CN211856606U - Simulated spot standard substance - Google Patents
Simulated spot standard substance Download PDFInfo
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- CN211856606U CN211856606U CN202020444769.3U CN202020444769U CN211856606U CN 211856606 U CN211856606 U CN 211856606U CN 202020444769 U CN202020444769 U CN 202020444769U CN 211856606 U CN211856606 U CN 211856606U
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Abstract
The utility model provides a simulation spot standard substance, standard substance includes the plastic film piece, evenly distributed has a plurality of hole sites on the plastic film piece, it has the spot of quantity determination to print in the hole site. The utility model discloses use the plastic film piece as the carrier, print the spot that quantity is confirmed at the specific hole site of plastic film piece, as simulation spot standard substance, the quantity value is reliable, and homogeneity and stability are good, provide the experiment basis for the formulation of the daily calibration work of instrument and national measurement calibration standard.
Description
Technical Field
The utility model belongs to the technical field of the analysis detects, a simulation spot standard substance is related to.
Background
The spot Enzyme-linked immunosorbent assay (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 count). The spot counting is the most important step of ELISPOT data processing, and the spot enzyme-linked immunoassay analyzer can fully automatically acquire, count and analyze spot images of all holes on an ELISPOT plate, so that the cellular immunity or humoral immunity function of an organism can be evaluated, and the spot enzyme-linked immunoassay analyzer is widely applied to the immunology-related fields of vaccine research, drug screening, infectious disease diagnosis and research, tumor immunity research and the like. It is worth mentioning that in the clinical evaluation of vaccines, a spot enzyme-linked immunoassay analyzer has been forcibly used in ELISPOT, which can effectively avoid deviation and increase objectivity, repeatability and counting speed. However, at present, no calibration method for a spot enzyme-linked immunoassay analyzer is available at home and abroad.
The cell preservation conditions are harsh, the stability is poor, and the requirements of standard substances are not met, so that the preparation of the standard substances for realizing quantity value transmission is difficult. Therefore, the spot enzyme-linked immunoassay analyzer calibration method is provided, which simulates a spot standard substance, is beneficial to providing an experimental basis for calibration work of a spot enzyme-linked immunoassay analyzer, and is established.
SUMMERY OF THE UTILITY MODEL
The not enough and actual demand to prior art, the utility model provides a simulation spot standard substance according to spot enzyme-linked immunoassay appearance's technical parameter, combines the cultivation growth characteristic of enzyme-linked spot, has developed simulation spot standard substance, and the quantity is reliable, and homogeneity and stability are good, are favorable to providing the experiment basis for spot enzyme-linked immunoassay appearance's calibration work.
In order to achieve the purpose, the method adopts the following technical scheme:
in a first aspect, the present invention provides a spot simulation standard substance, the standard substance includes a plastic film sheet, a plurality of hole sites are uniformly distributed on the plastic film sheet, and spots with determined quantity are printed in the hole sites.
The utility model discloses in, use the plastic film piece as the carrier, print the spot that quantity is confirmed at the specific hole site of plastic film piece, as simulation spot standard substance, the quantity value is reliable, and homogeneity and stability are good, provide the experiment basis for the routine calibration work of instrument and the formulation of national measurement calibration standard.
Preferably, 96 hole sites are uniformly distributed on the plastic film.
Preferably, the holes are distributed in an 8 × 12 arrangement.
Preferably, the well sites are arranged in a standard 96-well cell culture plate.
The utility model discloses in, set up the hole site into the arrangement mode unanimous with 96 hole cell culture plates of standard, compatible strong with the instrument, be suitable for popularization and application.
Preferably, the spots are located within the E3, B6, G6, E9 holes of the plastic film sheet.
The utility model discloses in, E3, B6, G6, E9 hole site are in the intermediate position of 96 hole cell culture boards, and the distance between each hole position is unanimous on the whole, has typicality, the utility model discloses print the simulation spot of different quantity at E3, B6, G6, E9 hole site, the spot number covers the number that often detects in the spot enzyme-linked immunoassay test, satisfies the calibration demand of spot enzyme-linked immunoassay appearance.
Preferably, the number of spots is 50 to 500, for example, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 spots may be provided.
According to the utility model discloses, the best spot in the spot enzyme-linked immunosorbent assay test detects the number and is 100 ~ 400, the utility model discloses a cover this scope, set up the quantity of spot into 50 ~ 500.
Preferably, the spots differ in number within the E3, B6, G6, E9 well sites.
Preferably, the number of the spots in the E3 hole site is 50-99, for example, 50, 60, 70, 80, 90 or 99, preferably 50.
Preferably, the number of the spots in the hole B6 is 100-199, such as 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 199, preferably 100.
Preferably, the number of the spots in the G6 hole site is 200-350, such as 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340 or 350, preferably 200.
Preferably, the number of the spots in the hole site of E9 is 351-500, such as 351, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500, preferably 500.
Preferably, the hole locations comprise positioning hole locations.
The utility model discloses in, set up the location hole site on the standard substance, after putting into the year thing storehouse of treating the calibration instrument, can confirm the position of standard substance in the instrument through the location hole site.
Preferably, the positioning hole locations are located 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 preferred technical scheme, the utility model provides a simulation spot standard substance, standard substance from the top has set gradually first protective layer, plastic film piece, white background paper and second protection frame layer.
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 for calibration of an instrument.
Preferably, the apparatus comprises a spot enzyme-linked immunoassay analyzer.
Compared with the prior art, the utility model discloses following beneficial effect has:
(1) the utility model discloses according to the technical parameter of spot enzyme-linked immunoassay appearance, combine the cultivation growth characteristic of enzyme-linked spot, designed simulation spot standard substance, appearance reference standard 96 hole cell culture boards, the outside includes first protective layer and second protective layer, and the inside includes high light-transmitting plastic diaphragm and white background paper, and first protective layer and second protective layer rely on the screw connection fastening, and the size is length 12.7cm, wide 8.5cm, high 1.3 cm;
(2) the utility model discloses the simulation spot standard substance of preparation, the quantity value is reliable, and homogeneity and stability are good, convenient to carry, and is good with the compatibility of instrument, and application scope is wide, has extensive application prospect in spot enzyme-linked immunosorbent assay's measurement calibration work, carries out experimental study to the measurement characteristic of instrument indicating value error and repeatability, provides the experiment basis for the formulation of the daily calibration work of instrument and national measurement calibration standard.
Drawings
FIG. 1 is a design drawing (front plan view) of a pattern simulating a speckle standard;
FIG. 2(A) is a drawing of a hole at E3, FIG. 2(B) is a drawing of a hole at B6, FIG. 2(C) is a drawing of a hole at G6, and FIG. 2(D) is a drawing of a hole at E9.
Detailed Description
To further illustrate the technical means and effects of the present invention, the present invention will be further described with reference to the following embodiments and accompanying drawings. It is to be understood that the specific embodiments described herein are for purposes of illustration only and are not to be construed as limitations of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
The main apparatus is as follows:
the wafer light shield image type coordinate measuring instrument (Shenzhen Limited) guarantees the traceability of instrument measurement through the calibration of a measuring mechanism, and before each use, the calibrated standard plate is used for confirmation to ensure that the instrument is in a normal working state.
Example 1 Structure of a simulated speckle Standard substance
And (3) designing a simulated spot standard substance graph by adopting AutoCAD according to the technical parameters of the spot enzyme-linked immunoassay analyzer, and finally imaging the simulated spot on the plastic film. Fig. 1 shows a design diagram (front plan view) of a pattern of a simulated speckle standard substance: (1) the size and the hole site of the plastic membrane 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) the holes A1, A12, H1 and H12 are positioning holes and are designed into a 'SIMT' shape; (4) the E3, B6, G6 and E9 wells were designed with 50, 100, 200 and 500 simulated spots of 50 μm diameter, with dimensions of 12.7cm long, 8.5cm wide and 1.3cm high, respectively.
Example 2 repeatability and reproducibility testing of the simulated spot standard detection method
The pictures of the holes of the standard substances E3, B6, G6 and E9 of the simulated spots shot by an image measuring machine are shown in FIG. 2(A), FIG. 2(B), FIG. 2(C) and FIG. 2(D), the spots in the holes are clearly visible and uniformly arranged, and are respectively 50, 100, 200 and 500 simulated spots with the diameter of 50 μm, so that the simulated spots are suitable for manual identification and counting.
Randomly extracting a block of simulated speckle standard substance candidate, respectively shooting E3, B6, G6 and E9 holes of the simulated speckle standard substance by using an image measuring machine, printing the holes on paper, detecting by adopting a manual identification and counting method, repeating for 7 times, and inspecting the repeatability of the method, wherein the precision is 0%.
Randomly extracting ten simulated spot standard substance candidates, respectively shooting E3, B6, G6 and E9 holes of the simulated spot standard substances by using an image measuring machine, printing the holes on paper, and detecting by adopting a manual identification counting method, wherein the reproducibility of the method is inspected, and the precision is (0-0.2)%.
Therefore, the repeatability and the reproducibility precision of the constructed simulated spot standard substance are high, and the requirement for developing the simulated spot standard substance is completely met.
Example 3 detection of homogeneity of a simulated spotted standard
200 simulated spot standard substance candidates prepared in the same batch are randomly extracted according to the regulations of 'general principle and statistical principle of standard substance definite value' of JJF 1343-0.05(14, 30), the batch of standard material was confirmed to be homogeneous.
Example 4 stability Studies of simulated Spot standards
Stability is an essential attribute of standard substances, and measures to maintain stability of candidates must be taken throughout the development of standard substances. Since the standard substances inevitably involved transportation during use, short-term and long-term stability studies were performed on the same batch of standard substances, respectively.
(1) Short term stability study
Short term stability studies employ simultaneous stability studies, i.e., all measurements of the stability study can be performed under repetitive conditions. Experimental arrangement two short-term stability studies, 3 standard substance candidates were randomly drawn as a transport sample for one time: the first time is 2 months Shanghai-Beijing high-speed railway round trip, and two city laboratories-high-speed railway stations are in automobile transportation; and in 7 months, the standard substance candidate is sent to Guangzhou city by adopting 'Yunyan express', and the receiver in Guangzhou city receives the standard substance candidate and then hands the standard substance candidate to be sent to Shanghai laboratory by 'Shunfeng' express. Both transports of the candidates were tested simultaneously with normal storage of the candidates for short term stability and each sample was assayed 3 times in duplicate.
The results were compared by t-test, and at 95% confidence level, neither was significantly different, and the simulated spot standard was stable under transport conditions, meeting the actual transport requirements.
(2) Long term stability study
Long term stability studies employ classical stability studies, i.e. samples prepared simultaneously are measured over time under the same conditions. And (3) randomly drawing 3 simulated spot standard substances on the same day after the preparation of the sample in 1, 2, 3, 6 and 12 months by adopting a principle of density before thinning, and repeatedly measuring each sample for 3 times to perform long-term stability investigation. Since no physical model can truly describe the abrasion mechanism of the spots in the simulated spot standard substance, the stability of the standard substance is evaluated by a linear model recommended by ISO guide 35.
As a result, the slope of each standard was not significant, indicating that the number of spots in the standard did not significantly increase or decrease within a predetermined period of time, and therefore, the sample was stable within 1 year when stored at room temperature (23. + -.1). degree.C. under a dark condition in a laboratory. At present, the stability test of the standard substance set is still under continuous monitoring.
Example 5 simulated spot standards assessment of quantitation and uncertainty
(1) Standard substance constant value
Three simulated spot standard substances are randomly extracted from the same batch of standard substances by 8 experimenters respectively, in order to avoid leading of a design value and ensure that the experimenters keep an unknown state of the design value, E3, B6, G6 and E9 holes of the simulated spot standard substances are respectively shot by an image measuring machine and printed on paper, and the detection is carried out by adopting a manual identification counting method, and each block is counted three times.
The results were examined by the Cocker test and the Grublas test, and the data of all units were retained and participated in the final rating, and the average and the total average of the counting results for each experimenter are shown in Table 1.
TABLE 1 simulated spot standard valuing results
(2) Uncertainty evaluation of standard substance quantitative result
The source of uncertainty for the simulated spot standard consisted of three parts: uncertainty due to inhomogeneity of the standard substance; uncertainty caused by variability of the standard substance over the life cycle; the uncertainty caused by the valuing process of the standard substance is mainly the uncertainty introduced by the statistics of the measured data. The statistical results are shown in Table 2.
TABLE 2 simulated spot standard uncertainty List
The uncertainty results are combined to obtain the fixed value result and uncertainty of the simulated spot standard substance, which are shown in Table 3.
TABLE 3 simulated spot standard quantitation results and uncertainty
Example 6 use of simulated speckle Standard substance in the calibration of instrumental measurements
In this embodiment, the successfully developed simulated spot standard substance is used to investigate the indication error and the repeatability measurement characteristic of the spot enzyme-linked immunoassay analyzer, and the results are shown in table 4 after experimental verification is performed on 10 instruments such as AID company, CTL company and the like, which proves the universality of the method, and the experimental scheme is as follows:
(1) error in indicating value
The error of the instrument is expressed as the relative error of the measured value of the spot simulation standard substance and the standard value. Starting the instrument, debugging according to the instruction, entering a normal working state, placing the spot simulation standard substance into a loading bin, fixing, respectively photographing and counting E3, B6, G6 and E9 holes, repeatedly measuring for three times, recording the measured value, 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.
In the formula, Δ N is a display error (%),
to measure the mean, NsIs a standard value.
(2) Repeatability of
Measurement reproducibility is expressed as the relative standard deviation of 6 measurements of the simulated spot standard. The instrument is debugged according to the use instruction, enters a normal working state, places the spot simulation standard substance into a loading bin and fixes, photographs G6 holes with the standard value of the simulation spot standard substance of 200 and counts 6 times, and calculates the measurement repeatability according to the formula (2).
Wherein RSD is relative standard deviation (%), N is number of measurements, and N isiIn the case of a single measurement, the measurement,
the average value was measured.
The error in the measurement and the reproducibility of the results obtained with 10 instruments are shown in Table 4.
TABLE 4 use of simulated speckle standard in instrument calibration
| Instrument for measuring the position of a moving object | Indicating value error (one) | Repeatability (%) |
| 1 | 1 | 0 |
| 2 | 0 | 0 |
| 3 | 2 | 0.3 |
| 4 | 0 | 0 |
| 5 | 1 | 0.2 |
| 6 | 0 | 0 |
| 7 | 1 | 0 |
| 8 | 1 | 0.2 |
| 9 | 1 | 0 |
| 10 | 2 | 0 |
To sum up, the utility model discloses simulate spot enzyme-linked immunosorbent assay cultured's spot, adopt AutoCAD design simulation spot figure, appearance reference standard 96 orifice plate, the outside includes first protective layer and second protective layer, inside includes white background paper and high light-transmitting plastic diaphragm, first protective layer and second protective layer rely on the screw connection fastening, successfully prepare and obtain simulation spot standard substance, the quantity value is reliable, homogeneity and stability are good, preserve under laboratory room temperature light-resistant condition, stability is more than 1 year, and convenient to carry, successfully use in the measurement calibration work of spot enzyme-linked immunoassay appearance, carry out experimental research to the measurement characteristic of instrument indicating value error and repeatability, the system of calibration work and national measurement calibration standard of instrument daily provides the experimental basis.
The applicant states that the present invention is described in detail by the above embodiments, but the present invention is not limited to the above detailed method, i.e. the present invention is not meant to be implemented by relying on the above detailed method. It should be clear to those skilled in the art that any improvement of the present invention, to the equivalent replacement of each raw material of the present invention, the addition of auxiliary components, the selection of specific modes, etc., all fall within the protection scope and disclosure scope of the present invention.
Claims (10)
1. The standard substance for simulating the spots is characterized by comprising a plastic film, wherein a plurality of hole sites are uniformly distributed on the plastic film, and spots with determined quantity are printed in the hole sites.
2. The standard substance of claim 1, wherein 96 holes are uniformly distributed on the plastic film;
the hole sites are arranged and distributed in an 8 multiplied by 12 mode;
the hole sites are arranged and distributed on a standard 96-hole cell culture plate.
3. The standard substance of claim 2, wherein the spots are located within the E3, B6, G6, E9 holes of the plastic film;
the number of the spots is 50-500;
the spots varied in number within the E3, B6, G6, E9 well sites.
4. The standard substance according to claim 3, wherein the number of the spots in the E3 pore site is 50-99;
the number of the spots in the hole B6 is 100-199;
the number of the spots in the G6 hole site is 200-350;
the number of the spots in the hole site E9 is 351-500.
5. The standard substance of claim 2, wherein the well locations comprise positioning well locations;
the positioning hole positions are located at A1, A12, H1 and H12.
6. The standard substance of claim 1, further comprising a protective layer.
7. The standard substance of claim 1, further comprising a background layer;
the background layer comprises white background paper.
8. The standard substance according to claim 6 or 7, wherein the standard substance is provided with a first protective layer, a plastic film, a white background paper and a second protective layer in this order from top to bottom.
9. The standard substance of claim 8, wherein the first and second protective layers are connected by a connecting means.
10. The standard substance according to claim 1, wherein the standard substance is shaped as a standard 96-well cell culture plate.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111289742A (en) * | 2020-03-31 | 2020-06-16 | 上海市计量测试技术研究院 | Simulated spot standard substance and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111289742A (en) * | 2020-03-31 | 2020-06-16 | 上海市计量测试技术研究院 | Simulated spot standard substance and application thereof |
| CN111289742B (en) * | 2020-03-31 | 2023-12-22 | 上海市计量测试技术研究院 | Spot-simulating standard substance and application thereof |
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