speciation Flashcards
what is speciation
establishment of species by origin by the use of analytical techniques
how is speciation done
sampling of tissue, food, fluid or other organic matter
what is the aim of speiciation
determine from a sample what species the sample came from
why is speciation important
food fraud
medical testing
forensics
protection of rare spieces
what types of approaches are used for speciation
utilise genetic differences between species via PCR or RT PCR - based on the gene
immunologic assays - ELISA, biosensors - Based on the gene product
food production is a —— pound industry
multi billion
what type of legislation is in place to protect the consumer
extensive
when were food laws passed about food adulteration
recently
how can we now detect and monitor food fraud
recent technological advancments
what is adulterated foods
food containing poisons, colouring, other additives to disguise, inferior quality or where an important constituent has been substituted
what is the most common for of adulteration
substitution of cheaper ingredient for a more expensive one
when was food adulteration a main problem
19th century
what was tea normally padded out with
gun powder
what was the most adulterated staple
bread - add boiled potato, sawdust. chalk, clay, bone dust,
cream thickened with what
calf brains
name the methods of speciation
antibody based
DNA based
Infer-red spectroscopy
mass spectroscopy
why use an ELISA for speciation
widely used in food compared to other methods it is highly sensitive cheap rapid requires little training antibodies readily available
what is an ELISA
quantitative method used to measure antigens and antibodies
based on antigen/antibody interactions
antibodies produced in immune resoponce
antibodies are specific to a particular antigen
what is an antibody
glycoprotein that recognise and bind to a particular antigen with very high specifity
advantages of polyclonal antibodies
broad specify
cheap
disadvantages of polyclonal
finate supply
advantages of monoclonal antibodies
narrow specifity
continuous supply
disadvantages of monoclonal antibodies
expensive
advantages of recombinant antibodies
narrow specify
genetic manipulation
disadvantages of recombinant antibodies
expensive
poor stability
what is a recombinant antibody
bacteria
fungi
plants
what is a monoclonal antibody
mouse
rat
hybridoma cells
what is a polyclonal antibody
rabbit sheep goat house chicken
advantages of ELISA
qualitative and quantitative detect antigen and how much is present can detect in serum, urine, saliva etc detect antibodies to indicate disease (HIV) can also use for environmental testing
what will a specific assay only detect
desired analyse - won’t give positive results with any other protein
what is the limit of detection
lowest amount of antigen needed to be detected
the lower the detection limit the —-
more sensitive the elisa
describe reactants in an ELISA
once bound stay attached to the solid surface.
washing steps- resulting in the removal of unbound reagents allow easy measurement of bound complex.
what are ELISA normally carried out in
96 well micro titre plate allowing large analysis of large number samples
what is the normal means of measurement in an ELISA
enzyme labelled component
normally peroxidase or phosphate conjugates
simple ELISA is —-
direct detection
Indirect ELISA is —
indirect detection
sandwich ELISA is —-
direct detection
competitive ELISA is
both direct and indirect
what are the disadvantages of an simple ELISA
primary antibody, must be labelled with enzyme or other label
uses a lot of antibody, unsatisfactory when primary antibody is in short supply/ expensive
describe advantages of simple ELISA
quick
simple
few steps
avoids potential cross reactivity which may occur when using secondary antibodies
advantages of indirect ELISA
labelled secondary antibody
increases signal strength as several labelled secondary antibodies can bind to primary
a single second antibody can be used with variety of primary antibodies made in same species
secondary antibody is species specific
disadvantages of indirect ELISA
more steps than direct
potential cross reactivity of secondary antibody
describe direct sandwich ELISA
antibody imobilised into plate to capture antigen and extract from a sample
antigen can be directly detected using labelled primary antibody or indirectly using primary antibody followed by labelled secondary antibody
advantages of sandwich ELISA
highly specific
used to extract minute amounts of antigen which is too small to be directly absorbed onto micro titre surface
highly sensitive
Disadvantages of sandwich ELISA
require 2 antigen specific antibodies
many steps involved
common problems associated with ELISA include
non specific binding - can be due to other substance in sample, limited by blocking plate
interference binding - due to other substance in sample
problems with reagents
- antibodies
- many buffers used
