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(2)-ELISA.-ENZYME LINKED IMMUNOSORBENT ASSAY

(2)-ELISA.-ENZYME LINKED IMMUNOSORBENT ASSAY

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IMMUNOLOGICAL IMMUNOLOGICAL TECHNIQUES TECHNIQUES P289 P289
For recognizing and quantifying For recognizing and quantifying antigens in tissues or fluids antigens in tissues or fluids man man y immunological techniques utili y immunological techniques utili ze the exquisite specificity of the ze the exquisite specificity of the antigen-antibody bond antigen-antibody bond. .
Cell populations can be identified Cell populations can be identified and and characterized by their surface marke characterized by their surface marke rs, using the techniques of immunoflu rs, using the techniques of immunoflu orescence or immunohistochemistry orescence or immunohistochemistry
Cell populations can be Cell populations can be isolated isolated 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 (FACS FACS), ), panning and density-dependent panning and density-dependent centrifugations. centrifugations.
The principle assays for lympho The principle assays for lympho cyte function cyte function are by antibody or are by antibody or cytokine production, by prolifer cytokine production, by prolifer ation in response to antigen, or ation in response to antigen, or by cytotoxicity. by cytotoxicity.
*ANTIGEN-ANTIBODY INTERACTION ANTIGEN-ANTIBODY INTERACTION 1. Precipitation reactions 1. Precipitation reactions 2. Haemagglutination and complement 2. Haemagglutination and complement fix fix ation ation 3. Direct and indirect 3. Direct and indirect immunofluorescence immunofluorescence 4. Immunoassay 4. Immunoassay 5. Immunoblotting 5. Immunoblotting and immunoprecipitation and immunoprecipitation
Immunochemical techniques Immunochemical techniques The study of antibodies(and some ot The study of antibodies(and some ot her immunologically important molecule her immunologically important molecule s such as complement components) is kno s such as complement components) is kno wn as immunochemistry wn as immunochemistry. .Such methods a Such methods a re known as immunochemical techniques. re known as immunochemical techniques.
Antibodies Antibodies Antibodies are a group of globular proteins know Antibodies 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 larg of 25,000 and is common to all classes, whereas the larg er(heavy) chain has a molecular weight of 50,000-77,00 er(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 forces and non-covalent forces.
Fab.(Fragment-antigen binding): Fab.(Fragment-antigen binding): The part of an antibody molecule which conta The part of an antibody molecule which conta ins the antigen-binding site, consisting of a light ch ins the antigen-binding site, consisting of a light ch ain and part of the heavy chain; it is produced by e ain 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 C for binding to antibody receptors on cells and the C 1q component of complement. 1q component of complement.
Complement Complement 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 pathway The 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 pathway Alternative 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.
Further reading: Further reading: Porter RR, Reid, KBM. The biochemistry of com Porter RR, Reid, KBM. The biochemistry of com plement. plement. Nature Nature 1978; 1978; 275 275: 699-704. : 699-704. Reid KBM, Porter RR. The proteolytic activatio Reid KBM, Porter RR. The proteolytic activatio n systems of complement. n systems of complement. Annu Rev Biochem Annu Rev Biochem 198 198 1; 1; 50: 50: 433-464. 433-464. Reid KBM, Day AJ. Structure-function relations Reid KBM, Day AJ. Structure-function relations hips of the complement components. hips of the complement components. Immunol To Immunol To day day, 1989; , 1989; 10: 10: 177-180. 177-180. Campbell RD, Law SKA, Reid KBM, Sim RB. St Campbell RD, Law SKA, Reid KBM, Sim RB. St ructure, organisation, and regulation of the comp ructure, organisation, and regulation of the comp lement genes. lement genes. Annu Rev Immunol Annu Rev Immunol 1988; 1988; 6: 6: 161-19 161-19 5 5.
Polyclonal and monoclonal antibodies Polyclonal and monoclonal antibodies Usually, many different antibodies, Usually, many different antibodies, recognising sever recognising sever al different epitopes al 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 fr sponse is described as polyclonal, as antibody is derived fr om more than one clone of B lymphocytes and shows heter om more than one clone of B lymphocytes and shows heter ogeneity in the amino acid sequences of the antigen-bindin ogeneity in the amino acid sequences of the antigen-bindin g immunoglobulins present. g immunoglobulins present. However, more recently, methods have been develop However, more recently, methods have been develop ed for deriving monoclonal antibodies, which are derived f ed for deriving monoclonal antibodies, which are derived f rom a single B cell clone and show identical amino acid seq rom a single B cell clone and show identical amino acid seq uence. Monoclonal antibody preparations show homogene uence. Monoclonal antibody preparations show homogene ous characteristics(including specificity and avidity for ant ous characteristics(including specificity and avidity for ant igen, i.e. they igen, i.e. they recognise a single epitope recognise a single epitope). ).
Production of antibodies Production 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 recognis recognis e proteins and peptides e proteins and peptides. . In some cases, particular parts of the antige In some cases, particular parts of the antige n produce very potent immune responses and su n produce very potent immune responses and su ch epitopes are known as immunodominant. Im ch epitopes are known as immunodominant. Im munogenicity tends to increase with size; munogenicity tends to increase with size; protein protein s with a molecular weight > 10,000 s with a molecular weight > 10,000 are usually i are usually i mmunogenic as long as they are recognised as fo mmunogenic as long as they are recognised as fo reign in responding animals. reign in responding animals.
For production of potent antibodies t For production of potent antibodies t hat perform well in immunochemical tech hat perform well in immunochemical tech niques, it is usually necessary to use an niques, it is usually necessary to use an ad ad juvant juvant 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 antigen forming 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.
Some commonly used adjuvants Some commonly used adjuvants P266 P266 *Freund’s complete adjuvant(FCA): Freund’s complete adjuvant(FCA): Mineral oil containing heat-killed mycobacteria Mineral oil containing heat-killed mycobacteria ( (Mycobacterium tuberculosis Mycobacterium tuberculosis), ), Used as emulsion with aqueous antigen Used as emulsion with aqueous antigen *Freund’s incomplete adjuvant(FIA): *Freund’s incomplete adjuvant(FIA): Mineral oil Mineral oil Used as emulsion with aqueous antigen Used as emulsion with aqueous antigen Alum, Bentonite, Quil A, MDP, MPL, Alum, Bentonite, Quil A, MDP, MPL, Bacillus pertussis Bacillus pertussis
Examples of immunization protocols that Examples of immunization protocols that have been used successfully have been used successfully P267 P267 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. Source Source: Reproduced from M.A.Kerr and R.Thorpe, : Reproduced from M.A.Kerr and R.Thorpe, I I mmunochemistry Labfax mmunochemistry Labfax(1994), Bios Scientific, Oxfor (1994), Bios Scientific, Oxfor d. d.
Antibodies and sera Antibodies and sera Rabbit polyclonals: Rabbit polyclonals: rabbit antisera rabbit antisera to huma to huma n Factor H, bovine Factor H and human n Factor H, bovine Factor H and human  2 21,and t 1,and t o the fusion protein GST-5 o the fusion protein GST-5th th domain of domain of  2 21 were a 1 were a vailable in our laboratory. vailable in our laboratory. Affinity purfied rabbit IgG anti-human Affinity purfied rabbit IgG anti-human  2 21 1 was made by passing 2ml of was made by passing 2ml of rabbit antiserum rabbit antiserum on a on a column(2ml volume) of column(2ml volume) of  2 21-Sepharose 1-Sepharose(1mg (1mg  2 21 co 1 co valently attached per ml of Sepharose). Bound ant valently attached per ml of Sepharose). Bound ant ibodies were eluted with ibodies were eluted with 3M MgCl 3M MgCl2 2, 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)
Production of monoclonal antibodies Production 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-secreti antibody-secreti ng lymphocytes ng 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 culture survive 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 pr uction of large quantities of monoclonal antibodies by pr oducing continously growing(immortal) cell lines that sec oducing continously growing(immortal) cell lines that sec rete antibody efficiently. These involve generation of hyb rete 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.
Monoclonal antibody production Monoclonal antibody production antigen antigen PEG PEG Spleen Spleen cells cells Cell fusion Cell fusion myeloma myeloma Culture in Culture in HAT HAT Myeloma cells Myeloma cells are killed by HAT. are killed by HAT. Spleen cells Spleen cells die off gradually. die off gradually. Only Only fused cells fused cells survive. survive. Test for Test for antibody-positive antibody-positive wells wells Clone antibody producers Clone antibody producers Cryopreserve Cryopreserve
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 r antigen. 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 li myeloma cell li ne ne 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 th omotes 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 mi HAT is a mi xture of xture of hypoxanthine, hypoxanthine, aminopterin aminopterin and and thymidine thymidine. . Ami Ami nopterin nopterin is powerful toxin which blocks a metabolic pat is powerful toxin which blocks a metabolic pat hway. This pathway can be bypassed if the cell is provid hway. This pathway can be bypassed if the cell is provid ed with the intermediate metabolites ed with the intermediate metabolites hypoxanthine hypoxanthine and and t t hymidine. hymidine.
Thus Thus spleen cells spleen cells can grow in HAT medium, but the can grow in HAT medium, but the mye mye loma cells loma 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 s efect 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 cells myeloma cells an an d d fused cells fused cells. The . The spleen cells spleen cells die in culture naturally after 1-2 die in culture naturally after 1-2 weeks and weeks and myeloma cells myeloma cells are killed by the HAT. are killed by the HAT. Fused cells Fused cells sur sur vive however, as they have the immortality of the vive however, as they have the immortality of the myeloma myeloma and and the metabolic bypass of the the metabolic bypass of the spleen cells spleen 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 cells spleen 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 immunoassay solid-phase immunoassay) and if posi ) and if posi tive the cultures are cloned by plating out so that there is only o tive 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 antibody monoclonal antibody. .
Bioscience Reports 3, 1119-1131(1983) Monoclonal antibodies against the complement control protein Factor H Monoclonal 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 Methods Materials and Methods Monoclonal antibodies: Monoclonal antibodies: Factor H was purified as described previously(R.B. Si Factor 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 Hsin ed for the presence of anti-Factor H antibodies according to the method of Hsin g g et al et 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 cells NS-O myeloma cells a a s described previously(Galfre s described previously(Galfre et al et 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 radioimmunoassay radioimmunoassay, and two , and two hybrid cultures producing antibodies were cloned by limiting dilution. These cl hybrid cultures producing antibodies were cloned by limiting dilution. These cl ones are referred to as ones are referred to as MRC OX23 MRC OX23 and and MRC OX24 MRC OX24. For . For large-scale productio large-scale productio n n of monoclonal antibodies, 5x10 of monoclonal antibodies, 5x106 6 cells of each hybrid cell line were injected cells of each hybrid cell line were injected int int raperitoneally raperitoneally 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 ant previously. Two weeks after injection of cells, ascites fluid was collected and ant ibodies were purified by ibodies were purified by affinity chromatography affinity chromatography on agarose-protein A(Sigma). on agarose-protein A(Sigma).
Dorothy Crowfoot Hodgkin(1910-1994) Dorothy Crowfoot Hodgkin(1910-1994) Vitamin B12 Vitamin 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.
Purification and fragmentation of immunoglobulins Purification and fragmentation of immunoglobulins Methods of immunoglobulin purification from Methods of immunoglobulin purification from the mixtures of proteins found in serum, culture supernata the mixtures of proteins found in serum, culture supernata nt and ascites include : nt and ascites include : * *precipitation techniques precipitation techniques (exploit differential solubility characteristics of antibodies and other proteins); * * ion-exchange chromatography ion-exchange chromatography (exploits charge diffe rences between immunoglobulins and other proteins); *gel filtration *gel filtration (separates proteins according to size) * *affinity chromatography affinity chromatography(exploits a specific interactio n between antibody and a molecule which it binds).
Ligands for affinity chromatography Ligands for affinity chromatography Ligand type Ligand type Example(s) Example(s) Antibody purification Antibody purification Hapten Hapten DNP(dinitrophenol) DNP(dinitrophenol) Antibodies that bind hapt Antibodies that bind hapt en en Antigen Antigen Haemoglobin, factor VII Haemoglobin, factor VII I I Antibodies of a single Antibodies of a single specificity specificity Bacterial immunoglobulin Bacterial immunoglobulin binding protein binding protein Protein A, protein G, Protein A, protein G, protein L protein L Most IgG subclasses from Most IgG subclasses from many species. Some k chai many species. Some k chai ns from many species ns from many species Anti-immunoglobulin antib Anti-immunoglobulin antib odies odies Goat anti-human IgG an Goat anti-human IgG an tibodies tibodies Class and /or species-speci Class and /or species-speci fic immunoglobulin fracti fic immunoglobulin fracti on on Lectins Lectins Jacalin Jacalin Mannan-binding protein Mannan-binding protein Human IgA Human IgA Mouse IgM Mouse IgM
The immuno-double-diffusion technique The 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-anti antigen-anti body body 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 serum serum. Th . Th is technique has been extended to the examinati is technique has been extended to the examinati on of the relationship between different antigen on of the relationship between different antigen s. s.
Thanks a lot! Thanks a lot!
Immunoblotting Immunoblotting The methods described so far are parti The methods described so far are parti cularly useful for measuring levels of certai cularly 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 pr is necessary to identify and characterize pr eviously eviously unknown antigens unknown antigens from a from a complex complex mixture mixture, in which case , in which case immunoblotting immunoblotting is v is v ery useful. ery useful.

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(2)-ELISA.-ENZYME LINKED IMMUNOSORBENT ASSAY

  • 1.
  • 2.
    For recognizing andquantifying For recognizing and quantifying antigens in tissues or fluids antigens in tissues or fluids man man y immunological techniques utili y immunological techniques utili ze the exquisite specificity of the ze the exquisite specificity of the antigen-antibody bond antigen-antibody bond. .
  • 3.
    Cell populations canbe identified Cell populations can be identified and and characterized by their surface marke characterized by their surface marke rs, using the techniques of immunoflu rs, using the techniques of immunoflu orescence or immunohistochemistry orescence or immunohistochemistry
  • 4.
    Cell populations canbe Cell populations can be isolated isolated 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 (FACS FACS), ), panning and density-dependent panning and density-dependent centrifugations. centrifugations.
  • 5.
    The principle assaysfor lympho The principle assays for lympho cyte function cyte function are by antibody or are by antibody or cytokine production, by prolifer cytokine production, by prolifer ation in response to antigen, or ation in response to antigen, or by cytotoxicity. by cytotoxicity.
  • 6.
    *ANTIGEN-ANTIBODY INTERACTION ANTIGEN-ANTIBODY INTERACTION 1.Precipitation reactions 1. Precipitation reactions 2. Haemagglutination and complement 2. Haemagglutination and complement fix fix ation ation 3. Direct and indirect 3. Direct and indirect immunofluorescence immunofluorescence 4. Immunoassay 4. Immunoassay 5. Immunoblotting 5. Immunoblotting and immunoprecipitation and immunoprecipitation
  • 7.
    Immunochemical techniques Immunochemical techniques Thestudy of antibodies(and some ot The study of antibodies(and some ot her immunologically important molecule her immunologically important molecule s such as complement components) is kno s such as complement components) is kno wn as immunochemistry wn as immunochemistry. .Such methods a Such methods a re known as immunochemical techniques. re known as immunochemical techniques.
  • 8.
    Antibodies Antibodies Antibodies are agroup of globular proteins know Antibodies 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 larg of 25,000 and is common to all classes, whereas the larg er(heavy) chain has a molecular weight of 50,000-77,00 er(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 forces and non-covalent forces.
  • 10.
    Fab.(Fragment-antigen binding): Fab.(Fragment-antigen binding): Thepart of an antibody molecule which conta The part of an antibody molecule which conta ins the antigen-binding site, consisting of a light ch ins the antigen-binding site, consisting of a light ch ain and part of the heavy chain; it is produced by e ain 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 C for binding to antibody receptors on cells and the C 1q component of complement. 1q component of complement.
  • 12.
    Complement Complement The complement systemis 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 pathway The 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 pathway Alternative 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: PorterRR, Reid, KBM. The biochemistry of com Porter RR, Reid, KBM. The biochemistry of com plement. plement. Nature Nature 1978; 1978; 275 275: 699-704. : 699-704. Reid KBM, Porter RR. The proteolytic activatio Reid KBM, Porter RR. The proteolytic activatio n systems of complement. n systems of complement. Annu Rev Biochem Annu Rev Biochem 198 198 1; 1; 50: 50: 433-464. 433-464. Reid KBM, Day AJ. Structure-function relations Reid KBM, Day AJ. Structure-function relations hips of the complement components. hips of the complement components. Immunol To Immunol To day day, 1989; , 1989; 10: 10: 177-180. 177-180. Campbell RD, Law SKA, Reid KBM, Sim RB. St Campbell RD, Law SKA, Reid KBM, Sim RB. St ructure, organisation, and regulation of the comp ructure, organisation, and regulation of the comp lement genes. lement genes. Annu Rev Immunol Annu Rev Immunol 1988; 1988; 6: 6: 161-19 161-19 5 5.
  • 15.
    Polyclonal and monoclonalantibodies Polyclonal and monoclonal antibodies Usually, many different antibodies, Usually, many different antibodies, recognising sever recognising sever al different epitopes al 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 fr sponse is described as polyclonal, as antibody is derived fr om more than one clone of B lymphocytes and shows heter om more than one clone of B lymphocytes and shows heter ogeneity in the amino acid sequences of the antigen-bindin ogeneity in the amino acid sequences of the antigen-bindin g immunoglobulins present. g immunoglobulins present. However, more recently, methods have been develop However, more recently, methods have been develop ed for deriving monoclonal antibodies, which are derived f ed for deriving monoclonal antibodies, which are derived f rom a single B cell clone and show identical amino acid seq rom a single B cell clone and show identical amino acid seq uence. Monoclonal antibody preparations show homogene uence. Monoclonal antibody preparations show homogene ous characteristics(including specificity and avidity for ant ous characteristics(including specificity and avidity for ant igen, i.e. they igen, i.e. they recognise a single epitope recognise a single epitope). ).
  • 16.
    Production of antibodies Productionof 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 recognis recognis e proteins and peptides e proteins and peptides. . In some cases, particular parts of the antige In some cases, particular parts of the antige n produce very potent immune responses and su n produce very potent immune responses and su ch epitopes are known as immunodominant. Im ch epitopes are known as immunodominant. Im munogenicity tends to increase with size; munogenicity tends to increase with size; protein protein s with a molecular weight > 10,000 s with a molecular weight > 10,000 are usually i are usually i mmunogenic as long as they are recognised as fo mmunogenic as long as they are recognised as fo reign in responding animals. reign in responding animals.
  • 17.
    For production ofpotent antibodies t For production of potent antibodies t hat perform well in immunochemical tech hat perform well in immunochemical tech niques, it is usually necessary to use an niques, it is usually necessary to use an ad ad juvant juvant 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 antigen forming 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 usedadjuvants Some commonly used adjuvants P266 P266 *Freund’s complete adjuvant(FCA): Freund’s complete adjuvant(FCA): Mineral oil containing heat-killed mycobacteria Mineral oil containing heat-killed mycobacteria ( (Mycobacterium tuberculosis Mycobacterium tuberculosis), ), Used as emulsion with aqueous antigen Used as emulsion with aqueous antigen *Freund’s incomplete adjuvant(FIA): *Freund’s incomplete adjuvant(FIA): Mineral oil Mineral oil Used as emulsion with aqueous antigen Used as emulsion with aqueous antigen Alum, Bentonite, Quil A, MDP, MPL, Alum, Bentonite, Quil A, MDP, MPL, Bacillus pertussis Bacillus pertussis
  • 19.
    Examples of immunizationprotocols that Examples of immunization protocols that have been used successfully have been used successfully P267 P267 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. Source Source: Reproduced from M.A.Kerr and R.Thorpe, : Reproduced from M.A.Kerr and R.Thorpe, I I mmunochemistry Labfax mmunochemistry Labfax(1994), Bios Scientific, Oxfor (1994), Bios Scientific, Oxfor d. d.
  • 20.
    Antibodies and sera Antibodiesand sera Rabbit polyclonals: Rabbit polyclonals: rabbit antisera rabbit antisera to huma to huma n Factor H, bovine Factor H and human n Factor H, bovine Factor H and human  2 21,and t 1,and t o the fusion protein GST-5 o the fusion protein GST-5th th domain of domain of  2 21 were a 1 were a vailable in our laboratory. vailable in our laboratory. Affinity purfied rabbit IgG anti-human Affinity purfied rabbit IgG anti-human  2 21 1 was made by passing 2ml of was made by passing 2ml of rabbit antiserum rabbit antiserum on a on a column(2ml volume) of column(2ml volume) of  2 21-Sepharose 1-Sepharose(1mg (1mg  2 21 co 1 co valently attached per ml of Sepharose). Bound ant valently attached per ml of Sepharose). Bound ant ibodies were eluted with ibodies were eluted with 3M MgCl 3M MgCl2 2, 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 monoclonalantibodies Production 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-secreti antibody-secreti ng lymphocytes ng 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 culture survive 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 pr uction of large quantities of monoclonal antibodies by pr oducing continously growing(immortal) cell lines that sec oducing continously growing(immortal) cell lines that sec rete antibody efficiently. These involve generation of hyb rete 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 production Monoclonalantibody production antigen antigen PEG PEG Spleen Spleen cells cells Cell fusion Cell fusion myeloma myeloma Culture in Culture in HAT HAT Myeloma cells Myeloma cells are killed by HAT. are killed by HAT. Spleen cells Spleen cells die off gradually. die off gradually. Only Only fused cells fused cells survive. survive. Test for Test for antibody-positive antibody-positive wells wells Clone antibody producers Clone antibody producers Cryopreserve Cryopreserve
  • 23.
    Animals(usually mice orrats) are immunized with Animals(usually mice or rats) are immunized with antigen. Once the animals are making a good antibody r antigen. 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 li myeloma cell li ne ne 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 th omotes 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 mi HAT is a mi xture of xture of hypoxanthine, hypoxanthine, aminopterin aminopterin and and thymidine thymidine. . Ami Ami nopterin nopterin is powerful toxin which blocks a metabolic pat is powerful toxin which blocks a metabolic pat hway. This pathway can be bypassed if the cell is provid hway. This pathway can be bypassed if the cell is provid ed with the intermediate metabolites ed with the intermediate metabolites hypoxanthine hypoxanthine and and t t hymidine. hymidine.
  • 24.
    Thus Thus spleen cells spleencells can grow in HAT medium, but the can grow in HAT medium, but the mye mye loma cells loma 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 s efect 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 cells myeloma cells an an d d fused cells fused cells. The . The spleen cells spleen cells die in culture naturally after 1-2 die in culture naturally after 1-2 weeks and weeks and myeloma cells myeloma cells are killed by the HAT. are killed by the HAT. Fused cells Fused cells sur sur vive however, as they have the immortality of the vive however, as they have the immortality of the myeloma myeloma and and the metabolic bypass of the the metabolic bypass of the spleen cells spleen 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 cells spleen 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 immunoassay solid-phase immunoassay) and if posi ) and if posi tive the cultures are cloned by plating out so that there is only o tive 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 antibody monoclonal antibody. .
  • 25.
    Bioscience Reports 3,1119-1131(1983) Monoclonal antibodies against the complement control protein Factor H Monoclonal 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 Methods Materials and Methods Monoclonal antibodies: Monoclonal antibodies: Factor H was purified as described previously(R.B. Si Factor 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 Hsin ed for the presence of anti-Factor H antibodies according to the method of Hsin g g et al et 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 cells NS-O myeloma cells a a s described previously(Galfre s described previously(Galfre et al et 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 radioimmunoassay radioimmunoassay, and two , and two hybrid cultures producing antibodies were cloned by limiting dilution. These cl hybrid cultures producing antibodies were cloned by limiting dilution. These cl ones are referred to as ones are referred to as MRC OX23 MRC OX23 and and MRC OX24 MRC OX24. For . For large-scale productio large-scale productio n n of monoclonal antibodies, 5x10 of monoclonal antibodies, 5x106 6 cells of each hybrid cell line were injected cells of each hybrid cell line were injected int int raperitoneally raperitoneally 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 ant previously. Two weeks after injection of cells, ascites fluid was collected and ant ibodies were purified by ibodies were purified by affinity chromatography affinity chromatography on agarose-protein A(Sigma). on agarose-protein A(Sigma).
  • 27.
    Dorothy Crowfoot Hodgkin(1910-1994) DorothyCrowfoot Hodgkin(1910-1994) Vitamin B12 Vitamin 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 fragmentationof immunoglobulins Purification and fragmentation of immunoglobulins Methods of immunoglobulin purification from Methods of immunoglobulin purification from the mixtures of proteins found in serum, culture supernata the mixtures of proteins found in serum, culture supernata nt and ascites include : nt and ascites include : * *precipitation techniques precipitation techniques (exploit differential solubility characteristics of antibodies and other proteins); * * ion-exchange chromatography ion-exchange chromatography (exploits charge diffe rences between immunoglobulins and other proteins); *gel filtration *gel filtration (separates proteins according to size) * *affinity chromatography affinity chromatography(exploits a specific interactio n between antibody and a molecule which it binds).
  • 34.
    Ligands for affinitychromatography Ligands for affinity chromatography Ligand type Ligand type Example(s) Example(s) Antibody purification Antibody purification Hapten Hapten DNP(dinitrophenol) DNP(dinitrophenol) Antibodies that bind hapt Antibodies that bind hapt en en Antigen Antigen Haemoglobin, factor VII Haemoglobin, factor VII I I Antibodies of a single Antibodies of a single specificity specificity Bacterial immunoglobulin Bacterial immunoglobulin binding protein binding protein Protein A, protein G, Protein A, protein G, protein L protein L Most IgG subclasses from Most IgG subclasses from many species. Some k chai many species. Some k chai ns from many species ns from many species Anti-immunoglobulin antib Anti-immunoglobulin antib odies odies Goat anti-human IgG an Goat anti-human IgG an tibodies tibodies Class and /or species-speci Class and /or species-speci fic immunoglobulin fracti fic immunoglobulin fracti on on Lectins Lectins Jacalin Jacalin Mannan-binding protein Mannan-binding protein Human IgA Human IgA Mouse IgM Mouse IgM
  • 36.
    The immuno-double-diffusion technique Theimmuno-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-anti antigen-anti body body 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 serum serum. Th . Th is technique has been extended to the examinati is technique has been extended to the examinati on of the relationship between different antigen on of the relationship between different antigen s. s.
  • 37.
  • 40.
    Immunoblotting Immunoblotting The methods describedso far are parti The methods described so far are parti cularly useful for measuring levels of certai cularly 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 pr is necessary to identify and characterize pr eviously eviously unknown antigens unknown antigens from a from a complex complex mixture mixture, in which case , in which case immunoblotting immunoblotting is v is v ery useful. ery useful.