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 2.ELISA

[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 measured.ELISA 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 100μL of solution to each well, then

put it in a humidified box under 37℃ for 45min.

5. Add secondary Ab: Discard the solution in ELISA plate and wash the plate 3 times. Then add 100μL of HRP-labeled secondary Ab to each well and put it in a humidified box under 37℃ for 30min.

6. Showing color: Discard the solution in ELISA plate and wash the plate 3 times. Then add 100μL of substrate solution to each well. Show color in dark for10min

[result]

the No.1 well is blank, No.2 is negative well, No.3 is positive well, No.4 is test well. The No.3 and No.4 became yellow while No.1 and No.2 is colorless. The color of the No.4 well is deeper than the No.3.

[analysis]

The result in No.4 well is positive because it become yellow,which means there are antibodies in the No.4 well also indicates that SRBC-

immunized mice had produced antibodies. The color of test well (No.4 well) is deeper than the positive control well(No.3 well), so antibodies amount in test serum is more than that in the positive control.

Test3. Hypersensitivity Test of Guinea Pig

[principles]

Hypersensitivity refers to the self tissue injury and/or biological disorder when the sensitized individual contacts the same allergen again. Hypersensitivity is the abnormal or pathologic immune response. According to the mechanisms of hypersensitivity, it could be divided into four types, typeⅠ、Ⅱ、Ⅲ and Ⅳ. Type I hypersensitivity is also called immediate type hypersensitivity or anaphylaxis. The antigen leads to hypersensitivity is called allergen. The antibody take part in anaphylaxis is mainly IgE.

Allergen-induced period：In type I hypersensitivity, allergen enters body for the first time and stimulates plasma cells to produce IgE. IgE could bind to the cell surface FcεR of the mast cells or basophils.

Allergen-stimulated period ：When the same allergen enters the sensitized body again, allergen could bind to the IgE on the cell surface. Then a series of reaction happen, which lead to the cell activation and biological mediators being released from these cells. These mediators lead to the attack of type I hypersensitivity.

Effect period：histamine is the main mediator that has important biological effects. The rapid release of histamine could increase vascular permeability, lead to vasodilatation, promote smooth muscle contraction and increase secretion of glands. Histamine could lead to the low blood

pressure, dyspnea, bellyache, anaphylactic shock, and even death.

[methods]

1.Allergen-induced injection: 2 weeks before test, inject diluted egg albumin 1ml to guinea pig subcutaneously.

2.Allergen-stimulated injection: Immobilize the guinea pig. Give an intracardiac injection of 1 ml diluted egg albumin. Release the guinea pig and observe its response.

[result]

In a few minutes after the allergen-stimulated injection,the Dyspnea convulsion appeared.The guinea pig began to Scratch its nose. About 10 minutes the guinea pig died.

 

第二篇：ELISA实验报告

酶联免疫吸附测定实验报告

ELISA

一 实验原理

酶联免疫吸附测定(enzyme-linked immunosorbent assay，简称ELISA)是在免疫酶技术(immunoenzymatic techniques)的基础上发展起来的一种新型的免疫测定技术。ELISA过程包括：抗原(抗体)吸附在固相载体上——包被，加待测抗体(抗原)，加相应酶标记抗体(抗原)，生成抗原(抗体)-待测抗体(抗原)-酶标记抗体的复合物，在于该酶的底物反应生成有色产物。然后借助分光光度计的光吸收计算抗体(抗原)的量。待测抗体(抗原)的定量与有色产生成正比。

ELISA常用的有4种方法，分别是直接法、间接法、双抗体夹心法和竞争法。在我们的实验中，采用的是间接法。主要步骤有：将抗原吸附于固相载体表面；加抗体，形成抗原-抗体复合物；加酶标抗体；加底物(测定底物的降解量=抗体量)。

二 实验用品及器具

1 聚苯乙烯微量细胞培养板(平板，96孔)

2 酶联免疫检测仪

3辣根过氧化物酶羊抗兔IgG

4 包被液

0.05mol/L 碳酸缓冲液(pH 9.6)

Na2CO3 0.15g

NaHCO3 0.293g

蒸馏水稀释至100mL

5 稀释液(PBS-Tween)

NaCl 8g

KCl 0.2g

KH2PO4 0.2g

Na2HPO4 ·10H2O 2.9g

6 Tween-20,0.5mL

蒸馏水加至1000mL

7 洗涤液：同稀释液

8 封闭液：质量分数为0.5%的BSA(用PBS配制)

9 邻苯二胺溶液(底物)

配置：0.1mol/L柠檬酸(2.1g/100ml)，取6.1ml

0.2mol/L Na2HPO4·12H2O(7.163g/100ml)，取6.4ml

加蒸馏水12.5ml

取邻苯二胺8mg(溶解)

临用前加30%(体积分数)H2O240μL

10 终止液：2mol/L H2SO4

三 实验步骤

1 在培养板的B2-B10、C2-C10、D2-D10号的well中分别加入0.025mg/ml的含Human IgG的包被液各200μL，在37℃的环境中存放30min

2 取出培养板，将包被液倒出，用PBS-T洗涤三次，每次每格200μL

3 洗涤之后加入含0.5%BSA的PBS(封闭液)，每格200μL，在37℃的环境中存放30mim

4取出培养板，将封闭液倒出，用PBS-T洗涤三次，每次每格200μL

5 将浓度为1:400的Rabbit-α-human IgG antiserum溶液(一抗)稀释为浓度为1:800、1:1600、1:3200、1:6400、1:12800、1:25600、1:51200的溶液，一次加入B2-B9、C2-C9、D2-D9，每格200μL，在B11、C11、D11中加入1:400的溶液200μL，在37℃的环境中存放1h

6取出培养板，将溶液倒出，用PBS-T洗涤三次，每次每格200μL

7 在B2-B11、C2-C11、D2-D11中分别加入Goat-α-rabbit IgG-HRP(浓度为1:20000)溶液(二抗)，每格200μL，在37℃的环境中存放30min

8取出培养板，将溶液倒出，用PBS-T洗涤三次，每次每格200μL

9 每格加入200μL邻苯二胺溶液进行反应，约5min后加入终止液(2mol/L的H2SO4溶液)

10 将培养板放入酶联免疫检测仪内进行分析，得出数据

四 实验数据

数据图表如下:(从图表中可以看出，在1:400到1:51200浓度的范围内，数据基本上是呈线性变化的)

由图线可知，在误差范围内，吸光度与被检血清浓度接近正比关系。这与公式：“吸光度 = e×光径×浓度”是吻合的。

五 实验后感想

这是我进入大学以来真正意义上的第一个实验，虽然整个实验的过程可以说有些枯燥乏味，因为大部分的时间都在等待中度过，但是不得不说，在这次实验中还是收获了不少的东西。

首先，这次实验所涉及的内容是我以前完全没有接触过的。在中学里学到的知识也许只能勉强解释一下抗原抗体是什么，是怎么工作的，但是在定量检测的这一块却从来没有接触过。所以这一次的实验也算是给自己开了开眼界，增长了知识。也许在以后的学习中，可能也会用到相关的知识，毕竟我是学生物医学工程的，以后难免会遇到一些有关的问题，这也算是一次提前学习吧。

其次，相对于其他实验来讲，像这种类型的实验，对于每种试剂的量的要求是相当高的，也就是说，可能只是手轻轻一抖，整个实验就必须得重新来过，所以这也是一个对自己的耐心、细心程度的锻炼。

做实验，团队合作是必不可少的，一个人做一个实验，总会出现这样那样的问题，导致实验失败，而两人的团队合作，就会使错误发生的概率降到很低，从而大大增加了实验的效率。

最后还要感谢辛苦的老师和助教们。在这次实验中，让我见识了一位最可爱的生物老师——余老师，在她不到一个小时的讲解时间里，都不知道被我们的笑声打断多少次了，幽默风趣乐观大概是我能想到的描述余老师最准确的词了。还有几位不辞辛劳一直在我们四周帮助我们的助教们，谢谢你们，让我有了一次如此精彩的经历。