Microplate Reader
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The document describes the operation, calibration, and maintenance of a microplate reader used for ELISA tests, which are employed to detect antibodies or antigens in samples. Key aspects include the reader's functionality based on spectrophotometer principles, required environmental conditions, and detailed steps for calibration and maintenance. It also addresses common faults and their troubleshooting measures to ensure accurate readings.
Microplate Reader
- 1. Microplate Reader It is a device which used to read the result of ELISA test utilizing theory of spectrophotometer. ELISA-(Enzyme-Linked Immunosorbent Assay) Biochemical technique used mainly in Immunology to detect the presence of an antibody or an antigen in a sample. Also called Photometric Microplate reader, ELISA reader
- 2. Theory of operation Microplate reader is specialized spectrophotometer which facilitates readings on a wide range of wavelengths, generally between 400 to 750 nm. Beer Lambert law, A = absorption ε = absorption coefficient l = path length c = concentration I0 = reference light intensity I = light intensity c l A light beam, passing through the sample has a diameter ranging between 1 to 3 mm. Then detection system detects the light coming from the sample, amplifies the signal and determines the sample’s absorbance.
- 4. Internal Parts Light Source ELISA Plate Power supply
- 5. Installation A clean, dust free environment. A stable work table away from equipment that vibrates (centrifuges, agitators).
- 6. calibration Calibration plates are equipped with at least three predetermined optic density values within the measurement ranges; low, medium, and high value. Place the calibration plate on the equipment and carry out a complete reading. Verify if there are differences in the readings obtained from well to well. If this is the case, invert the plate (180°) and repeat the reading to rule out that differences are attributed to the plate itself. Identify the reader requires calibration. If so, proceed with the calibration according to manufacturer.
- 7. Maintenance Basic : Daily Preventive : Quarterly Review that optical sensors of each channel are clean. 2. If dirt is detected, clean the light emitters and the sensors with a small brush. 3. When the daily operations begin, let the reader warm up for 30 minutes. Next, do a blank reading and then read a full plate of substrate. The readings must be identical. 4. Examine the automatic drawer sliding system. It must be smooth and constant. 1. 1. 2. 3. 4. Verify the stability of the lamp. Use the calibration plate conducting readings with intervals of 30 minutes with the same plate. Compare readings. There must be no differences. Clean the detectors’ optical systems and the lighting systems. Clean the plate drawer. Verify the alignment of each well with the light emission and detection systems.
- 8. Basic fault Identification The reader gives a reading that does not make sense 2. The reader’s readings vary from row to row. 3. The reader displays unexpected variation in the optical density readings. 1. 4. The reader displays a gradual increase or decrease from column to column. 5. Low reproducibility. 6. Misaligned light beam.
- 9. 1.0 The reader gives a reading that does not make sense …? The illumination lamp is out of service. Replace the lamp with one with the same characteristics as the original.
- 10. 2.0 The reader’s readings vary from row to row. • Dirty optical sensors. • The illumination system’s lenses or parts are dirty. • Lack of calibration in one or more channels. Clean the sensors. Clean the lighting system’s lenses. Verify the calibration of each one of the channels.
- 11. 3.0 The reader displays unexpected variation with different wavelengths. The reader’s lamp is unstable. Replace the lamp with one that has similar characteristics as the original.
- 12. 4.0 The reader displays a gradual increase or decrease from column to column. Inappropriate calibration of the plate’s controlling motor. Calibrate the plate’s controlling system so that at each step the wells remain exactly aligned with the lighting system.
- 13. 5.0 Low reproducibility. • Sample homogeneity. Mix the reagents before use. Allow these to equilibrate to room temperature. •Incorrect pipetting procedure. Ensure pipette’s tips are changed between samples and that excessive liquid inside is removed Check the calibration. •Reader not calibrated. •Reading without sufficient warm up time. •Expired reagents. •Insufficient or Inefficient washing Wait until the reader has warmed up to its operating temperature.
- 14. 6.0 Misaligned light beam. •The reader was transferred or moved without using Call the specialized service technician. the necessary precautions. •The light source has been changed and the replacement has not been installed or aligned incorrectly Align according to manufacturer instructions
- 15. Thank you.