Given the continued and alarming increase in both cases and deaths due COVID-19 around the world, scientists are still in search of effective tools for diagnosis of infection. One important way of testing for severe acute respiratory syndrome coronavirus 2 (SARS-cov-2) – serology, but this requires a supply of high-quality recombinant viral antigens.
A recent study published in the preprints server medRxiv* in July 2020 shows that it is indeed possible. The development of this inexpensive, but reliable and sensitive test could change the face of preventive measures for COVID-19 limited resources.
SARS-coronavirus-2 spike antigen is one of the most important viral antigens, with researchers focusing on it for the production of vaccines, antivirals and serological tests. Serology is important to track cases, contacts to display, epidemiological surveillance and identification of asymptomatic patients, and identify the mechanism of antiviral immunity.
In Spike protein consists of two subunits, S1 and S2, which mediate receptor binding and membrane fusion, respectively. This allows the virus to penetrate the cell. This protein is a target for neutralizing antibodies, and the most common serological tests for SARS-cov-2 is an enzyme immunosorbent analysis (ELISA) – based protein.
This test is popular for its simplicity and cross-reactivity against the spike proteins of other coronaviruses in the current edition. Moreover, its intensity corresponds to the level of neutralizing antibodies.
Study: characterizing the low-cost IFA
This study was based on the identification of the key features that will allow you to develop an inexpensive test enzyme immunoassay, based on the immunological reaction of antibodies with protein. The goal was to produce a test that will allow large-scale screening for infection in low-income.
Researchers have focused mainly on reducing the cost of manufacturing test by developing optimum means of production of the antigen and making the necessary changes in the method of collecting antigen and sample processing. As a result, they were able to develop a test that will cost less than 50 cents per sample.
To Produce Recombinant Antigen
The researchers cultured SARS-coronavirus-2 protein is in a stable condition prior to the merge in cell culture using a method that allows protein to be expressed stably and as a constitutive feature. It was through the integration of the transgene into the genome of cells in culture.
This adaptation leads to increased scalability, and low cost recombinant proteins. They also used methods of cotransfection gene’s, as well as open source plasmid, which has a selection handle to avoid having to wait for a synthetic gene that needs to be built and sent through the pandemic-disrupted supply chain. Thus, the result is a recombinant cell line expressing high levels of protein were produced within 24 days of transfection and still shows steady expression up to 100 days.
This achievement gives the opportunity to “develop a less expensive, long lasting batch refeed or perfusion technology for cell culture,” according to researchers. They also succeeded in finding an inexpensive nutrient media to support cell growth and high level production of protein. Thus, they have developed a workflow using low-cost methods to achieve the goal of increasing the density of cells in culture with high s secretion of the protein.
Affinity purification chromatography (AC) used resin was more expensive than originally planned ultrafiltration/diafiltration filter (UF/DF), but has become necessary because of failure of the latter to remove smaller contaminants protein. However, they found that it can be used for more than 30 cycles, which reduces the cost of its use.
Then they created an ELISA to detect anti-s antibodies in human serum, plasma, and eluted whole blood samples, called s-ufrj website IFA. They set the amount of highly purified protein (from AC) needed to ensure legible results between negative and positive samples, 150 NG.
Evaluation of sensitivity and specificity
Then the website ufrj Elisa was used to test 210 is negative and positive serum samples, 66 samples from 38 symptomatic COVID-19 patients, 124 samples from the pandemic, and 20 of COVID-19 negative people. They received 122 out of 124 negative samples, the specificity 98%. In addition, the samples 53/66 were positive for IgG, to reduce the sensitivity to 80%.
For comparison, obtained from a commercial IgG rapid diagnostic tests (RDTs), as approved by the Brazilian regulatory health organization, conducted by the Agency only 46% sensitivity.
Then they re-IgG-negative samples from S website ufrj ELISA IgM rapid test. They found that most samples that were negative for IgG and negative for IgM and IgG positive in the first test was IgM positive result of the proximate analysis as well. They, therefore, came to the conclusion that the two false-negative results from symptomatic patients, might have been samples collected early in the disease.
The increase in false positives with increasing duration of symptoms
When the website ufrj ELISA sample results were compared against the duration since symptom onset, they were more likely to be positive, as the duration increased, as a result of seroconversion some individuals who were PCR-positive, negative, scored on IFA for the first time, but positive for anti-s IgG antibodies for the second time. The level of seroconversion to Anti-s IgG antibodies with this test increased from 42% to 100% in direct proportion to the time of onset of symptoms, and from the tenth day, he was consistently above 90%.
An important finding was that the real test detects seroconversion earlier than the rapid test, which had a peak detection rate of 71%, even 20 days from the onset of symptoms.
They also tested the neutralizing ability COVID-19 patients with neutralization of plaque (MFN). Samples with high anti-s IgG antibodies titer was the highest titer of neutralization.
Simplify collection and storage of blood
The researchers also sought to overcome the traditional bottleneck of sample collection and processing in the clinical laboratory under refrigerated storage. They created a simple system for the collection of fingerprick blood in filter paper strips. The use of dried blood spot on filter paper showed comparable results of testing serum.
Dried blood samples (DBS) obtained by a prick of the finger with commercially available devices for piercing. 2.5 cm (W) x 7.5 cm (l) paper filter with three spots of blood from the same volunteers and a commercially available paper hole punching devices have been used DB (arrow), which eluted in the blood for ELISA testing.
Thus, low cost of consumables, along with labor, transportation, and equipment costs, all must fit in a half dollar per test, which is approximately 200 times less than the tests currently used in the United States. Another advantage is that spots of blood in sealed plastic bags can be stored for 2 months at least, but still return accurate results serological.
Thus, this study concludes: “on the S website ufrj enzyme immunoassay, including the use of eluates from whole blood pricking her finger as samples, allows a wide serological survey of the population, irrespective of their geographical and socio-economic aspects”. It will be invaluable for the formation of public health strategies to prevent further waves of the pandemic.
medRxiv publishes preliminary research reports that are not reviewed and therefore should not be considered as a convincing guide to clinical practice/behavior, health-related, or be considered as reliable information.