Antibodies are proteins produced during organic structures defense mechanism against foreign antigens and they are driven from plasma cells. In the event of an immune response B lymphocytes initiate the production of IgM antibody. In comparing to other Ig ‘s IgM is the largest and earliest antibody available in response to an antigen ( Bailey & A ; Johnson, 2006 ) . The big construction of this antibody is because it consists of an extra sphere in its changeless country ( Overfield et al, 2007 ) . This antibody has a polymeric construction & A ; it consists of heavy and light ironss. The adhering between two heavy ironss or between heavy and light ironss is facilitated via the disulphide bond. IgM antibody has a pentameric construction dwelling of five fractional monetary units. These fractional monetary units are joined together via a disulphide bond which occurs between the Fc part and the intersubunit, interasubunit- J concatenation. Two fab antigen binding sites are available on each IgM monomer and since IgM has a pentameric construction ten Fab antigen binding sites are available that can potentially interact with 10 antigens ( Overfield et al, 2007 ) ( Khurana, 2006 ) . The initial purpose of this practical was to detect if ruddy blood cell antigens can interact with IgM anti-D ( Rh ) antibody and conditions as a effect of this interaction agglutination occurs. The 2nd purpose was to detect weather dithiothreitol ( DTT ) cut downing agents is capable of changing the construction of IgM antibody at different concentration hence impacting the degree of agglutination and eventually to detect if indirect anti-IgM antibody is capable of easing agglutination. The big and pentameric construction of IgM antibody can potentiate the possibility of its interaction with ruddy blood cell antigens ensuing in formation of agglutination.
Material & A ; Method
For instructions on how to carry on the experiment with the relevant stuffs used please refer to the practical agenda. The concentrations of DTT added to the nine tubings where as followers ( 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009 & A ; 0.01 ) .
Degree of agglutination associated with the concentration of DTT
Concentration of DTT
( Mol/L )
( + ) ( – ) 2nd observation antibody labelling
Table 1: The above tabular array illustrates the figure of tubings labelled from 1-10 and the concentrations of DTT in ( Mol/L ) . As illustrated in the above tabular array the control tubing which is tube 1 lacked DTT while tubings which were numbered as ( 2, 3, 4, 5, 6 & A ; 7 ) consisted of different concentrations of DTT as shown here ( 0.001, 0.002, 0.003, 0.004, 0.005, 0.006 & A ; 0.007 ) . Harmonizing to the first observation consequences tubes numbered 1-7 expressed marks of agglutination as indicated by a positive mark ( + ) . Alternatively tubes numbered ( 8, 9 & A ; 10 ) which had the undermentioned DTT concentrations ( 0.007, 0.008 & A ; 0.009 ) expressed no indicants of agglutination hence they were marked as negative ( – ) . Due to clip restrictions consequences for the 2nd antibody labelling could non be obtained.
The strength of agglutination in these tubings depended on the concentration of DTT. The control tubing which is tube 1 is DTT deficient which is accompanied with agglutination. Tubes labelled 2-7 express different concentrations of DTT get downing from the lowest hence intensifying easy. In these tubings agglutination is still observed since the consequence of DTT is still non strong plenty to interrupt the bonds expressed in IgM antibody while as the concentration of DTT escalates further in tubes 8-10 agglutination is non evidenced. DTT is a cut downing agent capable of interceding intersubunit and interasubunit-J concatenation cleavage hence easing IgM fractional monetary unit ( Aµ2I»2 ) synthesis ( Kownatzki & A ; Drescher, 1973 ) . As the concentration of DTT escalates its capableness to interrupt these bonds with greater strength additions as seen in tubes 8-10 taking to greater IgM fractional monetary unit formation ( Aµ2I»2 ) and lessens the possibility of antigen antibody interaction hence deficiency of agglutination. In add-on DTT affects the construction of IgM heavy and light ironss by forestalling them from blossoming and causes this concatenation excessively separate consequently taking to agglutination lack. A uninterrupted rise in DTT concentration as evidenced in tubes 8-10 causes a diminution the chance of disulphide bonds from restarting their map in IgM antibody ( Valetti & A ; Sitia, 1994 ) . Harmonizing to the survey conducted by ( Marrodan et al, 2001 & A ; Morris et Al, 1974 ) DTT cut downing agent restrains agglutination from happening by easing the disulphide bond located in the IgM antibody to interrupt. In add-on the 19 S IgM antibody is cleaved by DTT into a 7S fractional monetary unit. The 7S antibody fractional monetary units are rendered incapable of keeping IgM antibody ‘s map and therefore wo n’t be able to interact with ruddy blood cell antigens taking to deficiency of agglutination ( Knight, 1978 ) .
Due to clip restriction for the experiment consequences for the 2nd antibody labelling could non be obtained. Harmonizing to ( Overfield et al, 2007 ) the missing agglutination as a effect of DTT consequence can be reversed by adding anti-IgM antibody hence marks of agglutination will look but the extent of agglutination will depend on whether the IgM antibody fractional monetary units have maintained their ability to adhere to ruddy blood cells antigen or due to high degree of DTT concentration they have been wholly deformed.
Harmonizing to the survey conducted by ( Emmerich et al, 2006 ) IgM antibody can be used in the diagnosing of Lassa virus infection which is extremely prevailing in Western African patients. This diagnosing is achieved via utilizing rearward enzyme immunoassay ( ELISA ) technique to place anti-Lassa IgM antibody. The consequence of this survey implemented that via utilizing rearward ELISA in 20 patients with mark of febrility high degree of anti-Lassa IgM antibody was diagnosed bespeaking the presence of the Lassa virus. In a survey conducted by ( Varsano et al, 1995 ) the presence of IgM antibody against respiratory syncytial virus antigen ( RSV ) was examined in 145 patients via utilizing the ELISA technique. Harmonizing to the consequence of this survey ELISA-IgM antibody sensing is a extremely efficient method in the diagnosing of RSV at early phase of the disease. In another survey by ( Tsuda et al, 2001 ) polymerase concatenation reaction ( PCR ) technique was used to observe for the presence of IgM antibody against TT virus ( TTV ) in the diagnosing of human circovirus. The consequence of this experiment suggests that healthy voluntaries were faulty of anti-TTV IgM antibody whereas infected persons showed marks of its presence suggesting that this method is good for diagnosing intents of human circovirus.
Immunoglobulin cleavage can be triggered via the action of different enzymes or chemicals. Papain is an enzyme that cleaves IgG antibody into three sections of FC, heavy and light ironss. Furthermore IgM antibody can be cleaved by pepsin enzyme either into an antibody that weights less accompanied with FC fragments ( Rudmann, 2005 ) ( Svehag et al, 1969 ) . Protease enzyme is driven from Neisseria gonorrhoeae bacteriums capable of spliting IgA antibody ( Pouedras et al, 1992 ) . Harmonizing to ( Akesson et al, 2006 ) streptococci pyogenes bacteriums is responsible in interceding diseases such as gotonsillitis, blood poisoning and it intervenes its action by doing IgG antibody cleavage via utilizing an enzyme called Ides. The action of this virus is to see that the antibody is unavailable to destruct the bacterium. Furthermore trypsin is another enzyme capable of spliting IgM antibody at temperature above 50 C taking to different FC fragment synthesis ( Andrew et al, 1970 ) .
Normally ruddy blood cell antigens are capable of interacting with IgM antibody ensuing in agglutination while in the presence of DTT cut downing agent this binding is inhibited taking to deficiency of agglutination. The extent of this suppression will depend on the concentration of DTT and the extend of IgM J concatenation, interchain & A ; intrachain cleavage via DTT. The greater the concentration of DTT the stronger its consequence is on this concatenation which lessens the likeliness of this concatenation recovering their binding capacity hence their ability to recover antigen binding activity. The construct of antigen antibody binding can be used for the diagnosing intent of many diseases.