biopharmaceutical products derived from endocrine and immune system ii

Question 1

what are immunoassays

Answer 1

assays that deploy Ab to detect and quantify a specific analyte

Question 2

what are the components of immunoassays

Answer 2

Ab against biomolecule of interest

Question 3

what are the properties of the Ab

Answer 3

either poly/monoclonal, usually raised in animal

Question 4

what are the methods used to detect Ag-Ab binding

Answer 4

through markers or agglutination/hemagglutination

Question 5

what are the types of markers used in different immunoassays

Answer 5

radioactive immunoassays use radioactive labels while enzyme immunoassays use enzymes

Question 6

what are the advantages and disadvantages of using radioactive labels

Answer 6

more sensitive than enzymes in recognising Ab-Ag binding but is generally unsafe

Question 7

how are enzymes used to detect Ag-Ab binding in immunoassays

Answer 7

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

Question 8

what is witnessed in agglutination/hemagglutination and what does it indicate

Answer 8

turbidity which indicated clumping of RBC

Question 9

what is the principle behind solid phase enzyme immunoassays

Answer 9

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

Question 10

what is the buffer solution often used for washing

Answer 10

phosphate buffer saline (PBS)

Question 11

what does lower absorbance of coloured product indicate

Answer 11

lower absorbance means lesser enzymatic activity means more Ag in sample and less of enzyme linked Ag binded to Ab

Question 12

what are the types of ELISA

Answer 12

direct, indirect, sandwich

Question 13

describe the steps for direct ELISA

Answer 13

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

Question 14

describe the steps for indirect ELISA

Answer 14

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

Question 15

describe the steps for sandwich ELISA

Answer 15

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

Question 16

what are the advantages of using ELISA

Answer 16

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

Question 17

what are the disadvantages of using ELISA

Answer 17

false positive and false negative results

Question 18

what can contribute to false positive results in ELISA

Answer 18

use of polyclonal Ab which may cross react with multiple epitopes coming from look alike Ag

inadequate blocking due to amount of bound Ab&raquo_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

Question 19

what can be done to tackle inadequate blocking in ELISA which causes false positive results

Answer 19

include blocking step using bovine serum albumin (BSA)

Question 20

what can contribute to false negative results in ELISA

Answer 20

primary and/or secondary Ab and enzymes denatured as they are protein in nature

Question 21

what is the principle behind hemagglutination

Answer 21

ability of viral particle to interact with RBC through a viral surface glycoprotein called hemaglutinin -> used to diagnose/ quantify enveloped virus in a sample

Question 22

what causes hemagglutination

Answer 22

presence of virus causes clumping of RBC forming a lattice instead of a nice full red dot

Question 23

what is the requirements in order for agglutination to occur

Answer 23

no RBC needed as long as either Ag or Ab or both are particulate in nature (semisolid/solid)

Question 24

how to ensure Ag/ Ab or both are particulate in nature

Answer 24

by being conjugated to a solid particle

Question 25

what will be witnessed if both Ag and Ab are particulate in nature

Answer 25

visible agglutination represented as cloudiness and turbidity more intense

Question 26

what will be witnessed if there are Ab, virus and RBC

Answer 26

hemagglutination inhibited -> full red dot observed

Question 27

how does passive agglutination work in pregnancy test kits

Answer 27

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)

Question 28

how does inhibition of agglutination work in pregnancy test kits

Answer 28

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)

Question 29

how does inhibition of agglutination work in HbA1c kits

Answer 29

latex coated with anti-HbA1c mouse monoclonal Ab with agglutinator, absorbance measured at 531nm

Question 30

what is the agglutinator in HbA1c kits

Answer 30

synthetic polymer containing multiple copies of the immunoreactive portion of HbA1c

Question 31

what is HbA1c

Answer 31

glycated hemoglobin

Question 32

how is HbA1c formed

Answer 32

sugar molecule chemically attached to hemoglobin without requiring an enzyme through a condensation reaction between glucose and amino end of beta chain in Hb

Question 33

how does insulin regulate blood glucose

Answer 33

by promoting shift of glucose from circulatory compartment to intracellular compartment

Question 34

how is insulin produced

Answer 34

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

Question 35

how many AA does preproinsulin have

Answer 35

23 AA

Question 36

how many AA in A and B chain of insulin

Answer 36

A chain has 21 AA, B chain has 30 AA

Question 37

what are the AA difference between porcine and human insulin

Answer 37

one AA difference at B30

Question 38

what are the AA difference between bovine and human insulin

Answer 38

three AA difference at A8, A10, B30

Question 39

what is the problem with slight AA differences to human insulin

Answer 39

can evoke immune responses like lipoatrophy and allergic reaction

Question 40

what is the process of producing a recombinant human insulin

Answer 40

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

Question 41

does insulin want to exist as a dimer or monomer

Answer 41

do not want to exist as monomer

Question 42

what are the properties of insulin

Answer 42

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

Question 43

why can insulin form dimers or oliglomerise

Answer 43

due to favourable hydrophobic interactions

Question 44

which form of insulin enters bloodstream to interact with receptors

Answer 44

monomer form of insulin

Question 45

what are examples of rapid acting insulin analogues

Answer 45

lispro, aspart, glulisine

Question 46

what are examples of intermediate acting insulin analogues

Answer 46

NPH (isophane insulin)

Question 47

what are examples of long acting insulin analogues

Answer 47

glargine, detemir

Question 48

what form does rapid acting insulin analogues exist as

Answer 48

monomer

Question 49

what is NPH

Answer 49

cloudy suspension of insulin and protamine, co crystallined with Zn in neutral pH using phosphate buffer

Question 50

what is the property of protamine

Answer 50

highly basic 30 AA peptide, its basicity allows it to react well with insulin

Question 51

what is the combination NPH can be made with regular insulin

Answer 51

70/30 or 50/50

Question 52

how would NPH be mixed with insulin

Answer 52

via homogenisation in acidic pH -> undergo hexamerisation with Zn in neutral pH -> crystallisation to form protamine

Question 53

how often are premixed insulin taken

Answer 53

BD before mealtime

Question 54

what is the structural modifications done to achieve glargine

Answer 54

alpha chain: GlyA21 attached to AsnA21 beta chain: 2 Arg residues attached to C terminus

Question 55

how does the structural modifications affect glargine

Answer 55

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

Question 56

why does a higher pI indicate higher solubility at acidic pH

Answer 56

protein solubility lowest at its pI

Question 57

what pH is glargine produced at and why

Answer 57

pH 4 to stabilise insulin hexamer which results in prolonged and predictable absorption from SC tissues

Question 58

what is the structural modifications done to achieve detemir

Answer 58

beta chain: ThrB30 deleted, C14 fatty acid covalently attached to LysB29

Question 59

what does attachment of C14 fatty acid to LysB29 provide

Answer 59

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

Question 60

how does the property of subQ tissues allow for binding of detemir

Answer 60

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