Monday, June 3, 2019
Complement Serum Activity by Lysing Sheep Erythrocytes
Complement Serum Activity by Lysing Sheep ErythrocytesIntroductionThe Immune dodge is a series of complex processes which has evolved to protect the automobile trunk from attack by foreign pathogens. These pathogens argon suitable 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 accommodative (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 world-class line of defence while adaptive immunity is the 2nds line and thus takes longer to act (Clancy, 1998). The equilibrate system is part of the immune system and cease be bought into action by the adaptive system if required. Complement is a group of protein s working together within the immune system once stirred up 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 instigate the inflammatory response. Within the backup 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 equilibrize has been triggered through the classical pathway. When the antibody binds to the antigen on the egress of the erythrocyte, a complement off trash 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 u nderstanding of how the complement immune system functions. It can also addition our understanding of autoimmunity and perhaps lead to ways in which the effects of immunity can be prolong 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 apply 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 turn up how efficiently the complement component of the immune system is working to defend their bodies.AimsTo determine complement serum activity by lysing sheep erythrocytesTo determine the multitude of complement required for 50% lysis.Materials20 Cuvettes 1.0ml20 test pipework plastic disposableAutomatic pipette 200-1000 l 6 tipsAutomatic pipette 0-200 l 6 tipsWater bath at 37CSpectrophotometer Test tube rackCentrifugeIce bucket IceMethodbackwash 4ml of erythrocyte suspension three times with barbitone saline solution.Prepare a 6% stock solution of erythrocytesIn one test tube mix3.0ml of sheep anti-erythrocyte antiserum, diluted 1/503.0ml of the 6% SRBCMix and gently by capping and inverting several timesIncubate at 37C for 15min in the water bath, mix every 5min.Set up the test tubes on ice in duplicates and labelAdd the reagents in order as shown in table 1 belowIncubate the tubes for 60 minutes at 37C mixing gently every 15minutesPlace the tubes on ice and then centrifuge at 200g for 10 minutes at 4CRemove the samples and put into cuvettes and read the absorbance at 541nm, with ammonia solution as blank record the results in a table.ResultsDiscussionWhen carrying out the experiment raw data was recorded, and presented in table 1. However the results obtained during the practical were non used as the erythrocytes lysed before complement was added and consequently com plement activity could not be observed as adding complement to lysed cells is not able to let out results, therefore the ideal data provided was used and analysed.From table 1 it is clear that absorbance levels increased as serum spate increased, this is imputable 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 energizing 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 workable 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 par to determine the actual value of 50% lysis. The equation was provided by the lecturer.Van Krogh equationx= k y 1/n100-yWherex= amount of complement (ml of undiluted serum)y= proportion of cells lysedk=50% unit of complimentn=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 t he 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 search other area of the subject at hand such as factors which affect the performance of complement like temperature or PH. This assay is a well-grounded 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 isnt 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). f inaleOverall 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.