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Immunoelectrophoresis provided a better immunologic analysis of a composite of antigens such as serpent venoms. ( Grabar and Williams,1955 ) . In the survey of Chippaux and Guyffon ( 1991 ) , immunological composing of an antivenom can be checked by immunoelectrophoresis against the venom used for immunisation or against another venom. In Myanmar, ASV has ne’er been produced from sheep antecedently, although it can be used as alternate beginning of ASV production. This was the first clip of ASV produced from sheep commercially. In this survey, immunological composings of sheep ASV was compared with standard Equus caballus ASV.

In the present survey, immunoelectrophoretic form of Equus caballus ( equid ) ASV produced 11 precipitin lines ( line no.1 to 11 ) when reacted with Russell ‘s viper venom ( RVV ) . Among them, 7 precipitin lines ( line no.1 to 7 ) moved toward anode, 1 remained ( line no.8 ) at the site of application and 3 ( line no.9,10 and 11 ) moved toward cathode. This was agree with the consequence of Htin-Aung ( 2009 ) , in his survey, he found merely 11 precipitin lines, out of which 8 moved to anode, 2 remained at the site of application and 1 moved to cathode. However, Khin- Aung- Cho ( 1979 ) found that Equus caballus ASV produced 13 precipitin lines with RVV and most of the precipitin lines moved towards anode site.

When compared to immunoelectrophoretic form of sheep ( ovine ) ASV, it produced merely 8 precipitin lines ( line no 1 to 8 ) when reacted with RVV. Among them 5 precipitin lines ( line no 1 to 5 ) moved toward anode, 1 remained ( line no 6 ) at the site of application and 2 ( line no 7 and 8 ) moved toward cathode. These 8 precipitin lines are fall ining each other independently and clearly from precipitin lines of Equus caballus ASV ( i.e. sheep ASV lines no 1 and 2 with Equus caballus ASV line no 2 and 3, sheep ASV line no 3,4 and 5 with Equus caballus ASV line no. 5,6 and 7, Sheep ASV line no. 6 with Equus caballus ASV line no.8, sheep ASV line no.7 and 8 with Equus caballus ASV line no.9 and11 ) . It is extremely likely that these precipitin lines could be ascribed as common antibodies. Horse ( equid ) ASV has more 3 antibody constituents ( line no. 1, 4 and 10 ) than sheep ASV. Two of these antibodies moved toward the anode site and one moved toward the cathode site. Harmonizing to Kabat and Mayer ( 1969 ) , negative charged, alkalic protein moved toward the anode and positive charged, acerb protein moved toward cathode site. Therefore, sheep ASV has 3 antibodies fewer than standard Equus caballus ASV to respond two negative charged, alkalic protein and one positive charged, acerb protein of Russell ‘s viper venom.

However, these two anti-snake venoms had the same invivo neutralisation authority consequence in mice deadliness trial ( MPF production record ) . Therefore, 3 antibody constituents that were missed in sheep ASV can non be the deadly protective antibody.

In decision, immunoelectrophoretic form of sheep ( ovine ) ASV was determined by immunoelectrophoresis method. Most of the common antibodies in the sheep ASV and standard Equus caballus ASV were same. However, Immunoelectrophoresis method could demo the antigen-antibody precipitin bands merely and could non place losing antibodies in sheep ASV. Therefore, farther survey was required to find the losing antibodies in item. It should be done by fractional process of the venom constituents by gel filtration column chromatography and each fraction should be checked with immunoblot method by utilizing sheep ASV.

7.2 Invitro neutralisation authority check by Double immunodiffusion

Ouchterlony dual immunodiffusion with Sewell titration method can observe sum of Ig quantitatively ( Harboe and Agnette, 1973 ) . Well no.1 contain increasing sum of venom concentrations ( 0.2 milligram to 1.8 milligrams ) with changeless sum of ASV. Well no.2 contain Russell ‘s viper venom merely to observe extra antivenom. Well no.3 contain ASV merely to observe extra venom.

At all venom concentrations 0.2 milligrams, 0.4 milligram and 0.6 milligram, distinguishable precipitin lines formed between good no.1 and good no.2. These consequence showed that tested ASV has extra sum of antibody after neutralizing the venom degree 0.2, 0.4 and 0.6 milligram. These extra antivenom ( antibody ) was detected by venom ( antigen ) in good no.2. Precipitin lines were more distinguishable at 0.2 me degree and go swoon to 0.4mg and 0.6 milligram because extra antibody was higher at 0.2 milligrams degree and go lower at 0.4 milligram and 0.6mg degree. Precipitin lines formed between good no.2 and good no.3 was normal venom ( antigen ) and antivenom ( antibody ) reaction. No precipitin lines between good no. 1 and good no. 3 showed tested ASV could wholly neutralize the tried venom and indicated no extra venom ( antigen ) .

At venom concentration 0.8 milligram degree, swoon precipitin lines formed between good no.1 and good no.2. This can besides be interpret as tried ASV has extra sum of antivenom ( antibody ) after neutralizing the venom degree 0.8 mg. No precipitin lines formed between good no. 1 and good no.3 showed no extra venom ( antigen ) .Faint precipitin lines showed lesser extra antivenom ( antibody ) when compared to 0.2mg,0.4mg and 0.6mg. Precipitin lines formed between good no.2 and good no.3 was normal venom ( antigen ) and antivenom ( antibody ) reaction.

At venom concentration 1.0mg milligram degree, there was no precipitin lines between good no1 and good no.2, well no.2 and good no.3 in home base no.3 ( batch-J090001 ) , plate no.5 ( batch-A10001 ) and plate no.6 ( batch-N10003 ) . This consequences showed no extra antivenom ( antibody ) and no extra venom ( antigen ) at 1.0 milligram venom concentration degree. It can besides be say that tested ASV could wholly neutralize tried venom sum 1.0mg and no extra venom and extra antivenom was noted. Precipitin lines formed between good no.2 and good no.3 was normal venom ( antigen ) and antivenom ( antibody ) reaction. This was agree with the invivo neutralisation trial consequence of MPF which mentioned that 1 milliliter of ASV can neutralize 1 milligram of Russell ‘s viper venom. However, swoon precipitin lines were still present between good no1 and good no.2 in home base no.7 ( batch-J10002 ) and plate no.8 ( batch-E11004 ) . This swoon precipitin lines indicate that few sum of extra antivenom ( antibody ) was still present at 1.0mg venom concentration degree.

At venom concentration 1.2 milligram degree, there was no precipitin lines between good no1 and good no.2, well no.2 and good no.3 in Plate no.3 ( batch- J090001 ) , plate no.7 ( batch-J10002 ) and plate no.8 ( batch-E11004 ) . However, weak precipitin line between good no.1 and good no.3 in Plate no.5 ( batch-A10001 ) and plate no.6 ( batch no-N10003 ) . These consequences showed that tested ASV could neutralize 1.2 milligram of venom wholly and partly. This swoon precipitin lines between good no.1 and good no.3 indicate that few sum of extra venom ( antigen ) was present at 1.2mg venom concentration degree. Precipitin lines formed between good no.2 and good no.3 was normal venom ( antigen ) and antivenom ( antibody ) reaction. This consequence may be possible because MPF added allowance antibody titre at concluding dilution of antivenom. Tun-Pe ( ) proved that ASV produced from MPF has batch to batch fluctuation.

At venom concentrations 1.4mg, 1.6mg and 1.8mg, distinguishable precipitin lines formed between good no.1 and good no.3. These consequence showed that tested ASV has extra sum of venom ( antigen ) after neutralizing the venom degree 1.4, 1.6 and 1.8 milligram. It can be interpret that tested ASV could non neutralize the venom concentration degree 1.4mg, 1.6mg and 1.8 mg. Precipitin lines were more distinguishable from 1.4 milligrams to 1.8mg because extra venom ( antigen ) which could non neutralized by tried ASV become increased. Precipitin lines formed between good no.2 and good no.3 was normal venom ( antigen ) and antivenom ( antibody ) reaction.

Harmonizing to the consequences, invitro neutralisation authority check of sheep ( ovine ) ASV could be determined as 1 milliliter of antivenom could neutralize 1.0mg of Russell ‘s viper venom. Batch to batch fluctuation of authority check was besides noted. It besides found that invitro authority assay consequence are non rather different from invivo neutralisation assay consequence. Therefore, invitro neutralisation authority check by dual immunodiffusion with Sewell titration can be used as alternate assay method for invivo neutralisation mice deadliness trial. It was really inexpensive, easy to execute and no particular equipments are needed. However, it can non observe deadly factor, hemorrhagic factor, mortification factor, oedema factor individually and WHO did non urge as concluding authority check. It besides has another drawback that it needs long incubation period such as preincubation clip for venom-antivenom mixture was 48 hour and incubation clip for sample burden was 18 hour. Entire clip require for each trial was 3 yearss ( 72 hour ) . Therefore, it can non be used for determing of ASV authority desperately. However, this preincubation period can be reduced to one hr because most of the preincubation clip in invivo neutralisation trial is 30 proceedingss. Further survey was required to cut down the clip period.

For the production propose and carnal ethical issue, it can be used as initial authority assay method for antivenom and immune serum. By utilizing this method, Htin Aung ( 2009 ) proved that ASV ( equid ) produced for Russell & A ; acirc ; ˆ™s viper can traverse neutralize the venom of green cavity viper ( Trimeresurus erythrurus ) . Khin-Aye-Nyein ( 2006 ) besides used this method to find neutralisation authority check of cobra antivenom ( Naja kaouthia ) and krait antivenom ( Bungarus fasciatus ) against ptyalizing cobra venom ( Naja siamensis ) .

7.3 Invitro authority check by individual radial immunodiffusion

In Australia, radial immunodiffusion home base check method is used to measure the initial authority of antisnake venom plasma alternatively of utilizing mice or guinea pig deadliness trial ( WHO, 2006 ) . In the present survey, individual radial immunodiffusion method was used to find authority of sheep ( ovine ) ASV. It showed that diameter of precipitin rings were enlarged with increasing venom concentrations ( 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 ) . Five batches of sheep ( ovine ) ASV was done and standard linear curve was drawn. Five batches of Equus caballus ( equid ) ASV was besides done to compare sheep ASV.

In decision, the freshly developed Ovine Anti-snake venom produced from Myanmar Pharmaceutical Factory can respond and, neutralize the venom of Russell & A ; acirc ; ˆ™s viper ( Daboia siamensis ) invitro.

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