Dimension Vista® System Principles of Vista® Chemistry LOCI® Online Training

Define non-competitive, competitive, and homogeneous immunoassays Describe how LOCI® technology detects immune complexes Describe how LOCI® test methods are processed and where the assay measurement takes place on the Dimension Vista® System Welcome to the LOCI® online education support tool.  After completing this course, you'll be able to: Select Next to continue. Bienvenido al curso de formación on-line de descripción del hardware del Analizador RAPIDPoint ® 500. El Analizador RAPIDPoint ® 500 está diseñado para usarse en un entorno de laboratorio o cerca el paciente, con la finalidad de realizar análisis de los gases, electrolitos, metabolitos, nBili, la hemoglobina total, y derivados de la hemoglobina en las muestras de sangre total arterial, venosa y capilar. Congratulations. You’ve completed the LOCI® Online Education Support Tool. In this course, you’ve learned how the Dimension Vista® System performs LOCI® test methods. Here’s a brief review: Define non-competitive, competitive, and homogeneous immunoassays LOCI® technology enables the Dimension Vista® System to perform non-competitive and competitive homogeneous immunoassays. In a non-competitive immunoassay, antigen in the test sample binds to receptors on two different monoclonal antibodies. Immune complexes that form “sandwich” the antigen between antibodies. In a competitive immunoassay, antigen in the test sample binds to biotinylated monoclonal antibody. Immune complexes form. The reactants are then incubated with antigen analog-coated synthetic particles that compete with antigen in the test sample for “unoccupied” binding sites on the biotinylated monoclonal antibody. Homogeneous immunoassays do not require separation of immune complexes from unbound reactants before measurement. These immunoassays are generally much easier and faster to perform than heterogeneous immunoassays, which require a washing step to separate immune complexes from unbound reactants before measurement. Describe how LOCI® technology detects immune complexes LOCI® advanced chemiluminescence represents an important advance in immunochemistry because it pairs a highly sensitive immunoassay with a patented, state-of-the-art detection method. The key reactants shown here are described below.   Chemibeads-Chemibeads (1) are coated with monoclonal antibodies (2) that are specific for the analyte (3) being tested. The analyte serves as the antigen in the reaction. Chemibeads are also coated with a substance that generates chemiluminescence when in close proximity to singlet oxygen. Biotinylated antibodies—Non-competitive LOCI® immunoassays use a second monoclonal antibody (4) specific for the analyte being tested. Biotin(5) is attached to this "biotinylated" antibody. During incubation at 37°C, Ab + Ag + Ab immune complexes form, where antigen is "sandwiched" between monoclonal antibodies. In the laboratory, biotin is used to physically bridge complexes-with the help of streptavidin (6). Streptavidin-Streptavidin has a unique property-it readily binds to the biotin portion of biotinylated antibodies to form bead-pair immune complexes consisting of Chemibeads + Sensibeads (7). Sensibeads-Sensibeads are coated with streptavidin as well as a photosensitive dye. When illuminated at 680 nm, Sensibeads generate singlet oxygen, which diffuses into nearby Chemibeads and triggers a chemiluminescent reaction. The LOCI® reader measures the intensity of chemiluminescence at 612 nm. The signal is proportional to the concentration of analyte in the test sample! Describe how LOCI® test methods are processed and where the assay measurement takes place on the Dimension Vista® System Examples of LOCI® test methods include CTnI, MMB, MYO, NT proBNP, FT3, and FT4. For LOCI® test methods that use non-competitive immunoassays formats, the Dimension Vista® System performs the following tasks: LOCI® reactions take place in reaction vessels. The cuvette wheel rotates to move the reaction vessel to a reagent probe, which dispenses reagent from a Flex® reagent cartridge into the reaction vessel. The cuvette wheel rotates to move the reaction vessel to the sample probe, which dispenses up to 20 µL sample from an aliquot plate well into the reaction vessel. After incubation, the cuvette wheel rotates to move the reaction vessel to the LOCI® reader. Following a final incubation, the LOCI® reader uses an LED light source to flash the reaction solution at 680 nm, which releases singlet oxygen from Sensibeads. Chemibeads in close proximity to singlet oxygen generate chemiluminescence. The LOCI® reader alternates between flashing the reactants at 680 nm to release singlet oxygen, and reading the generated chemiluminescent signal at 612 nm. The LOCI® reader performs up to four flash/read cycles, depending on the test method. No washing or separation steps to remove unbound reactants are necessary before measurement! The signal that is measured is proportional to the concentration of analyte in the test sample. Select Next to continue.   LOCI® technology enables the Dimension Vista® System to perform non-competitive and competitive homogeneous immunoassays. In the Nephelometry online training course, you learned some basic facts about immunoassays performed in the clinical laboratory. Here’s a quick review: Antibodies are used as reagents to measure analytes in body fluids because of their unique property of recognizing and distinguishing closely related antigens. During the immunoassay, the analyte to be measured usually serves as the antigen and binds to specific antibody in the reagent to form immune complexes. Depending on the detection method, the concentration of the analyte being measured is either directly or indirectly proportional to the concentration of immune complexes that form.   Inmunology Review II Learn about the formats used by the different LOCI® test methods. Which LOCI® test methods use non-competitive versus competitive homogeneous immunoassay formats?   Non-competitive immunoassays Competitive immunoassays CTnI MMB MYO NT proBNP FT3 FT4 When complete, select the X in the upper-right corner to close the window and continue. Immunology Review Types of Immunoassays Learn about non-competitive and competitive homogeneous immunoassays. Checklist TitleChecklist TypeChecklist ContentImmunology Review Types of ImmunoassaysHTMLSelect each checkbox to learn more about immunoassays. What is a non-competitive (sandwich) immunoassay?HTML  In non-competitive immunoassays: Analyte in the test sample serves as the antigen and is incubated with two reagents that each contain a monoclonal antibody. In the example shown here, one of the monoclonal antibodies is free, and the other is attached to a synthetic particle. Each monoclonal antibody has receptors for a specific site on the antigen—the two different monoclonal antibodies do not compete for the same binding sites on the antigen. During the reaction, antigen binds to receptors in both antibodies. Immune complexes that form "sandwich" the antigen between antibodies. In this example, the amount of antigen in the sample is directly proportional to the concentration of synthetic-bound immune complexes that form. Key: 1. Free monoclonal antibody  2. Antigen (analyte in sample)  3. Monoclonal antibody bound to synthetic particleWhat is a competitive immunoassay?HTML  In competitive immunoassays: Analyte in the test sample serves as the antigen and is incubated with reagent containing biotinylated monoclonal antibody. During the reaction, antigen binds to antibody receptors to form immune complexes. The reactants are also incubated with antigen analog-coated synthetic particles that compete with antigen in the test sample for "unoccupied" binding sites on the biotinylated monoclonal antibody. In this example, the amount of antigen in the sample is indirectly proportional to the concentration of synthetic-bound immune complexes that form. Key: 1. Biotinylated monoclonal antibody  2. Antigen (analyte in sample)  3. Antigen analog bound to synthetic particleHeterogeneous & homogeneous immunoassaysHTML  Heterogeneous immunoassays require a washing step before measurement to separate immune complexes from unbound reactants remaining in the sample. After washing, only bound reactants remain, as shown in the example above. Homogeneous immunoassays—such as LOCI® test methods—do not require separation of immune complexes from unbound reactants before the immune complexes are measured. These immunoassays are generally much easier and faster to perform! Key: 1. Free monoclonal antibody  2. Antigen (analyte in sample)  3. Monoclonal antibody bound to synthetic particle  4. Unbound reactants removed during washing step When complete, select the X in the upper-right corner to close the window and continue. Now that you can visualize what is happening during non-competitive and competitive homogeneous immunoassays, let’s learn how LOCI® advanced chemiluminescence detects immune complexes.   What is the LOCI® detection method? Learn how LOCI® technology detects immune complexes to measure analytes. Checklist TitleChecklist TypeChecklist ContentWhat is the LOCI® detection method?HTMLSelect each checkbox to learn more about LOCI® technology components. Key LOCI® ReactantsHTML LOCI® advanced chemiluminescence involves the reactants shown above. Click each checkbox on the left to learn the function of each component used during the immunoassay. Key 1. Chemibead  2. Monoclonal antibody  3. Antigen (analyte in sample)  4. Biotinylated antibody  5. Biotin  6. Streptavidin  7. SensibeadChemibeadsHTML Non-competitive (sandwich) immunoassays use method-specific synthetic Chemibeads that are coated with monoclonal antibodies specific for the analyte being tested. Key 1. Chemibead  2. Monoclonal antibodyBiotinylated AntibodiesHTML  Immunoassay reactants also include the analyte being tested, which serves as the antigen (Ag), as well as a second monoclonal antibody (Ab) specific for the analyte being tested. Biotin is attached to this antibody—it's a biotinylated antibody! During incubation at 37° C, Ab + Ag + Ab immune complexes form, where antigen is "sandwiched" between monoclonal antibodies. Key 1. Chemibead  2. Monoclonal antibody  3. Antigen (analyte in sample)  4. Biotinylated antibody  5. BiotinSensibeads + StreptavidinHTML  Immunoassay reactants also include Sensibeads coated with streptavidin. Sensibeads contain a photosensitive dye. Streptavidin is a small protein with unique biochemical properties, such as its high affinity to bind to biotin. When added to the assay, streptavidin quickly binds to the biotin portion of the biotinylated antibody to form bead-pair immune complexes. Key 1. Chemibead  2. Monoclonal antibody  3. Antigen (analyte in sample)  4. Biotinylated antibody  5. Biotin  6. Streptavidin  7. SensibeadGenerating ChemiluminescenceHTML  When illuminated at a 680 nm wavelength, Sensibeads generate singlet oxygen, which diffuses into nearby Chemibeads and triggers a chemiluminescent reaction. The resulting signal is measured at 612 nm and is proportional to the concentration of analyte being tested. Key 1. LED light source  2. Singlet oxygen released from Sensibead  3. Chemiluminescence generated from Chemibead When complete, select the X in the upper-right corner to close the window and continue. Now that you understand how LOCI® technology detects immune complexes, let’s go inside the Dimension Vista® System to see how a sample with a LOCI® test request is processed—you can see this only in the virtual laboratory! LOCI® Measurements on the Dimension Vista® System Learn more about LOCI® measurements on the Dimension Vista® System. Slide NumberText BlocksCalloutsAudio ScriptImage File1Welcome to the Virtual Chemistry Lab. Let's learn how LOCI® test methods are processed and where the assay measurement takes place on the Dimension Vista® System. Select Next to continue.Note: If audio does not automatically start, select the play arrow in the top left to begin.Welcome to the Virtual Chemistry Lab! Let’s learn how LOCI™ test methods are processed and where the assay measurement takes place on the Dimension Vista® System. 2Getting Started LOCI® test methods are performed using disposable reaction vessels. Select Next to continue.LOCI™ test methods are performed using disposable reaction vessels.3Pipetting Reagent To get started, the reagent probe dispenses reagent from a Flex® reagent cartridge into a reaction vessel, which is loaded into the cuvette ring. The cuvette ring holds up to 94 reaction vessels. Select Next to continue.Select each number to review the corresponding text.Note: If audio does not automatically start, select the play arrow in the top left to begin.CalloutsReagent Probe Reaction Vessel Cuvette RingTo get started, the reagent probe dispenses reagent from a Flex® reagent cartridge into a reaction vessel, which is loaded into the cuvette ring. The cuvette ring holds up to 94 reaction vessels. 4Transporting Reaction Vessel The cuvette ring rotates to move the reaction vessel into position near the aliquot plate. Select Next to continue.Select each number to review the corresponding text.CalloutsCuvette RingAliquot PlateNext, the cuvette ring rotates to move the reaction vessel into position near the aliquot plate.5Pipetting Sample Sample probe 1 dispenses sample from the aliquot plate well into the reaction vessel. The simplicity of the homogeneous immunoassay format requires less sample volume—up to a maximum of only 20 µL! Select Next to continue.Select each number to review the corresponding text.CalloutsSample Probe 1Aliquot PlateSample probe 1 dispenses sample from the aliquot plate well into the reaction vessel. The simplicity of the homogeneous immunoassay format requires less sample volume-only 20 microliters! 6Forming Immune Complexes Immune complexes form as analyte in the sample reacts simultaneously with monoclonal antibody attached to Chemibeads, and with “free” biotinylated monoclonal antibody. Key Chemibead Monoclonal antibody Antigen (analyte in sample) Biotinylated antibody Biotin Note: Not all Chemibeads are functionalized with antibody. Select Next to continue.Slide QuestionAnswer TextWhat occurs in the reaction vessel during this incubation period?The antigen (analyte) is sandwiched between two monoclonal antibodies-this is an example of a non-competitive immunoassay! Immune complexes form as analyte in the sample reacts simultaneously with monoclonal antibody attached to Chemibeads, and with “free” biotinylated monoclonal antibody.7Forming Bead Immune Complexes After the initial incubation period, Sensibeads coated with streptavidin are added to the reaction vessel. Streptavidin quickly binds to the biotin portion of the biotinylated antibody to form bead-pair immune complexes. Key Chemibead Monoclonal antibody Antigen (analyte in sample) Biotinylated antibody Biotin Streptavidin Sensibead Select Next to continue,After the initial incubation period, Sensibeads coated with streptavidin are added to the reaction vessel. Streptavidin quickly binds to the biotin portion of the biotinylated antibody to form bead-pair immune complexes. 8Almost Showtime. The reaction vessel now has all the LOCI® reactants... ...so the cuvette ring rotates to move the reaction vessel to the LOCI® reader. Select the play arrow to begin video.  Select Next to continue.CalloutsLOCI® reader The reaction vessel now has all the LOCI™ reactants, so the cuvette ring rotates to move the reaction vessel to the LOCI™ reader.9Releasing Singlet Oxygen Following a final incubation, the LOCI® reader uses an LED light source to flash the reaction solution at 680 nm, which releases singlet oxygen from Sensibeads. No washing or separation steps to remove unbound reactants are necessary before measurement-this feature allows faster turnaround times for test results! Key LED light source Singlet oxygen released from Sensibeads Select Next to continue.Following a final incubation, the LOCI™ reader uses an LED light source to flash the reaction solution at 680 nanometers, which releases singlet oxygen from Sensibeads. No washing or separation steps to remove unbound reactants are necessary before measurementthis feature allows faster turnaround times for test results! 10Generating Chemiluminescence Chemibeads react with singlet oxygen and generate chemiluminescence at 612 nm-the LOCI® reader measures light at this wavelength, too! Key LED light source Singlet oxygen released from Sensibeads Chemiluminescence generated from Chemibeads Select Next to continue.Slide QuestionAnswer TextWhy is it OK to skip a wash phase and leave unbound sample reactants in the reaction vessel during measurement?Chemiluminescence is generated only if both Sensibeads and Chemibeads are in close proximity to each other-such as in bead-pair immune complex formations! So, unbound reactants play no role in the amount of chemiluminescence measured.Chemibeads react with singlet oxygen and generate chemiluminescence at 612 nm-the LOCI™ reader measures light at this wavelength, too!11Generating Results Key components of the LOCI® reader include an LED, which generates light, and a channel photo multiplier (CPM), which measures emitted light. To determine the final result, the LOCI® reader alternates between... Flashing the reactants at 680 nm to release singlet oxygen, and Reading the generated chemiluminescent signal at 612 nm ...for up to four flash/read cycles-depending on the test method. After the assay is completed, the reaction vessel is discarded. Select Next to continue.Select each number to review the corresponding text. CalloutsLEDChannel Photo Multiplier (CPM)Key components of the LOCI™ reader include an LED, which generates light, and a channel photo multiplier (or CPM), which measures emitted light. To determine the final result, the LOCI™ reader alternates between flashing the reactants at 680 nm to release singlet oxygen, and reading the generated chemiluminescent signal at 612 nm for up to four flash/read cycles-depending on the test method. After the assay is completed, the reaction vessel is discarded. 12You've just learned how LOCI® test methods are processed and where the assay measurement takes place on the Dimension Vista® System.When complete, select the X in the upper-right corner to close the window and continue. Congratulations! You’ve just learned how LOCI™ test methods are processed and where the assay measurement takes place on the Dimension Vista® System.