Detection is carried out by the addition of a substrate that can generate a color. The substrate for HRP is hydrogen peroxide and results in a blue color change. ALP measures the yellow color of nitrophenol after room temperature incubation periods of 15to 30 minutes and usually uses P-Nitrophenyl-phosphate pNPP as its substrate. The wells are washed two or more times during each wash step, depending on the specific protocol being followed.
In the ELISA protocol, usually, a serial dilution of concentrations is placed in the wells of the plate. After the results are measured, a standard curve from the serial dilutions data is plotted with a concentration on the x-axis using a log scale and absorbance on the y-axis using a linear scale.
The first binding step involves adding antigen to the plates, which is incubated for one hour at 37 degrees C or can be incubated at 4 degrees C overnight.
Once the incubation step is completed, the next step is to wash the plates of any potential unbound antibody and block any unbound sites on the ELISA plate using agents like BSA, ovalbumin, aprotinin, or other animal proteins. This second step is important because it prevents the binding of any non-specific antibodies to the plate and minimizes false-positive results.
After adding the buffer, the plate is rewashed, and a selected enzyme-conjugated primary detection antibody is added. The plate is further incubated for one hour. The color change of the sample occurs by either the hydrolysis of phosphate groups from the substrate by AP or by the oxidation of substrates by HRP.
Its disadvantages include its low sensitivity compared to the other types of ELISA and its high cost of reaction. Indirect ELISA requires two antibodies, a primary detection antibody that sticks to the protein of interest and a secondary enzyme-linked antibody complementary to the primary antibody. The primary antibody is added first, followed by a wash step, and then the enzyme-conjugated secondary antibody is added and incubated.
After this, the steps are the same as the direct ELISA, which includes a wash step, the addition of substrate, and detection of a color change. It is also less expensive and more flexible due to the many possible primary antibodies that can be used.
The only major disadvantage with this type of ELISA is the risk of cross-reactivity between the secondary detection antibodies. The buffer washes are carried out for at least hours at room temperature. Finally, the plate is washed with PBS once again before the addition of the antigen. The antigen of interest is then added to the plates to bind to the capture antibody and incubated for 90 min at 37 degrees C.
The plate is rewashed, and the primary detection antibody is then added to the plate and incubated for another 1 to 2 hours at room temperature, followed by a buffer wash. Then the secondary enzyme-conjugated antibody is added and incubated for another 1 to 2 hours. The plate is rewashed, and the substrate is added to produce a color change.
This type of ELISA utilizes two specific antibodies, an enzyme-conjugated antibody and another antibody present in the test serum if the serum is positive. Combining the two antibodies into the wells will allow for a competition for binding to antigen. The presence of a color change means that the test is negative because the enzyme-conjugated antibody bound the antigens not the antibodies of the test serum. The absence of color indicates a positive test and the presence of antibodies in the test serum.
However, the benefits are that there is less sample purification needed, it can measure a large range of antigens in a given sample, can be used for small antigens, and has low variability.
Enzyme-linked immunosorbent assays are applied in many diagnostic tests. Factors that can interfere with appropriate ELISA testing can occur at any phase of the testing process, beginning with specimen collection. The quality and integrity of the assay plate, coating buffer, capture antibody, blocking buffer, target antigen, detection antibody, enzyme conjugate, washes, substrate, signal detection can all interfere with proper ELISA testing.
The quantitative concentration results are plotted and compared to a standard curve. The semiquantitative results compare the intensity of the signals, which can compare relative antigen levels in a sample. Once color changes are measured from the assay, the results are graphed either on paper or software. Typically, the graph compares optical density to log concentration, which gives a sigmoidal curve.
Known concentrations give the graph's standard curve, and measurement of unknowns can then occur when sample values are compared to the linear portion of the graphed standard curve. ELISAs can be used in many settings, including rapid antibody screening tests for Human immunodeficiency virus HIV , detection of other viruses, bacteria, fungi, autoimmune diseases, food allergens, blood typing, presence of the pregnancy hormone hCG, laboratory and clinical research, forensic toxicology and many other diagnostic settings.
Diagnosis requires further testing by Western blot due to potential false positives. All sera yielded a negative result in the blocking ELISA with a blocking value much lower than the cutoff value.
Thus, non-specific positive swine sera were clearly discriminated from the ASFV-positive sera, suggesting that the established blocking ELISA has a satisfactory analytical specificity. Reproducibility determines whether an entire experiment or study can be reproduced.
In this study, an intra-assay CV ranging from 1. As shown in Figure 5 , the antibody response against p30 protein was detected seroconversion as early as 10 Dpi in two out of five pigs and the p30 response peaked around 20 Dpi.
Kinetics of antibody response in serum from ASFV-infected pigs. Serum samples were collected from six pigs infected by ASFV at 0, 5, 10, 15, and 20 days post inoculation.
Sensitivity assay. ASFV causes a serious deterioration and incalculable adverse economic impact around the world especially in China which has the largest pig industry 13 , 16 , Currently, there is no vaccine or other treatments available for ASFV. The principal strategy for control remains early detection, quarantine, and depopulation of affected herds.
Cost-effective detection strategies are needed for conducting high-throughput surveillance 2 , 7 , 24 — Although the seroconversion time was later than the virus genome-detected time, no significant symptoms were found in the variant strain-infected animals, and the infected pigs underwent intermittent detoxification In addition, in several areas Africa and Europe , many pigs or wild boar survived infection and presented no clinical signs of ASFV at the time of samplings, without the presence of ASFV attenuated variants 36 — 38 ; thus, we need to accurately detect the antibody of the animal to be tested to facilitate the determination of the infection of the pigs.
Therefore, a sensitive and reliable serological diagnostic assay is required, so as laboratories can effectively and quickly detect ASFV infection 18 , ELISA is considered a common tool to carry out serological surveillance. The other is blocking or competitive ELISA, where virus-specific antibodies in samples react with antigens to block or compete with the binding of a mAb to the antigens.
The specificity of iELISA is generally influenced by high background due to the non-specific reaction of serum antibodies to contaminant antigens in the tests The ELISA is a rapid, economical, and sensitive diagnostic method for screening large numbers of sera for antibodies.
Additionally, the specificity of this method is supposed to be even higher due to the usage of mAbs. Blocking ELISAs were widely used for a broad range of applications concerning serological diagnosis of various diseases in different animal species 40 , Based on the selected cutoff value of It has been reported that intrinsic and external factors, such as autoantibodies, sample quality, and sample storage conditions, can affect the serologic testing Physical and chemical parameters can also affect the test results in the laboratory, such as hemolysis and lipemia In our study, of the two false-negative serum samples and the one false-positive sample, these three serum samples were confirmed as negative by IFA.
It detected an increasing trend of antibody response against p30 protein through the time course of the study 0—20 dpi. The detection of seroconversion at 10 dpi was consistent with the findings in previous studies 23 , 43 — 45 , so this detection method can be used as an early detection kit.
The maximum dilution of the ASFV-positive standard serum and the ASFV-positive standard serum against the CD2v-negative one at different dilutions were and , respectively, indicating that the pbased blocking ELISA was highly sensitivity and it can detection for variant strains. Furthermore, with the emergence of domestic attenuated strains and atypical clinical symptoms, antibody detection methods can be used as an effective means to detect infections.
The antibody detection methods can be used to screen ASF antigen—antibody double-negative pigs upon introducing pigs into farm. Due to its simplicity concerning the coating antigen production, easiness to perform, and low cost, the test will be a useful tool for field surveillance and epidemiological studies in swine herd.
The non-invasive test for a complete epidemiological investigation in the field is very important, especially ASF. In subsequent studies, an attempt should be made to establish an antibody detection method for oral fluid. This study prepared three monoclonal antibodies against the structural p30 protein of ASFV, and their diagnostic application was investigated. The p30 mAb-based blocking ELISA developed in this study demonstrated a high repeatability with maximized diagnostic sensitivity and specificity in laboratory settings.
All authors have critically read and edited the manuscript. This project was funded by the Epidemiological characteristics and evolution of African swine fever virus and early detection technology and the research and application of key technology against African Swine Fever ABA The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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This article was submitted to Veterinary Infectious Diseases, a section of the journal Frontiers in Veterinary Science. Received Sep 22; Accepted Nov 5. The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Introduction African swine fever ASF , caused by African swine fever virus ASFV , is a highly contagious hemorrhage lethal disease of domestic and wild pigs and is responsible for serious economical losses, international trading, and adverse sociophysical impacts 1 — 6.
Materials and Methods Production of Recombinant p30 in Escherichia coli The ASFV CPL bp gene sequence from positive samples during the surveillance was used for the preparation of p30 recombinant protein fragments. Open in a separate window. Figure 1. Figure 2. Table 1 Identification of subclasses of p30 monoclonal antibodies.
Figure 3. Figure 4. Figure 5. Figure 6. Discussion ASFV causes a serious deterioration and incalculable adverse economic impact around the world especially in China which has the largest pig industry 13 , 16 , Conclusion This study prepared three monoclonal antibodies against the structural p30 protein of ASFV, and their diagnostic application was investigated.
Funding This project was funded by the Epidemiological characteristics and evolution of African swine fever virus and early detection technology and the research and application of key technology against African Swine Fever ABA Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher's Note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.
References 1. Transb Emerg Dis. Zakaryan H, Revilla Y. African swine fever virus: current state and future perspectives in vaccine and antiviral research. Vet Microbiol. African swine fever virus: an emerging DNA arbovirus. Publication types Research Support, Non-U. Gov't Research Support, U. Gov't, Non-P.
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