1. Immunoassay
28/02/2019

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

3. 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

4. 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

5. 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

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

7. 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.

8. 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

9. The AQT90 wheels Storage wheel for test cartridges1. 3. 2.
Storage wheel for test cartridges
Incubation wheel where the tests are performed
Process wheel of the optical unit for read-out

10. 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

11. 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

12. Detection of resolved-fluorescence
Fluorescence from chelate has very long lifetime
Measurement is performed at 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

13. 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
Full moon
Starlight
0.000,
AQT detection level
< 0.000,001
AQT excitation level like a flash light
AQT excitation level
150 k

14. Immunoassay
28/02/2019