How the test is performed The goal of an ELISA is to find out if a particular protein is present and how much does it contain in a sample (3). There are a few ways for performing an ELISA. Here is one of the commonly used methods and the one significant to detecting HIV antibodies:
ELISAs are performed in 96-well plates which allow high throughput results (2-3). The bottom of each well is coated with prepared HIV protein to which will bind the antibody (diagram 1) (2-3).
Entire blood is allowed to clot and the cells are centrifuged out to obtain the clear serum with antibodies which is called the primary antibodies (2). The serum is incubated in a well, and each well contains a different serum (diagram 2) (2-3).
The antibodies of the patient in the serum. If the patient is HIV positive, then this serum will contain antibodies to HIV, and those antibodies will bind to the HIV antigens on the plate (2).
The serum is removed after a period of time, and weakly adherent antibodies are washed off with a series of buffer rinses (9). A secondary antibody is added to each well to detect the bound antibodies (diagram 3) (2-3).
Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies (2).
An enzyme such as peroxidase or alkaline phosphatise is attached to the secondary antibody (3). These enzymes can metabolize colourless substrates called chromagens into colour products (diagram 4) (2,9).
After an incubation period, the secondary antibody solution is removed and weakly adherent ones are washed off as before (3). The final step is the adding up of the enzyme substrate and the production of colour product in wells with secondary antibodies bound (3,9).
When the enzyme reaction is complete, the entire plate is placed into a plate reader and the optical density is determined for each well (3). The amount of colour produced is correspondent to the amount of primary antibody bound to the proteins on the bottom of the wells (diagram 5) (9).
Limitations of this test A negative aspect of the ELISA is the occurrence of false positive or false negative result. As the Virtual Medical Centre and Stowell stated an example such that the antibodies induced by a recent flu injection can cause a positive result and so although ELISA is an important diagnostic test for HIV, in various cases it is usually followed up by a confirmatory test (7,9). False positives may also occur if you have an underlying condition such as Lupus or rheumatoid disease (7). However, a negative result does not always mean there is no infection as some antibodies are not produced at once following infection (9). The Virtual Medical Centre reported that the antibodies to HIV do not appear in blood until 6 weeks after exposure to the virus so it is recommended that if the individual suspect he or she has been exposed then the test should be repeated after 6 weeks to 6 months (7).
Other assays are routinely performed to detect HIV There are a number of tests besides ELISA can find antibodies or genetic material (RNA) to the HIV virus. These tests include the most common examination used that is a Western blot test. A positive ELISA test is always followed by a Western blot test and a positive Western blot confirms an HIV infection (6). A negative Western blot test means the ELISA test was a false positive test (4,6). However, negative tests do not rule out HIV infection (1,4). There is a period called the window period-the time between HIV infection and the appearance of anti-HIV antibodies that can be measured, thus a negative HIV ELISA and Western blot will not rule out HIV infection (6,8). In addition, the Polymerase Chain Reaction (PCR) finds either the RNA of the HIV virus or the HIV’s DNA in white blood cells infected with the virus (8). The PCR test is very useful to find a very recent infection, determine if an HIV infection is present when antibody test results were uncertain, and screen blood or organs for HIV before donation (8). However, PCR test is not done as regularly as antibody assay because it requires technical skill and expensive equipment (4,8). In addition, the Indirect Fluorescent Antibody (IFA) test also detects HIV antibodies (8). It is used to confirm the results of an ELISA just like a Western blot test. But it is more expensive than a Western blot test and not frequently used (8).
Causes and Treatments of Dengue fever
Dengue fever is caused by positive-stranded RNA virus of the family Flaviviridae; genus Flavivirus. Dengue virus is transmitted to humans by a mosquito vector Aedes aegypti belonging to genus Aedes of family Culicideae. 50 -100 million peoples are affected by dengue virus . Indian scientists have develop a vaccine for the prevention of the dengue fever. Dengue Virus Genome Contain 10 Viral Protein C,prM,E,NS1.NS2A,NS2B,NS3,NS4A,NS4B,NS5. vector population suppression ,replacement are genetic approaches for control of dengue. In the population suppression vector number is reduced to minimize disease transmission and population replacement of wild-type vector was replaced with genetically modified vector incapable of transmitting the disease. Sterile Insect Technique (SIT) is a particular type and environmentally benign way to suppress the insect population, it is based on collective farming, and radiation-mediated sterilization, and large number of male insects are release. The release of insects carrying a dominant lethal (RIDL) offers a resolution to many of the weaknesses of traditional SIT which has narrow its application in mosquitoes while sustaining the efficacy ecologically friendly and specific types. RNA interference is a mechanism of gene control sequence after transcriptional selectively. RNA viruses Interference (RNAi) pathway is one of the most critical components of the innate immune system of insects that can frustrate a variety of RNA virus such as flavivirus. From all the genetic based method RIDL techniques is most advance.
Introduction: Dengue fever is a viral disease transmitted by the rapid development of Aedes aegypti. (McCall and Kittayapong 2006) originally innate to West Africa and spread in the whole world (Antonelli, Clayton et al.) 50 -100 million peoples are affected by dengue virus (Wilke and Marrelli 2012). Dengue virus belongs to the Flaviviridae family. Dengue virus has four major serotypes but antigenically distinct single stranded RNA viruses . The four serotypes of dengue viruses cause disease. The severe form of the disease, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DHF / DSS), can lead to internal bleeding and even death. (Malavige, Fernando et al. 2004; Olson, Alphey et al. 2006)
There is no specific therapeutic drug available for dengue fever. Indian scientists have develop a vaccine for the prevention of the dengue fever animal trials of this vaccine have yielded good results but it is not yet commercialize. By Using yeast “pastoris Pichia” they have created a non-infectious dengue fever virus like particles made of only large DENV” protein envelope ” to active virus-specific immunity. (Thisyakorn 2013).
Dengue virus is 50-nm containing lipid membrane.180 identical copies of envelop protein is present at the surface of viral membrane short transmembrane segment. The virus genome is of about 11000 bases that translates into a single large polyprotein that is cleaved into 3 structural and 7 non-structural genes and short non-coding regions is also present on both the 5′ and 3′ ends The structural proteins are the capsid (C) protein, the envelope (E) glycoprotein and the membrane (M) protein. (Alcaraz-Estrada, Yocupicio-Monroy et al. 2010) (Nukui, Tajima et al. 2006)
The increase of vectors in world is reason of emergence of diseases in new areas. therefore, there is need to control mosquito. Bed nets, space spraying are largely ineffective against this day biting mosquito. Therefore, there is a need to develop useful strategy to overcome dengue fever. Dengue control can be reduced or elimination of the vector population. The population vector can be reduced or eliminated based on genetic methods. There are two strategic objectives to control dengue by genetically modified mosquitoes.(McCall and Kittayapong 2006; Olson, Alphey et al. 2006)
vector population suppression
In the population suppression vector number is reduced to minimize disease transmission and population replacement of wild-type vector was replaced with genetically modified vector incapable of transmitting the disease.(Antonelli, Clayton et al.) suppression techniques and replacement of population are not mutually exclusive and can be implemented in conjunction with other approaches. (Olson, Alphey et al. 2006; Organization 2009) For suppression of the vector population SIT (sterile insect technique) SIT is a specific control environment of the species method in which large numbers of sterile males are release these mate with wild female reduces the reproductive capacity of mosquito. (Coleman and Alphey 2004; Helinski, El-Sayed et al. 2006) SIT mosquito control approaches have been tense and still planned .Reduction mating competitiveness, and sterile female does not reduce the population ,fertility of irradiated males residual is the limitation of the approaches to mosquito control SIT. In SIT male mosquitoes are about liberation are not taking blood meals, and lethality performance so early larval death is density dependent reduction occur. (Yakob, Alphey et al. 2008; de Valdez, Nimmo et al. 2011; Lacroix, McKemey et al. 2012) RIDL (Release of Insects carrying a Dominant Lethal) strategy to reduce these limitations by recombinant DNA technology. In RIDL male n female mosquitoes are genetically modified approaches. (Coleman and Alphey 2004; Atkinson, Su et al. 2007; Phuc, Andreasen et al. 2007) OX513A RIDL bisex is a strain. The males are released to mate with wild females. The offspring of these mating die due to late larvae or pupae. Thus the target population can be reduced. (Wallace 2013)
In female transgenic mosquitoes OX3604C that genes are present to conduct flightless female-tetracycline-repressible phenotype. flightless females can not mate with a male. (de Valdez, Nimmo et al. 2011; McGraw and O’Neill 2013) And for substituting additional vector population or foreign genes are inserted to prevent the transmission vector of the disease. Foreign gene can be a gene Antipathogen, or a combination of transgenic approaches that will reduce or eliminate the transmission of disease from vectors to human.(Basu and Bhattacharya 2014)
Genetic approaches leading to vector population suppression:
GMV-based development of Sterile-Insect Technique (SIT):
The sterile insect technique (SIT) is a specific method of species and environmentally benign control the insect population that relies on mass rearing and release of sterile insects.(Wilke and Marrelli 2012) These released insects compete for mates with wild females. Wild female pairing to a sterile male has no or fewer children, so that the population tends to decrease. If enough sterile insects are released for a sufficient period, the target population is controlled or even eradicated locally(Wallace 2013). SIT has been used successfully for over 50 years to control the entire area and / or elimination of several important agricultural pests and disease vectors, including the Mediterranean fruit fly, screwworm. (Coleman and Alphey 2004)Although conducted a series of tests in the 1970s, with some success, there is today no SIT programs on large-scale operation against mosquito species. (Yakob, Alphey et al. 2008; Lacroix, McKemey et al. 2012; Mwasunda October, 2012) Ae. aegypti is a, mosquito species fit for mass rearing. This species also appears to be reasonably uniform over large areas, without the problems of sub-species and barriers to mating.
SIT has many advantages as compare to traditional strategies:
it is species specific and to stay away from The harmful effects of pesticides on non-target species.
The mating behavior of male insects is highly efficient as a substitute of relying on Human rights activists.
wild insects Density are condensed that’s why it works better (Coleman and Alphey 2004)
LIMITATION of SIT:
The main problem of SIT is the loss of competitiveness mating for the wild type due to sterilization of insects by irradiation.
For mosquitoes, releasing only male is considered essential, since the sterile females bite and can thus transmit the disease, while male mosquitoes do not bite. So SIT including large-scale, gender-based separation . pupal size can constantly give people male-only population for release.
A key difficulty for mosquito SIT is sterilization. Irradiation of pupae appear to harm insects; irradiation as adults is less harmful but operationally much harder. Some trials used sterilizing chemicals such as thiotepa, which was effective for sterilization, but led to trace contamination with this mutagenic chemical.
Another problem for mosquito SIT relates to the biology of the mosquito population. Unlike agricultural pests against which the main SIT programs are directed at present, mosquito populations can be regulated primarily by density-dependent effects, in which a very fruitful population is maintained at a level stable for limited resource constraints,
others problems of SIT is fitness costs and operational difficulty of irradiation.(Coleman and Alphey 2004; Olson, Alphey et al. 2006; Wilke and Marrelli 2012)
Release of Insects Carrying a Dominant Lethal Gene:
Insects are designed to carry a dominant lethal genetic system can be used to replace the need for radiation sterilization in a similar SIT program. In this RIDL is replaced engineered lethal dominant. At the same time RIDL and SIT, and some or all of the descendants of people die as a result of the release of inheriting one or more dominant lethal mutations, so that the population tends to decline. (Atkinson, Su et al. 2007; Yakob, Alphey et al. 2008; Massonnet-Bruneel, Corre-Catelin et al. 2013) Mechanism: Male mosquitoes have developed genetically modified to contain lethal gene, in order to combat the spread of dengue fever.
LA513 is piggyBac transposon-based non-independent from 8.4 kilobytes. Insert LA513 transposon within aeydes mosquitoes by genetic engineering techniques to produce toxins in the larvae of mosquitoes stage “under normal condition, and thus cause the death of the larva. Transposon LA513 mainly containing DsRed2 gene that is to engage in the modified identify genetically by red fluorescence due to the expression of that gene. and tTAV gene (tetracycline -repressible transcriptional activator) tTAV gene is under the control of its binding site, titO, and minimal promoter of Drosophila HSP70, and 3 ‘UTR sequence of the fruit fly FS (1) K10. tTAV bind with its binding site titOs and gives a very high level of expression of tTAV which is toxic under normal conditions. In The presence of tetracycline tTAV connect with them. This model does not bind tTAV with titO and do not even lead to the expression of more tTAV therefore this construction gives a very high level expression of tTAV in the absence of tetracycline, but gives low expression in the presence of tetracycline. The high level expression of tTAV may be due to the interaction with the VP16 domain transcription factor that provides a key to the building of this deadly system. (Massonnet-Bruneel, Corre-Catelin et al. 2013)
When genetically modified mosquitoes are released in the wild and mate with wild female offspring to inherit this trait lethality. The resulting offspring die before reaching the age of majority and the local mosquito swarms fall.(McGraw and O’Neill 2013)
Oxitec is a British company is working to find innovative solutions to control insects using biotechnology and genetics in advance (called RIDL) placed Egyptian genetically modified Aedes this product and is named as a OX513A bisex RIDL strain. (Massonnet-Bruneel, Corre-Catelin et al. 2013)
Aeydes mosquito, one of the most important carrier of the dengue fever,and decreased by 80% in the 2010 trial of these genetically modified mosquitoes in the Cayman Islands. Using genetically modified male aegypti we can reduce or eliminate the population of mosquitoes that specie, and thus we can control the transmission of dengue virus to humans, which causes dengue fever.
Population replacement techniques:
Population replacement techniques involve the insertion of additional or foreign genes to prevent the mosquitoes from transmitting the disease. The techniques may include inserting an antipathogen gene, an immune system upregulator, or a combination of transgenic approaches that will ultimately result in the reduction or elimination of disease transmission from mosquito to human. To assess whether a mosquito has reduced disease transmission capability, the virus titer in the experimental transgenic mosquitoes midgut and salivary glands can be measured and compared to a wildtype control.
At least three genetic-transformation systems have been described and used successfully in Ae. aegypti to generate GMVs. These transformation systems are based on the Class II TEs Mos1 (Mariner), Hermes and piggyBac . Mos1 and piggyBac are the most commonly used TEs mos1 for generating GMVs.
Evidence of rnai mechanism in mosquitoes:
List the RNA’s strategy Interference with the recurrence of dengue virus can operate Effective in mosquitoes and mosquito cells. The first evidence is recombinant Sindbis viruses having DENV-2-prM sequence was transformed in to in adult female mosquitoes. Sindbis viruses is single-stranded RNA virus. During replication it is convert in to double stranded RNA and intermediary expressing DENV-2 prM that is activate the RNAi mechanism . Combination of DENV-2 and the recombinant SINV having DENV-2-prM sequence slabs DENV replication in mosquitoes.
in mosquito cell culture Expression of a dsRNA hairpin structure is a strong Catalyst of the RNAi mechnism. 567 nucleotide of the prM sequence from DENV-2 in a sense orientation and first 290 nt repeated in antisense orientation is made hairpin structure .cloned this into plasmid after transcription DENV-2 specific dsRNA is formed that stop the DENV-2 replication.
transfection of C6/36 (Aedes albopictus) with dsRNA of arbovirus genome.these are the evidence which shows that Aedes species have RNAi mechanism resemble to other organisms.
RNAi Mechnism :
In RNAi mechanism dsRNA is integrate in The cytoplasm of the cell. RNase III enzyme Dicer is responsible to digest dsRNA in a small interfering RNA 21-23 BP (RNAs siRNAs). These double stranded siRNAs having two nucleotides over hangs at both of the 3’ends. Then siRNAs is unwounded in a single stranded by the enzyme complex called the RNA-induced silencing complex,(RISC).one strand act as RISC-targeting co-factor. siRNA having RISC binds with complementary mRNA. cleavage in the middle of the siRNA binding region by RISC is occur .then mRNA Is degrade by natural endogenous mechanisms.
RNAi mechanism for dengue control:
Sánchez-Vargas and collegues proved that RNAi paly a important role in the reduction of dengue infection. AAV (adeno-associated virus )siRNA infected the dendritic cells and decrease Dengue infection in human.
Mosquito cell line transformed culture (C6 / 36, A. albopictus) by way of plasmid Which contain the inverted sequence of PRM gene of dengue And form dsRNA 290-BP. These transformed cells contain resistance to the dengue virus 2. When these cells are infected with dengue virus -2 viral RAN collection is not occur. These transformed mosquitoes cells Containing all the machines of RNAi. This finding proved that RNAi play a important role in reduction of dengue virus 2.
Transposon ,that contain the inverted repeat sequence of prM gene of dengue virus is transformed in mosquito embryo along with carboxypeptidase A Promoter .after the intake of bloodmeal of mosquitoes dsRNA 578-BP is produce. When genetically modified Mosquito Carb 77 stuck With DENV2 having bloodmeal dsRNA is expressed along virus replication. thus preserving the Spreading of the virus from the host to the salivary Glands. RNAi mechanism is accountable for the resistance of DENV2.
SiRNA-mediated silencing of receptors and Facility clathrin – mediated endocytosis can prevent entry of Dengue virus .The proliferation of HepG2 cells, reducing the Virus. inhibit the dengue fever to develop into serious forms .Specified cellular genes involved in endocytosis operations And the dynamics of the cell structure, the task of infection DENV. SiRNA directing genes linked with clathrin mediated endocytosis.
Villegas-Rosales et al. projected three siRNAs that have ability to silence the four DENV serotypes genome By pointing NS4B and NS5 sequences . SiRNA along with RNAi self-processing machinery have a role in the preclusion of unadorned dengue infection. In the development of new therepuetical drugs RNAI can play an imporatn role .