Complex Of Antigens Such As Snake Venoms Biology Essay

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Immunoelectrophoresis provided a better immunologic analysis of a complex of antigens such as snake venoms.(Grabar and Williams,1955). In the study of Chippaux and Guyffon (1991), immunological composition of an antivenom can be checked by immunoelectrophoresis against the venom used for immunization or against another venom. In Myanmar, ASV has never been produced from sheep previously, although it can be used as alternative source of ASV production. This was the first time of ASV produced from sheep commercially. In this study, immunological compositions of sheep ASV was compared with standard horse ASV.

In the present study, immunoelectrophoretic pattern of horse (equine) 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 result of Htin-Aung (2009), in his study, he found only 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 horse ASV produced 13 precipitin lines with RVV and most of the precipitin lines moved towards anode site.

When compared to immunoelectrophoretic pattern of sheep (ovine) ASV, it produced only 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 joining each other independently and distinctly from precipitin lines of horse ASV (i.e. sheep ASV lines no 1 and 2 with horse ASV line no 2 and 3, sheep ASV line no 3,4 and 5 with horse ASV line no. 5,6 and 7, Sheep ASV line no. 6 with horse ASV line no.8, sheep ASV line no.7 and 8 with horse ASV line no.9 and11). It is highly probable that these precipitin lines could be ascribed as common antibodies. Horse (equine) ASV has more 3 antibody components (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. According to Kabat and Mayer (1969), negative charged, alkaline protein moved toward the anode and positive charged, acid protein moved toward cathode site. Therefore, sheep ASV has 3 antibodies fewer than standard horse ASV to react two negative charged, alkaline protein and one positive charged, acid protein of Russell's viper venom.

However, these two anti-snake venoms had the same invivo neutralization potency result in mice lethality test (MPF production record). Therefore, 3 antibody components that were missed in sheep ASV cannot be the lethal protective antibody.

In conclusion, immunoelectrophoretic pattern of sheep (ovine) ASV was determined by immunoelectrophoresis method. Most of the common antibodies in the sheep ASV and standard horse ASV were same. However, Immunoelectrophoresis method could show the antigen-antibody precipitin bands only and could not identify missing antibodies in sheep ASV. Therefore, further study was required to determine the missing antibodies in detail. It should be done by fractionation of the venom components by gel filtration column chromatography and each fraction should be checked with immunoblot method by using sheep ASV.

7.2 Invitro neutralization potency assay by Double immunodiffusion

Ouchterlony double immunodiffusion with Sewell titration method can detect amount of immunoglobulin quantitatively (Harboe and Agnette, 1973). Well no.1 contain increasing amount of venom concentrations (0.2 mg to 1.8 mg) with constant amount of ASV. Well no.2 contain Russell's viper venom only to detect excess antivenom. Well no.3 contain ASV only to detect excess venom.

At all venom concentrations 0.2 mg, 0.4 mg and 0.6 mg, distinct precipitin lines formed between well no.1 and well no.2. These result showed that tested ASV has excess amount of antibody after neutralizing the venom level 0.2, 0.4 and 0.6 mg. These excess antivenom (antibody) was detected by venom (antigen) in well no.2. Precipitin lines were more distinct at 0.2 me level and become faint to 0.4mg and 0.6 mg because excess antibody was higher at 0.2 mg level and become lower at 0.4 mg and 0.6mg level. Precipitin lines formed between well no.2 and well no.3 was normal venom (antigen) and antivenom(antibody) reaction. No precipitin lines between well no. 1 and well no. 3 showed tested ASV could completely neutralize the tested venom and indicated no excess venom(antigen).

At venom concentration 0.8 mg level, faint precipitin lines formed between well no.1 and well no.2. This can also be interpret as tested ASV has excess amount of antivenom (antibody) after neutralizing the venom level 0.8 mg. No precipitin lines formed between well no. 1 and well no.3 showed no excess venom(antigen).Faint precipitin lines showed lesser excess antivenom(antibody) when compared to 0.2mg,0.4mg and 0.6mg. Precipitin lines formed between well no.2 and well no.3 was normal venom (antigen) and antivenom(antibody) reaction.

At venom concentration 1.0mg mg level, there was no precipitin lines between well no1 and well no.2, well no.2 and well no.3 in plate no.3(batch-J090001), plate no.5(batch-A10001) and plate no.6(batch-N10003). This results showed no excess antivenom (antibody) and no excess venom (antigen) at 1.0 mg venom concentration level. It can also be say that tested ASV could completely neutralize tested venom amount 1.0mg and no excess venom and excess antivenom was noted. Precipitin lines formed between well no.2 and well no.3 was normal venom (antigen) and antivenom (antibody) reaction. This was agree with the invivo neutralization test result of MPF which mentioned that 1 mL of ASV can neutralize 1 mg of Russell's viper venom. However, faint precipitin lines were still present between well no1 and well no.2 in plate no.7(batch-J10002) and plate no.8(batch-E11004). This faint precipitin lines indicate that few amount of excess antivenom (antibody) was still present at 1.0mg venom concentration level.

At venom concentration 1.2 mg level, there was no precipitin lines between well no1 and well no.2, well no.2 and well no.3 in Plate no.3(batch- J090001), plate no.7(batch-J10002) and plate no.8(batch-E11004). However, faint precipitin line between well no.1 and well no.3 in Plate no.5 (batch-A10001) and plate no.6 (batch no-N10003). These results showed that tested ASV could neutralize 1.2 mg of venom completely and partially. This faint precipitin lines between well no.1 and well no.3 indicate that few amount of excess venom (antigen) was present at 1.2mg venom concentration level. Precipitin lines formed between well no.2 and well no.3 was normal venom (antigen) and antivenom(antibody) reaction. This result may be possible because MPF added allowance antibody titer at final dilution of antivenom. Tun-Pe ( ) proved that ASV produced from MPF has batch to batch variation.

At venom concentrations 1.4mg, 1.6mg and 1.8mg, distinct precipitin lines formed between well no.1 and well no.3. These result showed that tested ASV has excess amount of venom (antigen) after neutralizing the venom level 1.4, 1.6 and 1.8 mg. It can be interpret that tested ASV could not neutralize the venom concentration level 1.4mg, 1.6mg and 1.8 mg. Precipitin lines were more distinct from 1.4 mg to 1.8mg because excess venom(antigen) which could not neutralized by tested ASV become increased. Precipitin lines formed between well no.2 and well no.3 was normal venom (antigen) and antivenom (antibody) reaction.

According to the results, invitro neutralization potency assay of sheep(ovine) ASV could be determined as 1 mL of antivenom could neutralize 1.0mg of russell's viper venom. Batch to batch variation of potency assay was also noted. It also found that invitro potency assay result are not quite different from invivo neutralization assay result. Therefore, invitro neutralization potency assay by double immunodiffusion with Sewell titration can be used as alternative assay method for invivo neutralization mice lethality test. It was very cheap, easy to perform and no special equipments are needed. However, it cannot detect lethal factor, haemorrhagic factor, necrosis factor, oedema factor separately and WHO did not recommend as final potency assay. It also has another drawback that it needs long incubation period such as preincubation time for venom-antivenom mixture was 48 hr and incubation time for sample loading was 18 hr. Total time require for each test was 3 days (72 hr). Therefore, it cannot be used for determing of ASV potency urgently. However, this preincubation period can be reduced to one hour because most of the preincubation time in invivo neutralization test is 30 minutes. Further study was required to reduce the time period.

For the production propose and animal ethical issue, it can be used as initial potency assay method for antivenom and immune serum. By using this method, Htin Aung (2009) proved that ASV (equine) produced for Russell’s viper can cross neutralize the venom of green pit viper (Trimeresurus erythrurus). Khin-Aye-Nyein (2006) also used this method to determine neutralization potency assay of cobra antivenom(Naja kaouthia) and krait antivenom (Bungarus fasciatus) against spitting cobra venom(Naja siamensis).

7.3 Invitro potency assay by single radial immunodiffusion

In Australia, radial immunodiffusion plate assay method is used to assess the initial potency of antisnake venom plasma instead of using mice or guinea pig lethality test (WHO, 2006). In the present study, single radial immunodiffusion method was used to determine potency 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 horse (equine) ASV was also done to compare sheep ASV.

In conclusion, the newly developed Ovine Anti-snake venom produced from Myanmar Pharmaceutical Factory can react and, neutralize the venom of Russell’s viper (Daboia siamensis) invitro.

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.