Identifying Target Proteins: Serology & Monoclonal Antibodies - Prof. Dinender Singla, Study notes of Molecular biology

Download Identifying Target Proteins: Serology & Monoclonal Antibodies - Prof. Dinender Singla and more Study notes Molecular biology in PDF only on Docsity! 1. Immunological Diagnostic Procedures a. ELISA i. Direct ELISA 1. No secondary Ab necessary ii. Indirect ELISA 1. Bind the sample being tested for the presence of a specific molecule or organism to a solid support, such as a plastic microtiter plate, which usually contains 96 sample wells. Wash the support to remove unbound molecules 2. Add a marker-specific Ab (primary Ab directed against the target antigen) to the bound material, and then wash the support to remove unbound primary Ab 3. Add a second Ab (secondary Ab) that binds specifically to the primary Ab and not to the target molecule a. Bound (conjugated) to the secondary Ab is an enzyme that can catalyze a reaction that converts a colorless substrate into a colored product b. Wash the mixture to remove any unbound secondary Ab-enzyme conjugate 4. Add the colorless substrate 5. Observe or measure the amount of colored product iii. Sandwich ELISA 1. Plate is coated with a capture Ab 2. Sample is added, and any Ag present binds to capture Ab 3. Detecting Ab is added, and binds to Ag 4. Enzyme-linked secondary Ab is added, and binds to detecting Ab 5. Substrate is added, and is converted by enzyme to detectable form iv. Variations 1. Abs attached to well a. Patient sample added containing Ag, Ag will bind to the Ab in the well b. Peroxidase-conjugated antibody added c. To identify the presence of target protein in a sample 2. Serology a. Bacterial Ag affixed to substrate Patient serum added b. If Ab v. Ag present, it will stay in the well c. Peroxidase-conjugated anti-human IgG Ab added color reaction = antibody in serum recognizes Ag d. Testing for Ab production in response to antigen 3. Seronegative a. No specific antibodies to a pathogen (no prior exposure) 4. Seropositive a. Production of specific antibodies to a pathogen 5. Seroconversion a. Switch from seronegative to seropositive 2. Monoclonal Antibodies a. Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent cell b. Each Ab binds only one specific antigen c. Types i. Murine monoclonal Abs ii. Chimeric and humanized monoclonal Abs iii. Human monoclonal Abs 1. Produced by merging the DNA that encodes the binding portion of the monoclonal mouse Ab with human Ab-producing DNA d. HAT Procedure (Hypoxanthine, Aminopterin, Thymidine) i. Steps: 1. Immunize Mouse --> 2. Isolate Spleen cells + Myeloma Cells (HGPRT-) 3. Nonfused Spleen Cells (S); Nonfused Myeloma Cells (M); Fused Spleen-Spleen cells (S-S); Fused Myeloma-Myeloma Cells (M-M); Fused Hybrid Spleen-Myeloma Cells (S-M)--> 4. Addition of HAT Medium--> a. M and M-M cannot grow; S and S-S cannot divide; OR b. S-M grow and divide i. Survive in HAT because the spleen cell contributes a functional HGPRT, which can utilize the exogenous Hypoxanthine in the medium even though purine production by means of dihydrofolate reductase is blocked by aminopterin, and because the cell division functions of the myeloma cell are active e. Production and Screening of Mabs i. Spleen cells from a mouse that was immunized with a specific antigen are isolated and fused in culture with myeloma cells that do not produce antibody chains ii. Fused cells are selected for the ability to grow on HAT medium, which contains previously mentioned iii. Cells that produce a specific antibody the immunizing Ag (hybridomas) are identified by an immunoassay and individually subcultured iv. A hybridoma, which grows in culture and secrets a single type of antibody molecules, is the source of a monoclonal antibody f. Targets for Diagnostic Mabs i. Polypeptide Hormones 1. Chorionic gonadotropin 2. Growth hormone 3. Luteinizing hormone 4. Follicle-stimulating hormone 5. Thyroid-stimulating hormone 6. Prolactin ii. Tumor Markers 1. Carcinoembryonic antigen 2. Prostate-specific antigen 3. Interleukin-2 receptor 4. Epidermal growth factor receptor iii. Cytokines 1. Interleukins 1-8 2. Colony-stimulating factor iv. Drug Monitoring 1. Theophylline 2. Gentamicin 3. Cyclosporin v. Miscellaneous Targets 1. Thyroxine 2. Vitamin B12 3. Ferritin 4. Fibrin Degradation Products 5. Tau protein vi. Infectious Diseases 1. Chlamydia 2. Herpes 3. Rubella 4. Hep B 5. Legionella 6. HIV 3. Colored Fluorescence Proteins a. GFP i. 238aa long photoprotein ii. Isolated from jellyfish Aequorea victoria b. RFP i. Isolated from Discosoma coral ii. Production of monomeric red fluorescent protein requires 33 mutations c. Luciferase i. Come from lux genes; firefly luc genes ii. Includes five genes (luxCDABE) iii. Peaks at 490nm iv. Genes A and B are responsible for generating the light signal 4. DNA Diagnostic Systems a. General Laboratory Nucleic Acid Hybridization Scheme i. Bind ssDNA (target) to a membrane support ii. Add single-stranded labeled DNA (probe) under appropriate conditions of temperature and ionic strength to promote base paiting between the probe and the target DNAs iii. Wash the support to remove excess unbound labeled probe DNA iv. Detect the hybrid sequences that form between the probe and target DNA b. Chemiluminescence i. Biotin-labeled probe is bound to the target DNA ii. Streptavidin is bound to the biotin molecules iii. Biotin-labeled AP binds to Streptavidin iv. AP converts the substrate into a light-emitting product v. E, Biotin; AP, alkaline phosphate c. Molecular Beacon i. Nonradioactive method for detecting specific sequences of nucleic acids ii. Typlical molecular beacon probe is 25nt long 1. 15nt in the middle are complementary to the target DNA and are designed so that this single- stranded molecles does not form a structure in which these nucleotides base pair with one another 2. This enzyme generates (2'-5') linked oligonucleotides-->activates later cellular endoribonuclease-- >cleaves viral RNA f. Pharmaceuticals i. Human Growth Hormone 1. Produced in E. coli, approved for human use 2. Also binds to the prolactin receptor a. Site-specific mutagenesis to prevent this binding ii. Tumor Necrosis Factor α 1. Anti-tumor agent 2. Toxic and non-specific a. TNFα fusion construct with a tumor cell targeting peptide iii. Interleukins 1. Control inflammatory responses a. Hard to administer as therapeutics 2. Probiotic administration of IL-10 to control Crohn's disease 2. Human Growth Hormone (hGH) a. Somatotropin; 191-amino acid; 22,125 Da b. Native and Modified i. hGH treatment is given to the chronic renal insufficiency and Turner's syndrome ii. Oligonucleotide-directed mutagenesis (Hist 18., -21, and Glu-174) was altered used to alter hGH so that it no longer bound to the prolactin receptor but retained its specificity for the growth hormone receptor iii. Limitation of hGH native; cross reactivity with other cell tyes iv. Every day therapy is required, inconvenient and expensive c. Growth Hormone Binding i. Derivatization of growth hormone by coupling it to a protein of the growth hormone receptor using a 20- amino acid peptide 1. Monomeric derivative 2. Dimeric derivative 3. Monomeric derivative bound to a growth hormone receptor 4. Stays in the system for longer time of periods a. Therapy required every 10 days 3. Tumor Necrosis Factor (TNF) Alpha a. Tumor Necrosis Factor Alpha i. Anti-tumor agent ii. Toxic and non-specific 1. TNFα fusion construct with a tumor cell targeting peptide iii. Addition of 6-amino acids didn't alter receptor binding site; however, enhanced antitumor activity and demonstrated less toxicity 4. Enzymes a. Cystic Fibrosis i. Most common Caucasian genetic disease ii. Results from loss of chloride channel function in plasma membrane iii. Alters the physiology of the mucus layer in the lungs iv. Generates massive lung functions 1. Breathing difficulties due to pathogen biofilm formation v. Neutrophils (phagocytes) recruited vi. Neutrophils and pathogens both killed 1. Huge amounts of DNA released 2. Extremely viscous mucus 3. Breathing difficulties b. DNase I i. Hydrolyzes long polymeric DNA into short oligomers ii. Purified enzyme delivered by an inhaled mist into lungs of CF patients 1. Decreases viscosity of the mucus 2. Relieves most severe smptoms iii. Problem: lysed neurophiles release actin 1. Actin binds an inhibits Dnase I (reduces effectiveness) c. Alginate Lyase i. Alginate 1. Polysaccharide polymer produced by seaweed and many bacteria 2. Chains of β-D-mannuronate and α-L-Guluronate 3. Crosslinking with Ca2+ and metal ions -->elastic gel ii. Pseudomonas aeruginosa ("mucoid" strains) 1. Produces copious amounts of alginate in biofilms 2. Increases viscosity of mucus in CF patients iii. Flavobacterium sp. 1. Produces an alginate lyase 2. Cloned and expressed in E. coli iv. Alginate Lyase-Producing Clone 1. An alginate lyase-producing clone from a clone bank of a Flavobacterium so. In E. coli 2. The alginate that is present in the growth medium is digested by alginase secreted by E. coli clones so that the alginate in the vicinity of this colony is not cross-linked when Ca2+ is added v. Recombinate Flavobacterium alginate lyase protein precursor in E. coli 1. A 6-kDa peptide is removed from the N-terminus of the 69-kDa precurosor to yield a 63-kDa protein that can depolymerize alginate from both seaweed and bacteria 2. A second cleavage even converts the 63-kDa protein to a 23-kDa protein that is active againsr seaweed alginate and a 40-kDa protein that hydrolyzes bacterial alginate vi. DNA Construct Encoding the 40 kDa Alginate Lyase 1. Bacillus subtilis expression vector 2. The leader peptide from a B. subtilis alpha-amylase gene is fused to the N-terminus of the lyase coding sequence 3. Under control of the penicillinase promoter 4. Produces activate lyase in medium d. Enzymatic Conversion of Phenylalanine i. A disease phenylketonuria results from the impaired function of enzyme PHENYLALANINE HYDROXYLASE ii. Treatment strategy-conrolled diet of PHENYLANALINE or SEARCH for another enzyme 5. Intestinal Interleukin-10 Secreting Bacteria on Inflammatory Bowel Disease in Mice a. Interleukins i. Control inflammatory responses in Crohn disease and Ulcerative colitis ii. Hard to administer as therapeutics because IL-10 is not clinically acceptable as this needs to be injected in frequent injections iii. Prebiotic (Lactococcus lactis) administration of IL-10 to control Crohn Disease Data Mouse IL-10 Deficient mouse Ulcerative Colitis Mouse +5% Dextran Sulfate (which inflames the intestine) Ulcerative colitis Mouse IL-10 deficient mouse + engineered IL-10 producing L. lactis Prevented onset of ulcerative colitis Mouse +5% dextran sulfate + engineered IL-10 producing L. lactis 50% reduction in ulcerative colitis 6. Problems with monoclonal Abs a. Generated by mouse fusion cell lines i. B Cells (Ab production) and B cell myelomas (growth) ii. Problem = Mouse antibody cross-reactivity in humans iii. Answer: Human/mouse fusion cell ines 1. Chromosomes of human B cells are unstable in fusion cell lines 2. Ethical to intentionally inject humans and collect splenic cells? 3. Human myelomas cannot replace mouse B cell myelomas iv. An alternate v. Chimeric or Humanized Mab 7. Structure of Ab a. H and L contains contain variable (VL and VH) and constant (CL, CH1, CH2, and CH3) domains with their CDRs (CDR1, CDR2, and CDR3) b. The Fv, Fab, and Fc portions of an antibody molecule are delineated c. The N-terminal (NH2) and C-Terminal (COOH) ends of each polypeptide chain are indicated d. CDR: Complementarity-Determining Region 8. Engineered Chimeric Ab a. The VL and VH DNA regions from the immunoglobulin L and H genes that encode part of a mAb were substituted or the VL and VH DNA regions of a human Ig molecule b. The product of the constructed gene is a chimeric (partially humanized) Ig with the antigen-binding specificity of the mAb and both lowered immunogenicity in humans and human Fc effector capabilities 9. Engineered Humanized Ab a. The CDTs (CDR1, CDR2, and CDR3) from the genes for H and L Ig chains of the mAb replace the CDRs of the genes for a human antibody b. The product of this constructed gene is an Ig with the antigen-binding specificity of the mAb and all the other properties of a human antibody molecule 10. Human Monoclonal Antibodies and Geno Mouse a. Mouse Ab genes are inactived by specific deletions in Embryonic Stem (ES) cells, which are subsequently used to generate transgenic mice unable to make Abs b. The human genes encoding Ig light and heavy chains are introduced on a YAC into mouse ES cells c. These cells are used to generate transgenic mice able to synthesize both mouse and human Abs d. The mice generated from these two types of manipulation are cross-bred, and mice that can synthesize only human Igs are selected, immunized, and used to make hybridomas producing human Abs 11. Generation of Combinatorial Library of VL and VH regions of the Ab in E. coli a. Steps i. Extract mRNA from isolated lymphocytes ii. Convert mRNA to cDNA iii. Amplify H and L chains by PCR iv. Cut with specific set of restriction endonucleases 1. Clone H chain sequences into "H Chain" bacteriophage vector; and 2. Clone L chain sequences into "L chain" bacteriophage vector v. Combine H and L chains into one bacteriophage vector vi. Screen plaques from Ag binding vii. Excise H and L chains as part of a plasmid viii. Transform E. coli with plasmid-H-L chain DNA construct ix. Harvest Ag-binding Fv fragment from E. coli 12. DNA Constructs of an Fv Combinatorial Gene Library a. Portions of the cDNAs of the L(A) and H(B) chains are separately cloned into bacteriophage λ vectors b. Each of these libraries is digested with EcoRI and then fragments from the H chain library are ligated to the fragments from the L chain library 13. Fv Ab Combinatorial Library in the Bacteriophage M13 a. The cDNAs for the VL and VH regions are amplified by PCR and then ligated to DNA that encodes a short linker peptide b. Each single-chain antibody DNA construct is cloned into gene 3 of bacteriophage M13 c. There are three copies of the phage gene 3 protein, which is a phage surface protein, per M13 bacteriophage 14. Construction of a Large Library of Single-Chain Ab a. B cells from several nonimmunized individuals were collected and pooled, the mRNA was isolated and used to program the synthesis of cDNA oligonucleotide primers containing DNA sequences that included small portions of the framework region sequence were added to the cDNA preparation, and all six CDRs were amplified separately by PCR b. The amplified CDRs from all six PCRs were mixed together with oligonucleotides encoding the framework regions and the linker, and genes encoding the variable L and H domains were synthesized by overlap extension PCR 15. Mab-Based Drug Delivery Systems a. Cancer "Vaccines": Immunotoxins i. AB Toxins 1. Catalytic A subunit often causes death 2. Cell binding B subunit responsible for host cell specificity ii. Immunotoxins 1. Catalytic A subunit causes cell death 2. Recombinant "B" subunit antibody that recognizes cancer protein ligand for an upregulated cancer receptor 16. Antibody as Therapeutic a. Antifibrin antibody, monoclonal Ab, that is specific for the fibrin found in blood clots, is coupled to plasminogen activator (PA) b. After the complex binds to the fibrin of a blood clot, the plasminogen activator causes plasmin to accumulate in the vicinity of the clot c. The plasmin then degrades the clot 1. Antisense RNA a. A specified mRNA which prevents translation of the protein, antisense RNA b. To be an effective therapeutic agent, an antisense RNA must bind to a specified mRNA and prevent translation of the protein c. One that base pairs with a specific mRNA is called an antisense oligonucleotide d. Inhibition of translation of specific mRNAs by antisense (As) nucleic acid molecules i. The promoter and polyadenylation regions are marked p and pa respectively; the intron is indicated by the letter A; and the exons are indicated by numbers (1 and 2) 1. cDNA (AS gene) is cloned into an expression vector in reverse orientation, and the construct is transfected into a cell, where the AS RNA is synthesized a. The AS RNA hybridizes to the target mRNA, and translation is blocked 2. An AS oligonucleotide is introduced into a cell, and after it hybridizes with the target mRNA, translation is blocked e. cDNA for human insulin-like growth factor 1 (ILGF-1) cloned on a vector in the antisense orientation under the transcriptional control of a metallothionein promoter (MTp) i. Following transfection into tumor-causing cells, low levels of ZnSO4 are added, the cells have decreased tumorigenicity 2. Antisense Oligonucleotides (ON) and their Selection