ELISA-英文版实验报告

Test1. Production of antibody

[principle]

1.

In

the

special

humoral

immune

response,

antigen

can

stimulate

immune

system

to

produce

specific

antibodies.

Each

antigen

molecule

has

several

different

antigenic

determinants,

so

it

can

be

recognized

by

different

B

cells

and

then

these

B

cells

will

produce

different

specific

antibodies that can bind with epitopes specifically. The immunity serum

produced

by

using

antigen

to

immune

animal

is

a

mixture

of

many

different

antibodies,

called

polyclonal

antibody.

The

most

common

method to produce polyclonal antibody is using pure antigen to immune

animal.

2.

The

factors

influencing

producing

of

antibodies

is

related

to

antigen

purity, animal, amount of antigen, ways of injection, times of immunity,

etc.

Primary immune animal, the antigen enter animal for the first time, the

body

will

produce

a

small

amount

antibodies

after

a

latency,

but

when

second injection antigen to the animal, there will be a fast response to the

antigen and a lot antibodies will be produced. So in this test, use antigen

to immune mice 3 times, then we can detect antibodies in the serum.

[methods]

Preparation of antigen:

1.

add 3 ml N.S to tube containing SRBC and mix them.

2.

centrifuge 2000rmp, 5 min.

3.

discard the supernant

4.

repeat above operation.

5.

Use N.S to dilute and make 20%

、

40% SRBC solution.

6.

Take 0.1ml 40% to immunize the mice subcutaneously.

7.

Do the secondary and third injection after every week.

Test

[principle]

1.

Immunological

labeling

techniques:

Ag-Ab

reactions

are

combined

with

labeling

techniques.

Known

Ag

or

Ab

is

labeled

with

radioisotope,

enzyme

or

fluorescein

and

unknown

Ab

or

Ag

are

indirectly

detected

by

labeled

molecules.

It

can

divided

into

Radioimmunoassay(RIA),

Immunofluorescence

technique

and

Enzyme

Immunoassay.

The

Direct

labeling

techniques

are

to

label

each Ag and Ab, the indirect labeling techniques are to label secondary

Ab.

2.

Enzyme immunoAssay (EIA): Ag-Ab reactions with enzyme-labeled

Ag

or

Ab.

Usually

using

horseradish

peroxidase

(HRP)

，

alkaline

phosphatase (AP) to label Ag or Ab. It can be divided into ELISA and

immunochemistry.

3.

ELISA:

Unknown Ab or Ag in blood or culture medium are detected

by

enzyme-labeled

Ag

or

Ab.

It

can

be

divided

into

3

methods,

Indirect

ELISA(Known

Ag,

enzyme-labeled

secondary

Ab

to

detect

unknown Ab ),Sandwich ELISA and Competitive ELISA.

First, the known antibody or antigen is fixed on a solid carrier. Then a

sample and the corresponding enzyme-linked Ab/Ag are added to bind

to

the

Ag/Ab

attached

to

the

solid

carrier.

The

resulting

Ag- Ab

complex and the excess Ab/Ag are separated by washing. Finally the

substrate

is

added

and

catalysis

cause

a

color

change

which

can

be

has the advantages of high sensitivity and specificity.

It combines the

specificity of the Ab-Ag reaction with the catalysis

of enzymes.

[methods]

1.

Preparation

of

Ag:

add

3ml

N.S

to

tube

containing

defibrated

SRBC and mix. Then centrifuge 2000rmp, 5 min. add 3ml N.S to

the tube and mixed, then take 2 ml packed SRBC. Add 2ml DDW

and shatter SRBC and dilute with coating buffer in a ratio of 1:400.

2.

Coating Ag:

Add 100μL of Ag to each well of ELISA plate

, then

use N.S to wash for five times.

3.

Prepare

the

serum:

remove

the

eyeball

of

the

SRBC-immunized

mice and collect the blood into Ep tube.

Wait for about 5 minute

then

centrifuge

at

12000rmp

for

10min.

take

the

supernant

and

dilute the serum 1:10.

4.

Add test serum: sign and add 100

μL of solution

to each well, then