Elisa - a Rapid Immunochemical Assay

Detection of Anti-BSA in Serum A, B and C samples, and Determination of Anti-BSA Amount in Rabbit Anti-Serum, Normal Rabbit Serum, Purified Bound and Unbound Fractions along with Different Types of Control by Employing Indirect ELISA.

Introduction

B cells participation in the adaptive immunity by producing antibodies is often measured by analysing the distinct antibody produced in the blood or plasma during humoral immune response. Antibody molecules has a high affinity to their corresponding antigen and hence, detection and measurement of protein or antigen by using antibodies is very easy due to their specificity. An assay which directly detects the binding of antibody to antigen is said to detect primary interaction. In addition, secondary interaction assay is one which further measures the amount of antibody present by the change which is induced in the physical state of the antigen for e.g. precipitation or clumping.

ELISA is a rapid immunochemical assay that involves an enzyme that catalyzes a biochemical reaction which involves an antibody or antigen (immunologic molecules). The ELISA is a fundamental tool of clinical immunology, and is used as an initial screen for pathogens detection (Ma et al. 2014). Based on the principle of antibody-antibody interaction, in this assay we can easy visualisation the results and can be completed without the use of radioactive materials. The ELISA technique is the first and most basic assay to determine if an individual is positive for a selected pathogen, such as HIV and Hepatitis B or C, or bacteria (Yolken 1980). The applications of immunoassays are extended to other fields such as infectious diseases, autoimmune diseases, cancer, degenerative diseases, haematology and pharmacology (Erdile, Smith & Berd 2001). Other uses of the ELISA include detection of prohibited drugs such as cocaine and methamphetamines (Lewis et al. 2003). Applications have not been confined to human health care. Many applications have been described in veterinary medicine, agriculture, environmental health and the food industry (Asensio et al. 2008).

There are four main types of ELISA namely direct, indirect, sandwich and competitive. A direct ELISA is the simplest and quickest ELISA which uses a single primary antibody that is linked with an enzyme whereby this complex reacts directly with the antigen which is fixed to the plate (Gretchen & McNellis 1998). After washing, the substrate is added to give a colour change which can be measured spectrophotometric ally. Direct ELISAs are also used in immunehistochemical staining. On the other side, indirect ELISA is the most widely used whereby the antigen is fixed onto the plate, followed by the addition of a primary antibody (Gretchen & McNellis 1998). Consequently, an antibody-enzyme complex is added which has an affinity for the primary antibody. The indirect ELISA is commonly used for detecting HIV antigen in serum of patients. A positive ELISA test is always followed by a Western blot test and a positive Western blot confirms an HIV infection.

Moreover, sandwich ELISAs include the immobilisation of antibodies to the microtitre plate (Ma et al. 2014). The antigen is then loaded into the plate. A secondary antibody/enzyme complex is added before finally adding a substrate to catalyse the colour change. The sandwich ELISA is normally used to detect viruses in plants. On the other side, competitive ELISA was the first reported method which involves the immobilisation of antigen onto the microtitre plate, followed by a blocking step where a concentrated solution of a protein such as serum albumin is used to coat the remaining plastic surface to prevent false positive results (Perrett et al. 2010). Antibody-enzyme conjugate and antibodies are added to different wells of the plate. The conjugate binds to the immobilized antigen, but so does the unconjugated antibody, and the binding of each is mutually exclusive. Subsequently, there is the addition of substrate to initiate the enzymatic reaction. The rate of product release, or the accumulation of product after a fixed time, is determined. Reactions to which conjugate was added will have the highest absorbance whilst those lacking conjugate or controls will yield the lowest absorbance.

Therefore, this experiment will make use of the indirect ELISA where the antigen (BSA) is immobilised onto the wells and different types of serum samples as well as positive and negative controls will be added. Consequently, there will be the addition of antibody-Horseradish peroxidase complex which will bind to the antigen-antibody complex. The, ABTS substrate is to be added whereby the bound enzyme will react with substrate to produce a coloured precipitate. The colour change will be measured by a spectrophotometer which will record the absorbance of the wells. The intensity of the colour is directly proportional to the quantity of bound conjugate and activity of the enzyme on the plate.

Materials and Method

Part I: Screening test for detection of Antibody in culture media by ELISA

Materials and Equipment

1. 96-well microtiter plate
2. Purified antigen (BSA)
3. Carbonate-bicarbonate buffer pH 9.6
4. PBS-Tween (0.15M Phosphate buffer saline containing 0.5% Tween)
5. Anti rabbit- HRP conjugate
6. ABTS solution
7. 0.5M oxalic acid
8. Water bath
9. ELISA plate Reader/Filter (405nm)
10. Serial serum samples (A, B & C) from various sources
11. Normal serum (-ve control)
12. Anti-BSA serum (+ve control)

Method

The 96-well microtiter plate was already coated with the BSA antigen and washed five times with PBS-Tween. The fifth wash was discarded. The positive and negative controls was added to column 1 in rows A and B respectively. Serum samples A, B and C were added in duplicates to column 1 in according rows. Fifty microliters of PBS-Tween were loaded to all the other wells. In order to perform serial dilutions, 50 µL sample was transferred by using a multichannel auto pipette from well 1 to well 2 of all eight rows, and mixed by pipetting up and down. The process is repeated until column 11 where 50 µL was removed from well 11 and discarded, so as column 12 only contained 50 µL of PBS-Tween to act as ‘No Serum’ control. The microtiter plate was incubated at 37oC for 30 minutes.