complement serum activity by lysing sheep erythrocytes

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Introduction

The Immune system is a series of complex processes which has evolved to protect the body from attack by foreign pathogens. These pathogens are able to enter our body through the skin or lining of the internal organs. The immune system is able to protect us from intracellular and extracellular organisms as well as from ourselves, stopping malignancies and autoimmune diseases from spreading in our bodies (Bastian, 1993). There are two lines of defence, the adaptive (specific) and innate (non-specific immunity), though both are united in their goal to destroy pathogens they have different ways to tackle this. Innate immunity is the 1st line of defence while adaptive immunity is the 2nds line and thus takes longer to act (Clancy, 1998). The complement system is part of the immune system and can be bought into action by the adaptive system if required. Complement is a group of proteins working together within the immune system; once stimulated by one of many triggers, proteases begin to cleave protein in the system, bringing a cascade of enzyme reactions in order to fight off foreign pathogens and activate the inflammatory response. Within the complement cascade there are many proteins that play a role but C3 is a protein critical to the effector functions of the system (Abbas, 1994).

There are many paths for immune mediated lysis and the one we will be looking at is intravascular haemolyse and occurs when the complement has been triggered through the classical pathway. When the antibody binds to the antigen on the surface of the erythrocyte, a complement component triggers the membrane attack complex to form pores in the cell membrane resulting in cell lysis (Chapel, 1990). The intensity and speed at which cells lyse is dependent upon the rate at which the complement cascades to enable complete cell lysis.

Experiments like these are able to provide us with an understanding of how the complement immune system functions. It can also increase our understanding of autoimmunity and perhaps lead to ways in which the effects of immunity can be prolonged or inhibited according to the disease. Systemic Lupus Erythematosus (SLE) is an autoimmune disease, in which complement is analysed, as getting SLE is dependent upon the gene which is responsible for producing MHC, a component used in haemolysis (American, 1993), patients with other immunological disorders can require their complement activity to be monitored and thus this assay would be able to show how efficiently the complement component of the immune system is working to defend their bodies.

Aims

To determine complement serum activity by lysing sheep erythrocytes

To determine the volume of complement required for 50% lysis.

Materials

20 Cuvettes 1.0ml

20 test tube plastic disposable

Automatic pipette 200-1000 µl & 6 tips

Automatic pipette 0-200 µl & 6 tips

Water bath at 37oC

Spectrophotometer

Test tube rack

Centrifuge

Ice bucket & Ice

Method

Wash 4ml of erythrocyte suspension three times with barbitone saline solution.

Prepare a 6% stock solution of erythrocytes

In one test tube mix

3.0ml of sheep anti-erythrocyte antiserum, diluted 1/50

3.0ml of the 6% SRBC

Mix and gently by capping and inverting several times

Incubate at 37oC for 15min in the water bath, mix every 5min.

Set up the test tubes on ice in duplicates and label

Add the reagents in order as shown in table 1 below

Incubate the tubes for 60 minutes at 37oC mixing gently every 15minutes

Place the tubes on ice and then centrifuge at 200g for 10 minutes at 4oC

Remove the samples and put into cuvettes and read the absorbance at 541nm, with ammonia solution as blank record the results in a table.

Table : Tubes for Assay

VOLUME OF REAGENT (ml)

TUBE NUMBER:

1

2

3

4

5

6

(Blank)

Buffer (ml)

1.10

1.05

1.00

0.90

0.80

1.20

Guinea-pig Serum (ml)

Initial dil. 1:30

(check this has been diluted already!!)

0.10

0.15

0.20

0.30

0.40

0.00

Sensitised erythrocytes (ml') suspension

(as prepared in part II above; NOTE this will be given you)

0.30

0.30

0.30

0.30

0.30

0.30

Results

Discussion

When carrying out the experiment raw data was recorded, and presented in table 1. However the results obtained during the practical were not used as the erythrocytes lysed before complement was added and therefore complement activity could not be observed as adding complement to lysed cells is not able to produce results, therefore the ideal data provided was used and analysed.

From table 1 it is clear that absorbance levels increased as serum volume increased, this is due to the fact that as volumes of complement increase more red blood cells are lysed which in turn allows haemoglobin to be let out, this is of a dark colour and as more cells are lyses the darker the resulting sample will be, and so the absorbance as read the spectrophotometer will increase. After the guinea pig serum has been mixed with the sensitised erythrocytes, it produces anti-body coated cells with complement attaching to the antibody, and activating this attracts the MAC molecules to take action and lyse the cell (Kuby, 1994). Following the pattern seen in table one table 3 shows a progressive % lysis of cells as the volume of serum is increased, however for the 100% lysis an ammonia buffer was used to ensure that all cells are lysed during the experiment.

Further to this graph 1 produced a sigmoid curve, from which it was possible to estimate CH50. However calculating the 50% lysis from this graph is not very accurate. Thus a log graph 2 was constructed, with the use of van Krogh equation to determine the actual value of 50% lysis. The equation was provided by the lecturer.

Van Krogh equation:

x= k[ y ]1/n

100-y

Where:

x= amount of complement (ml of undiluted serum)

y= proportion of cells lysed

k=50% unit of compliment

n=inclination of graph (ideally 0.2)

This resulted in table 4 giving a volume of 133.5 CH50/ml. However when calculating CH50 the x values were all in the negative. Moreover, it was not possible to compare data sets obtained against ideal data as the experiment did not yield results due to lysis of erythrocytes before complement was added. This could have occurred due to improper pipetting, handling or transporting of the cells as shaking them too much could have lysed them due to shock, as the cells were sensitized and thus prone to quick lysis. Further to this it was reported by Inglis, et al, 2007, that the use of erythrocytes from different sheep can yield inaccurate results and thus produce different CH50. Although there are many inaccuracies present within the experiment, it also gives scope to further improve the method as well as explore other area of the subject at hand such as factors which affect the performance of complement like temperature or PH. This assay is a good way to measure the activity of the immune system within patients, such as patients with LSE as mentioned earlier, other patients with low immunity can also be tested to see how the complement system is or isn't aiding their recovery, thus steps can be taken by medical professionals to either boost or monitor the progress of the patients immunity as fundamentally the immune system is required to work at its optimum to keep humans and animals from dying of disease(Inglis,et al, 2007).

Conclusion

Overall this experiment has shown how complement is important in aiding white blood cells to lyse foreign bodies. Though in the experiment carried out the blood cells lysed before complement was added the method was presented and the ideal set of data, showed what results should have been obtained. Also the hypothesis that as the complement concentration increases so will the absorbance proved positive.

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