ELISA(酶联免疫吸附测定)实验报告

ELISA

Lin Chengyu Bio 04 2010030007

Experiment Date: 2012-03-12 Submitting Date: 2012-03-21

1Introduction

1.1Background information

ELISA (Enzyme-linked Immunosorbent Assay) is a solid-phase assay for antibodies

employing ligands labeled with enzymes which is widely used for immunological assays.

This technique can be applied to detect antigens or antibodies for qualitative or

quantitative purpose. Since enzyme reactions are very well known amplification

processes, the signal is generated by enzymes which are linked to the detection reagents

in fixed proportions to allow accurate quantification.1

1.2Major principles

Figure 1 Schematic diagram of ELISA2

Figure 2 Procedure of indirect ELISA3

As shown in Figure 1 & 2, the general procedure of indirect ELSIA is to: incubate the

plate well with antigen, wash off unbounded antigen, incubate with 1st antibody, wash

off unbounded 1st antibody, incubate with labeled 2nd antibody, wash off unbounded 2nd

antibody, incubate with enzyme substrate solution, and detect optical density or other

index showing enzyme activity.

2Experiment Operation

2.1Antigen coating

(1)Prepare an antigen solution in coating buffer (human IgG at 0.025mg/ml);

(2)Pipette 200 μl antigen solution to each well (Row: B~G; Column: 2~10; Column 11

is negative control without antigen) of the microtiter plate;

(3)Incubate the plate at 37 ℃for 30 min;

(4)Remove the antigen solution;

(5)Wash each well with 200 μl with PBS-T for 3 times;

(6)Block each well (Row: B~G; Column: 2~11) with 200 μl 0.5% BSA-PBS, and

incubate the plate at 37 ℃for 30 min;

(7)Remove the blocking solution;

(8)Wash each well with 200 μl with PBS-T for 3 times.

2.2Primary antibody reaction

(1)Dilute the primary antibody (rabbit-anti-human IgG antiserum) in PBS-T for

different dilution (from 1:400 to 1:51,200 in 2-folds dilution);

(2)Add 200 μl diluted antibody solution to each well following Table 1;

Table 1 Scheme to add primary antibody

(3)Incubate the plate at 37 ℃for 1 hour;

(4)Remove the primary antibody solution;

(5)Wash each well with 200 μl PBS-T for 3 times.

2.3Application of secondary antibody

(1)Dilute the peroxidase conjugated secondary antibody (Goat-anti-rabbit IgG-HRP) in

PBS-T at the dilution of 1:20,000 and 1:40,000;

(2)Add 200 μl secondary antibody solution to each well following Table 2;

(3)Incubate the plate at 37 ℃for 1 hour;

(4)Remove the secondary antibody solution;

(5)Wash each well with 200 μl PBS-T for 3 times.

2.4Substrate development

(1)Add 200 μl substrate solution to each well (Row: B~G ,Column: 2~11);

(2)Incubate for approximately 3 min;

(3)Add 50 μl 2 M H2SO4 to each well to terminate the reaction;

(4)Measure optical density at 490 nm.

3Raw data and its processing

3.1Raw data

3.2Data processing

Set Row B, C, and D as Group I, and Row E, F, and G as Group II. The processed data

is shown in Table 4

Table 4 Processed data: optical density of each group

Set different dilutions of primary antibody as x axis, optical density as y axis, draw

Figure 3 to illustrate their relation.