PMB: Rapid microbial detection Assays 10 Flashcards
What is an example of a rapid microbial Assay?
What is it used for?
- Immunodiagnostic assay
- Used to identidy or detect presences of pathogens
- Useful for clinical/ pharmaceutical samples
What do immunodiagonostic assays rely on/ work?
- Require the presence of an AB. The immunodiagnostic assay recoginizes an antigen on a pathogen
Benefits of an immunodiagnostic assay?
- They do not need to cultivate/ grow the microbe or pathogen. This means that detection of the organism is quicker and therefore treatment is more rapid
What does the potential of immunodiagnostic assays depend on?
the whole process relies on the quality of the detection level. Potiental for detection is dependent upon AB specificity. The better the specificity of AB. the more useful for detection. In some cases ABs can be fairly general so this would not be useful
Give examples of Immunodiagnostic assays:
- Aggulation Assays
Describe how aggulation assays work
In this method there is interaction between ABs and antigens on pathogens which leads to clumping.
This is due to there being several antigen recoginition sites on the AB and multiple antigens binding sites on the outside of the pathogens. ABs bind to more than one antigen and antigens bind to more than one AB
What are benefits of the aggulation method?
- Very quick - few mins get results
- Cheap to perform
- Useful for lots of different pathogens e.g. MRSA
- Highly specific - if you get the right AB
How does latex agglutination tests work?
- Latex beads are coated with AB. These AB coated particles are mixed/inoculates with the clinical sample (e.g. urine, serum). Serial diultions are formed with saline.
- Each diltution is checked for agglutination
- If the antigen is present then the latex suspension aggultinates.
- Clumps indicate a postiive reaction
What is the detection limit for latex agglutination assays?
0,1 - 0.2 ug/ml
What does ELISA stand for?
Why is this known as an indirect method?
Why is this the most common method?
- Enzyme-linked imunosorbent Assay
- Uses enzymes attached to ABs as a indirect detection method
- It is most common immunodiagnostic assay because it is highly sensitive
What enzymes are commonly used in ELISA and what is the detection limit?
peroxidase or alternatively B-galactosidase
Detection limit about 1 ng/ml of antigen
How does the ELISA method work?
- TMB acts as a donar of hydrogen atoms
- Hydrogen peroxidase reduced to water by peroxidase enzymes e.g. horeseradish peroxidase.
- After the reaction - turns blue
How is the organism detected in ELISA? What method is best and why
- Can sometimes be done visually
- but better to use spectrophotometer:
- Better detection levels
- Allows for quantification
- Allows automation
Describe how a direct ELISA is carried out
- In this method the antigen is sandwitched between to different ABs i.e. primary and secondary ones
- The AB which recognises the antigen is added to the reaction vessel. And any excess (non-attached) AB is washed away
- The sample to be analysed is added and any antigen present will be bound and trapped. Excess AB washed away
- The AB containing the attached enxyme is added to the reaction vessel and also binds to the antigens. Excess AB is washed away]
- The reaction can now be perfomed and a coloured product si formed. this is measured and is directly proportional to the amountof antigwen in the orginal sample
How does an indirect ELISA assay work and how is it carried out? Examples
- Remember that patients will already be producing ABs against the pathogen.
- There is strips which contain the antigen which screens for the ABs in the serum
- An example of this test is the HIV test by Alere
- Attach antigen to the reaction vessel or strip
- The serum is then added
- Incubate and wash
- Add ant-human antibody
- Detect with enzyme
The quantity of detection will be propotyional to the level of AB in the serum
