Iycee Charles de Gaulle Summary Determination Of Antibody Specificity By Elisa Biology Essay

Determination Of Antibody Specificity By Elisa Biology Essay

The purpose of the research lab experiment was to follow the class of an immunization. And utilizing the trial proctor the alterations in the antibody titer ( figure of antibodies produced ) during the class of the immunization and find weather the antibody extracted from the 3rd immunization was specific to human albumen.

Introduction: Antibodies are a critical portion of the immune response, protecting animate beings against foreign affair. Antibodies are produced by B lymph cells that are activated in response to foreign molecules ( antigens ) . Any molecule that triggers an immune response is called an antigen. The organic structure ‘s recognises its ain molecules as “ ego ” and onslaughts anything foreign “ non ego ” . Antibodies produced in the immune response travel all over the organic structure and bind to the antigen that stimulated its production. The binding of an antigen and antibody is called an immune composite. ( Brooker 2007 )Antibody construction and specificity: Antibodies besides known as Igs recognize and bind antigens. The acknowledgment of a peculiar antigen is indispensable for a specific immune response.

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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 bring forth plasma cells. Plasma cells so secrete big sums of antibodies that have the same binding specificity as the original B cell receptor. The binding of an Ig to an antigen is extremely specific and has a batch to make the construction of the Ig. The basic construction of an Ig consists of 2 indistinguishable visible radiation ironss and 2 indistinguishable heavy ironss held together by disulphide bonds. There are five distinguishable categories of Ig that differ in size, charge, aminic acid sequence and saccharide content.

These can be farther divided in to subclasses ( Isotypes ) based on the construction of the heavy concatenation. All isotopes except ( IgD ) are bifunctional. They recognize and bind the antigen and so advance its violent death or remotion by the activation of effector mechanisms. Different Ig categories and bomber categories activate different effector mechanisms.Non particular ( Innate ) unsusceptibility and Specific ( acquired ) unsusceptibility: The immune system is divided in to two types. Non-specific ( innate ) unsusceptibility and Specific ( acquired ) unsusceptibility. Both systems work together to bring forth an efficient immune response. All animate beings are born with non-specific ( Innate ) unsusceptibility.

The organic structure ‘s defense mechanisms are present from birth and support against foreign molecules irrespective of the specific individuality. Alternatively nonspecific defense mechanisms recognise common conserved belongingss of the molecule judging it to be foreign. The opposition to a peculiar Antigen does non increase on exposure to it the 2nd clip. So we can state unconditioned unsusceptibility is present all the clip, it is non antigen particular and has no memory. Non-specific unsusceptibility besides includes the organic structure ‘s outer barriers ( tegument ) and mucus membranes. Cells besides take portion in non-specific ( innate ) unsusceptibility as cellular barriers apposing infections. These include Phagocytes, Non-phagocytes and Natural killer cells. ( Campbell and Reece )Specific ( acquired ) unsusceptibility is found in craniates merely.

Unlike non-specific ( innate ) unsusceptibility specific ( acquired ) unsusceptibility develops merely after the organic structure is exposed to foreign substances. Specific ( acquired ) unsusceptibility is antigen specific and contains memory cells. Cells of the immune system foremost encounter the antigen and subsequently recognize it to be attacked. Thus the 2nd exposure to the antigen manufacturers a more improved and efficient response.

( Brooker )Lymphatic system: The cells responsible for supplying specific unsusceptibility are Lymphocytes ( B cells and T cells ) . Both sets of cells originated from root cells in bone marrow. The B cells mature within the bone marrow and T cells mature within the Thymus secretory organ. Lymphocytes continuously circulate between lymphatic variety meats, the blood and all the tissues of the organic structure. On meeting an antigen an immune response is invoked.

The migration of lymph cells to an country where an encroacher has been detected greatly increases. This increases the opportunities of a lymph cell meeting an antigen it specifically recognizes. ( Brooker 2008 ) ( Campbell and Reece )Clonal choice theory: Lymphocytes are responsible for Immune acknowledgment in Specific ( acquired ) immune responses. They achieve this by clonal choice. As we have learnt each lymph cell recognises merely one specific antigen. When a lymph cell comes in to reach with a specific antigen it recognizes it and is induced to proliferate quickly. The proliferating cells produce effecter cells and memory cells. Therefore over the following few yearss the organic structure produces a sufficient sum of ringers ( effecter cells ) of the original lymph cell that came into contact with the antigen for a successful immune response.

The memory cells remain. This procedure of clonal choice is operation in both sets of lymph cells ( B cells and T cells ) ( Immune 200? brostoff )Principals of ELISA and the immunization agenda: Enzyme-linked immnosorbent check ( ELISA ) is an immunological technique designed to observe antibodies or antigens by the usage of enzymes. An antigen is incubated on to a fictile home base and becomes absorbed to the base of it.

Any free antigen is so washed off. Test antibody is added which binds to the antigen. A ligand that can observe the presence of an antibody is coupled to a enzyme such as peroxidise. This binds the trial antibody and after the free ligand is washed off the edge ligand is visualised by adding a colorless substrate that acted on by the enzyme linked to the ligand to bring forth a colored terminal merchandise. The sum of trial antibody is measured by measuring the sum of coloured terminal merchandise by optical denseness scanning home base.

( immune )To fix the antibody for the Elisa trial to be carried out a coney was inoculated with human albumen raising an immune response, this was the trial. Another coney was inoculated with phosphate buffer solution this was used as a control. After 10 yearss of vaccination blood was extracted from both coneies and the antiserum was collected. This process was carried out three times across 3 hebdomads. 6 sets of antisera were prepared, three from the trial coney ( anti- albumen antiserum ) and three from the control coney ( non-immune antiserum ) .

In the ELISA trial Human albumen was incubated on to the base of polyvinyl chloride ( PVC ) home base. Sets of bleeds at assorted dilutions from both the trial and control coney where added. The primary binding was monitored by utilizing a secondary antibody ( horseradish peroxidise conjugulated secondary antibody ) . The antibody concentrations at half maximum antigen binding were taken as an estimation of antibody titer.Experiment 1 appraisal of antibody titer

Table of Raw informations

Table1

Antibody dilutions

Optical density 450nm

1

2

3

4

5

6

7

8

9

10

11

12

1/2500

A

2.

1992.0112.1572.3122.2162.3580.1510.1620.

1810.1700.2540.

243

1/5000

Bacillus

2.0331.7062.1412.0852.1712.

2770.1320.1300.1420.1370.

2050.185

1/10000

C

1.5861.7712.

1752.1082.2712.2300.1560.

1600.1410.1450.1530.131

1/25000

Calciferol

1.7641.4711.

9261.9942.0602.0680.

1500.1400.0720.

1330.1320.134

1/50000

Tocopherol

1.5861.4731.9151.8811.

9552.0090.2140.

1650.1470.1280.1150.178

1/100000

F

1.5181.

0921.5151.3871.7951.

8360.1940.1540.1320.1480.1380.

128

1/200000

Gram

0.8110.7881.3831.3371.6781.6450.1490.

1450.1440.1310.1350.131

1/400000

Hydrogen

0.

5260.5351.1380.9361.4841.3110.1770.1730.

1400.1440.1140.

160

Bleed

Bleed 1

Bleed 2

Bleed 3

Bleed1

Bleed2

Bleed3

Immune ( I ) NonImmune ( NI )

( I )

( I )

( I )

( NI )

( NI )

( NI )

Example computation for expected mean

Dilution

log10 Dilution

( I ) Optical density at 450nm

( NI ) Absorbance at 450nm

Corrected mean

250025003.3983.398( A1 + A2 ) /2( 2.199+2.

011 ) /2- ( A7 + A8 ) /2- ( 0.151+0.162 ) /2Corrected mean1.949Experiment 1: Corrected mean optical densityBleed1 corrected average optical densityTable1.1

Dilution

log10 [ Dilution ]

Corrected mean

2500

3.3981.

949

5000

3.6991.739

10000

41.521

25000

4.

3981.473

50000

4.6991.34

100000

51.

131

200000

5.3010.653

400000

5.6020.384Bleed 2: corrected mean optical densityTable1.

2

Dilution

log10 [ Dilution ]

Corrected mean

2500

3.3982.059

5000

3.6991.974

10000

41.

999

25000

4.3981.858

50000

4.6991.761

100000

51.

311

200000

5.3011.223

400000

5.6020.895Bleed 3: corrected mean optical densityTable 1.3

Dilution

log10 [ Dilution ]

Corrected mean

2500

3.3982.

039

5000

3.6991.976

10000

42.

109

25000

4.3981.931

50000

4.6991.836

100000

51.683

200000

5.3011.

529

400000

5.6021.261Figure 14Table1.4

Bleed

Titer

Bleed 11.601Bleed 21.668Bleed 31.723Experiment 2 finding of antibody specificity

Table of Raw informations

Table2

Optical density 450nm

Albumin species on home base

Human

caprine animal

coney

homo

caprine animal

coney

Antibody dilutions

1

2

3

4

5

6

7

8

9

10

11

12

1/2500

A

2.

3772.2221.2641.2190.3600.2690.

2650.3720.1770.1950.

3280.324

1/5000

Bacillus

2.4462.1851.0740.

8940.3100.2840.1880.

1590.1000.1670.1660.

153

1/10000

C

2.2822.0630.

7040.6800.2740.2490.1460.1530.1440.1120.

1510.190

1/25000

Calciferol

2.0151.

8650.4220.4050.2310.2220.1330.1800.1130.

0900.1710.187

1/50000

Tocopherol

2.0431.8100.3280.

2480.2420.2050.1250.1070.

1170.1080.1880.143

1/100000

F

1.5891.5980.3100.

2290.5110.2290.2580.2810.1590.1630.

1590.326

1/200000

Gram

1.4681.3220.2120.2530.2340.2120.

1300.1550.1030.

0880.1590.175

1/400000

Hydrogen

1.2221.

1830.1800.1980.2130.1920.1250.1110.

1020.1040.1390.

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 optical densityHuman albumin average optical densityTable2.1

Dilution

log10 [ Dilution ]

Corrected mean

2500

3.3981.981

5000

3.6992.142

10000

42.023

25000

4.

3981.784

50000

4.6991.811

100000

51.

478

200000

5.3011.253

400000

5.6021.085Goat Albumin corrected average optical densityTable 2.2

Dilution

log10 [ Dilution ]

Corrected mean

2500

3.

3981.056

5000

3.6990.851

10000

40.564

25000

4.

3980.312

50000

4.6990.176

100000

50.109

200000

5.3010.137

400000

5.6020.

086Rabbit albumin corrected average optical densityTable 2.3

Dilution

log10 [ Dilution ]

Corrected mean

2500

3.3980.000

5000

3.6990.

137

10000

40.091

25000

4.3980.048

50000

4.6990.058

100000

50.

128

200000

5.3010.056

400000

5.6020.041Figure 24Table2.4

Albumin species

Titer

Human1.687Goat1.

347coney1.179

Discussion

Experiment 1

Figure 1 is a graph demoing the addition in the figure of antibodies across the immunization agenda. The antibody titer of bleed 1 is 1.601 the antibody titer for bleed 2 is 1.668 and for bleed 3 it is 1.723. The addition from in antibody production from bleed 1 to shed blood 2 ( 0.067 ) is greater than the addition of antibody production from bleed 2 to shed blood 3 ( 0.

055 ) . This can be explained by the development of the immune response of the coney. Measures of antibody titer 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 clip the coney has come into contact with human albumen.Bleeds 2 and 3 represent the specific 9aquirred immune response in the coney. Due to the old invasion of human albumin the coney 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 albumen. When human albumen is encountered once more memory cells that specifically recognise it proliferate quickly organizing big Numberss of ringers and effector cells greatly heightening the immune response ( clonal choice ) . Thus the immune response is greater and more efficient. In Figure 1 Bleed 3 shows that the antibody production is making its upper limit after the immunization.

This is indicated by the fact that there merely a little addition from bleed 2 to shed blood 3.Switch in Antibody category during immune responseImmunogenicity of albumen & A ; Polyclonal nature of antiserum

Experiment 2

Figure2 shows the responsiveness of bleed 3 with human, caprine animal and coney albumen.We know bleed 3 contains a high figure of anti human albumen antibodies so it is non surprising when we see that human albumen has the highest titer for the antibody ( 1.687 ) . However it is surprising to see that there is adhering with caprine animal albumen and non every bit much but still evident with coney albumen.

This can be explained by cross responsiveness and the preservation of albumen across animate beings. Immunogenicity is besides a cardinal point to analyze while looking at these consequences.

Albumin preservation

Cross reactivty

Poly clonal and mony conal

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

Development of an immune response & A ; how consequences relate to this

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

Switch in Antibody category during immune response

Immunogenicity of albumen & A ; Polyclonal nature of antiserum

Decision

refrences