment has selected for use the common a few strain phytoplanktons in China coastal waters as the reference algae, they are: revolve chain Chaetoceros (Chaetoceros curvisetus), weak Chaetoceros (Chaetoceros debilis), very thin Chaetoceros (Chaetoceros gracilis), miniature Chaetoceros (Chaetoceros minutissimus), middle Skeletonemacostatum (Skeletonema costatum), Thalassiosira nordenskioldi Cleve (Thalassiosira nordenskioldi), membranous boat-shaped algae (Navicula membranacea), the pseudo-rhombus algae (Pseudo-nitzschia pungens) of spine, Nitzschia closterium minutissima (Nitzschia closterium), Michaelis unarmored dinoflagellate (Gymnodinium mikimotoi), unarmored dinoflagellate (Gymnodinium sp.), red englena (Gymnodinium sanguineum), Alexandrium tamarense (Alexandrium tamarens), minisize Prorocentrum (Prorocentrum minimum), ocean Prorocentrum (Prorocentrum micans), Prorocentrum donghaiense (Prorocentrum donghaiense), they all separate from natural sea-water, by obtaining pure culture at microscopically picking single algae cell repeatedly.Cultivate and collect these algaes by embodiment 1 described method.
Carrying out the sero-fast specificity of anti-Heterosigma akashiwo by the described indirect enzyme-linked immunosorbent assay step of claim identifies.All algaes after the microscopic counting, are got same cell amount (2.0 * 10 with 1ml phosphate buffered saline(PBS) resuspension
4) be suspended in phosphate buffered saline(PBS) (final volume 1ml), as negative control, data result is listed in table 1 with the phosphate buffered saline(PBS) solution that does not contain frustule.As can be seen from the table, Heterosigma akashiwo light absorption value/negative control light absorption value (P/N)＞2.1, the P/N value of other marine algas illustrate that all below 1.5 anti-Heterosigma akashiwo antiserum has very high specificity, can discern Heterosigma akashiwo single-mindedly, therefore can be used for the qualitative detection of Heterosigma akashiwo.
Therefore, for certain sample to be checked, carry out the indirect enzyme-linked immunosorbent assay reaction with the described step of present embodiment, if the P/N value greater than 2.1, promptly has the existence of Heterosigma akashiwo in this sample of decidable; If this sample is a pure culture, then this pure culture of decidable is a Heterosigma akashiwo.The advantage of this qualitative determination method is because use is the selectivity antiserum of Heterosigma akashiwo, therefore whether to have Heterosigma akashiwo in the test sample very exactly.
The quantitative measurement of 3. pairs of Heterosigma akashiwos of embodiment
1. press embodiment 1 described method and cultivate and collect Heterosigma akashiwo.
2. to contain 0,38,192,960,4800,24000,120000, the Heterosigma akashiwo cell standard solution of 600000 cells/ml and detected sample solution coated elisa plate, every hole 100 microlitres, 37 ℃ of incubations sealed with 10% skimmed milk power solution after 20 minutes, 37 ℃ of incubations were washed plate after 20 minutes, add anti-Heterosigma akashiwo serum dilution (1: 15000), hatch after 15 minutes for 37 ℃ and wash plate, add ELIAS secondary antibody solution (goat anti-rabbit igg-horseradish peroxidase, dilute 12000 times), hatched 15 minutes for 37 ℃, add enzyme reaction substrate TMB matrix liquid after washing plate, hatch adding 2mol/L sulfuric acid cessation reaction after 10 minutes, read the light absorption value at each 450nm place, hole on the plate.
3. typical curve: the common logarithm value with frustule number in the Heterosigma akashiwo standard model solution is a horizontal ordinate, with the absorptance (%) of standard model, i.e. standard model hole light absorption value A/A
0* 100, be ordinate, make typical curve.Common logarithm value with algae cell density in the Heterosigma akashiwo standard model solution is a horizontal ordinate, with the Logit value of absorptance, i.e. ln[A/ (A
0-A)], be ordinate, set up the straight-line equation of typical curve linear detection range.Its linear equation is ln[A/ (A
0-A)]=0.7927 * log algae cell density-5.1664, related coefficient is R=0.9954, linear detection range is between 192～480000 cells/ml.
4. quantitative Analysis and analysis:
Light absorption value according to testing sample, at first calculate absorptance separately, then calculate the Logit value of absorptance, the linear equation of substitution typical curve, obtain corresponding log algae cell density value, can calculate the density of Heterosigma akashiwo cell in the testing sample easily, the density of Heterosigma akashiwo also can be obtained a result through simple computation in the corresponding original water sample.
Embodiment 4. uses the actual quantification of this law to seawater sample
According to embodiment 3 described methods, 5 parts of seawater samples have been carried out the detection by quantitative of Heterosigma akashiwo cell.To these 5 parts of seawater samples, after detecting with indirect enzyme-linked immunosorbent assay method and microscopic counting respectively, the results are shown in table 2.Through comparative analysis, find that the result of indirect enzyme-linked immunosorbent assay method and microscopic counting detection is identical substantially.
The sero-fast cross reaction of each marine alga of table 1 and anti-Heterosigma akashiwo detects
| The marine alga title | Light absorption value | The P/N value |
| Heterosigma akashiwo revolves the pseudo-rhombus algae of the weak Chaetoceros Chaetoceros gracilis miniature Chaetoceros Skeletonema Costatum Thalassiosira nordenskioldi Cleve of chain Chaetoceros membranous boat-shaped algae spine Nitzschia closterium minutissima Michaelis unarmored dinoflagellate unarmored dinoflagellate red englena Alexandrium tamarense minisize Prorocentrum ocean Prorocentrum Prorocentrum donghaiense negative control | 0.403 0.103 0.121 0.117 0.108 0.114 0.129 0.131 0.092 0.058 0.131 0.097 0.104 0.094 0.115 0.074 0.113 0.085 | 4.7 1.2 1.4 1.4 1.3 1.3 1.5 1.5 1.1 0.7 1.5 1.1 1.2 1.1 1.4 0.9 1.3 1.0 |
The testing result of Heterosigma akashiwo density in table 2 seawater sample.Sample is that the axenic culture of Heterosigma akashiwo adds the natural sea-water dilution and forms in the table.The measurement result of density (individual cells/ml) is the mean+SD of 6 parts of parallel sample measurement results.
| Sample number into spectrum | The indirect enzyme-linked immunosorbent assay measurement result | The microscopic count result |
| 1 2 | (6.4±1.1)×10 2 (2.0±0.2)×10 3 | (7.5±0.4)×10 2 (2.6±0.4)×10 3 |
| 3 4 5 | (6.1±0.6)×10 3 (1.7±0.2)×10 4 (4.9±0.5)×10 6 | (6.6±0.2)×10 3 (1.5±0.3)×10 4 (5.3±0.1)×10 6 |
The present invention has successfully prepared the antibody of anti-Heterosigma akashiwo, and has set up the indirect enzyme-linked immunosorbent assay detection method, detects Heterosigma akashiwo but use this method qualitative, quantitative ground.The inventor uses the high specific polyclonal antibody that the Heterosigma akashiwo cell is carried out the indirect enzyme-linked immunosorbent assay qualitative and quantitative detection in the world first, and has obtained result preferably.The advantage of this method: can carry out simultaneously quantitatively and qualitative detection, both know whether have Heterosigma akashiwo to exist in the sample to be checked, can know simultaneously also how many numbers of this algae has by this method.The tangible advantage of another of this method is that whole process can be finished within several hours fast, and testing result is more accurate simultaneously.
Claims (5)
1. the indirect enzyme-linked immunosorbent assay method of a detection by quantitative Heterosigma akashiwo, it is characterized in that, antibody based on the identification Heterosigma akashiwo, at first with Heterosigma akashiwo standard model and sample coated elisa plate to be checked, hatching the back for 37 ℃ seals with skimmed milk power solution, add the anti-Heterosigma akashiwo antiserum of specificity dilute solution after washing plate, wash plate after hatching for 37 ℃, add ELIAS secondary antibody solution, hatch for 37 ℃, add enzyme reaction substrate after washing plate, hatch the back and add cessation reaction liquid cessation reaction; Use the light absorption value in each hole on the microplate reader assay plate then, through after the data-switching, draw the typical curve that Heterosigma akashiwo detects, its linear range is carried out quantitatively the Heterosigma akashiwo in the testing sample through calculating linear equation.
2. the indirect enzyme-linked immunosorbent assay method of detection by quantitative Heterosigma akashiwo according to claim 1 is characterized in that, below does further to limit by step:
(1) with Heterosigma akashiwo standard model and sample coated elisa plate to be checked, every hole 100 microlitres, 37 ℃ were sealed with 10% skimmed milk power solution after 20-40 minute, wash plate after 20-40 minute for 37 ℃, add the anti-Heterosigma akashiwo antiserum of specificity dilute solution, hatch after 10-20 minute for 37 ℃ and wash plate, add ELIAS secondary antibody solution, hatched 10-20 minute for 37 ℃, add enzyme reaction substrate after washing plate, hatch and add cessation reaction liquid cessation reaction after 10-15 minute;
(2) with the light absorption value in each hole on the microplate reader assay plate, calculate corresponding absorptance % then, computing formula is A/A
0* 100%, A
0Be saturated light absorption value, value 3.5, A is the light absorption value in each hole, then calculate the Logit value of each hole absorptance, with be ordinate, common logarithm value with algae cell density in the standard model gradient dilution liquid is a horizontal ordinate, draw out the typical curve of Heterosigma akashiwo detection by quantitative, find out its range of linearity and calculate the Logit value of absorptance and the straight line correlation equation of the common logarithm value of corresponding algae cell density, linearly dependent coefficient must be more than 0.99, according to the linear equation that obtains, the Logit value of substitution testing sample hole absorptance, promptly obtain the common logarithm value of corresponding algae cell density, through exponent arithmetic finish to Heterosigma akashiwo in the testing sample quantitatively.
3. the indirect enzyme-linked immunosorbent assay method of detection by quantitative Heterosigma akashiwo according to claim 1, it is characterized in that, the antibody of described identification Heterosigma akashiwo, it can discern Heterosigma akashiwo specifically, obtains via Heterosigma akashiwo cellular immunity animal used as test.
4, the indirect enzyme-linked immunosorbent assay method of detection by quantitative Heterosigma akashiwo according to claim 1, it is characterized in that, described with Heterosigma akashiwo standard model coated elisa plate, be meant: with the gradient dilution solution coated elisa plate that contains the Heterosigma akashiwo cell through accurate light microscopic counting.
5, the indirect enzyme-linked immunosorbent assay method of detection by quantitative Heterosigma akashiwo according to claim 1, it is characterized in that, described with Heterosigma akashiwo sample coated elisa plate to be checked, be meant: with natural sea-water sample to be checked or natural sea-water sample concentration solution coated elisa plate.
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