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Particle counting immunoassay
- 2. TABLE OF CONTENTS 01 02 03 04 Introduction Detailed Explanation Application Future Aspects and Current advances 01
- 3. Introduction ● Immunoassays assays are one of the most commonly used diagnosis tools in biotechnology. ● One of the most accurate immunoassays is the Particle counting immunoassay ● Today we will discuss in what is so special in this immunoassay technique. ● But before that let’s get into simpler terms first such as the Immunoassay and describe the traditionally used ones.
- 4. What is an Immunoassay? ● An immunoassay is an analytical technique which uses naturally occurring reagents known as antibodies for the selective determination of sample components ● Immunoassays are commonly used in a wide variety of areas, especially in biochemistry and clinical chemistry
- 5. Why use an Immunoassay? ● Immunoassays are inexpensive to perform ● They are specific in their target and are highly selective ● Low limits of detection ● Have High throughput ;can be repeated in one batch. ● Applicable to the determination of a wide range of compounds.
- 6. Antibodies An antibody (Ab), or immunoglobulin (Ig), is a member of a family of glycoproteins that make up part of the body’s immune system. It is the body’s self defense mechanism against viruses and bacteria. Basic Structure The antibody consists of four polypeptides-two identical heavy chains (H) and two identical light chains (L) connected by disulfide bonds. These are arranged in a “Y”-shaped structure ending with two identical sites that recognize and bind a given foreign agent or antigen
- 7. Chemiluminescent Immunoassay Counting Immunoassay Enzyme Immunoassay Radio Immunoassay Fluro immunoassay Types of Immunoassays
- 8. Particle counting Immunoassay ● It combines basic ELISA tests or bead based immunoassays with additional steps to give us a better range of detection ● A capture antibody is coated to either a plate or bead, the plate capture is passive adsorption ● Sample is added to the capture antibody ● Next step is incubation and washing.
- 9. Cont... ● Then the second antibody(the detection antibody) directly conjugated with a fluorophore is added to the assay ● Further incubation and washing is done. ● This is the same as the ELISA protocol so far. ● An extra elution step is added which releases the antibodies ● This elluate is then transferred to another plate which contains a neutralisation buffer. ● This eluate is then aspirated into a detection system which is flow based.
- 10. Cont... ● The sample runs through a narrow capillary, the detection occurs in a 5 by 10 micron confocal laser focused at that point. ● The laser excites the fluorophore tag on the detection antibody and reports detected events.
- 12. Cont... ● Sample matrices include serum, plasma, urine with no minimal dilution. ● Capture surface can be either plates or magnetic beads or polystyrene beads. ● Detection antibody tags:Streptavidin Alexa Fluor or Directly conjugated antibody. ● The samples can be run or neat or diluted with the dilution depending on the analyte in the required range.
- 13. Advantages ● Very specific gives better results than ELISA as it eliminates matrix interferences and also the detected light is specific to the tag recording the data at (2pg/ml) ● Reduced sample size of 10-100 microlitres. ● Unquantifiable proteins can be quantified because of enhanced detection ratio ● Solves the problem of agglutination by giving a consistent signal ● Cost effective in the long run.
- 24. Thank You.