Biochemical assays.pptx

different types assays for qualitative analysis

1. Presented By Parimal Hadge PE/2019/311 PC-611 BIOCHEMICAL ASSAYS

2. Contents Introduction Various types of assays Principle of Glutathione assay ELISA Summary 1/27/2021 2

3. An assay is an analytical measurement procedure defined by a set of reagents that produces a detectable signal, allowing a biological process to be quantified. The quality of an assay is defined by the robustness and reproducibility of this signal in the absence of any test compounds or in the presence of inactive compounds. Assay quality depends on Type of signal measured (absorbance, fluorescence, luminescence, radioactivity, etc.) Reagents Reaction conditions Analytical and automation instrumentation Statistical models for the data analysis 1/27/2021 Garbison , K.E., et al 2004. Assay guidance manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences . 3 INTRODUCTION

4. 1/27/2021 4 A biochemical assay is an analytical in vitro procedure used to detect, quantify and/or study the binding or activity of a biological molecule. INTRODUCTION

5. 1/27/2021 5 Cell death and survival assay Annexin V and Propidium Iodide Assay TUNEL Assay Hoechst 3342 and Propidium Iodide Assay Caspase 3 Immunodetection Assay MTS Assay LDH Release Assay Senescence Assay Clonogenic Assay MTT assay Lowry method Bradford method Becinilic acid method DCFDA Lipid peroxidation assay Glutathione assay DPPH Assay Antioxidant assays Protein estimation assay

6. The assay is based on the glutathione recycling system by DTNB and glutathione reductase. DTNB and glutathione (GSH) react to generate 2-nitro-5-thiobenzoic acid which has a yellow color . The generated GSSG can be reduced back to GSH by glutathione reductase, and GSH reacts with DTNB again to produce more 2-nitro-5-thiobenzoic acid. Therefore, the recycling system dramatically improves the sensitivity of total glutathione detection. can quantify glutathione from 1-100 ng/well in a 200 μ L reaction. For detecting lower glutathione concentrations, such as in blood samples, increasing reaction time will generate stronger signal. 1/27/2021 6

7. The assay is based on the reaction of GSH with DTNB (also known as Ellman’s reagent) that produces the TNB chromophore, which has a maximal absorbance at 412 nm, and oxidized glutathione–TNB adduct (GS–TNB). The rate of formation of TNB, measured at 412 nm, is proportional to the concentration of GSH in the sample. The disulfide product (GS–TNB) is then reduced by GR in the presence of NADPH, recycling GSH back into the reaction. Because GR reduces the GSSG formed into 2GSH, the amount of glutathione measured represents the sum of reduced and oxidized glutathione in the sample ([GSH]total =[GSH] + 2 × [GSSG]). The rate of change in absorbance (ΔA412 nm min–1) is made to be linear for the convenience and consistency of measurement, and is linearly proportional to the total concentration of GSH. The concentration of an unknown sample is determined by calculating from the linear equation or the regression curve generated from several standards of GSH. 1/27/2021 7

8. 1/27/2021 8 SAMPLE PREPARATION Homogenize the tissue in 0.6% sulfosalicylic acid–Triton-X solution. Transfer the clear supernatant to a new tube, and use it for the total GSH assay. Centrifuge the homogenized tissue sample at 8000 g for 10 min at 2–4 °C

9. ELISA method was pioneered largely by the Swiss scientists Engvall,and Perlmann developed the ELISA method in 1971 by modifying the RIA method The enzymes that can be employed in ELISA include beta galactosidase , glucose oxidase, peroxidase, and alkaline phosphatase. The enzyme-substrate reaction is usually completed within30–60 min. The reaction can be stopped using sodium hydroxide ( NaOH ), hydrochloric acid ( HCl ) or sulfuric acid (H2SO4). The results are read on a spectrophotometer and at 400–600 nm depending on the characteristics of the conjugate used. 1/27/2021 Aydin , S., 2015. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides , 72 , pp.4-15. 9 ENZYME LINKED IMMUNOSORBENT ASSAY ENZYME SUBSTRATE CHROMOGEN STOPPER Alkaline phosphatase P-NPP P-NPP+ Diethandamine+MgCl2 1M NaOH Horse radish peroxidase H2O2 Tetramethybenzidine+phosphate-citrate buffer 1M H2SO4 Horse radish peroxidase H2O2 O-Phenylenediamine+HCl 1M HCl

10. ELISA Method used to detect the presence of antigens, cytokines and other secreted factors in the culture medium The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Detection by checking absorbance at 450 nm 1/27/2021 10

11. PLATE PREPARATION 1/27/2021 11

12. ASSAY PROCEDURE 1/27/2021 12

13. 1/27/2021 13 ADVANTAGES OF ELISA

14. A biochemical assay is an analytical in vitro procedure used to detect, quantify and/or study the binding or activity of a biological molecule. These assay should be robustness and reproducible. There are number of biochemical assay available so far for the qualitative and qualitative analysis of biological samples. The selection of suitable assay depend on Type of signal (absorbance, fluorescence, luminescence, radioactivity, etc.) Reagents available Reaction conditions Analytical and automation instrumentation Statistical models for the data analysis 1/27/2021 14 SUMMARY

15. 27/01/2021 15 THANK YOU