Instructions for working with the Eliza Reader device
Instructions for working with the Eliza Reader device
Target
The purpose of this guide is to describe the process of doing the work and the quality control of the ELISA reader.
2- Scope of application
This instruction applies to the Department of Hormone Studies.
3- Responsibility for implementation
He is responsible for implementing this executive method with the technical staff of the Department of Hormoneology and is responsible for quality assurance.
4- Definitions
does not have
5- Description of actions
Principles:
Immunosuppressive methods:
In cases where the antigen-antibody reaction is not visible, these reactions must be revealed in a way that led to the emergence of a new generation of methods called labeled Immunoassay. In these reactions, the antibody or antigen is marked by the material
- Radioimmunoassay: Antibody or antigen immunodeficiency is measured using radioisotope materials.
Radio Immunoassay (RIA): The antigen is labeled.
Immunoradiometric Assay (IRMA): The antibody is labeled.
Immunosuppressive enzyme: An enzyme is used to label.
Enzyme Immunoassay (EIA): The antigen is labeled.
Immunoenzymometric Assay (IEMA): The antibody is labeled.
ELISA is the common name for such commonly used methods, in other words, enzyme immunoassay evaluations in which antibodies or antigens are coated on a solid phase are known as ELISA.
The basis of the reaction:
Indirect ELISA:
It is used in serum samples to determine specific antibodies or antibody titration.
In response, the antigen is attached to the wall of the wells (made of polystyrene). An antibody sample is then added to the wells.
After adding the sample and during the incubation period, washing is done and then the enzyme antimony globulin marked with the enzyme is added to the well. (Depending on what class of antibody is important for the measurement, the type of antigen used is also different, for example, IgG is used for IgG).
The specificity of the method depends on the antigen shortened in the wells.
Dilute the sample antigen by sampling diluent to prevent non-specific adsorption of the proteins in the serum and to prevent the occupation of the connection points of the sample antigen.
Sandwich ELISA:
This method is divided into two categories:
A) Ag Capture or Ab Sandwich method
The most common method is ELISA, and an antigen (which must have two different antigenic regions, such as TSH, LH, FSH, PSA, and hCG) is placed between two specific antibodies. An antibody is trapped in the well to trap the antigen, and the second antibody, labeled with the enzyme, acts as an identifier.
B) Antibody Capture method
This method is divided into two categories
1) Direct Ab Capture or Ag Sandwich method
In this method, a shortened antigen is used to trap a specific antibody. The marking antigen is then added to the enzyme to the environment and attached to it via another antibody arm (Fab). As a result, the specific antibody is sandwiched between two antigens (such as the HBs Ab kit).
1) Indirect Ab Capture or Indirect Ag Sandwich method
The anthomen of globulin is cut into wells and trapped by adding an antibody sample, and then a safety complex is formed by adding a specific antigen to the environment. The label-specific antibody against the antigen is then used as an identification system.
Competitive or restraining bonus:
In the above methods, the basis for measuring competition is between two antibodies or two antigens (one of which is a marker).
Competitive method: Both stamped and non-stamped analyzes are added to the system together.
Inhibition or blocking method: Analyze is added first and after a period of incubation the marker analyte is added.
A) Competitive or inhibitory alloy for antigen:
The labeling antigen and the antigen in the sample competed for binding to a specific antibody shortened in the well.
The curve of this method is inverse, meaning that the marker analysis is less attached to the antibody in the presence of large amounts of non-marker analysis in the sample. Quantitative luminescence method is used.
B) Competitive or inhibitory analysis for antigen:
The labeling antibody and the antibody in the sample competed for binding to a truncated antigen in the well. The curve of this method is inverse, meaning that the marker analysis is less attached to the antibody in the presence of large amounts of non-marker analysis in the sample. The most obvious example of this method is the HBc Ab test.
This device is able to read monochromatic or micro-matte plates with 4 filters 405/450/492/630 nm. The advantage of reading in two-wave systems is that they have the advantage of correcting the absorption of light and in them the defects caused by the optical system, changes from well to well and the final volume of the wells are automatically eliminated. This filter is made up of Differential Filters.
In reader devices, the choice of wavelength is selected by filters or grades (there are special structures that, when light is shone on them, only light with a certain wavelength is emitted from them).
- This device accepts all standard micro plates with round and smooth wells. Also, this device has the ability to read the plate in the direction of (1-12 and (A-H) and after reading the plate, it sends the results to the printer for printing.
- This device is able to read the sample of patients in different ways to facilitate the use and reduce errors. This program is recorded in the device's memory, which is accessible by the keys on the keyboard.
Instructions
another way:In checking that the device is linear, the amount of content inside the wells must be the same (because the path light path of different wells must be the same).
First, dissolve potassium dichromate reagent (dissolve 3.1 g of potassium dichromate in 800 ml of distilled water, then add 3.3 g of potassium to it and increase the volume to one liter). Ratio of 1.2, 1.4, 1.8) and then calibrated with a 200 l sampler, 200 l of each dilution (not diluted as 1/1 dilution) is added to 3 wells successively (from Each dilution is poured into three wells) then in Absorbance Mode,
We read their absorption with the initial 450 nm filter and the 630 nm differential filter and compare it with the expected values obtained from the average optical absorption of the first 3 wells (which are obtained directly from the OD reading of the main reagent).
To obtain the Expected value in each digit, we first consider the average light absorption obtained for the original solution as the base number, and then multiply the dilution coefficient for different dilutions.
To get the desired range in each dilution, we use the following formula:
Expected value ± (1% of the expected value + 0.01A)
For example, if the dilution of 1.4 is the expected number of 0.438, the acceptable range is calculated as follows:
0.438 ± (0.01 x 0.438 +0.01) = 0.438 ± 0.015
As a result, the acceptable range for dilution is 1.4, 0.424 - 0.454.
TEST 186
We can also use Test # 186 to check the filters. If the number obtained is between 2-10, the performance of the filters is appropriate. Otherwise, send the device to the support company for service.
To perform the test, first turn on the device and leave it for 15 minutes to warm up, and then after the word selection mode appears, press the Test button, then enter the number 186 and press Enter. After a few seconds, four numbers appear for filters 0, 1, 2, 3. If these numbers are between 10-2, it indicates the health of the filters (in new filters this number is closer to 10 and in older filters it is closer to 2 Is).
Photometric accuracy control:
The photometric accuracy test is used to determine whether the maximum amount of light absorption occurs at a certain wavelength, in other words, the purpose of determining the difference between actual absorption and observed absorption. Photometric accuracy depends on the lamp's ability to provide maximum photoelectric radiation, the type and quality of the monochromator (monochromatic).
To check the photometric accuracy of the solution of potassium dichromate alloy potassium (40 mg of potassium dichromate in 800
Dissolve one milliliter of distilled water, then add 3.3 grams of potash and increase the volume to one liter). With a 200 l sampler (which has been carefully controlled qualitatively and its CV is below 3%), we pour it directly into 5 wells from the above reagent and read it in Absorption Mode with the initial filter of 405 nm and the differential filter of 630 nm absorption and then We calculate its average. The number obtained should be in the range of 0.235 ± 0.040 or 0.195 - 0.275.
Repetition control:
To find out if there is any disturbance in the dirty sensors, the lenses of the device and the improper calibration of one or more channels of the device or the instability of the device's lamp. In this case, read OD at least 8 times at different concentrations and then calculate the CV (Coefficient Variation) (less than 3% is acceptable).
To perform this control, first make a 1.2 dilution of the reagent prepared to control the linearity and then add 200 ml of the original reagent and diluted reagent to 8 wells and absorb it in Absorption Mode with primary filter 405 nm and differential filter 630 nm. We read it and then calculate the mean, standard deviation (SD) and its change coefficient.
method:
- Turn on the Power button to select Select Mode.
- Press the Poly button and select the desired wavelength (2 Enter for 405 nm wavelength and 4 Enter for 490 nm wavelength)
- Then enter the number of standards and press Enter.
- Enter twice in a row.
- Enter the values of the standards and after each Enter.
- Then it asks if the standards and tests are Duplicate or not, then we press the No button, then we press Enter again and then we press the Read button.
- If we have the desired program in the memory, press the A button, enter the program number and press the Read button.
- After reading the plate, we turn off both the device and the printer.
Eliza Reader calibration control
1- Pour the same color solution in the wells and read their light absorption. Then remove the plates and rotate 180 degrees again and re-absorb the light. If the light absorption is the same in both cases, the device is calibrated.
2- To check the calibration of the motor that moves the plate, an empty plate can be used. In this case, if there is no significant difference between the absorption of the light read for the first column and the last column, it can be seen that the engine is healthy and calibrated.
Error sources in ELISA tests:
1- Failure to observe the method mentioned in the brochure (the manufacturer can change the method of work without prior notice).
2 - Open the plate containing the plate (immediately after leaving the refrigerator): The cold plate turns the water vapor in the air into small droplets that will settle on the walls of the wells. This causes the sample to be diluted in some tests where the sample size is small (eg 10 microliters).
The method of reading the device is as follows.
1- Absorbance mode (ABS KEY)
2- Single Calibrator Mode (STAND KEY)
3- Cut Off Mode (C.OFF KEY)
4-% Absorbance Multi - Point Mode (% ABS KEY)
5- Point –to-point mode (PGM KEY)
6- Best Fit By Polynomial Regression (POLY KEY)
7- Best Fit By Linear Regression (REGR KEY)
- Pre-saved programs can be requested by the user using the test list at any time.
- The device available in this section is set to read the kit in A-H direction, which can be changed using the 8 × 12 key on the keyboard.
To use the Point To poit mode (PGM KEY) program:
First, press the 5 (PGM) key, different filters appear on the screen, select the appropriate filter according to the type of test, and press the Enter key, then for the Blank option, depending on the type of test, key 1 (yes) or key Select 0 (NO), then enter the number of calibrators and the value of each and press Enter.
If the calibrators and specimens are used individually or in pairs, use the keys 0 and 1 to make the correct choice. Finally, press the Read key. To determine the position of the last well, we use the 9th key. It may work when working with the Stat Fax 2100 device. Error messages appear on the screen of the device. For how to fix this error, refer to the device's manual.
Required checks:
In order to control the performance of the device's lamp and filters, we must follow the instructions below.
To do this, periodically (once every 2 weeks) press the Self CK key so that the device automatically checks the output light intensity of the rotation of the filters, the XY movement of the Alphanumeric display display, and the printer, and the result Displays.
The following messages will be seen:
Lamp out ok or Lamp out put low
Photometer operation ok
Plate transporter ok
Mechanism failure
After the check is found, the printer will record the results. The System Check Ok message indicates that the device is ready for use. The device must be checked periodically and periodically when the device is returned from service. Manual for the device, see section 2.4.5 of Flags and Error messages, or contact support company.
Linearity
In addition to the above, the linearity of the device should be evaluated on a monthly basis. The purpose of this test is to determine the device's ability to comply with Beer's Law, according to which the concentration of the solution is directly proportional to the amount of light absorbed.
Note that the linear control in the ELISA device is an important parameter in the efficiency of the above device because the control of photometric accuracy can be controlled to a large extent by setting different standards in each work run, but if the linear device has problems, there are huge differences in answers. Achieved.
Linearity loss at high concentrations usually indicates the presence of stray light, which can be due to the deterioration of the filters. To check the linearity of the device, you must first select a solution with a maximum light absorption at a wavelength of 450 nm or close to it (a picric acid solution is recommended) and pour the plate into all wells and read the plate at a wavelength of 450 nm.
The result of optical absorption of all wells, if the filter is healthy, should be> 3.0, then once again we read the optical absorption without the plate. In such cases, the optical absorption for all wells should be between ± 0.01. Call.
4 - Be careful that the desired test affects hemolysis?
Hemoglobin has a peroxidizing activity due to its protein nature, and also due to the presence of iron and can accelerate the reaction rate of hydrogen peroxide and chromogen, and falsely increase the absorption of light.
Hemoglobin, on the other hand, alters the response of antigens and antibodies and increases the time it takes for the reaction to balance (such as Free T4, which is falsely reduced).
5- Are we allowed to use plasma and if we use plasma, do we need a special anticoagulant?
In these cases, you should refer to the kit brochure and carefully check the type of sample recommended. What types of plasma and anticoagulants can be used to obtain plasma? Plasma made of sodium citrate is not suitable for diluting plasma.
EDTA anticoagulant as a metal ion chlorinator can inhibit zinc (Zn) as a cofactor of the alkaline phosphatase enzyme.
Sodium fluoride, which is used as a sugar preservative, can inhibit the activity of urease enzyme, so it interferes with the measurements that urease enzyme has been used as a marker.
Sodium azide is a potent inhibitor of the enzyme peroxidase, and sodium azide should never be used in the measurement of those whose enzymes indicate peroxidase.
6- When painting, the plates should be at a temperature of 18-25 25 C and not exposed to cold and hot winds, because the ambient temperature can change the speed of the painting reaction, and as a result, lead to more or less painting. Disabling the positive and negative controls invalidate all work done.
7- Error during incubation time (time error is more in short-term incubation). During the TMB (Tetramethylbenzenidine) incubation phase - which is the substrate and is converted to an enzyme by color - aluminum foil should not be used at all to cover the surface of the plate.
8- Observance of Soak Time causes non-specific connections to be removed from the wells. Failure to do so will result in a background color throughout the worktop wells, and if the test lacks a blank well, this will lead to false answers. This time is in the kit and varies from 30 seconds to a few minutes.
9- After reading the OD plates, if the OD of the well is high, it is better to pay attention to the color of the well, and if the color of the well is not bold, it may be due to sticking a opaque substance such as surgical glove powder on the back of the plate. Clean the lint behind the plate.
10. There should be no interruption between the measurement steps as this may lead to the evaporation of the contents of the wells.
11- Washing problems: Washing is done to separate the compounds attached to the bottom of the well from the compounds that are not connected. Apart from the washing solution compounds, other points should also be considered.
Washing problems are difficult to diagnose and can be accidentally identified with readings that are too high or too low. Fixing this problem is recalibrating the washing machine. The washing solution is usually thicker and must be diluted in the laboratory.
If the dilution is not done properly and the solution is too concentrated, it will lead to the destruction and separation of the bonded molecules. On the contrary, reducing the ability of the washing solution due to heavy dilution will not separate the non-specific bonds and create high background adsorption. Be.
The washing step should be done at least three times and the excess solution should be drained or aspirated. Finally, the excess solution inside the well should be emptied by tapping on the surface of a paper towel or damp cloth.
If the washing solution has a bubble, prevent it from entering the wells, as this bubble reduces the contact surface of the wash solution with the well and reduces the washing effects. The pH of distilled water used for washing solution, if it is too acidic or alkaline, can affect the antigen-antibody bonds and affect the measurement.
12- In case of automatic washing with the washer device, make sure that the rate of pouring the solution and aspiration of the solution is adjusted.
13- Lipemia in measuring the analyzes of a hydrophobic molecule because the distribution of the analysis between the two phases of hydrophobic and hydrophilic is disrupted due to the presence of lipids. Also, in some analyzes that bind to the carrier protein, the presence of free fatty acids may interfere with the binding of the analyte to the carrier protein and cause a false increase in the amount of free analyte. (Especially thyroid and steroid hormones). Therefore, it is necessary to fast during these tests.
14- Re-melting of the serum affects some analyzes (it is ineffective in progesterone and testosterone).
15- It is better to press the sampler piston only down until the first pause to empty the extraction volume. If the piston is lowered to the second pause, it will cause a bubble to interfere with the measurement system. Detectors and samples should not be thrown into the well from above. The tip of the sampler should not come into contact with the bottom of the well. Do not contact the wall of the well with a sharp angle.Control and maintenance:
Keep the device in a dry environment and in a dust-free environment. The appropriate conditions are 35-18 degrees and the humidity should be less than 85%. Other chemicals and abrasive cleaners are not recommended.
Service and repair:
Refer to the attached user manual.
Safety considerations for working with the device:
Refer to the attached user manual.
