This document discusses various types of biochemical assays and provides details on glutathione and ELISA assays. It defines an assay as an analytical measurement procedure that produces a detectable signal to quantify a biological process. Different types of assays are described including enzymatic, antioxidant, cytotoxic, and ELISA assays. The principles of glutathione and ELISA assays are explained, covering reaction components, conditions, and instrumentation. ELISA assays offer benefits like sensitivity, specificity, practicality, and enabling analysis of multiple samples quickly.

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
3/30/2022 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
3/30/2022
Garbison, K.E., et al 2004. Assay guidance manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences.
3
INTRODUCTION

4. 3/30/2022 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.
Enzymatic assay
Antioxidant
Analytical assay
Neuro-inflammation assay
Neurotransmitter assay
Cytotoxic assay
Enzyme linked immunosorbent assay
Chemiluminescent assay
Fluorescent assay
Cell death and survival assay
Kinetic assay
INTRODUCTION

5. 3/30/2022 5
Cell death and survival assay
1. Annexin V and Propidium
Iodide Assay
2. TUNEL Assay
3. Hoechst 3342 and Propidium
Iodide Assay
4. Caspase 3 Immunodetection
Assay
5. MTS Assay
6. LDH Release Assay
7. Senescence Assay
8. Clonogenic Assay
9. MTT assay
1. Lowry method
2. Bradford method
3. Becinilic acid method
1. DCFDA
2. Lipid peroxidation assay
3. Glutathione assay
4. 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 μL reaction.
 For detecting lower glutathione concentrations, such as in blood
samples, increasing reaction time will generate stronger signal.
3/30/2022 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.
3/30/2022 7

8. 3/30/2022 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 8000g 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.
3/30/2022
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
3/30/2022 10

11. PLATE PREPARATION
Aspiration with wash buffer (400μl )
Blocking with 300μl of reagent diluent
Aspiration with wash buffer (400μl )
coating of 96 well plate with 100μl of Capture antibody
3/30/2022 11

12. ASSAY PROCEDURE
Determine the optical density of each well immediately, using a microplate reader.
Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
Add 100 μL of Substrate Solution. Incubate for 20 minat RT. Avoid placing the plate in direct light.
Aspiration with wash buffer (400μl)
Add 100 μL of the of Streptavidin-HRP. Cover the plate and incubate for 20 mins at RT. Avoid placing the
plate in direct light.
Aspiration with wash buffer (400μl )
100 μL of the Detection Antibody in Reagent Diluent, incubate 2 hr at RT
Aspiration with wash buffer (400μl )
100 μL of sample or standards in Reagent Diluent Cover with an adhesive strip and incubate 2 hours at
room temperature.
3/30/2022 12

13. In recent years, ELISA test has been used very commonly in peptide and protein analyses
Sensitive and specific test
It has a wide area of application due to its ease of use and speed
It is more practical as there is no need to study two serum samples
long lifespan of the reagents used
Being free of radiation risks associated with waste substances
Enabling the analyses of multiple samples in a short time period in diagnosis and research laboratories
3/30/2022 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
3/30/2022 14
SUMMARY

15. 30/03/2022 15
THANK YOU