Determination Of Antibody Specificity Biology Essay

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The purpose of this report is to monitor changes in the titre of antisera during an immunisation schedule and also to determine whether the antisera, taken after the third immunisation, are specific to human albumin. The results are obtained over a 2 week period.

The immune system has the following tasks, the detection and inactivation of infectious agents gaining access to the body (viruses, bacteria, fungi, protozoa and worms) or their toxins. Detection and killing of virus infected body cells. Detection and killing of cancer cells. There are many factors which influence immunogenicity, this includes the contribution of the Immunogen Foreignness . The immune system usually discriminates between self and non-self such that only foreign molecules are immunogenic. In general, the more distantly related two species are, the greater the immunogenicity of a molecule from one species will be when exposed to the other. A graft from an unrelated human will be rejected within about 2 weeks unless immunosuppressive drugs are used. An exception: some self tissues (like corneal tissue and sperm) are never encountered by the immune system during the development of tolerance to self-antigens

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In general, the larger the molecule the more immunogenic it is likely to be, however, there is no absolute size above which a substance will be immunogenic. Chemical Composition - In general, the more complex the substance is chemically the more immunogenic it will be. This includes the whether it is primary, secondary, tertiary structure in the case of proteins of whether is it. Physical form - In general particulate antigens are more immunogenic than soluble ones and denatured antigens more immunogenic than the native form. Degradability - Antigens that are easily phagocytosed are generally more immunogenic. This is because for most antigens (T-dependant antigens) the development of an immune response requires that the antigen be phagocytised, processed and presented to helper T cells by an antigen presenting cell (APC).

There are two defences, the innate immune system which responds to many features on a pathogen and the adaptive immune system responding to specific features (Stryer 2006) differences between the innate immunity and the adaptive (aquired immune system) are that the Innate immunity is formed from the physical barriers, whereas acquired immunity requires recognition specificity to foreign (non-self) substances and results in antibodies production.

Innate immune system often provides defence to an organism through barriers, this includes Skin- provides a physical barrier, White blood cells, Epithelia (mucous covered)- In areas of diffusion e.g. gut, lungs. Epithelia allows for easy diffusion, the mucous also collects pathogens. HCl in stomach- Denatures enzymes of pathogen these result in different functions which aim to avoid infection Blood-clotting- prevents loss of blood as well as protection from pathogens. Rapidly regenerating surfaces such as skin have a short life span so therefore reduce the risk of infection

Flow of urine/tears and coughing and vomiting. As well as the cellular and humoral defences of lysosomes, stomach acid and (Toole, 2004) .

However, a pathogen is still able to enter in which case another form of defence occurs. Neutralise toxins, prevents multiplying of pathogen, kills pathogen, removal of remains. This is referred to as phagocytosis. Innate immunity is Inbuilt to resist infection it is Present from birth, not antigen-specific, not enhanced by second exposure, has no memory, uses cellular and humoral components is poorly effective without adaptive immunity and is also involved in the triggering and amplification of adaptive immune responses. (Toole, 2004)

The different types of immunity can be split into Natural and artificial immunity. Natural Immunity required from normal life processes. E.g. having had the disease before, whereas Artificial inducing immunity (immunisation). Acquired from deliberate exposure to the antigens/antibodies. (Toole, 2004)

Active Immunity resulting from an individuals own immune system, rather than an outside source (generally long lasting). There is a build up own defence of antibodies. Natural active- Results from an infection, memory cells are made and can fight the infection in future times. With Artificial active immunity an Immune response is induced (immunisation) without suffering symptoms of the disease. Antigens injected or orally taken with allows the body to make memory cells of the disease as well as short term B cells. (vaccination). (Toole, 2004) The vaccine may be living attenuated forms- alive but harmless in causing symptoms antigens still recognised though. (measles. Tb)

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Dead micro-organisms. Killed but still possess antigens have been used for vaccination against many deadly micro-organisms.

Adaptive immunity

Immunity established to adapt to infection and is learnt by experience it is linked with pathogen-specific immunity and is enhanced by second exposure since it builds up memory to invading pathogens if exposed again. Adaptive immunity Uses cellular and humoral components, however, it Is poorly effective without innate immunity.

Table 1: Shows the differences between immune response to primary and secondary exposure.

Primary response

Secondary response

Slow

fast

Low levels of antigen in serum

High levels of antigen in serum

Short-lived

Long-lived

Mainly IgM

Mainly IgG

Low affinty

High affinity

(Table references from Toole, 2004)

Figure 1: Shows the difference in antibody concentration and time taken for a response between primary and secondonary exposure.

(Adapted from year 1 immunology lectures)

In this report the antigen (Human albumin) was used a foreign substance capable of inducing an immune response. This was obtained from blood serum which is blood plasma minus fibrinogens (and other clotting proteins)

During Week 1: A PVC plate is provided on which 1 µg albumin is placed. 6 antiserum samples are provided, 3 of which have been collected from the 1st, 2nd and 3rd immunisation with human albumin (test). (bleeds 1,2,3). The other 3 are collected after the 1st, 2nd and 3rd immunisation with phosphate buffered saline (control). The 6 anti-sera have been diluted 1/1000.

The 3 bleeds are prepared into the following dilutions containing PBS and marvel-:

Table 1: Shows the dilution factor

Dilution factor

2500

5000

10000

25000

50000

100000

200000

400000

Antibody Dilution

Antiserum volume

(PBS/ marvel)

1 / 2500

200µl of stock

300 µl

1 / 5000

250µl of 2500 dilution

250 µl

1 / 10,000

250µl of 5000 dilution

250 µl

1 / 25,000

200µl of 10,000 dilution

300 µl

1 / 50,000

250µl of 25,000 dilution

250 µl

1 /100,000

250µl of 50,000 dilution

250 µl

1 / 200,000

250µl of 100,000 dilution

250 µl

1 / 400,000

250µl of 200,000 dilution

250 µl

For bleed 3 double the volume is used compared with bleeds 1+2 as bleed 3 is also used in experiment 2. The concentration however remains the same.

Week 2:

All wells are flooded with PBS, once dried the secondary antibody is added (horseradish peroxidise conjugated goat anti-rabbit Ig). This is left to incubate for 1 hour at 37oC .

(rcn.com/jkimball.ma.ultranet/BiologyPages)

The drawing illustrates the activation of the one B cell in a pool of B cells whose BCR is specific for an epitope (small dark spheres) on the antigen.

This is known as clonal selection because it is the antigen that selects particular lymphocytes for clonal expansion. This leads to the production of a vast number of plasma cells which secrete antibodies. (rcn.com/jkimball.ma.ultranet/BiologyPages)

Clonal selection begins during the early stages of development In the fetus lymphocytes are being produced and collide with other body cells If the lymphocytes and these cells 'fit' then they die or are suppressed. So they remaining lymphocytes will only recognise 'foreign' cells rather than own body cells. (Toole 2004). This occurs in hemapoeitic stem cells which may differentiate into lymphocytes which contain the various antigen receptor points. Only will these cells remain and circulate in plasma until an invading pathogen comes into contact with it.

(chemistry.umeche.maine.edu)

From figure it shows the 2 heavy chains are composed of four domains (blue) and the two light chains having two domains are assembled by disulfide bonds (red) . The antigen binding sites are formed by the N-terminal ends of the light and heavy chains This is where the variation in structure occurs that allows antibodies to recognize and bind tightly different antigens (chemistry.umeche.maine.edu)

An antigen is recognised as non-self by the immune system, thus provoking an immune response. They are proteins on the invading cell, or cancer cells wall and trigger the production of anti-bodies. Anti-bodies also known as immunoglobulins (Ig) are made by B lymphocytes.

They react with the antigen by attaching to them with a lock and key type fit (similar to enzyme specificity). They are very specific to antigen due to the high abundance of proteins and also due to the 2 binding sites- these differ between antibodies and are known as the variable region. Differ due to a difference in amino acid sequence on the binding site, this results in a specific shape which will match a specific antigen. (Toole 2004)

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The rest of the molecule is known as the constant region. This is the same for all antibodies and is the part which binds to the receptors on the phagocyte, making phagocytosis easier.

The adaptive immune response which involves antibodies is part of the Humoral Immune Response which is immunity from antibodies produced by lymphocyte. This process is aided by a CD 4 T-helper 2 cell. Secreted antibodies bind to antigens on the surfaces of invading microbes (such as viruses or bacteria), which signals to other cells in the body such as natural killer cells which will destroy them.

ELISA

Enzyme-Linked ImmunoSorbant Assay has many useful implications from medicine

e.g. Allergy Tests, Prostate Cancer, HIV test to the environment in the measure of environmental pollutants in water. ELISA is to determine if a particular protein is present in a sample and if so, how much. There are two main variations on this method: you can determine how much antibody is in a sample, or you can determine how much protein is bound by an antibody. (www.bio.davidson.edu). For this report the antibody concentration is of interest. The wells are coated with the appropriate antigen then the solution or serum containing antibodies is added. Once bound to the loose antigens an enzyme-conjugated anti-immunoglobulin is added which consists of an antibody against the antibodies being tested. (users.rcn.com). for example antibodies from another specie, in this case antibodies from a goat and rabbit are conjugated to the enzyme. After washing away with unreacted reagent, the substrate is added. The absorbance level is proportional to the amount of enzyme-labelled antibodies bound therefore the concentration of antibodies is assessed. (users.rcn.com)

Results

Week 1

Table 1 Shows the absorbance at 450nm and log dillution for the immune response to the first exposure of human albumin (bleed 1). As shown with for immune (control) and non-immune (test) rabbit serum.

Antibody

Dilution

Log 10 Dilution

Absorbance at 450 nm

Average (M1-M2)

human albumin antisera (I)

Non-immune antisera

(NI)

Bleed1 (1)

Bleed1 (2)

Mean

Beed1 (1)

Bleed1 (2)

Mean

2500

3.398

1.582

1.589

1.5855

0.193

0.143

0.168

1.4175

5000

3.699

1.494

1.502

1.498

0.162

0.137

0.1495

1.3485

10000

4

1.471

1.537

1.504

0.134

0.154

0.144

1.36

25000

4.398

1.207

1.205

1.206

0.159

1.158

1.1585

1.0475

50000

4.699

0.933

1.09

1.0115

0.16

0.121

0.1405

0.871

100000

5

0.622

0.754

0.688

0.15

0.152

0.151

0.537

200000

5.301

0.446

0.487

0.4665

0.112

0.127

0.1195

0.347

400000

5.602

0.244

0.265

0.2545

0.132

0.142

0.137

0.1175

Table 2

Shows the absorbance at 450nm and log dillution for the immune response to the second exposure of human albumin (bleed 2). As shown with for immune (control) and non-immune (test) rabbit serum.

Dilution factor

Log 10 Dilution

Absorbance at 450 nm

Average (M1-M2)

Anti-human albumin antisera

Non-immune antisera

Bleed2 (1)

Bleed2 (2)

Mean

Bleed2 (1)

Bleed2 (1)

Mean

2500

3.398

1.653

1.715

1.684

0.177

0.211

0.194

1.49

5000

3.699

1.651

1.658

1.6545

0.176

0.172

0.174

1.4805

10000

4

1.535

1.577

1.556

0.163

0.179

0.171

1.385

25000

4.398

1.482

1.52

1.501

0.105

0.104

0.1045

1.3965

50000

4.699

1.263

1.409

1.336

0.136

0.162

0.149

1.187

100000

5

1.214

1.151

1.1825

0.132

0.12

0.126

1.0565

200000

5.301

0.935

1.098

1.0165

0.121

0.126

0.1235

0.893

400000

5.602

0.655

0.684

0.6695

0.124

0.114

0.119

0.5505Table 3 Shows the absorbance at 450nm and log dilution for the immune response to the third exposure of human albumin (bleed 3). As shown with for immune (control) and non-immune (test) rabbit serum.

Antibody

Dilution

Log 10 Dilution

Absorbance at 450 nm

Average (M1-M2)

Anti-human albumin antisera

Non-immune antisera

Bleed3 (1)

Bleed3 (2)

Mean

Bleed3 (1)

Bleed3 (2)

Mean

2500

3.398

1.684

1.635

1.6595

0.414

0.319

0.3665

1.293

5000

3.699

1.675

1.718

1.6965

0.227

0.191

0.209

1.4875

10000

4

1.587

1.706

1.6465

0.167

0.203

0.185

1.4615

25000

4.398

1.434

1.579

1.5065

0.182

0.174

0.178

1.3285

50000

4.699

1.48

1.491

1.4855

0.163

0.176

0.1695

1.316

100000

5

1.322

1.314

1.318

0.134

0.146

0.14

1.178

200000

5.301

1.057

1.169

1.113

0.114

0.103

0.1085

1.0045

400000

5.602

0.842

0.837

0.8395

0.11

0.126

0.118

0.7215

Week 2

Table 4-Absorbance at 450nm for the response to rabbit albumin from rabbit serum after being exposed to human albumin for the third time

Antibody

Dilution

Log 10 Dilution

Absorbance at 450 nm of human albumin

Average (M1-M2)

Anti-human albumin antisera (I)

Non-immune antisera

(NI)

Test 1

Test 2

Mean

Test

Test 2

Mean

2500

3.398

1.673

1.741

1.707

0.296

0.235

0.2655

1.4415

5000

3.699

1.634

1.584

1.609

0.231

0.239

0.235

1.374

10000

4

1.533

1.506

1.5195

0.236

0.203

0.2195

1.3

25000

4.398

1.283

1.281

1.282

0.208

0.185

0.1965

1.0855

50000

4.699

1.246

1.247

1.2465

0.183

0.168

0.1755

1.071

100000

5

0.967

0.952

0.9595

0.164

0.166

0.165

0.7945

200000

5.301

0.841

0.806

0.8235

0.258

0.255

0.2565

0.567

400000

5.602

1.089

1.209

1.149

0.241

0.27

0.2555

0.8935

Table 5-Absorbance at 450nm for the response to goat albumin for rabbit serum after the third exposure to human albumin

Antibody

Dilution

Log 10 Dilution

Absorbance at 450 nm of goat albumin

Average (M1-M2)

Anti-human albumin antisera (I)

Non-immune antisera

(NI)

Test 1

Test 2

Mean

Test 1

Test 2

Mean

2500

3.398

0.735

0.719

0.727

0.165

0.197

0.181

0.546

5000

3.699

0.549

0.57

0.5595

0.157

0.173

0.165

0.3945

10000

4

0.457

0.475

0.466

0.188

0.162

0.175

0.291

25000

4.398

0.366

0.329

0.3475

0.143

0.143

0.143

0.2045

50000

4.699

0.264

0.255

0.2595

0.141

0.139

0.14

0.1195

100000

5

0.255

0.225

0.2375

0.153

0.137

0.145

0.0925

200000

5.301

0.218

0.226

0.222

0.128

0.231

0.1795

0.0425

400000

5.602

0.407

0.389

0.398

0.18

0.174

0.177

0.221

Table 6-Absorbance at 450nm for the response to third exposure of human albumin for immune and non-immune goats

Antibody

Dilution

Log 10 Dilution

Absorbance at 450 nm of rabbit albumin

Average (M1-M2)

Anti-human albumin antisera (I)

Non-immune antisera

(NI)

Test 1

Test 2

Mean

Test 1

Test 2

Mean

2500

3.398

0.272

0.387

0.3295

0.216

0.246

0.231

0.0985

5000

3.699

0.278

0.308

0.293

0.201

0.207

0.204

0.089

10000

4

0.243

0.264

0.2535

0.201

0.216

0.2085

0.045

25000

4.398

0.23

0.241

0.2355

0.194

0.195

0.1945

0.041

50000

4.699

0.205

0.206

0.2055

0.175

0.155

0.165

0.0405

100000

5

0.217

0.214

0.2155

0.173

0.177

0.175

0.0405

200000

5.301

0.205

0.286

0.2455

0.205

0.214

0.2095

0.036

400000

5.602

0.383

0.347

0.365

0.296

0.262

0.279

0.086

Volume content for each dilution:

Antibody Dilution

Antiserum volume

(PBS/ marvel)

1 / 2500

200µl of stock

300 µl

1 / 5000

250µl of 2500 dilution

250 µl

1 / 10,000

250µl of 5000 dilution

250 µl

1 / 25,000

200µl of 10,000 dilution

300 µl

1 / 50,000

250µl of 25,000 dilution

250 µl

1 /100,000

250µl of 50,000 dilution

250 µl

1 / 200,000

250µl of 100,000 dilution

250 µl

1 / 400,000

250µl of 200,000 dilution

250 µl

Graph 1: Shows the results from experiment 1. Series 1,2,3 refer to the bleeds. See also graph 2 and 3.

Results from the titre values for each bleed.

Bleed

Titre

1

70,795

2

251,189

3

398,107

Discussion

Results show that the absorbance for bleed 3 is higher than for bleeds 1 and 2. This supports the understanding that after exposure to an antigen memory cells. Memory B cells are activated/re-activated upon further exposure to specific pathogens of which memory cells have been produced. In this case the human albumin acts the same as an invading pathogen since the antigens cause an immune response.

Upon first exposure to human albumin Plasma cells or B-cells are secreted which produced antibodies these only survive a few days but during which can produced up to 2000 antibodies every second. (Toole 2004) This is referred to as the primary immune response. Memory cells; survive much longer, do not produce antibodies directly but circulate in the blood plasma until they will re-encounter the antigen they are matched with, once they do they begin to divide into more plasma and memory cells, prepared for the infection. This is known as the secondary immune response. This is quicker and has greater effect, eliminating the pathogen even before Individuals show symptoms. This can explain why a majority of us only encounter chickenpox and measles once in our lifetime, since the pathogens causing these are quickly indentified and vast amounts of plasma cells and memory cells are formed. (Toole 2004)

The immune system recognises self and non-self therefore only foreign molecules are immunogenic. The rabbit obtained antibodies recognise the human albumin as more foreign since these are the most distantly related species, compared with goat serum. Rabbits are known to mount a strong immune response against foreign antibodies. Z. An (2009) therefore the antibodies produce a greater response to exposure to a human antigen. The antibodies and antigen in the case of the rabbit are from the same species therefore giving the lowest response.

Clonal selection theory is also supported with the results since the rabbit antibody and rabbit antigen gave the lowest immune response producing the lowest quanity of antibodies as reflected in the low absorbance. Therefore we can say the absorbance is low due to the antibodies present not recognising rabbit albumin as foreign since these lymphocyte producing antibodies would have been destroyed once bound to a cell in the same organism, giving rise to only non-self recognising antibodies, hence the slight immune response shown.

In the primary response the major class of antibody produced is IgM whereas in the secondary response it is IgG, this is perhaps shown with the increased affinity to antibodies. After a class switch has occurred in the immune response, somatic mutations occur which fine tune the antibodies to be of higher affinity. (pathmicro.med.sc.edu)

Immunogenicity of albumin & Polyclonal nature of antiserum

In conclusion bleed 3 has a higher immune response as predicted since it has built up an adaptive immune response to the antigen, producing memory cells

Rabbit is less immunogenic since the antigen causes less of an immunogenic response since the antigen isn't seen as such as 'foreign' body by the rabbit's immune system. A human antigen however is much more 'foreign' and therefore causes more of a response. This supports the prediction that the bonding of albumin is more specific to humans.