ELISAfor the detection of Antibodies to Ebola Virus
Sincethe occurrence of the high-mortality Ebola epidemics, itsepidemiology and ecology have been considerably studied. However, thestudies have proved challenging since there is a lack of effectivetools of serology that can be used to survey individuals andpopulations that have shown previous or past infections. The Ebolavirus has a single-stranded RNA genome that is non-infectious andwill only spread by directly coming in contact with body fluids orblood of a person with a developed symptom for the disease. Severaltests have been used to identify the disease. However, the IFAT(Indirect Fluorescent Antibody Test) has been the most frequentlyused test for the Ebola virus because it has shown excellentsensitivity for the antibodies. The test has been increasinglycriticized because of the lack of specificity of populations and theincapability of result comparisons with other useful techniques.
Othercurrent tests that are being done include the ELISA (Enzyme-LinkedImmunosorbent Assay) and the RIPA (radioimmunoprecipitation assay).They are commonly used to screen antibodies of many viral diseases,but the mostly used is he ELISA because of several advantages itexhibits the test is very quick and is safe with zero harmfulness,it can also be used in a varied group of tests on humans and animals.Also, it uses both antibodies and antigens such that even a smallconcentration can be utilized because it captures both antibodiesantigens. This paper is going to discuss the ELISA test process, itscorrelation, necessity and the sufficiency in the detection of theEbola virus.
TheEnzyme-Linked Immunosorbent Assay is a frequently used technique inbiomedicine to detect specific antibodies and antigens in any givensamples. The technique is also able to quantify the antigens andantibodies in the samples given. The process relies on the concept ofimmunology where antigens bind to specific antibodies. ELISA allowsfor the detection of small numbers of antigens, for example,peptides, hormones, proteins, or the antibodies in a sample. it isalso referred to as the EIA or the Enzyme Immunoassay and during thetest, the antigen is required to be immobilized to a hard surface andthen an antibody is complexed with it in the presence of an enzyme.To achieve detection, the enzyme conjugated activity is assessedduring the incubation period until the product has been produced andmeasured.
Materialsand method
Forthe viral antigens, cultured tissue cells infected with the Ebolavirus were used. The viral antigens of the IgG test were thenextracted using the basic detergent buffer. All these cells wereplaced in roller bottles and allowed to continue spreading till thecytopathic effects were achieved. The remaining cells that wereattached to the roller bottles’ walls were then scraped off using arubber policeman. Afterward, the supernatant and the cells werecentrifuged for 10 minutes at 4°C to separate them. The cells werethen washed and pelleted then the cell pellet was hung in boratesaline using a vortex.
Forthe sera, animals with previous EBO virus infections wereexperimentally used for the IgM and IgG tests. Three animals wereused, two infected with the EBO virus and the Simian HemorrhagicFever Virus (SHFV)while the last one was infected with anotherisolated EBO virus but not contaminated with any SHFV.
Procedurefor the ELISA Test
Thistest is straight forward and requires no special preparation. Thereare also no risks, but when performing it, one should take a lot ofcare for any infections, bruising or bleeding more than usual in thecase of drawing blood for the sample. Sometimes if the patient hashad trouble drawing blood, he or she might feel faint at first(Ksiazek, 1999).
Aftercollecting and preparing the samples to be tested, positive andnegative controls were then added to test for the availability of thevirus in the samples. Immediately, all the reagents were added to thepolyvinyl chloride microtiter plate’s wells in volumes of 100µL.This process allowed for the antibodies and antigens to be absorbedto wells overnight. After this, 100µL of diluted serum samples werepipetted then mixed and incubated in a humid 37°C chamber for 60minutes. The samples were then aspirated and washed thrice withPhosphate-buffered saline. After the process, 100µL of the antibodylabeled #1 was added, mixed gently and incubated at 37°C for aboutan hour.
Again,the sample was washed and aspirated thrice with the washer buffer and100µL of the detection antibody labeled #2 was added. It was thenmixed gently and incubated in the humid chamber for 30 minutes at37°C. during the third step, the mixture was again aspirated andwashed three times with the wash buffer and 100µL of the enzymesubstrate added to it. It was then mixed gently and incubated for 15minutes in the humid chamber at 37°C. a blue color was thenobserved to appear in the positive samples and the positivecalibrators. Further to the procedure, 100µL of the stop solutionwas pipetted into each and mixed gently. The blue color was observedto start turning yellow. The plates were then read in aspectrophotometer, and the OD (optical density) values at 410nm werecaptured in a microcomputer.
Results
Thesera used (IgG) were for persons who had previously been exposed andinfected with the Ebola virus. The antibodies were detectable, andtheir titers stayed moderate at 800-1600. The antibodies (IgM) wereof primates that were experimentally infected with the EBO virus.However, no IgM antibodies had been detected but for one animal whoseantibody was detected and the levels were adjusted to 410nm.
Conclusion
theevidence from the results indicates that the ELISA test is useful indetecting both IgG and IgM antibodies and that it is a useful testfor investigating the Ebola virus. The data strongly indicates thatit is an accurate test that gave adjusted OD values. Thus, ELISApromises to be an excellent serological tool that will help withinvestigations involving epidemiology and epizootiology.
ComparingELISA to IFAT (Indirect Fluorescent Antibody Test) data, it isapparent that ELISA has several advantages. IFAT test is the mostpreferred use by many investigators when dealing with outbreaks ofepidemiological diseases. However, the ELISA test showed an enduringsensitivity and specificity of the IgG ELISA for the Ebola virusthis makes it a perfect test for the investigation of the ecology ofEbola and other related viruses. The other advantage of the ELISA isits ease to be performed thus making it a scalable test that can bedone widely. Another tool it provides to investigators is thecapability to detect a virus-specific antigen, nucleic acids, andpolymerase chain reactions. This would make it very easy and quick toidentify recent or acute cases of Ebola. It is therefore believedthat the specificity of the ELISA test would make it the bestalternative to the IFAT test for the Ebola Virus IgG antibodies (Ganet al., 2013).
References
Gan,S. & Patel, K. (2013). Enzyme Immunoassay and Enzyme-LinkedImmunosorbent Assay. JournalOf Investigative Dermatology,133(9),1-3. http://dx.doi.org/10.1038/jid.2013.287
Ksiazek,T., West, C., Rollin, P., Jahrling, P., & Peters, C. (1999).ELISA for the Detection of Antibodies to Ebola Viruses. TheJournal Of Infectious Diseases,179(s1),S192-S198. http://dx.doi.org/10.1086/514313