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pr3152 > biopharmaceutical products derived from endocrine and immune system ii > Flashcards

biopharmaceutical products derived from endocrine and immune system ii Flashcards

1
Q

what are immunoassays

A

assays that deploy Ab to detect and quantify a specific analyte

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2
Q

what are the components of immunoassays

A

Ab against biomolecule of interest

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3
Q

what are the properties of the Ab

A

either poly/monoclonal, usually raised in animal

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4
Q

what are the methods used to detect Ag-Ab binding

A

through markers or agglutination/hemagglutination

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5
Q

what are the types of markers used in different immunoassays

A

radioactive immunoassays use radioactive labels while enzyme immunoassays use enzymes

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6
Q

what are the advantages and disadvantages of using radioactive labels

A

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

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7
Q

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

A

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

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8
Q

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

A

turbidity which indicated clumping of RBC

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9
Q

what is the principle behind solid phase enzyme immunoassays

A

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

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10
Q

what is the buffer solution often used for washing

A

phosphate buffer saline (PBS)

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11
Q

what does lower absorbance of coloured product indicate

A

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

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12
Q

what are the types of ELISA

A

direct, indirect, sandwich

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13
Q

describe the steps for direct ELISA

A

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

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14
Q

describe the steps for indirect ELISA

A

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

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15
Q

describe the steps for sandwich ELISA

A

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

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16
Q

what are the advantages of using ELISA

A

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

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17
Q

what are the disadvantages of using ELISA

A

false positive and false negative results

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18
Q

what can contribute to false positive results in ELISA

A

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

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19
Q

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

A

include blocking step using bovine serum albumin (BSA)

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20
Q

what can contribute to false negative results in ELISA

A

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

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21
Q

what is the principle behind hemagglutination

A

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

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22
Q

what causes hemagglutination

A

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

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23
Q

what is the requirements in order for agglutination to occur

A

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

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24
Q

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

A

by being conjugated to a solid particle

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25
Q

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

A

visible agglutination represented as cloudiness and turbidity more intense

26
Q

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

A

hemagglutination inhibited -> full red dot observed

27
Q

how does passive agglutination work in pregnancy test kits

A

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)

28
Q

how does inhibition of agglutination work in pregnancy test kits

A

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)

29
Q

how does inhibition of agglutination work in HbA1c kits

A

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

30
Q

what is the agglutinator in HbA1c kits

A

synthetic polymer containing multiple copies of the immunoreactive portion of HbA1c

31
Q

what is HbA1c

A

glycated hemoglobin

32
Q

how is HbA1c formed

A

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

33
Q

how does insulin regulate blood glucose

A

by promoting shift of glucose from circulatory compartment to intracellular compartment

34
Q

how is insulin produced

A

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

35
Q

how many AA does preproinsulin have

A

23 AA

36
Q

how many AA in A and B chain of insulin

A

A chain has 21 AA, B chain has 30 AA

37
Q

what are the AA difference between porcine and human insulin

A

one AA difference at B30

38
Q

what are the AA difference between bovine and human insulin

A

three AA difference at A8, A10, B30

39
Q

what is the problem with slight AA differences to human insulin

A

can evoke immune responses like lipoatrophy and allergic reaction

40
Q

what is the process of producing a recombinant human insulin

A

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

41
Q

does insulin want to exist as a dimer or monomer

A

do not want to exist as monomer

42
Q

what are the properties of insulin

A

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

43
Q

why can insulin form dimers or oliglomerise

A

due to favourable hydrophobic interactions

44
Q

which form of insulin enters bloodstream to interact with receptors

A

monomer form of insulin

45
Q

what are examples of rapid acting insulin analogues

A

lispro, aspart, glulisine

46
Q

what are examples of intermediate acting insulin analogues

A

NPH (isophane insulin)

47
Q

what are examples of long acting insulin analogues

A

glargine, detemir

48
Q

what form does rapid acting insulin analogues exist as

A

monomer

49
Q

what is NPH

A

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

50
Q

what is the property of protamine

A

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

51
Q

what is the combination NPH can be made with regular insulin

A

70/30 or 50/50

52
Q

how would NPH be mixed with insulin

A

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

53
Q

how often are premixed insulin taken

A

BD before mealtime

54
Q

what is the structural modifications done to achieve glargine

A

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

55
Q

how does the structural modifications affect glargine

A

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

56
Q

why does a higher pI indicate higher solubility at acidic pH

A

protein solubility lowest at its pI

57
Q

what pH is glargine produced at and why

A

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

58
Q

what is the structural modifications done to achieve detemir

A

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

59
Q

what does attachment of C14 fatty acid to LysB29 provide

A

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

60
Q

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

A

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

pr3152 (6 decks)

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