Changes which occur in the titre of antisera

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The aims of this experiment are to monitor any changes which occur in the titre of antisera during an immunisation schedule. The second aim is to determine whether or not the antisera taken up after the 3rd immunisation is specific only to human albumin. Both of these aims will be examined with the use of ELISA.

Introduction:-

The immune system has evolved to protect multicellular organisms; organisms which have more than 1 cell, from pathogens (Kindt et al, 2004).The response of the multicellular organism to infection include a non specific response and a specific response, also known as adaptive immune response. The function of the non specific responses is to prevent entry of disease causing agents into the body and to stop the spreading of infectious agents if they were to gain entry into the body (Cummings, 2003, Kindt et al, 2004). Some examples of disease causing agents are Bacteria, viruses and parasites.

This applies for the immune responses in invertebrates. Vertebrates also depend on this non specific mechanism, but they also possess a more sophisticated defence network called the Adaptive immune response. The innate immune response ultimately triggers the adaptive immune response and they work accordingly to eliminate any pathogens (Cummings, 2003). The adaptive immune response is highly specific to that particular pathogen which induced it and can supply long lasting protection, unlike the innate immune response. There are 2 classes of the adaptive immune response; i)Antibody response where the antibodies are produced by B cells ii) Cell mediated where T cells recognise cell associated antigens. An Antigen is any foreign molecule that can trigger an adaptive immune response. Most antigens are either protein molecules or can be large polysaccharides. The cell mediated response can be associated with the Clonal Selection theory (Brooker et al,2008).

Clonal selection theory is when Lymphocytes, a type of white blood cell, encounter a foreign molecule and it replicates itself with the cells expressing the same receptor. These are called clones and the formation of these clones is called clonal selection (Brooker, 2008, Kindt et al, 2004). This process of clonal selection needs the function of helper T cells which secrete cytokines (chemicals that initiate cell division) (Brooker et al, 2008). This process is a very important immunological process and determines which B and T lymphocytes will be cloned in vast numbers to fight a particular antigen (Kindt et al, 2004). This is the body's way of inhibiting and preventing infection by producing vast amounts of a particular lymphocyte which have antibodies on their surface ([1]). The basic concept of the Clonal selection theory is that an antigen interacts with a receptor on the lymphocyte, activating the cell and causing it to divide. The division of the cell produces clones of itself whose receptors bind to that exact antigen. The cells then begin to differentiate into effector cells and memory cells. When that antigen has been eliminated by the effector cells, the immune response stops while the Memory cells remain (De Girolamo, 2010, Immunology lecture, Nottingham Trent University, Kindt et al, 2004)

Antibodies are protein molecules known as immunoglobulins which are produced by B cells and have a role in inactivating any foreign molecules by binding to them forming an antigen-antibody complex (Brooker et al, 2008, Cummings, 2003). They are Y-shaped molecules and are secreted by plasma cells. They are made up of 4 polypeptide subunits: 2 heavy chains and 2 short chains. A hinge region which is composed of 2 disulfide bonds, allow the flexibility of the molecule and separates the light chains the top half of the heavy chains. The five classes of immunoglobulins are IgM, IgG, IgE, IgA and IgD. Each of these classes possesses unique structure, function and size. The most abundant in mammals are IgG and IgM. These two immunoglobulin provide most of the specific immunity against non-self species(Brooker et al, 2008). A typical antibody structure is shown below.

Figure 1 shows the structure of an antibody. The disulfide bridge shown at the centre of the molecule is known as the hinge region. ([2]).

A significant feature of immunoglobulins is their 'variable region' which varies amongst different B cells. This region is the site of the antibody which recognises specific antigens (Brooker et al, 2008). The variable regions on the immunoglobulins are the top halves of the heavy and light chains and they are the specific antigen binding sites. Antibody specificity is a property of antibodies which allow them to react with certain antigenic molecules and not others (Kindt et al, 2004, Keren et al, 1992).

Enzyme-linked immunosorbent assay (ELISA) is a technique used for detecting antigens or antibodies using an enzyme (Kindt et al, 2004). It works by the enzyme, which is attached to an antibody, reacting with a colourless substrate to produce a coloured product of the reaction. This test can determine the unknown concentration of a sample by producing a curve based on known concentration of antibody or antigen. This principle applies to one of the aims of this experiment and will be shown in the results section (Kindt et al, 2004). It has a wide variety of applications such as for use in Medicine, Veterinary, Pharmaceutical, Environmental use and use in lifestyle such as pregnancy tests (De Geralomo, 2010).

A vaccination is the administration of a vaccine to help produce immunity to a particular disease. A vaccine is an attenuated stain which contains disease causing microorganism for example Polio (Kindt et al, 2004). An Immunisation Schedule or Vaccine schedule is a series of vaccinations over a period of time to help prevent the effects of infection and to allow the body's immune system to handle the immune response accordingly. When a vaccination is given for the first time, they enter the blood interacting the body's tissue. The foreign antigen encounters Macrophages, which are non specific, and they engulf it. A chemical signal is then transmitted to Helper T cells that antigens are invading ,which then convey to the B-cells to proliferate into plasma cells to produce antibody against the weakened pathogen administrated as a vaccine and activate T cells to attack and destroy the antigen (Kindt et al, 2004). After a vaccination is given the memory cells of the immune system will 'remember' that specific disease causing antigen, and will have a more effective response with vast numbers if that antigen were to present itself again in the body by producing high levels of antibodies ([3]).

Results for Experiment 1:-

Table.1 shows the mean absorbencies for the duplicate data for Bleed 1

Dilution

Log10[dilution]

Immune Abs@450nm

Subtract

(-)

Non Immune Abs@450nm

Corrected mean Abs

2500

3.397

(A1+A2)/2

2.264

-

(A7+A8)/2

0.1305

2.1335

5000

3.698

(B1+B2)/2

1.2415

-

(B7+B8)/2

0.1105

1.131

10000

4

(C1+C2)/2

1.457

-

(C7+C8)/2

0.108

1.349

25000

4.397

(D1+D2)/2

1.406

-

(D7+D8)/2

0.0905

1.3155

50000

4.698

(E1+E2)/2

1.2525

-

(E7+E8)/2

0.12

1.1325

100000

5

(F1+F2)/2

1.099

-

(F7+F8)/2

0.096

1.003

200000

5.301

(G1+G2)/2

1.018

-

(G7+G8)/2

0.0885

0.9295

400000

5 .602

(H1+H2)/2

0.9755

-

(H7+H8)/2

0.1035

0.872

Table.2 shows the mean absorbencies for the duplicate for bleed 2

Dilution

Log10[dilution]

Immune Abs@450nm

Subtract

(-)

Non Immune Abs@450nm

Corrected mean Abs

2500

3.397

(A3+A4)/2

4.108

-

(A9+A10)/2

0.348

3.76

5000

3.698

(B3+B4)/2

3.923

-

(B9+B10)/2

0.28

3.64

10000

4

(C3+C4)/2

3.854

-

(C9+C10)/2

0.314

3.54

25000

4.397

(D3+D4)/2

3.454

-

(D9+D10)/2

0.247

3.21

50000

4.698

(E3+E4)/2

3.248

-

(E9+E10)/2

0.245

3.00

100000

5

(F3+F4)/2

2.558

-

(F9+F10)/2

0.231

2.33

200000

5.301

(G3+G4)/2

1.556

-

(G9+G10)/2

0.216

1.34

400000

5.602

(H3+H4)/2

1.275

-

(H9+H10)/2

0.205

1.07

Table. 3 shows the mean absorbancies for the duplicate for bleed 3

Dilution

Log10[dilution]

Immune Abs@450nm

Subtract

(-)

Non Immune Abs@450nm

Corrected mean Abs

2500

3.397

(A5+A6)/2

4.13

-

(A11+A12)/2

0.479

3.651

5000

3.698

(B5+B6)/2

4.028

-

(B11+B12)/2

0.345

3.683

10000

4

(C5+C6)/2

3.828

-

(C11+C12)/2

0.303

3.525

25000

4.397

(D5+D6)/2

3.554

-

(D11+D12)/2

0.243

3.311

50000

4.698

(E5+E6)/2

3.282

-

(E11+E12)/2

0.22

3.062

100000

5

(F5+F6)/2

2.663

-

(F11+F12)/2

0.207

2.456

200000

5.301

(G5+G6)/2

2.508

-

(G11+G12)/2

0.198

2.31

400000

5.602

(H5+H6)/2

1.793

-

(H11+H12)/2

0.203

1.59

Estimation of antibody titre:

To find an estimation for the antibody titre we must work out the Half maximal antigen binding for bleeds 1,2 and 3. This can be worked by finding the maximum Mean Corrected absorbance for each and then dividing this by 2 to find 50% maximal antigen binding. A graph has been plotted showing all three curves representing each of the 3 bleeds. The calculations below show how It can be worked out:-

Bleed 1

Half Maximal antigen binding is

2.1335/2 = 1.06675

I found this value on the y-axis and using a ruler drew a dotted line across parallel to the x axis until it met the curve for bleed 2. I then drew a dotted line from this point downwards parallel to the y-axis and found where it met the x-axis.

∴ The value for half maximal antigen binding was 4.8375

Titre = Inv Log10 [Half max antigen binding]

Titre = Inv Log10 [4.8375] = 68785.99123

Table. 4 shows results for Experiment 1 with estimation of antibody titres.

Bleed

Half Max Antigen Binding

Titre

1

4.84

68786

2

5.16

145378

3

5.24

172783Bleed 2

The same concept applies as mentioned above.

3.76/2 = 1.88

∴ Half Maximal antigen binding is 5.1625

Titre= Inv Log10 [5.1625] = 145378.4386

Bleed 3

3.683/2 = 1.8415

∴ Half maximal antigen binding is 5.2375

Titre= Inv Log10[5.2375] = 172782.5981

Graph 1- The graph shows that the mean absorbencies for Bleed 2 and Bleed 3 are significantly higher than bleed 1. This will be discussed in the discussion part of this report. The curve for Bleed 3 is declines slightly at a higher Dilution compared to bleed 2. Bleeds 2 and 3 also showed the higher values for half maximal antigen binding, so therefore their antisera had contained more antibodies.

Experiment 2:-

Table. 5 shows the mean absorbencies for Human Albumin

Dilution

Log10[Dilution]

Immune Abs@450nm

Subtract

(-)

Non Immune Abs@450nm

Corrected mean Abs

2500

3.397

(A1+A2)/2

1.62

-

(A7+A8)/2

0.19

1.43

5000

3.698

(B1+B2)/2

1.76

-

(B7+B8)/2

0.14

1.62

10000

4

(C1+C2)/2

1.62

-

(C7+C8)/2

0.117

1.51

25000

4.397

(D1+D2)/2

1.49

-

(D7+D8)/2

0.1205

1.37

50000

4.698

(E1+E2)/2

1.22

-

(E7+E8)/2

0.12

1.10

100000

5

(F1+F2)/2

1.11

-

(F7+F8)/2

0.11

1.00

200000

5.301

(G1+G2)/2

0.81

-

(G7+G8)/2

0.12

0.69

400000

5.601

(H1+H2)/2

0.89

-

(H7+H8)/2

0.130

0.79

Table.6 shows the mean abosorbancies for goat

Dilution

Log10[Dilution]

Immune Abs@450nm

Subtract

(-)

Non Immune Abs@450nm

Corrected mean Abs

2500

3.397

(A3+A4)/2

0.73

-

(A9+A10)/2

0.659

0.071

5000

3.698

(B3+B4)/2

0.4915

-

(B9+B10)/2

0.114

0.3775

10000

4

(C3+C4)/2

0.381

-

(C9+C10)/2

0.1065

0.2745

25000

4.397

(D3+D4)/2

0.2655

-

(D9+D10)/2

0.097

0.1685

50000

4.698

(E3+E4)/2

0.2175

-

(E9+E10)/2

0.105

0.1125

100000

5

(F3+F4)/2

0.195

-

(F9+F10)/2

0.0965

0.0985

200000

5.301

(G3+G4)/2

0.1615

-

(G9+G10)/2

0.1045

0.057

400000

5.601

(H3+H4)/2

0.1515

-

(H9+H10)/2

0.114

0.0375

Table.7 shows mean absorbencies for rabbit

Dilution

Log10[Dilution]

Immune Abs@450nm

Subtract

(-)

Non Immune Abs@450nm

Corrected mean Abs

2500

3.397

(A5+A6)/2

0.182

-

(A11+A12)/2

0.167

0.015

5000

3.698

(B5+B6)/2

0.1825

-

(B11+B12)/2

0.16

0.022

10000

4

(C5+C6)/2

0.176

-

(C11+C12)/2

0.137

0.039

25000

4.397

(D5+D6)/2

0.178

-

(D11+D12)/2

0.14

0.038

50000

4.698

(E5+E6)/2

0.163

-

(E11+E12)/2

0.1205

0.043

100000

5

(F5+F6)/2

0.165

-

(F11+F12)/2

0.1295

0.035

200000

5.301

(G5+G6)/2

0.157

-

(G11+G12)/2

0.156

0.00

400000

5.601

(H5+H6)/2

0.138

-

(H11+H12)/2

0.13

0.00

Antibody Titre for Ex 2

Human: 1.62/2 = 0.81 Table 8 shows the final Titre values for

each species.

Specie

Half Max Antigen Binding

Titre

Human

5.225

167880

Goat

3.54

2985

Rabbit

3.475

2512

Half antigen maximal binding 5.225

Titre = Inv Log10 [5.225] = 167880

Goat: 0.3775/2 = 0.18875

Half antigen maximal binding 3.54

Titre = Inv Log10 [3.54] = 2985

Rabbit: 0.046/2 = 0.023

Half antigen maximal binding 3.475

Titre = Inv Log10 [3.475] = 2512

Graph 2: These curves show the antibody specificity of Human, Rabbit and Goat antisera by the use of ELISA. The curve for human albumin intake showed a far greater Mean converted absorbancie as compared to the Goat and Rabbit. By looking at the curve it is obvious that the human had a far greater specificity to the albumin compared to the goat and rabbit. This is proven by the very high number of antibodies which were present in the titre for Human albumin intake and the reduced numbers of antibodies present in the goat and rabbit.

Discussion:

From the results of the first experiment where changes were monitored in the titre of antisera during the immunisation schedule, it appeared that the amount of antibody present in the antisera had increased from Bleed 1 to Bleed 3. This response would be expected, as mentioned in my introduction, due to the fact that after the first vaccination the rabbit's body would have gained a 'memory' for that particular antigen, so would produce a higher quantity of antibody next time if that antigen were to be present again ([3]).

The results of Ex 1 can also be linked to the Clonal selection theory where lymphocytes have multiplied in number by cloning themselves when encountering an antigen, in this case human albumin. Clonal selection theory would therefore result in a higher number of antibodies in the body as the particular B cell will be cloned and the plasma cells will produce antibodies more quickly and in larger numbers to deal with the antigen (Girolamo, 2010,; Cummings, 2003). In the early stages of the immune response an antibody to fight the antigen would have appeared in the serum, Bleed 1. This antibody would mainly have been IgM, but eventually IgG would start to appear and this would dominate the IgM response (Keren et. al., 1992). Looking at the graph for experiment 1, the curve for bleed 1 represents IgM, but then for bleed 2 IgG antibodies have been released in high quantities and then bleed 3 shows some more IgG antibodies have been produced. Immunogenicity is the ability of a substance, such as an antigen to provoke an immune response (Cummings, 2003).

In experiment 2 the human albumin had the greatest response and led to the highest quantity of antibody being produced. This is seen on the curve for Exp 2 and in the calculations above in the results section where 167880 estimated antibodies were present. The reason for such a high number of antibodies is because the rabbit had had 3 previous interactions with human albumin, so would have 'remembered' the foreign antigen and produced antibodies against it more effectively. There was a small response to the goat albumin; this may have been the primary immune response where a small number of antibodies had been produced by Plasma cells (Kindt, 2004). When the rabbit albumin had interacted there was a minimal/negligible immune response. The reason for a minor production of antibodies could be because the albumin had come from same species therefore maybe a low antibody count.

Conclusion:

The findings of experiment 1 showed us that the amount of antibodies in antisera of a rabbit increases during the course of an immunisation schedule with 3 vaccinations over 3 weeks.

In experiment 2 the results showed that the antiserum taken after the third immunisation was specific to the human immunogen as the titre for antibody was highest for human albumin. While a low concentration for goat albumin and negligible concentration for rabbit albumin.

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