CN111289742B - Spot-simulating standard substance and application thereof - Google Patents
Spot-simulating standard substance and application thereof Download PDFInfo
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- CN111289742B CN111289742B CN202010245056.9A CN202010245056A CN111289742B CN 111289742 B CN111289742 B CN 111289742B CN 202010245056 A CN202010245056 A CN 202010245056A CN 111289742 B CN111289742 B CN 111289742B
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- 239000000126 substance Substances 0.000 title claims abstract description 75
- 239000002985 plastic film Substances 0.000 claims abstract description 21
- 229920006255 plastic film Polymers 0.000 claims abstract description 21
- 238000002965 ELISA Methods 0.000 claims description 24
- 239000011241 protective layer Substances 0.000 claims description 17
- 238000004113 cell culture Methods 0.000 claims description 9
- 238000002834 transmittance Methods 0.000 claims description 4
- 230000005540 biological transmission Effects 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 abstract description 6
- 238000000034 method Methods 0.000 description 18
- 238000005259 measurement Methods 0.000 description 12
- 238000004088 simulation Methods 0.000 description 10
- 238000013461 design Methods 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 238000011835 investigation Methods 0.000 description 5
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 4
- 230000007774 longterm Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 238000001134 F-test Methods 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 238000004164 analytical calibration Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000556 factor analysis Methods 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a standard substance for simulating spots and application thereof. The invention takes the plastic film as a carrier, prints the spots with determined quantity on specific hole sites of the plastic film, is used as a standard substance for simulating the spots, has reliable quantity value and good uniformity and stability, and provides an experimental foundation for daily calibration work of instruments and the preparation of national metering calibration standards.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, relates to a simulated spot standard substance and application thereof, and particularly relates to a simulated spot standard substance and application thereof in calibration of a spot ELISA analyzer.
Background
Spot Enzyme-linked immunosorbent assay (Enzyme-linked immunospot, ELISPOT) detects antibody-secreting cells or cytokine-secreting cells at the single cell level and counts the number of spot-forming cells (i.e., spot counts). The spot counting is the most important step in ELISPOT data processing, and the spot ELISA analyzer can fully automatically collect, count and analyze spot images of all holes on an ELISPOT plate, so that the cellular immunity or humoral immunity function of an organism is evaluated, and the method is widely applied to the related immunology fields such as vaccine research, drug screening, infectious disease diagnosis and research, tumor immunity research and the like. It is worth mentioning that in clinical evaluation of vaccines, spot enzyme-linked immunosorbent assay has been mandatory for ELISPOT, which can effectively avoid bias and increase objectivity, repeatability and counting speed. However, no method for calibrating a spot ELISA analyzer is currently available at home and abroad.
Because the cell preservation condition is harsh, the stability is poor, and the cell preservation condition does not meet the requirements of standard substances, the cell preservation condition is difficult to be used for preparing the standard substances to realize the quantity value transmission. Therefore, the spot standard substance is simulated, an experimental basis is provided for the calibration work of the spot ELISA analyzer, and a current calibration method of the spot ELISA analyzer is established.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides the simulated spot standard substance and the application thereof, and develops the simulated spot standard substance according to the technical parameters of the spot ELISA analyzer and in combination with the culture growth characteristics of the ELISA spots, so that the simulated spot standard substance has reliable magnitude and good uniformity and stability, and is beneficial to providing an experimental foundation for the calibration work of the spot ELISA analyzer.
To achieve the purpose, the technical scheme is as follows:
in a first aspect, the invention provides a standard substance for simulating spots, the standard substance comprises a plastic film, a plurality of hole sites are uniformly distributed on the plastic film, and a certain number of spots are printed in the hole sites.
According to the invention, the plastic film is used as a carrier, the spots with determined quantity are printed on specific hole sites of the plastic film, and the spots are used as standard substances for simulating the spots, so that the quantity is reliable, the uniformity and the stability are good, and an experimental basis is provided for daily calibration work of an instrument and the preparation of national metering calibration standards.
Preferably, 96 hole sites are uniformly distributed on the plastic film.
Preferably, the hole sites are distributed in an 8×12 arrangement.
Preferably, the well sites are in a standard 96 well cell culture plate arrangement.
In the invention, the hole sites are arranged in a mode consistent with the standard 96-hole cell culture plates, so that the invention has strong compatibility with instruments and is suitable for popularization and application.
Preferably, the spots are located in the E3, B6, G6, E9 holes of the plastic film.
In the invention, the E3, B6, G6 and E9 hole sites are positioned in the middle position of the 96-hole cell culture plate, the distances among the hole sites are approximately consistent, and the method has the characteristic that different numbers of simulated spots are printed on the E3, B6, G6 and E9 hole sites, the number of the spots covers the number commonly detected in the spot ELISA test, and the calibration requirement of the spot ELISA analyzer is met.
Preferably, the number of spots is (50-500), for example, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500.
According to the present invention, the optimal number of spot detections in the spot ELISA assay is (100-400), and the number of spots is set to 50-500 in order to cover this range.
Preferably, the spots differ in number in the E3, B6, G6, E9 hole sites.
Preferably, the number of spots in the E3 hole site is (50-500), for example, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500, preferably 50.
Preferably, the number of spots in the B6 hole site is (50-500), for example, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500, preferably 100.
Preferably, the number of spots in the G6 hole site is (50-500), for example, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500, preferably 200.
Preferably, the number of spots in the E9 hole site is (50-500), for example, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500, preferably 500.
Preferably, the hole sites comprise locating hole sites.
In the invention, a positioning hole site is arranged on a standard substance, and after the standard substance is placed in a carrying bin of an instrument to be calibrated, the position of the standard substance in the instrument can be determined through the positioning hole site.
Preferably, the positioning hole sites are positioned at A1, A12, H1 and H12.
Preferably, the standard substance further comprises a protective layer.
Preferably, the standard substance further comprises a background layer.
Preferably, the background layer comprises white background paper.
As a preferable technical scheme, the invention provides a spot-simulating standard substance, which is sequentially provided with a first protective layer, a plastic film, white background paper and a second protective layer from top to bottom.
Preferably, the first protective layer and the second protective layer are connected by a connecting means.
Preferably, the standard substance is in the shape of a standard 96-well cell culture plate.
In a second aspect, the present invention provides the use of a standard substance as described in the first aspect in instrument metering calibration.
Preferably, the apparatus comprises a spot enzyme-linked immunoassay.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the technical parameters of the spot ELISA analyzer and combining with the culture growth characteristics of ELISA spots, the invention designs a standard substance for simulating spots, wherein the external shape of the standard 96-hole cell culture plate is referred to as a standard, the external shape of the standard 96-hole cell culture plate comprises a first protective layer and a second protective layer, the internal part of the standard 96-hole cell culture plate comprises a high-light-transmission plastic film and white background paper, the first protective layer and the second protective layer are fastened by means of screw connection, and the size of the standard substance is 12.7cm long, 8.5cm wide and 1.3cm high;
(2) The simulated spot standard substance prepared by the invention has the advantages of reliable quantity, good uniformity and stability, convenient carrying, good compatibility with instruments and wide application range, has wide application prospect in the metering calibration work of the spot ELISA analyzer, performs experimental study on the metering characteristics of instrument indication errors and repeatability, and provides an experimental basis for the daily calibration work of the instruments and the preparation of national metering calibration standards.
Drawings
FIG. 1 is a pattern design (front plan view) of a simulated speckle standard;
fig. 2 (a) is an E3 hole site picture, fig. 2 (B) is a B6 hole site picture, fig. 2 (C) is a G6 hole site picture, and fig. 2 (D) is an E9 hole site picture.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
The main instrument is as follows:
the traceability of the measurement of the instrument is ensured by the calibration of a metering mechanism, and the calibrated standard plate is used for confirmation before each use, so that the instrument is ensured to be in a normal working state.
EXAMPLE 1 preparation of mock speckle Standard substance
According to the technical parameters of the spot ELISA analyzer, adopting AutoCAD design to simulate spot standard substance patterns, and finally imaging the simulated spots on a plastic film. As shown in fig. 1, a pattern design diagram (front plan view) of the simulated speckle standard substance is shown: (1) The size and the hole position of the plastic film are consistent with those of a standard 96-hole plate; (2) The simulated spot holes are uniformly distributed on the plastic film according to the arrangement mode of the 96-hole plate; (3) A1, A12, H1 and H12 holes are positioning holes, and a SIMT word is designed; (4) The E3, B6, G6 and E9 wells were designed with 50, 100, 200 and 500 simulated spots of 50 μm diameter, 12.7cm long, 8.5cm wide and 1.3cm high, respectively.
Example 2 reproducibility and reproducibility of the method for detecting a simulated speckle Standard
The images of the holes of the simulated spot standard substances E3, B6, G6 and E9 are shot by an image measuring machine, as shown in fig. 2 (A), 2 (B), 2 (C) and 2 (D), spots in the holes are clearly visible, the arrangement is uniform, and 50, 100, 200 and 500 simulated spots with the diameters of 50 mu m are respectively arranged, so that the method is suitable for manual identification and counting.
Randomly extracting a piece of simulation spot standard substance candidate, respectively shooting E3, B6, G6 and E9 holes of the simulation spot standard substance by using an image measuring machine, printing on paper, detecting by adopting a manual identification counting method, repeating for 7 times, and checking the repeatability of the method with the precision of 0%.
Ten simulated spot standard substance candidates are randomly extracted, E3, B6, G6 and E9 holes of the simulated spot standard substances are respectively shot by an image measuring machine and printed on paper, detection is carried out by adopting a manual identification counting method, and the reproducibility of the method is inspected with the precision of (0-0.2%).
Therefore, the constructed simulated spot standard substance has higher repeatability and reproducibility precision, and completely meets the development requirement of the simulated spot standard substance.
Example 3 uniformity detection of simulated speckle Standard
200 blocks of simulated spot standard substance candidates prepared in the same batch are randomly extracted according to the rule of general principles and statistical principles of standard substance determination of JJF 1343-2012, 15 blocks of the candidate are randomly extracted, each block is counted three times, 3 repeated counting data are taken as a group, 15 groups of 45 data are taken as a whole, and a single factor analysis of variance (F-test) is adopted for uniformity test, and the result shows that under the 95% confidence level, the F value is smaller than F 0.05 (14, 30) proving the batchThe standard substances were all homogeneous.
Example 4 stability investigation of a simulated speckle Standard
Stability is a fundamental property of standard substances and measures to maintain stability of candidates must go through the whole process of standard substance development. Since the standard substance inevitably involves transportation during use, the present embodiment performs short-term and long-term stability investigation on the same batch of standard substance, respectively.
(1) Short term stability investigation
Short term stability studies employ simultaneous stability studies, i.e. measurements of all stability studies can be performed under repetitive conditions. The experiment was arranged for two short-term stability studies, with 3 standard substance candidates randomly extracted as one transport sample: the first time is 2 months of Shanghai-Beijing high-speed rail back and forth, and two city laboratories-high-speed rail stations are all transported by automobiles; and the standard substance candidate is sent to Guangzhou city by adopting 'Yunda express' for 7 months, and then the standard substance candidate is sent back to Shanghai laboratory by 'Shunfeng' express after being received by a receiver in Guangzhou city. Both transport candidates were tested simultaneously with normal storage candidates for short term stability, and each sample was assayed 3 times in duplicate.
The results were compared using t-test, and on a 95% confidence level, there was no significant difference between the two, and the simulated spot standard was stable under transportation conditions, meeting the actual transportation requirements.
(2) Long-term stability investigation
Long-term stability studies were performed using classical stability studies, i.e. samples prepared simultaneously were measured over time under the same conditions. The principle of density-first and then thinning is adopted, 3 simulated spot standard substances are randomly extracted on the day of sample preparation, 1, 2, 3, 6 and 12 months, each sample is repeatedly measured for 3 times, and long-term stability investigation is carried out. Since no physical model can truly describe the wear mechanism of the spots in the simulated spot standard substance, the stability of the standard substance is evaluated by using a linear model recommended by the ISO code 35.
As a result, the slopes of the respective standards were not significant, indicating that the number of spots in the standard had no significant tendency to rise or fall within a predetermined period of time, and therefore, the standard was stored at room temperature (23.+ -. 1) ℃ in the laboratory under a dark condition, and it was confirmed that the standard was stable within 1 year. At present, the stability test of the standard substance is still in continuous monitoring.
Example 5 evaluation of the fixed value and uncertainty of the Standard substance of the simulated spots
(1) Standard substance constant value
Three simulated spot standard substances are randomly extracted from the same batch of standard substances by 8 testers respectively, in order to avoid leading of design values, the testers are ensured to keep an unknown state of the design values, E3 holes, B6 holes, G6 holes and E9 holes of the simulated spot standard substances are respectively shot by an image measuring machine and printed on paper, detection is carried out by adopting a manual identification counting method, and each block is counted for three times.
The results were subjected to the Ke Kelun test and the gabplace test, and the data of all units were retained and participated in the final determination, and the average value and the total average value of the count results of each experimenter are shown in table 1.
TABLE 1 simulation of the fixed value results for the speckle Standard substance
(2) Uncertainty evaluation of standard substance fixed value result
The uncertainty source of the simulated spot standard consists of three parts: uncertainty caused by non-uniformity of the standard substance; uncertainty caused by variability of the standard substance during the expiration date; uncertainty caused by the process of determining the value of the standard substance is mainly uncertainty introduced by statistics of measured data. The statistical results are shown in Table 2.
TABLE 2 simulation of uncertainty list of speckle criteria
And combining the uncertainty results to obtain a fixed value result and uncertainty of the standard substance of the simulated spots, wherein the fixed value result and the uncertainty are shown in Table 3.
TABLE 3 simulation of the spot standard mass constant value results and uncertainty
Example 6 application of simulated speckle Standard in instrument metrology calibration
In the embodiment, the indication error and the repeatability measurement characteristic of the spot ELISA analyzer are inspected by using the successfully developed simulated spot standard substance, and experimental verification is carried out in more than 10 instruments such as AID company, CTL company and the like, the result is shown in Table 4, the universality of the method is proved, and the experimental scheme is as follows:
(1) Indication error
The error of the instrument is expressed as the relative error of the measured value and the standard value of the spot simulation standard substance. Starting an instrument, debugging according to a use instruction, entering a normal working state, putting a spot simulation standard substance into a carrying bin and fixing, photographing and counting holes E3, B6, G6 and E9 respectively, repeatedly measuring for three times, recording measured values, calculating the indication error of each calibration point according to a formula (1), and taking the maximum absolute value as the indication error of the instrument.
Wherein DeltaN is the error (%) of the indication,to measure the average value, N s Is the standard value.
(2) Repeatability of
Measurement repeatability is expressed as the relative standard deviation of 6 measurements of the simulated spot standard. The instrument is debugged according to the instruction book, enters a normal working state, puts the spot simulation standard substance into a carrying bin and fixes the spot simulation standard substance, photographs and counts 6 times the G6 hole with the standard value of 200 of the simulation spot standard substance, and calculates the measurement repeatability according to the formula (2).
Wherein RSD is the relative standard deviation (%), N is the number of measurements, N i For the value of the single measurement it is,for the measurement of the average value.
The error in the indication and the repeatability of the 10 instruments tested are shown in table 4.
Table 4 application of simulated speckle criteria in instrument calibration
In summary, the spot for simulating the spot ELISA reaction culture adopts the AutoCAD design to simulate the spot pattern, the appearance refers to a standard 96-pore plate, the outside comprises a first protective layer and a second protective layer, the inside comprises white background paper and a high-transmittance plastic film, the first protective layer and the second protective layer are fastened by means of screw connection, the simulated spot standard substance is successfully prepared, the quantity value is reliable, the uniformity and the stability are good, the simulated spot ELISA reaction culture spot is preserved under the condition of keeping away from light in a laboratory at room temperature, the stability is more than 1 year, the carrying is convenient, the simulated spot ELISA reaction culture spot is successfully applied to the measurement calibration work of a spot ELISA analyzer, the experimental study is carried out on the measurement characteristics of instrument indication errors and repeatability, and an experimental basis is provided for the daily calibration work of the instrument and the preparation of national measurement calibration standards.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (5)
1. The spot simulating standard substance for the spot ELISA analyzer is characterized by comprising a high-light-transmission plastic film, wherein a plurality of hole sites are uniformly distributed on the high-light-transmission plastic film, and a certain number of spots are printed in the hole sites;
the standard substance is sequentially provided with a first protective layer, a high-light-transmittance plastic film, white background paper and a second protective layer from top to bottom;
the shape of the standard substance is a standard 96-well cell culture plate;
96 hole sites are uniformly distributed on the high-light-transmittance plastic film;
the holes on the high-light-transmittance plastic film are distributed in an 8 multiplied by 12 arrangement mode;
the holes on the high-light-transmission plastic film are distributed in a standard 96-hole cell culture plate arrangement manner;
the number of spots in the hole sites is 50-500 independently;
the diameter of the spot in the hole site is 50 mu m;
the hole sites comprise positioning hole sites;
the positioning hole sites are positioned at A1, A12, H1 and H12.
2. The standard substance according to claim 1, wherein the spots are located in the E3, B6, G6, E9 holes of the high light transmission plastic film.
3. The standard substance according to claim 2, wherein the spots differ in number in the E3, B6, G6, E9 pore sites;
the number of the spots in the E3 hole site is 50-500;
the number of the spots in the B6 hole site is 50-500;
the number of the spots in the G6 hole site is 50-500;
the number of the spots in the E9 hole sites is 50-500.
4. The standard substance according to claim 1, wherein the first protective layer and the second protective layer are connected by a connecting means.
5. Use of a standard substance according to any one of claims 1-4 in a spot enzyme-linked immunoassay.
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| 李兰英.斑点酶联免疫分析仪校准用模拟斑点标准物质.上海计量测试.2020,第47卷(第4期),2-7. * |
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