Determination Of Antibody Specificity By Elisa Biology Essay

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The aim of the laboratory experiment was to follow the course of an immunisation. And using the test monitor the changes in the antibody titre (number of antibodies produced) during the course of the immunisation and determine weather the antibody extracted from the third immunisation was specific to human albumin.

Introduction: Antibodies are a vital part of the immune response, protecting animals against foreign matter. Antibodies are produced by b lymphocytes that are activated in response to foreign molecules (antigens). Any molecule that triggers an immune response is called an antigen. The body's recognises its own molecules as "self" and attacks anything foreign "non self". Antibodies produced in the immune response travel all over the body and bind to the antigen that stimulated its production. The binding of an antigen and antibody is called an immune complex. (Brooker 2007)

Antibody structure and specificity: Antibodies also known as immunoglobulins recognize and bind antigens. The recognition of a particular antigen is essential for a specific immune response. There are two types of molecules that are involved in a specific immune response. Immunoglobulins and T cell receptors TCRs. Immunoglobulins are expressed as cell surface receptors on B cells or found in serum secreted by plasma cells. When a B cell receptor comes into contact with an antigen it specifically recognizes the B cell is activated and begins to produce plasma cells. Plasma cells then secrete large amounts of antibodies that have the same binding specificity as the original B cell receptor. The binding of an immunoglobulin to an antigen is highly specific and has a lot to do the structure of the immunoglobulin. The basic structure of an immunoglobulin consists of 2 identical light chains and 2 identical heavy chains held together by disulphide bonds. There are five distinct classes of immunoglobulin that differ in size, charge, amino acid sequence and carbohydrate content. These can be further divided in to subclasses (Isotypes) based on the structure of the heavy chain. All isotopes except (IgD) are bifunctional. They recognize and bind the antigen and then promote its killing or removal by the activation of effecter mechanisms. Different immunoglobulin classes and sub classes activate different effecter mechanisms.

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Non specific (Innate) immunity and Specific (acquired) immunity: The immune system is divided in to two types. Non-specific (innate) immunity and Specific (acquired) immunity. Both systems work together to produce an efficient immune response. All animals are born with non-specific (Innate) immunity. The body's defences are present from birth and defend against foreign molecules regardless of the specific identity. Instead nonspecific defences recognise common conserved properties of the molecule judging it to be foreign. The resistance to a particular Antigen does not increase on exposure to it the second time. So we can say innate immunity is present all the time, it is not antigen specific and has no memory. Non-specific immunity also includes the body's outer barriers (skin) and mucus membranes. Cells also take part in non-specific (innate) immunity as cellular barriers apposing infections. These include Phagocytes, Non-phagocytes and Natural killer cells. (Campbell and Reece)

Specific (acquired) immunity is found in vertebrates only. Unlike non-specific (innate) immunity specific (acquired) immunity develops only after the body is exposed to foreign substances. Specific (acquired) immunity is antigen specific and contains memory cells. Cells of the immune system first encounter the antigen and later recognise it to be attacked. Thus the second exposure to the antigen producers a more improved and efficient response. (Brooker)

Lymphatic system: The cells responsible for providing specific immunity are Lymphocytes (B cells and T cells). Both sets of cells originated from stem cells in bone marrow. The B cells mature within the bone marrow and t cells mature within the thymus gland. Lymphocytes continuously circulate between lymphatic organs, the blood and all the tissues of the body. On encountering an antigen an immune response is invoked. The migration of lymphocytes to an area where an invader has been detected greatly increases. This increases the chances of a lymphocyte encountering an antigen it specifically recognizes. (Brooker 2008) (Campbell and Reece)

Clonal selection theory: Lymphocytes are responsible for Immune recognition in Specific (acquired) immune responses. They achieve this by clonal selection. As we have learnt each lymphocyte recognises just one specific antigen. When a lymphocyte comes in to contact with a specific antigen it recognizes it and is induced to proliferate rapidly. The proliferating cells produce effecter cells and memory cells. Thus over the next few days the body produces a sufficient amount of clones (effecter cells) of the original lymphocyte that came into contact with the antigen for a successful immune response. The memory cells remain. This process of clonal selection is operation in both sets of lymphocytes (B cells and T cells) (Immune 200? brostoff)

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Principals of ELISA and the immunisation schedule: Enzyme-linked immnosorbent assay (ELISA) is an immunological technique designed to detect antibodies or antigens by the use of enzymes. An antigen is incubated on to a plastic plate and becomes absorbed to the base of it. Any free antigen is then washed away. Test antibody is added which binds to the antigen. A ligand that can detect the presence of an antibody is coupled to a enzyme such as peroxidise. This binds the test antibody and after the free ligand is washed away the bound ligand is visualised by adding a colorless substrate that acted on by the enzyme linked to the ligand to produce a coloured end product. The amount of test antibody is measured by assessing the amount of colored end product by optical density scanning plate. (immune)

To prepare the antibody for the Elisa test to be carried out a rabbit was inoculated with human albumin invoking an immune response, this was the test. Another rabbit was inoculated with phosphate buffer solution this was used as a control. After 10 days of inoculation blood was extracted from both rabbits and the antiserum was collected. This procedure was carried out three times across 3 weeks. 6 sets of antisera were prepared, three from the test rabbit (anti- albumin antiserum) and three from the control rabbit (non-immune antiserum).

In the ELISA test Human albumin was incubated on to the base of polyvinyl chloride (PVC) plate. Sets of bleeds at various dilutions from both the test and control rabbit where added. The primary binding was monitored by using a secondary antibody (horseradish peroxidise conjugulated secondary antibody). The antibody concentrations at half maximal antigen binding were taken as an estimate of antibody titre.

Experiment 1 estimation of antibody titre

Table of Raw data

Table1

Antibody dilutions

Absorbance 450nm

1

2

3

4

5

6

7

8

9

10

11

12

1/2500

A

2.199

2.011

2.157

2.312

2.216

2.358

0.151

0.162

0.181

0.170

0.254

0.243

1/5000

B

2.033

1.706

2.141

2.085

2.171

2.277

0.132

0.130

0.142

0.137

0.205

0.185

1/10000

C

1.586

1.771

2.175

2.108

2.271

2.230

0.156

0.160

0.141

0.145

0.153

0.131

1/25000

D

1.764

1.471

1.926

1.994

2.060

2.068

0.150

0.140

0.072

0.133

0.132

0.134

1/50000

E

1.586

1.473

1.915

1.881

1.955

2.009

0.214

0.165

0.147

0.128

0.115

0.178

1/100000

F

1.518

1.092

1.515

1.387

1.795

1.836

0.194

0.154

0.132

0.148

0.138

0.128

1/200000

G

0.811

0.788

1.383

1.337

1.678

1.645

0.149

0.145

0.144

0.131

0.135

0.131

1/400000

H

0.526

0.535

1.138

0.936

1.484

1.311

0.177

0.173

0.140

0.144

0.114

0.160

Bleed

Bleed 1

Bleed 2

Bleed 3

Bleed1

Bleed2

Bleed3

Immune(I) NonImmune (NI)

(I)

(I)

(I)

(NI)

(NI)

(NI)

Example calculation for expected mean

Dilution

log10 Dilution

(I) Absorbance at 450nm

(NI) Absorbance at 450nm

Corrected mean

2500

2500

3.398

3.398

(A1 + A2)/2

(2.199+2.011)/2

- (A7 + A8)/2

-(0.151+0.162)/2

Corrected mean

1.949

Experiment 1: Corrected mean absorbance

Bleed1 corrected mean absorbance

Table1.1

Dilution

log10 [Dilution]

Corrected mean

2500

3.398

1.949

5000

3.699

1.739

10000

4

1.521

25000

4.398

1.473

50000

4.699

1.34

100000

5

1.131

200000

5.301

0.653

400000

5.602

0.384

Bleed 2: corrected mean absorbance

Table1.2

Dilution

log10 [Dilution]

Corrected mean

2500

3.398

2.059

5000

3.699

1.974

10000

4

1.999

25000

4.398

1.858

50000

4.699

1.761

100000

5

1.311

200000

5.301

1.223

400000

5.602

0.895

Bleed 3: corrected mean absorbance

Table 1.3

Dilution

log10 [Dilution]

Corrected mean

2500

3.398

2.039

5000

3.699

1.976

10000

4

2.109

25000

4.398

1.931

50000

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4.699

1.836

100000

5

1.683

200000

5.301

1.529

400000

5.602

1.261

Figure 1

4

Table1.4

Bleed

Titre

Bleed 1

1.601

Bleed 2

1.668

Bleed 3

1.723

Experiment 2 determination of antibody specificity

Table of Raw data

Table2

Absorbance 450nm

Albumin species on plate

Human

goat

rabbit

human

goat

rabbit

Antibody dilutions

1

2

3

4

5

6

7

8

9

10

11

12

1/2500

A

2.377

2.222

1.264

1.219

0.360

0.269

0.265

0.372

0.177

0.195

0.328

0.324

1/5000

B

2.446

2.185

1.074

0.894

0.310

0.284

0.188

0.159

0.100

0.167

0.166

0.153

1/10000

C

2.282

2.063

0.704

0.680

0.274

0.249

0.146

0.153

0.144

0.112

0.151

0.190

1/25000

D

2.015

1.865

0.422

0.405

0.231

0.222

0.133

0.180

0.113

0.090

0.171

0.187

1/50000

E

2.043

1.810

0.328

0.248

0.242

0.205

0.125

0.107

0.117

0.108

0.188

0.143

1/100000

F

1.589

1.598

0.310

0.229

0.511

0.229

0.258

0.281

0.159

0.163

0.159

0.326

1/200000

G

1.468

1.322

0.212

0.253

0.234

0.212

0.130

0.155

0.103

0.088

0.159

0.175

1/400000

H

1.222

1.183

0.180

0.198

0.213

0.192

0.125

0.111

0.102

0.104

0.139

0.185

Bleed

Bleed 1

Bleed 2

Bleed 3

Bleed1

Bleed2

Bleed3

Immune(I) NonImmune (NI)

(I)

(I)

(I)

(NI)

(NI)

(NI)

Experiment 2 corrected mean absorbance

Human albumin mean absorbance

Table2.1

Dilution

log10 [Dilution]

Corrected mean

2500

3.398

1.981

5000

3.699

2.142

10000

4

2.023

25000

4.398

1.784

50000

4.699

1.811

100000

5

1.478

200000

5.301

1.253

400000

5.602

1.085

Goat Albumin corrected mean absorbance

Table 2.2

Dilution

log10 [Dilution]

Corrected mean

2500

3.398

1.056

5000

3.699

0.851

10000

4

0.564

25000

4.398

0.312

50000

4.699

0.176

100000

5

0.109

200000

5.301

0.137

400000

5.602

0.086

Rabbit albumin corrected mean absorbance

Table 2.3

Dilution

log10 [Dilution]

Corrected mean

2500

3.398

0.000

5000

3.699

0.137

10000

4

0.091

25000

4.398

0.048

50000

4.699

0.058

100000

5

0.128

200000

5.301

0.056

400000

5.602

0.041

Figure 2

4

Table2.4

Albumin species

Titre

Human

1.687

Goat

1.347

rabbit

1.179

Discussion

Experiment 1

Figure 1 is a graph showing the increase in the number of antibodies across the immunisation schedule. The antibody titre of bleed 1 is 1.601 the antibody titre for bleed 2 is 1.668 and for bleed 3 it is 1.723. The increase from in antibody production from bleed 1 to bleed 2(0.067) is greater than the increase of antibody production from bleed 2 to bleed 3 (0.055). This can be explained by the development of the immune response of the rabbit. Measures of antibody titre in the blood serum show the difference in primary and secondary immune response. Bleed 1 represents the antibodies produced during the innate immune response as this is the first time the rabbit has come into contact with human albumin.

Bleeds 2 and 3 represent the specific 9aquirred immune response in the rabbit. Due to the previous invasion of human albumin the rabbit has produced B memory cells and is able respond quicker to the antigen. The specific acquired immune response relies on B and t cells produced after the initial exposure to human albumin. When human albumin is encountered again memory cells that specifically recognise it proliferate rapidly forming large numbers of clones and effecter cells greatly enhancing the immune response (clonal selection). Thus the immune response is greater and more efficient. In Figure 1 Bleed 3 shows that the antibody production is reaching its maximum after the immunisation. This is indicated by the fact that there only a slight increase from bleed 2 to bleed 3.

Switch in Antibody class during immune response

Immunogenicity of albumin & Polyclonal nature of antiserum

Experiment 2

Figure2 shows the reactivity of bleed 3 with human, goat and rabbit albumin.

We know bleed 3 contains a high number of anti human albumin antibodies so it is not surprising when we see that human albumin has the highest titre for the antibody (1.687). However it is surprising to see that there is binding with goat albumin and not as much but still apparent with rabbit albumin. This can be explained by cross reactivity and the conservation of albumin across animals. Immunogenicity is also a key point to study while looking at these results.

Albumin conservation

Cross reactivty

Poly clonal and mony conal

Lymphocytes with receptors that bind self antigens are eliminated early in development, assuring self tolerance.

Development of an immune response & how results relate to this

How development of immune response relates to the clonal selection theory (effector/plasma cells & memory cells)

Switch in Antibody class during immune response

Immunogenicity of albumin & Polyclonal nature of antiserum

Conclusion

refrences