PLANT IMMUNOLOGY Lecture 3 4 IMMUNODETECTION Antibodybased methods

PLANT IMMUNOLOGY Lecture 3, 4

IMMUNODETECTION • Antibody-based methods allowing the specific: • Detection • Quantification • Localisation • Of antigens by means of antibody binding

BASIC IMMUNO-DETECTION TECHNIQUES a) Precipitation and agglutination reactions b) Immuno diffusion tests c) Immuno electrophoresis d) Immuno electron microscopy e) Immuno capture reverse transcription technique f) Immuno capillary zone electrophoresis

PRECIPITATION AND AGGLUTINATION REACTIONS • Agglutination reaction and precipitation reaction have great importance in immunology as they are serological reactions that help in the detection of bacterial infection in the serum of a patient. • Major difference between precipitation and agglutination is the size of antigens involved. • Antigens are soluble in case of precipitation while they are insoluble in agglutination • Agglutination is more sensitive than precipitation.

PRECIPITATION REACTIONS • These are serological assays for the detection of immunoglobulin levels from the serum of a patient with infection. • The cross-linking of soluble antigen to create an insoluble precipitate that is visible. • Precipitation reactions depend on the formation of lattices and occur best when antigen and antibody are present in optimal proportions.

KEY POINTS • Precipitation assays are performed in semi-solid media such as agar or agarose where antibodies and antigens can diffuse toward one another and form a visible line of precipitation. • There are several precipitation methods applied in the diagnostic laboratory like single, double, and electroimmunodiffusion.

AGGLUTINATION • Agglutination reactions are used to assess the presence of antibodies in a specimen by mixing it with particulate antigens. • The interaction of particulate antigens (cells that carry antigens) with antibodies leads to agglutination reactions. • Agglutination reactions can be used to type blood cells for transfusion, to identify bacterial cultures, and to detect the presence and relative amount of specific antibody in a patient’s serum.

KEY POINTS • Agglutination reactions produce visible aggregates of antibodyantigen complexes when antibodies or antigens are conjugated to a carrier. • Carriers used in agglutination methods could be artificial (e. g. , latex or charcoal) or biological (e. g. , erythrocytes). • There are various methods of agglutination reactions that follow the same principle, but they differ in the elements they employ based on the desired endpoint and the main purpose of the test.

IMMUNODIFFUSION TESTS • Immunodiffusion is a diagnostic test which involves diffusion through a substance such as agar. • Theses are used: Ø To determine relative concentrations of Antibodies / Antigens. Ø To compare Antigens or Ø To determine the relative purity of an Antigen preparation. Ø For disease diagnosis. Ø Serological surveys.

• The combination of antibody (Ab) with antigen(Ag) is the fundamental reaction of immunology. • An antigen reacts with a specific antibody to form an antigen-antibody complex, the composition of which depends on the nature, concentration and proportion of the initial reactants

IMMUNODIFFUSION TECHNIQUES • Radial Immunodiffusion - A single diffusion technique where Ab is put into gel and Ag is measured by the size of a precipitin ring formed when it diffused out in all directions from a well cut into the gel. • Ouchterlony Double Diffusion - Both Ab and Ag diffuse from wells into a gel medium.

IMMUNOELECTROPHORESIS • Double-diffusion technique that utilizes electric current to enhance results. • Speed, Specificity. • Introduced by Grabar and Williams in 1953. • Combine immunodiffusion with electrophoresis • Can be used for semi-quantitaion of wide range of antigens • Qualitative • Antigen source: serum.

ELECTROPHORESIS TECHNIQUES • Electrophoresis separates molecules according to differences in their electrical charge. Ø Rocket Immunoelectrophoresis: Ø One dimension electroimmunodiffusion. • Electrophoresis is used to facilitate migration of antigen in agar - Ag diffuses out of the well: precipitation begins - Change in Ag concentration: dissolution and reformation of precipitate. • End result: precipitin line = conical-shaped, resembles a racket. • The height of the racket is measured - Height of the racket is directly proportional to the amount of antigen present. Ø Countercurrent Immunoelectrophoresis: Ag and Ab migrate toward each other by electrophoresis– Used only when Ag and Ab have opposite charges - + Ag Ab • Qualitative- Rapid. • Detection of Antinuclear- Ribonuclear protein.

USES • Immunodeficiencies can be detected by this procedure, if no precipitin band is formed for a particular Ig. • Overproduction of serum proteins. • Deficiencies in complement can also be detected. • Identification of monoclonal protein • May be used to identify urine proteins. • Testing normal & abnormal proteins in serum/urine. • Purity of Ag.

WHAT ARE PLANTIBODIES? • A plantibody is an antibody produced by genetically modified plants. • Antibodies are part of animal immune systems, and are produced in plants by transforming them with antibody genes from animals. • This was first done in 1989, with a mouse antibody made by tobacco plants. • Although plants do not naturally make antibodies, plantibodies have been shown to function in the same way as normal antibodies. • The term plantibody as well as the concept is trademarked by the company Biolex.

ANTIBODY FACTORIES • Plants are being used as antibody factories, using their endomembrane and secretory systems to produce large amounts of clinically viable proteins. • Antibodies can be expressed in plants as either full-length molecules or as smaller fragments. • Antibodies are produced in plants for both humans therapeutic purposes and for protection for plants against diseases.

FULL-SIZE MONOCLONAL ANTIBODIES RECENTLY PRODUCED IN TRANSGENIC PLANTS PLANT ANTIBODY TYPE (TARGET) PURPOSE TOBACCO Ig. G (NEMATODE) Plant pathogen resistance TOBACCO s. Ig. G/A (steptococcus mutans) Therapeutic (topical) SOYABEAN, RICE Ig. G ( herpes simplex virus) Therapeutic (topical) TOBACCO Ig. G (colon cancer) Therapeutic (systemic injection) ALFALFA Ig. G ( human Ig. G) Diagnostic TOBACCO Ig. G (rabies virus) Therapeutic (systemic injection) TOBACCO Ig. G (hepatitis B virus) Therapeutic TOBACCO Ig. G ( low mol. wt. phosphonate ester) Catalytic antibodies

METHODS FOR PLANTIBODY PRODUCTION • Using transformation and transient expression to introduce new genes into a host cell. • Targeting to the APOPLASM by the tagging with a small peptide sequence. • Signal molecules added to target the protein to the Endoplasmic Reticulum (E. R); to ensures correct folding of the protein. • Higher protein levels obtained in the E. R and the apoplasm as compared to the cytosol.

TRANSFORMATION TARGETING APOPLASM

GLYCOSYLATION • Glycosylation refers to the enzymatic process that attaches glycans to proteins, lipids, or other organic molecules. • It occurs in all higher eukaryotes in the golgi complex. • Glycans serve a variety of structural and functional roles in membrane and secreted proteins. • Glycosylation by plants differs to that found in mammalian cells. Plant glycans are smaller and have different terminal sugar residues. • It is thought this may have an immunogenic effect and have low antigen-binding affinity.

PURIFICATION OF PLANTIBODIES • Plantibodies can be purified cheaply in large quantities. • Transgenic seeds assure excellent storage properties and due to limited range of endogenous proteins in seeds, separation of plantibody is less complicated. • There is no risk of spreading animal diseases to humans as the antibodies are produced by plants • The production of large amounts of clinically viable protein especially Ig. A has many applications in medicine.

TECHNIQUES FOR PURIFICATION OF PLANTIBODIES • Filtration • Immunofluoresence • Chromatography • Diafiltration • Polymer fusion Evaluation techniques for the Plantibodies • RIA(Radioimmunoassay) Northern blot analysis • ELISA (Enzyme linked immunosorbent assay) Western blot analysis • Immunofluorescence Southern blot analysis

SECRETORY IGA • Most abundant Ig in th body…… protcts the mucosal surfaces. • Each component made by separate transgenic plant. Plants are then crossed to give multimeric molecules. • Protection to the hepatitus virus and other diseases.

WHICH PLANTS? • Plants such as tobacco are used to express plantibodies. • Plant seeds are an attractive storage organ for plantibodies. • Now being produced in corn.

PLANT APPLICATIONS

PROTECTION AGAINST PLANT PATHOGENS AND PESTS • Antibodies expressed in plants can protect them from pathogens and pests. • Termed IMMUNOMODULATION • Antibodies interfere with pathogens infectivity- they bind to the target pathogen antigen. • Block plant-pathogen infection

PROTECTION AGAINST NEMATODES • Nematodes cause huge crop damage. • Plants engineered to express Sc. Fv’s against nematode cellulases have been shown to inactivate the pests. • This technology could eradicate the need for toxic nematicides and fumigants that are currently in use.

WHAT HAS BEEN ACHIEVED SO FAR? • Currently many plantibody applications in clinical trails. • Humarised monoclonal plantibodies have proved effective against genital herpes. • Examples of applications from the treatment of gastro-intestinal disease to tooth decay. • A topical lubricant is under developemtn that could provide protection against STD’s and pregnancy.

CONCLUSION • Plants provide a cheap, efficient and safe system for the production of antibodies. • The progression of transgenic plant technology now has allowed for the progression of human life and other medicinal advancements. • It is projected that in the near future, many of the necessary human antibodies will have an origin as a plantibody.