biopharmaceutical products derived from endocrine and immune system ii Flashcards
what are immunoassays
assays that deploy Ab to detect and quantify a specific analyte
what are the components of immunoassays
Ab against biomolecule of interest
what are the properties of the Ab
either poly/monoclonal, usually raised in animal
what are the methods used to detect Ag-Ab binding
through markers or agglutination/hemagglutination
what are the types of markers used in different immunoassays
radioactive immunoassays use radioactive labels while enzyme immunoassays use enzymes
what are the advantages and disadvantages of using radioactive labels
more sensitive than enzymes in recognising Ab-Ag binding but is generally unsafe
how are enzymes used to detect Ag-Ab binding in immunoassays
enzymes and Ab are protein in nature where they have AA sequences and non reacting side chains, enzymes can be pegged to Ab by having reactions in the side chain of AA in Ab
what is witnessed in agglutination/hemagglutination and what does it indicate
turbidity which indicated clumping of RBC
what is the principle behind solid phase enzyme immunoassays
Ab being protein in nature can adsorb well onto plate surfaces -> Ab immobilised on solid surface -> incubate with known amounts enzyme linked Ag from lab and with sample containing Ag that are not enzyme linked -> enzyme linked and unlinked Ag will compete for binding to Ab -> wash away unbound Ag through washing with buffer solutions -> add enzyme substrate to determine the enzymatic activity by measuring absorbance of coloured product formed
what is the buffer solution often used for washing
phosphate buffer saline (PBS)
what does lower absorbance of coloured product indicate
lower absorbance means lesser enzymatic activity means more Ag in sample and less of enzyme linked Ag binded to Ab
what are the types of ELISA
direct, indirect, sandwich
describe the steps for direct ELISA
Ag bound to solid surface -> washing step -> add in known amount of enzyme linked primary Ab to bind with Ag -> washing step -> add in substrate -> measure absorbance of coloured product
describe the steps for indirect ELISA
Ag bound to solid surface -> washing step -> add in known amount of unlinked primary Ab to bind with Ag -> washing step -> add in known amount of enzyme linked secondary Ab that can recognise Fc domain of primary Ab -> washing step -> add in substrate -> measure absorbance of coloured product
describe the steps for sandwich ELISA
lab developed Ab bound to solid surface -> washing step -> add in Ag -> washing step -> add in primary Ab to recognise Ag -> washing step -> add in enzyme linked secondary Ab that recognises Fc domain of primary Ab -> washing step -> add in substrate -> measure absorbance of coloured product
what are the advantages of using ELISA
specific and sensitive (can be quantitative, semi-quanitative or qualitative) depending on specificity of Ab used, measuring of absorbance only requires a UV spectrophotometer which is easy and safe to use
what are the disadvantages of using ELISA
false positive and false negative results
what can contribute to false positive results in ELISA
use of polyclonal Ab which may cross react with multiple epitopes coming from look alike Ag
inadequate blocking due to amount of bound Ab»_space;> Ag from sample that leads to non specific binding to impurities to sample which can contribute to colour changes as well, dependent on Ag-Ab binding
inadequate washing
cross reactivity of secondary Ab
what can be done to tackle inadequate blocking in ELISA which causes false positive results
include blocking step using bovine serum albumin (BSA)
what can contribute to false negative results in ELISA
primary and/or secondary Ab and enzymes denatured as they are protein in nature
what is the principle behind hemagglutination
ability of viral particle to interact with RBC through a viral surface glycoprotein called hemaglutinin -> used to diagnose/ quantify enveloped virus in a sample
what causes hemagglutination
presence of virus causes clumping of RBC forming a lattice instead of a nice full red dot
what is the requirements in order for agglutination to occur
no RBC needed as long as either Ag or Ab or both are particulate in nature (semisolid/solid)
how to ensure Ag/ Ab or both are particulate in nature
by being conjugated to a solid particle
what will be witnessed if both Ag and Ab are particulate in nature
visible agglutination represented as cloudiness and turbidity more intense
what will be witnessed if there are Ab, virus and RBC
hemagglutination inhibited -> full red dot observed
how does passive agglutination work in pregnancy test kits
latex particles are coated with anti-hCG Ab -> if urine contains hCG -> agglutination occurs -> positive test (coloured dye added so that cloudiness seen as red line)
how does inhibition of agglutination work in pregnancy test kits
latex particles are coated with particulate anti-hCG Ab and standard amount of particulate hCG added which act as agglutinator -> if urine contains soluble hCG -> will compete for binding to anti-hCG Ab -> prevent agglutination -> positive result as seen from lower turbidity (because now binding is not between both particulates but one soluble one particulate)
how does inhibition of agglutination work in HbA1c kits
latex coated with anti-HbA1c mouse monoclonal Ab with agglutinator, absorbance measured at 531nm
what is the agglutinator in HbA1c kits
synthetic polymer containing multiple copies of the immunoreactive portion of HbA1c
what is HbA1c
glycated hemoglobin
how is HbA1c formed
sugar molecule chemically attached to hemoglobin without requiring an enzyme through a condensation reaction between glucose and amino end of beta chain in Hb
how does insulin regulate blood glucose
by promoting shift of glucose from circulatory compartment to intracellular compartment
how is insulin produced
beta cells in islets of langerhans of pancreas produces preproinsulin where there is C chain connecting A and B chain -> mature insulin has C chain cleaved and disulfide bonds formed between A and B chain
how many AA does preproinsulin have
23 AA
how many AA in A and B chain of insulin
A chain has 21 AA, B chain has 30 AA
what are the AA difference between porcine and human insulin
one AA difference at B30
what are the AA difference between bovine and human insulin
three AA difference at A8, A10, B30
what is the problem with slight AA differences to human insulin
can evoke immune responses like lipoatrophy and allergic reaction
what is the process of producing a recombinant human insulin
genes encode for A, B, C chain -> Met codon chemically (ATG start codon) synthesised at 5’ end of proinsulin cDNA which is the site of translation for a single polypeptide chain -> Met codon at N terminus removed by cyanogen bromide (CnBr) -> C chain removed by enzymatic action of trypsin/ carboxypeptidase -> disulfide bonds formed between A and B chains after rearrangement and folding -> yield human insulin
does insulin want to exist as a dimer or monomer
do not want to exist as monomer
what are the properties of insulin
intrinsic ability to form dimers/ oliglomerise, able to form Zn containing hexamer in presence of Zn which will dissociate into monomer at site of injection when injected conc falls to physiological levels
why can insulin form dimers or oliglomerise
due to favourable hydrophobic interactions
which form of insulin enters bloodstream to interact with receptors
monomer form of insulin
what are examples of rapid acting insulin analogues
lispro, aspart, glulisine
what are examples of intermediate acting insulin analogues
NPH (isophane insulin)
what are examples of long acting insulin analogues
glargine, detemir
what form does rapid acting insulin analogues exist as
monomer
what is NPH
cloudy suspension of insulin and protamine, co crystallined with Zn in neutral pH using phosphate buffer
what is the property of protamine
highly basic 30 AA peptide, its basicity allows it to react well with insulin
what is the combination NPH can be made with regular insulin
70/30 or 50/50
how would NPH be mixed with insulin
via homogenisation in acidic pH -> undergo hexamerisation with Zn in neutral pH -> crystallisation to form protamine
how often are premixed insulin taken
BD before mealtime
what is the structural modifications done to achieve glargine
alpha chain: GlyA21 attached to AsnA21
beta chain: 2 Arg residues attached to C terminus
how does the structural modifications affect glargine
changes the pI to 6.7 (compared to pI of insulin is 5.4) which makes glargine more soluble at acidic pH, less soluble at physiological pH
why does a higher pI indicate higher solubility at acidic pH
protein solubility lowest at its pI
what pH is glargine produced at and why
pH 4 to stabilise insulin hexamer which results in prolonged and predictable absorption from SC tissues
what is the structural modifications done to achieve detemir
beta chain: ThrB30 deleted, C14 fatty acid covalently attached to LysB29
what does attachment of C14 fatty acid to LysB29 provide
LysB29 is lipophilic so adding C14 fatty acid can increase hydrophobicity which allows for binding to serum albumin both at site of injection and upon gaining entry into circulation at site of injection, and slow dissociation in blood which leads to prolonged release
how does the property of subQ tissues allow for binding of detemir
vasculature and blood capillaries are very porous at both ends where higher hydrostatic pressure at arterial end moved albumin into interstitial fluid in SQ tissues
