Microbiology Flashcards
What does ELISA stand for?
What does ELISA detect?
Enzyme Linked Immunosorbent Assay
Technique used to detect specific molecules in samples
What are antibodies?
What do antibodies do?
Secreted by B-lymphocytes when matured to plasma cells
Recognise and bind to molecules (antigens) on foreign particles, marking them for destruction by T-lymphocytes
What are the uses of ELISA in veterinary medicine?
Disease detection
Detection of illegal drugs
Detection of hormones
What is antibody detecting ELISA?
Uses antigens to detect a specific antibody in sample
What is antigen ELISA?
What are the two types?
Uses specific capture antibodies to detect specific antigen in sample
Sandwich and competitive
What is sandwich ELISA?
What is competitive ELISA?
Uses an antibody pair to detect specific antigen
If antigen is small and has only one epitope the sample antigen is made to compete with a labelled antigen for antibody binding sites
What does epitope mean?
Binding site for antibody
How do rapid immunomigrations (RIM) work?
Proteins absorbed onto strip of tissue such as nitrocellulose
Sample applied to one end of strip and migrates along by capillary action
(sample may need to be pre-treated (eg. diluted) prior to application
Why in blood RIM tests to the lines show up as red?
Pigment in blood is red due to haemoglobin, therefore the line shows up red due to the pigment
What is agglutination?
What can it indicate?
Process where antibodies clump together cells or particles
Presence of antibodies against bacteria or red blood cells
What does latex bead agglutination assay detect?
Detect antibodies or antigens in body fluids including saliva, urine, cerebrospinal fluid, or blood
What is haemagglutination?
What common assay uses this?
Agglutination of red blood cells
Direct Coombs’ Tests - identifies the presence of non-agglutinating antibodies on the surface of erythrocytes
What must be included to make an immunoassay valid?
A positive and negative control
What is a positive control?
Confirms if the procedure is performing as intended
- allows for confidence in the diagnostic test results, confirms that negative results are accurate, and assists in any protocol adjustments or optimizations that may be necessary
What is a negative control?
Why is this useful for assays?
Known to not contain the biomolecule of interest
- adds validity to any positive results
It will show if any solutions of the assay are contaminated
In a sandwich ELSIA, what would happen if the blocking step was left out?
In a sandwich ELSIA, what would happen there was no washing steps after the detection antibody was added to the well?
All wells, including the negative control wells, would show uniform overdevelopment
Same answer again!
In antibody detecting ELISA what will the enzyme linked antibody attach to?
The constant region of the patient antibody
List the 5 steps involved in indirect ELISA (measure antibody concentrations in various types of samples):
List the 5 steps involved in sandwich ELISA (measure antigen concentrations in various types of sample):
Why are wells blocked in ELISA?
Blocked prior to sample addition to prevent non-specific binding
Is ELISA qualitative or quantitive?
Both
What is herd health?
How is it managed/maintained?
What is the objective?
Monitoring and control/prevention of (endemic) diseases of UK farmed species (Infectious diseases, metabolic diseases, etc), but also to optimse fertility, nutrition and production
- Monitoring and appraising; a continual active process (keep records)
- A regular veterinary visit
- An overview of preventive systems
To optimise cow health and welfare as well as farm sustainability and profitability
What is limit detection when talking about ELISA?
The minimal concentration that will be detected positive by the ELISA test (manufacturer should provide this number)
What is sensitivity when describing diagnostic tests?
What is the sensitivity of a veterinary diagnostic test?
What type of test characteristic is sensitivity?
How would you know a tests sensitivity?
The test’s ability to identify positive results
Probability of a positive test, given that the patient truly has the disease
(if the test had 95% sensitivity and 100 cows where infected 95 would test positive and 5 would be false negatives)
Prevalence-independent test characteristic
The manufacturer should provide the sensitivity of the test.
What is a tests specificity?
What is the specificity of a veterinary diagnostic test?
What type of test characteristic is specificity?
How would you know a tests specificity?
The test’s ability to identify negative results
The probability of a negative test given the patient if uninfected/healthy
(100 animals, 50 uninfected, specify of 98% = 49 of the 50 would test as negative and 1 would be a false positive)
Prevalence-independent test characteristic
The manufacturer should provide the specificity of the test.
What is seroconversion?
How can seroconversion be assed?
Interpret the serology results from paired samples:
Development of detectable specific antibodies to microorganisms in the blood serum as a result of exposure, infection or immunisation
Single sample cannot indicate when exposure occurred or if it was associated with a clinical disease so paired samples are taken 10-14 days apart (depends on disease)
If there is acute infectious disease suspected why would multiple paired sample be taken around 4 weeks apart (time really depends on pathogen)?
Due to expectation that a rising in antibody titre will be demonstrated
What values for IBR ELISA indicate negative, inconclusive and positive results?
What value of increase in a paired sample is considered significant?
0.15< = negative
0.15-0.25 = inconclusive
>0.25 = positive (previous exposure or recurrence)
Increase in more than 0.2
What values for RSV ELISA indicate negative, inconclusive and positive results?
What value of increase in a paired sample is considered significant?
0.15< = negative
0.15-0.24 = inconclusive
>0.24 = positive
Increase in more than 0.2
What values for BVD ELISA indicate negative, inconclusive and positive results?
0.2< = negative (no exposure of consistently infected)
0.2-0.3 = inconclusive
>0.3 = positive (previous exposure and seroconversion)
What values for mycoplasma bovis ELISA indicate negative, inconclusive and positive results?
What value of increase in a paired sample is considered significant?
0.25< = negative
0.25-0.29 = inconclusive
>0.29 = positive
Increase in more than 0.3
When screening using ELISA for IBR in cattle a few cattle are identified as latently infected (not an active infection), what prognosis would they be given?
(positive, inconclusive, negative?)
Seronegative
