Immunoassay 28/02/2019

Agenda Immunoassay - antibody and antigen AQT90 FLEX measuring cup The measurering proces The detection system

Antibodies An antibody is a protein of the immune system Produced as defense mechanism for foreign substances (antigens) for example bacteria or viruses High diversity: Composed of a constant and a variable region The immune system creates billions of different antibodies High specificity One antibody binds one antigen Strong binding affinity 1

Antigens An antigen is a substance which is foreign to the body It provokes an immune response with production of antibodies Antigens are typically proteins on the surface of virus and bacteria 1

Immunoassay An immunoassay is a biochemical test that measures the level of a substance in blood, using the reaction of an antibody to its antigen Quantitative measurement of antigen in blood sample via immunoassay Myo CRP TnI

Labeled tracer antibodies Immunoassay in AQT Antigen in blood sample Labeled tracer antibodies Incubation Sandwich complex Unbound tracer antibodies Capture antibodies

Production of cups Raw cup bought from Nunc (Polystyrene – MaxiSorp™) Coating with streptavidin Drying Coating with biotin linked capture antibodies Adding insulating layer and additives Adding tracer antibodies Packing Total production time (at Innotrac): 3 weeks Drying process very sensitive. If too long/intensive, there is a risk of cracks which can cause meltdown of the tracers into the capture phase.

Measuring process Aspirate an exact amount of sample (5-30 µL – parameter dependent) Dispense sample plus assay buffer into sample cup Incubate and mix/agitate at 37 ºC Wash - remove unbound antigens and tracer antibodies Dry the cup by heating lamp Measurement by light excitation and detection of emitted light

The AQT90 wheels Storage wheel for test cartridges 1. 3. 2. Storage wheel for test cartridges Incubation wheel where the tests are performed Process wheel of the optical unit for read-out

Detection of sandwich complex Detection method: Time-resolved fluorescence Detection of sandwich complex via detection of the label europium chelate Response curve Signal generated by label Photo-multiplier tube counts the number of photons (particles of light) emitted by Eu chelate. The concentration of the parameter in the blood is proportional to the signal generated by the label. Concentration of analyte

Unique fluorescence properties of europium Eu chelate is excited with light at a wavelength of 340 nm In response, Eu chelate emits light at a wavelength of 616 nm Conventional fluorescence Time-resolved fluorescence

Detection of resolved-fluorescence Fluorescence from chelate has very long lifetime Measurement is performed at 400-800 µs after excitation pulse At that time, short decay-time fluorescense is undetectable Measurement cycle lasts 1 ms, repeated 1000 times Total measurement time 1 sec Long decay-time fluorescence from europium chelate Short decay-time fluorescence

Detection - light levels Lux Bright sun 50 k – 100 k Hazy day 25 k – 50 k Cloudy bright 10 k – 25 k Cloudy dull 2 k – 10 k Very dull 100 – 2 k Sunset 1 - 100 Full moon 0.01 - 0.1 Starlight 0.000,1 - 0.001 AQT detection level < 0.000,001 AQT excitation level like a flash light AQT excitation level 150 k

Immunoassay 28/02/2019