(2)-ELISA.-ENZYME LINKED IMMUNOSORBENT ASSAY

(2)-ELISA.-ENZYME LINKED IMMUNOSORBENT ASSAY

1. IMMUNOLOGICAL IMMUNOLOGICAL
TECHNIQUESTECHNIQUES
P289P289

2. For recognizing and quantifying For recognizing and quantifying
antigens in tissues or fluidsantigens in tissues or fluids manman
y immunological techniques utiliy immunological techniques utili
ze the exquisite specificity of the ze the exquisite specificity of the
antigen-antibody bondantigen-antibody bond..

3. Cell populations can be identifiedCell populations can be identified and and
characterized by their surface markecharacterized by their surface marke
rs, using the techniques of immunoflurs, using the techniques of immunoflu
orescence or immunohistochemistryorescence or immunohistochemistry

4. Cell populations can be Cell populations can be
isolatedisolated according to their according to their
surface markers, by techniques surface markers, by techniques
which include fluorescence-which include fluorescence-
activated cell sorting (activated cell sorting (FACSFACS), ),
panning and density-dependent panning and density-dependent
centrifugations.centrifugations.

5. The principle assays for lymphoThe principle assays for lympho
cyte functioncyte function are by antibody or are by antibody or
cytokine production, by prolifercytokine production, by prolifer
ation in response to antigen, or ation in response to antigen, or
by cytotoxicity.by cytotoxicity.

6. *ANTIGEN-ANTIBODY INTERACTIONANTIGEN-ANTIBODY INTERACTION
 
1. Precipitation reactions1. Precipitation reactions
2.
 Haemagglutination and complement
2.
 Haemagglutination and complement
fixfix
ationation
3.
  Direct and indirect
3.
  Direct and indirect
immunofluorescenceimmunofluorescence
4.
  Immunoassay
4.
  Immunoassay
5.
  Immunoblotting
5.
  Immunoblotting
and immunoprecipitation and immunoprecipitation
  

7. Immunochemical techniquesImmunochemical techniques
The study of antibodies(and some otThe study of antibodies(and some ot
her immunologically important moleculeher immunologically important molecule
s such as complement components) is knos such as complement components) is kno
wn as immunochemistrywn as immunochemistry..Such methods aSuch methods a
re known as immunochemical techniques.re known as immunochemical techniques.

8. AntibodiesAntibodies
Antibodies are a group of globular proteins knowAntibodies are a group of globular proteins know
n as immunoglobulins.n as immunoglobulins.
The basic four-chain model for immunoglobulin The basic four-chain model for immunoglobulin
molecules is based on two distinct types of polypeptide molecules is based on two distinct types of polypeptide
chain.chain. The smaller(light) chain has a molecular weight The smaller(light) chain has a molecular weight
of 25,000 and is common to all classes, whereas the largof 25,000 and is common to all classes, whereas the larg
er(heavy) chain has a molecular weight of 50,000-77,00er(heavy) chain has a molecular weight of 50,000-77,00
0 and is structurally distinct for each class or subclass.0 and is structurally distinct for each class or subclass.
The polypeptide chains are linked together by covalent The polypeptide chains are linked together by covalent
and non-covalent forcesand non-covalent forces.

10. Fab.(Fragment-antigen binding):Fab.(Fragment-antigen binding):
The part of an antibody molecule which contaThe part of an antibody molecule which conta
ins the antigen-binding site, consisting of a light chins the antigen-binding site, consisting of a light ch
ain and part of the heavy chain; it is produced by eain and part of the heavy chain; it is produced by e
nzymatic digestion. (papain, pepsin)nzymatic digestion. (papain, pepsin)
Fc.(Fragment crystallisable):Fc.(Fragment crystallisable):
The portion of an antibody that is responsible The portion of an antibody that is responsible
for binding to antibody receptors on cells and the Cfor binding to antibody receptors on cells and the C
1q component of complement.1q component of complement.

12. ComplementComplement
The complement system is part of the innate The complement system is part of the innate
immune system. There are two main pathways immune system. There are two main pathways
for complement activation, the classical and for complement activation, the classical and
alternative pathway. alternative pathway. The classical pathwayThe classical pathway links links
the adaptive immune system, antibody, to the the adaptive immune system, antibody, to the
innate immune system, complement, by the innate immune system, complement, by the
binding to immune complexes of C1q. binding to immune complexes of C1q.
Alternative pathwayAlternative pathway activation is initiated when activation is initiated when
C3, activated by the ‘tick-over’pathway, deposits C3, activated by the ‘tick-over’pathway, deposits
on foreign surfaces, lacking regulatory molecules.on foreign surfaces, lacking regulatory molecules.

14. Further reading:Further reading:
Porter RR, Reid, KBM. The biochemistry of comPorter RR, Reid, KBM. The biochemistry of com
plement. plement. Nature Nature 1978; 1978; 275275: 699-704.: 699-704.
Reid KBM, Porter RR. The proteolytic activatioReid KBM, Porter RR. The proteolytic activatio
n systems of complement. n systems of complement. Annu Rev BiochemAnnu Rev Biochem 198 198
1; 1; 50:50: 433-464. 433-464.
Reid KBM, Day AJ. Structure-function relationsReid KBM, Day AJ. Structure-function relations
hips of the complement components. hips of the complement components. Immunol ToImmunol To
dayday, 1989; , 1989; 10:10: 177-180. 177-180.
Campbell RD, Law SKA, Reid KBM, Sim RB. StCampbell RD, Law SKA, Reid KBM, Sim RB. St
ructure, organisation, and regulation of the compructure, organisation, and regulation of the comp
lement genes. lement genes. Annu Rev ImmunolAnnu Rev Immunol 1988; 1988; 6:6: 161-19 161-19
55.

15. Polyclonal and monoclonal antibodiesPolyclonal and monoclonal antibodies
Usually, many different antibodies, Usually, many different antibodies, recognising severrecognising sever
al different epitopesal different epitopes on each antigen are present. Such a re on each antigen are present. Such a re
sponse is described as polyclonal, as antibody is derived frsponse is described as polyclonal, as antibody is derived fr
om more than one clone of B lymphocytes and shows heterom more than one clone of B lymphocytes and shows heter
ogeneity in the amino acid sequences of the antigen-bindinogeneity in the amino acid sequences of the antigen-bindin
g immunoglobulins present.g immunoglobulins present.
However, more recently, methods have been developHowever, more recently, methods have been develop
ed for deriving monoclonal antibodies, which are derived fed for deriving monoclonal antibodies, which are derived f
rom a single B cell clone and show identical amino acid seqrom a single B cell clone and show identical amino acid seq
uence. Monoclonal antibody preparations show homogeneuence. Monoclonal antibody preparations show homogene
ous characteristics(including specificity and avidity for antous characteristics(including specificity and avidity for ant
igen, i.e. they igen, i.e. they recognise a single epitoperecognise a single epitope).).

16. Production of antibodiesProduction of antibodies
Production of polyclonal antibodies(antisera)Production of polyclonal antibodies(antisera)
In general, most immunochemical methods In general, most immunochemical methods
are devised for use with antibodies that are devised for use with antibodies that recognisrecognis
e proteins and peptidese proteins and peptides..
In some cases, particular parts of the antigeIn some cases, particular parts of the antige
n produce very potent immune responses and sun produce very potent immune responses and su
ch epitopes are known as immunodominant. Imch epitopes are known as immunodominant. Im
munogenicity tends to increase with size; munogenicity tends to increase with size; proteinprotein
s with a molecular weight > 10,000s with a molecular weight > 10,000 are usually i are usually i
mmunogenic as long as they are recognised as fommunogenic as long as they are recognised as fo
reign in responding animals.reign in responding animals.

17. For production of potent antibodies tFor production of potent antibodies t
hat perform well in immunochemical techhat perform well in immunochemical tech
niques, it is usually necessary to use an niques, it is usually necessary to use an adad
juvantjuvant as part of the immunogen. Such su as part of the immunogen. Such su
bstances potentiate the immune response bstances potentiate the immune response
by by forming a slow-release depot of antigenforming a slow-release depot of antigen,,
by stimulating T cell help or by aiding an by stimulating T cell help or by aiding an
tigen presentation.tigen presentation.

18. Some commonly used adjuvantsSome commonly used adjuvants
  P266P266
*Freund’s complete adjuvant(FCA):Freund’s complete adjuvant(FCA):
Mineral oil containing heat-killed mycobacteriaMineral oil containing heat-killed mycobacteria
((Mycobacterium tuberculosisMycobacterium tuberculosis),),
Used as emulsion with aqueous antigenUsed as emulsion with aqueous antigen
*Freund’s incomplete adjuvant(FIA):*Freund’s incomplete adjuvant(FIA):
Mineral oilMineral oil
Used as emulsion with aqueous antigenUsed as emulsion with aqueous antigen
Alum, Bentonite, Quil A, MDP, MPL, Alum, Bentonite, Quil A, MDP, MPL,
Bacillus pertussisBacillus pertussis

19. Examples of immunization protocols that Examples of immunization protocols that
have been used successfullyhave been used successfully
P267P267
i.p., intraperitoneally; i.p., intraperitoneally;
s.c., subcutaneously; s.c., subcutaneously;
i.m., intramuscularly; i.m., intramuscularly;
i.v., intravenously; i.v., intravenously;
i.d., intradermally.i.d., intradermally.
SourceSource: Reproduced from M.A.Kerr and R.Thorpe,: Reproduced from M.A.Kerr and R.Thorpe, I I
mmunochemistry Labfaxmmunochemistry Labfax(1994), Bios Scientific, Oxfor(1994), Bios Scientific, Oxfor
d.d.

20. Antibodies and seraAntibodies and sera
Rabbit polyclonals:Rabbit polyclonals: rabbit antiserarabbit antisera to humato huma
n Factor H, bovine Factor H and human n Factor H, bovine Factor H and human 
221,and t1,and t
o the fusion protein GST-5o the fusion protein GST-5
thth
domain of domain of 
221 were a1 were a
vailable in our laboratory.vailable in our laboratory.
Affinity purfied rabbit IgG anti-human Affinity purfied rabbit IgG anti-human 
221 1
was made by passing 2ml of was made by passing 2ml of rabbit antiserumrabbit antiserum on a on a
column(2ml volume) of column(2ml volume) of 
221-Sepharose1-Sepharose(1mg (1mg 
221 co1 co
valently attached per ml of Sepharose). Bound antvalently attached per ml of Sepharose). Bound ant
ibodies were eluted with ibodies were eluted with 3M MgCl3M MgCl
22, pH 6.8, pH 6.8 and di and di
alysed into water, then into PBS-0.5mM EDTA.alysed into water, then into PBS-0.5mM EDTA.
From Bing Bin’s thesis(1999)From Bing Bin’s thesis(1999)

21. Production of monoclonal antibodiesProduction of monoclonal antibodies
Monoclonal antibodies can be especially useful for Monoclonal antibodies can be especially useful for
immunochemical methods. Such antibodies are secreted immunochemical methods. Such antibodies are secreted
by cloned, i.e. monoclonal cells, mature, by cloned, i.e. monoclonal cells, mature, antibody-secretiantibody-secreti
ng lymphocytesng lymphocytes from immunised animals can be cloned, from immunised animals can be cloned,
but these but these survive for only a very short period in culturesurvive for only a very short period in culture, ,
and therefore do not provide useful amounts of antibody.and therefore do not provide useful amounts of antibody.
However, procedures have been developed to allow prod However, procedures have been developed to allow prod
uction of large quantities of monoclonal antibodies by pruction of large quantities of monoclonal antibodies by pr
oducing continously growing(immortal) cell lines that secoducing continously growing(immortal) cell lines that sec
rete antibody efficiently. These involve generation of hybrete antibody efficiently. These involve generation of hyb
rid cells, transformation of lymphocytes with a virus, or rid cells, transformation of lymphocytes with a virus, or
recombinant DNA procedures.recombinant DNA procedures.

22. Monoclonal antibody productionMonoclonal antibody production
antigenantigenPEGPEG
Spleen Spleen
cellscells
Cell fusionCell fusion myelomamyeloma
Culture inCulture in HATHAT
Myeloma cellsMyeloma cells are killed by HAT.are killed by HAT.
Spleen cellsSpleen cells die off gradually.die off gradually.
OnlyOnly fused cellsfused cells survive.survive.
Test forTest for antibody-positiveantibody-positive wellswells
Clone antibody producersClone antibody producers CryopreserveCryopreserve

23. Animals(usually mice or rats) are immunized with Animals(usually mice or rats) are immunized with
antigen. Once the animals are making a good antibody rantigen. Once the animals are making a good antibody r
esponse their spleens are removed and a cell suspension esponse their spleens are removed and a cell suspension
is prepared.is prepared. These cells are fused with a These cells are fused with a myeloma cell limyeloma cell li
nene by the addition of by the addition of polyethylene glycol(PEG)polyethylene glycol(PEG) which pr which pr
omotes membrane fusion. Only a small proportion of thomotes membrane fusion. Only a small proportion of th
e cell fuse successfully.e cell fuse successfully. The fusion mixture is then set up The fusion mixture is then set up
in culture with medium containing “HAT”.in culture with medium containing “HAT”. HAT is a miHAT is a mi
xture of xture of hypoxanthine,hypoxanthine, aminopterin aminopterin and and thymidinethymidine.. AmiAmi
nopterinnopterin is powerful toxin which blocks a metabolic patis powerful toxin which blocks a metabolic pat
hway. This pathway can be bypassed if the cell is providhway. This pathway can be bypassed if the cell is provid
ed with the intermediate metabolitesed with the intermediate metabolites hypoxanthinehypoxanthine andand tt
hymidine.hymidine.

24. Thus Thus spleen cellsspleen cells can grow in HAT medium, but the can grow in HAT medium, but the myemye
loma cellsloma cells die in HAT medium because they have a metabolic d die in HAT medium because they have a metabolic d
efect and cannot use the bypass pathway. When the culture is sefect and cannot use the bypass pathway. When the culture is s
et up in HAT medium it contains et up in HAT medium it contains spleen cells,spleen cells, myeloma cellsmyeloma cells an an
d d fused cellsfused cells. The . The spleen cellsspleen cells die in culture naturally after 1-2 die in culture naturally after 1-2
weeks and weeks and myeloma cellsmyeloma cells are killed by the HAT. are killed by the HAT. Fused cellsFused cells sur sur
vive however, as they have the immortality of thevive however, as they have the immortality of the myeloma myeloma and and
the metabolic bypass of the the metabolic bypass of the spleen cellsspleen cells. Some of them will also . Some of them will also
have the antibody-producing capacity of the have the antibody-producing capacity of the spleen cellsspleen cells. Any w. Any w
ells containing growing cells are tested for the production of the ells containing growing cells are tested for the production of the
desired antibody(often by desired antibody(often by solid-phase immunoassaysolid-phase immunoassay) and if posi) and if posi
tive the cultures are cloned by plating out so that there is only otive the cultures are cloned by plating out so that there is only o
ne cell in each well. This produces a clone of cells derived from ne cell in each well. This produces a clone of cells derived from
a single progenitor, which is both immortal and a producer of a single progenitor, which is both immortal and a producer of
monoclonal antibodymonoclonal antibody. .

25. Bioscience Reports 3, 1119-1131(1983)
Monoclonal antibodies against the complement control protein Factor HMonoclonal antibodies against the complement control protein Factor H
E.SIM, M.S. PALMER, M.PUKLAVEC and R.B. SIM
MRC Immunochemistry Unit, Department of Biochemistry,MRC Immunochemistry Unit, Department of Biochemistry,
University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
Materials and MethodsMaterials and Methods
Monoclonal antibodies:Monoclonal antibodies: Factor H was purified as described previously(R.B. SiFactor H was purified as described previously(R.B. Si
m & DiScipio,1982). m & DiScipio,1982). Mice(Balb/c)Mice(Balb/c) were immunized with Factor H and sera test were immunized with Factor H and sera test
ed for the presence of anti-Factor H antibodies according to the method of Hsined for the presence of anti-Factor H antibodies according to the method of Hsin
g g et alet al.(1982). The mouse with the highest serum titre was re-injected with 20.(1982). The mouse with the highest serum titre was re-injected with 20g g
of Factor H and spleen cells were fused 4 days later with of Factor H and spleen cells were fused 4 days later with NS-O myeloma cellsNS-O myeloma cells a a
s described previously(Galfre s described previously(Galfre et alet al., 1977). The culture supernatants were tested ., 1977). The culture supernatants were tested
for the presence of antibodies against Factor H by for the presence of antibodies against Factor H by radioimmunoassayradioimmunoassay, and two , and two
hybrid cultures producing antibodies were cloned by limiting dilution. These clhybrid cultures producing antibodies were cloned by limiting dilution. These cl
ones are referred to as ones are referred to as MRC OX23MRC OX23 and and MRC OX24MRC OX24. For . For large-scale productiolarge-scale productio
nn of monoclonal antibodies, 5x10 of monoclonal antibodies, 5x10
66
cells of each hybrid cell line were injected cells of each hybrid cell line were injected intint
raperitoneallyraperitoneally into Balb/c mice which had been primed with pristance 3 weeks into Balb/c mice which had been primed with pristance 3 weeks
previously. Two weeks after injection of cells, ascites fluid was collected and antpreviously. Two weeks after injection of cells, ascites fluid was collected and ant
ibodies were purified by ibodies were purified by affinity chromatographyaffinity chromatography on agarose-protein A(Sigma). on agarose-protein A(Sigma).

27. Dorothy Crowfoot Hodgkin(1910-1994)Dorothy Crowfoot Hodgkin(1910-1994)
Vitamin B12Vitamin B12
The three-dimensional structure of the The three-dimensional structure of the
cofactor was determined by X-ray cofactor was determined by X-ray
crystallography by Dorothy Crowfoot crystallography by Dorothy Crowfoot
Hodgkin in 1956.Hodgkin in 1956.
From Principles of Biochemistry (II) Page 495.From Principles of Biochemistry (II) Page 495.

32. Purification and fragmentation of immunoglobulinsPurification and fragmentation of immunoglobulins
Methods of immunoglobulin purification from Methods of immunoglobulin purification from
the mixtures of proteins found in serum, culture supernatathe mixtures of proteins found in serum, culture supernata
nt and ascites include : nt and ascites include :
**precipitation techniquesprecipitation techniques (exploit differential solubility
characteristics of antibodies and other proteins);
* * ion-exchange chromatographyion-exchange chromatography (exploits charge diffe
rences between immunoglobulins and other proteins);
*gel filtration*gel filtration (separates proteins according to size)
**affinity chromatographyaffinity chromatography(exploits a specific interactio
n between antibody and a molecule which it binds).

34. Ligands for affinity chromatographyLigands for affinity chromatography
Ligand typeLigand type Example(s)Example(s) Antibody purificationAntibody purification
Hapten Hapten DNP(dinitrophenol)DNP(dinitrophenol) Antibodies that bind haptAntibodies that bind hapt
enen
AntigenAntigen Haemoglobin, factor VIIHaemoglobin, factor VII
II
Antibodies of a single Antibodies of a single
specificityspecificity
Bacterial immunoglobulin Bacterial immunoglobulin
binding proteinbinding protein
Protein A, protein G, Protein A, protein G,
protein Lprotein L
Most IgG subclasses from Most IgG subclasses from
many species. Some k chaimany species. Some k chai
ns from many speciesns from many species
Anti-immunoglobulin antibAnti-immunoglobulin antib
odies odies
Goat anti-human IgG anGoat anti-human IgG an
tibodiestibodies
Class and /or species-speciClass and /or species-speci
fic immunoglobulin fractific immunoglobulin fracti
onon
LectinsLectins JacalinJacalin
Mannan-binding proteinMannan-binding protein
Human IgAHuman IgA
Mouse IgMMouse IgM

36. The immuno-double-diffusion techniqueThe immuno-double-diffusion technique
By performing these reactions in agar gels By performing these reactions in agar gels
it is possible to distinguish separate it is possible to distinguish separate antigen-antiantigen-anti
bodybody reactions produced by different populatio reactions produced by different populatio
ns of antibody which are present in a ns of antibody which are present in a serumserum. Th. Th
is technique has been extended to the examinatiis technique has been extended to the examinati
on of the relationship between different antigenon of the relationship between different antigen
s.s.

37. Thanks a lot!Thanks a lot!

40. ImmunoblottingImmunoblotting
The methods described so far are partiThe methods described so far are parti
cularly useful for measuring levels of certaicularly useful for measuring levels of certai
n known antigens or antibodies, but often it n known antigens or antibodies, but often it
is necessary to identify and characterize pris necessary to identify and characterize pr
eviously eviously unknown antigensunknown antigens from a from a complex complex
mixturemixture, in which case , in which case immunoblottingimmunoblotting is v is v
ery useful.ery useful.