Current general health & safety procedures | My Assignment Tutor

BHS002-2, 27-2 COMPLEMENT PRACTICAL PROTOCOLDr Anna Furmanski1SAFETY INFORMATION:General notes:• Current general health & safety procedures in the UoB Life Sciences laboratories apply• NO PHONES or food/drink in the laboratory – this applies to everyone!• Cover any cuts and ensure you wash your hands before leaving the lab• Additional risks in this practical are as follows:o Biological material could transmit infectious agents and trigger hypersensitivityo Ammonia is irritant• Risks will be controlled/minimised by:o Use of dilute solutionso Use of appropriate PPE: gloves, lab coat and care when handling all substanceso Use of eye wash and/or soap and water to remove contaminant if requiredDETERMINATION OF SERUM COMPLEMENT ACTIVITY BY CALCULATION OF CH50Background information:• In a CH50 test, the complement activity of a blood serum sample is estimated by using sensitisederythrocytes (RBC) as target cells. If the classical complement cascade in the test serum sampleis functional, haemolysis will occur• Sheep RBC are coated with anti-sheep RBC antibodies to form immune complexes (Ag:Ab)• Test serum is added to the antibody-coated (sensitised) target erythrocytes. Any complementproteins in the serum will bind to the antibodies on the erythrocyte (the Ag:Ab complex) and initiatethe classical complement cascade. Membrane attack complexes (MAC) are formed in erythrocytemembrane, leading to lysis• Erythrocyte lysis releases haemoglobin (Hb) into the assay tube. Hb is coloured and the relativeabundance of Hb can be measured in each tube by spectrophotometry. Absorbance (Abs) isdirectly proportional to the extent of cell lysis• Hb content in each erythrocyte is a fixed, constant value. Therefore, a measurement of free Hbin the assay supernatant gives an indication of how many erythrocytes have been lysed.• The amount of serum (i.e. complement) added to Ab-coated cells is proportional to the degree ofcell lysis. This can be plotted in a dose-response graph, which takes the shape of a sigmoid curve(Figure 1). As the curve approaches maximum cell lysis, the curve begins to flatten. It is difficultto determine precisely the point at which total lysis (100%, called the CH100) occurs. It is easier touse the curve to define the point where 50% of the erythrocytes are lysed, called the CH50 point.Thus, one CH50 unit is the volume of serum required to cause lysis of 50% of the antibodycoated erythrocytes. CH50 is expressed as CH50 U/ml serum.• Diagnostic laboratories use the CH50 test to assess total complement activity in patient serum.Low or high readings indicate abnormalities in the complement cascade, which may contribute todisease pathogenesis. Complement defects are linked to recurrent infection, immune complexdisease and unwanted inflammation. Think about why!Key reading: Costabile M, Measuring the 50% Haemolytic Complement (CH50) Activity of Serum, J Vis Exp. 2010; (37): 1923BHS002-2, 27-2 COMPLEMENT PRACTICAL PROTOCOLDr Anna Furmanski2FIGURE 1: Lysis of antibody-coated sheep erythrocytes (sRBC) through complement activation.Sensitised RBC were subject to increasing volumes of test serum and the optical density of the resultingsupernatant was measured on a spectrophotometer. The steep ‘linear’ region of the curve is shown(triangle), the star represents a flattening of the curve. One CH50 unit is shown (red arrow).LABORATORY PRACTICAL TASK:Two patient serum samples along with a reference sample from healthy individual sera are to be assayedto analyse complement function. All sera will be assayed in duplicate.Separate CH50 assays are performed for Serum X, Serum Y and Reference Sera and you will thencalculate the number of CH50 units in 1ml of each serum sample.The CH50 assay is a major diagnostic test, used in the ‘real world’ to determine the activity of total serumcomplement. As you are working and during lab briefings, think about the immunology of what ishappening in each tube and consider why this test is important in diagnosis.10050% LYSISvolume of serum = one CH50 unitComplement added (arbitrary units)BHS002-2, 27-2 COMPLEMENT PRACTICAL PROTOCOLDr Anna Furmanski3ITEMS REQUIRED ReagentVolumeConcentrationAssay buffer50 mlpH 7.4 Buffer, containing Ca2+,Mg2+ and HEPESSheep erythrocytes in suspension (sheep RBC)13 mlWashed and resuspended in PBSat 5×108 cells/mlAnti-sheep RBC antibodies (Ab)13 ml1:50 dilution in PBSTest serum samples (X or Y) and reference sera2.5 ml1:100 dilution in PBSAmmonia solution.10 ml0.04% in distilled water KEEP REAGENTS ON ICE (except ammonia)OTHER MATERIALS & EQUIPMENT1. Ice tray/bucket + ice2. 1ml cuvettes3. 2ml pre-cooled eppendorf tubes, and one 15ml tube4. Tube rack for bench and water bath, to take 2ml tubes5. p100 or p200 pipette plus tips6. p1000 pipette plus tips7. Spectrophotometer8. Water bath and incubator, both at 37oC9. Eppendorf centrifugeBefore beginning check that:• you have read and understand the safety information• you have all the reagents and equipment listed above or that you know where they are or how theywill be provided• the water bath and incubator are at 37oC and that it contains the right test tube racks• the ice tray/bucket is full of ice and contains all the solutions and tubesBHS002-2, 27-2 COMPLEMENT PRACTICAL PROTOCOLDr Anna Furmanski4EXPERIMENTAL PROCEDUREImportant information:Erythrocytes (red blood cells, RBC):• can be viscous in solution and may prove difficult to pipette accurately – pipette slowly• quickly settle out of solution – mix well (but gently) before use• are fragile and will lyse if handled aggressively – mix gently by repeated inversion or slow repeatedpipettingYou are provided with a suspension of erythrocytes (5×108 cells/ml) – treat it gently!PART ONE: Sensitising erythrocytes with anti-erythrocyte antibodies1. Mix the following in a cold, labelled 15ml tube:3 ml anti-sheep RBC antibodies (Ab)3 ml of the sheep RBC suspension (sRBC)2. Check that the tubes are firmly capped. Mix gently by inverting the capped tube several times.3. Incubate at 37oC for 15 min in the water bath or incubator. Ensure each tube is appropriately labelledPART TWO: CH50 assayRemember: you will perform the assay for the Reference Sera, Serum X and Serum Y in duplicate1. Refer to Table 1. Prepare duplicates (two sets: set 1 [A] and a duplicate set 2 [B]) of numbered2ml tubes. Do this independently for each serum sample. Ensure all tubes are also labelled withyour initials so that you can identify them. Ensure you can identify which tube is which! You may findit useful to label tubes 1AX, 2AX, 3AX…1BX, 2BX, 3BX etc. 1AY, 2AY, 3AY…1BY, 2BY, etc. KEEPON ICE AT ALL TIMES2. Add reagents to your labelled tubes as described in the table below. Add ice-cold assay buffer first,followed by test serum and finally the Ab-coated (sensitised) erythrocytes that you prepared in PartOne. Ensure the caps are firmly closed and gently mix the tubes by inversion.TABLE 1: Set up for assay tubes for each sample as below(i.e. set up in duplicate for Reference Sera, Serum X, and Serum Y) VOLUME OF REAGENT (ml)TUBE NUMBER: (x2)(ie A1-7 and B1-7)1(Control)234567(100% Lysis)Assay buffer1.201.101.051.000.900.801.20ml ammonia solutionTest serum(pre-diluted)0.000.100.150.200.300.400.00Ab-coated (sensitised)RBC suspension0.300.300.300.300.300.300.30 BHS002-2, 27-2 COMPLEMENT PRACTICAL PROTOCOLDr Anna Furmanski53. Incubate the tubes in the incubator at 37oC for 60 minutes.PART THREE: Data collection4. Place the tubes on ice until a centrifuge is available5. Centrifuge at 300xg for 10 min at 4oC. While waiting, prepare cuvettes for each sample, ensuringthey are labelled6. Collect tubes from the centrifuge, taking care not to agitate them. Remove a sample of eachsupernatant (the liquid) into labelled cuvettes. Do not aspirate any of the debris from the bottom ofthe tubes. If you pipette or disturb debris/pelleted material or drop your tube it may need to be recentrifuged. Ask for assistance if you are unsure.7. Read the absorbance at 541nm, using 1 ml ammonia solution to ‘blank’ the spectrophotometer.Construct a suitable table of Results in your lab book8. Ensure you have recorded absorbance readings for all tubesPART FOUR: CALCULATIONS – in your own time!Perform all of the steps below separately for each serum.(1) Your no-serum control (tube 1) contains no complement protein and thus represents lysis caused byother factors such as rough handling. Subtract the absorbance value of the no-serum control fromthe absorbance values of tubes 2-7 before any other calculations are performed. Repeat this for thesecond (duplicate) set of tubes(2) Assume that tube 7 represents total (100%) lysis. For the first set of tubes (likely a single row onyour table), calculate the %lysis for each tube by dividing the absorbance values for each tube bythe absorbance for tube 7 (total lysis control), and multiply by 100 (to give % lysis). Repeat this forthe second (duplicate) set of tubes(3) Calculate the mean %lysis for each pair of duplicate tubes.(4) Using Excel, tabulate millilitres (ml) of undiluted serum against %lysis derived from your calculationsin the last step. Generate a graph and add a line (curve) through the pointsNOTE: To calculate the volume of undiluted serum added to each tube you need to account for thefact that the serum you added was already diluted (see pg3)(5) Clearly mark the volume of serum that triggers 50% cell lysis on your graph. This is equivalent toone CH50 Unit. You must perform this basic analysis.(6) Calculate the number of Units in 1ml of undiluted serum. Complement activity can be expressed asthe number of CH50 units per ml of serum (U/ml). You must perform this basic analysisData can be further analysed to obtain a more precise CH50 reading. There are several differentways to do this e.g. linear regression; or better by using the van Krogh equation, which logarithmicallytransforms the data. You can choose to do this if you wish to extend your analysis. The brief andtutorial will set out the expectation for what you MUST do.