QuantitativeELISADevelopmentforDetectionofInsulin.ppt

Quantitative ELISA Method Development for Detection of Insulin,Jingjing Wang,Purpose,Large molecule drug: analysis method Commercial market: method development,Mature skills on insulin ELISA kit PK-PD model,ELISA method,Insulin as the reference drug,Content,Structure and Function of Antibody Principle of ELISA Structure and Property of Insulin ELISA Method Development for Insulin Method Validation,Antibody,Antibodies are the antigen-binding and Y-shaped proteins present on the B-cell membrane and secreted by plasma cells.,Membrane-bound Antibody,Secreted-bound Antibody,Antigenic Specificity on B cells. B cell receptor (BCR),Humoral Immunity,BCR facilitates the activation of these cells and their subsequent differentiation into either antibody factories called plasma cell, or memory B cell that will survive in the body and remember that same antigen so the B cells can respond faster upon future exposure.,Structure and Function of Antibody (Ig G),V region: Highly variable sequence C region: Constant sequence Fab: Antigen-binding fragment Fc: Crystallizable fragment The antibody recognizes antigen. Each tip of the “Y“ of an antibody contains a paratope (a structure analogous to a lock) that is specific for one particular epitope (that is equivalent to a key) on an antigen, allowing these two structures to bind together with precision.,Fc,Fab,Principle of ELISA,ELISA: Enzyme-linked Immunosorbent Assay Specificity of antibodies or antigens Sensitivity of simple enzyme assays A high turnover number Qualitative analysis Quantitive analysis Indirect ELISA and Sandwich ELISA,Detecting Antibody,Detecting Antigen,Insulin (Antigen),Synthesized in pancreas within the -cells preproinsulin (109 amino acids) Proinsulin (86 amino acids, A-Chain, B-Chain, C-peptide) Insulin 5808 Da (51 amino acids, A-Chain, B-Chain) and C-peptide,Physiology effects regulates carbohydrate and fat metabolism causes cells to take up glucose , storing it as glycogen.,Development of Sandwich ELISA for Insulin,Basic procedures,Step 1 Coat the antibody onto the well (100 L, 4C , 16 h) Block and Wash (300 L) Step 2 Add samples or the target antigen (100 L, 25C , 2 h) Wash (300 L) Step 3 Add the detection antibody (100 L, 25C , 1 h) Wash (300 L) Step 4 Add substrate (100 L, 37C, 20 min) Step 5 Terminate the reaction and read the absorbance,Preliminary test on materials and reagents (Several controls),Optimization of coating antibody and detection antibody concentrations,Formal experiment,Materials and reagents (solid phase, coating antibody and detection antibody),Method validation (Accuracy, Specificity, Repeatability),Materials and reagents,Solid phase: polystyrene surface, 96 well plate. (Nunc, MaxiSorp),Very hydrophobic Low binding (100-200 ng of IgG /cm2),Modified to produce more COOH or OH,High binding (400 500 ng of IgG /cm2),Proteins Peptides longer than 15-20 amino acids Small molecule epitopes attached to a protein Bacteria and virus,Direct absorption to polystyrene,hydrophobic interactions van der Waals forces hydrogen bonding ionic interactions,better washing,Coating antibody: Insulin monoclonal antibody (Reactivity with rat) Clone number, specificity (insulin/proinsulin/C-peptide), reactivity (rat), application (ELISA), price (200 300 $/100 g ).,Detection antibody: anti-rat insulin monoclonal antibody labeled with enzyme,Horseradish peroxidase enzyme is widely used. TMB: tetramethyl benzidine OPD: Ortho-phenylenediamine (OPD) AP: Alkaline phosphatase p-Nitrophenylphosphate (pnpp),Preliminary test for reagent controls,Blank control wells receive buffer in place of test sample; to determine background absorbance levels in the absence of sample; Negative control wells receive negative sample, serum without insulin; to determine the matrix effect; serum pretreatment Conjugate control wells receive coating buffer solution without antibody; to ensure that non specific binding of the detection antibody to well surfaces; Substrate control wells receive BSA solution without detecting antibody; to ensure no other oxidases in reaction system,Coating antibody concentration: 5 g/ml Detection antibody concentration: 500 ng/ml All other conditions are referred to the common procedures.,Optimization of Antibody concentrations,Coating concentration: 0.1-15 g/ml (3 levels) Detection concentration: 500 10 ng/ml for monoclonal, (3 levels),To determine the coating concentration generating the best signal-to-noise ratio while maintaining low background in the blank rows.,Formal experiment Coating,Coating buffer: 50mM Carbonate pH 9.6; 10mM Tris pH 8.5; 10mM PBS pH 7.2, depending on the storage condition of antibody. (no detergents and extraneous proteins, pH pI) Coating concentration: depending on the optimized concentration. (100 L per well) Time and temperature: 4C , 16 -18 h Blocking: BSA 1% - 5% in PBS, 4C , 16 h or RT, 2 3 h, kept before used (Storage: fill the well with sucrose 2% in PBS and incubate 2 3 min; remove all liquid and dry 1 2 h in RT),Blocking,Wash,Wash buffer: Tween-20 or Triton X-100 0.01% to 0.05% in buffer, 300 L per well Wash 3 5 times after each incubation, and soak for 5 10 min every time. Do not allow the plate to dry.,Addition of Sample or Standard,Add biosamples or insulin standards prepared in buffer or in serum 100 L per well (duplicate) Keep a consistent incubation time between wells. Incubation: 2 h, 25C,Basic procedures,Step 1 Coat the antibody onto the well (100 L, 4C , 16 h) Block and Wash (300 L) Step 2 Add samples or the target antigen (100 L, 25C , 2 h) Wash (300 L) Step 3 Add the detection antibody (100 L, 25C , 1 h) Wash (300 L) Step 4 Add substrate (100 L, 37C, 20 min) Step 5 Terminate the reaction and read the absorbance with plate-reader,Plotting the data,Plotted with linear, y=ax2+bx+c, y=ax3+bX2+cx+d, or 4-parameter logistic nonlinear regression model,4-parameter logistic nonlinear regression model,Method Validation,Sensitivity, the lowest level of quantity will be detected by this assay. Selectivity, serum from different species containing insulin will be detected to evaluate its selective measurement. Precision within-batch precision and between-batch precision Recovery will be studied by adding insulin with a known concentration to serum samples from three rats and detecting the final concentration.,Ultra Sensitive Rat Insulin ELISA (EIA) Kit Catalog#90060 Ultra Sensitive Rat Insulin ELISA Kit,Low and High Range Rat Insulin Assay Using the Same Kit Sample Size : 5l Samples: Serum, plasma, or fluid Tests:96 wells (8 wells x 12 modules) Reagents:In liquid form (except standard) Assay Range: Low Range Assay: 0.1 - 6.4 ng/ml Wide Range Assay: 0.1 - 12.8 ng/ml* High Range Assay: 1 - 64 ng/ml * Intended for screening purpose Assay Time: Same day procedure Precision