GR1010129B - Diagnostic and therapeutic for the identification and treatment of sars-cov-2 - Google Patents

Abstract

The present invention provides methods and diagnostic kits for the detection or prediction of SARS-CoV-2. More particularly, the present invention provides for the diagnosis of SARS-CoV-2 by detecting in a human biological sample inhibition of protein translation by the phosphorylation of the eukaryotic initiation factor-2a (eIF-2a), and the formation of stress granules (SGs) known to be affected by Sars-CoV-2.

Description

DIAGNOSTIC FOR THE DETECTION OF SARS-COV-2

FIELD OF THE INVENTION

The present invention provides methods and diagnostic kits for the detection or prediction of SARS-CoV-2. In particular, the present invention provides for the diagnosis of SARS-CoV-2 by detecting in an isolated biological sample inhibition of protein translation by phosphorylation of eukaryotic protein synthesis initiation factor 2a (eIF-2a), and the formation of stress granules (SGs) which are known to be affected by sars-CoV-2.

HISTORY OF THE INVENTION

The recent outbreak of SARS-CoV-2, an infectious novel coronavirus that causes the disease COVTD-19, is spreading worldwide with devastating effects on the human population. This disease follows the emergence of SARS-CoV and MERS-CoV in 2002 and 2012, respectively.

The biology of coronaviruses has been explored over the past forty years leading to our current knowledge of viral entry pathways, viral receptors, cellular and organotropic (Rev Weiss and Leibowitz 2011, Weiss 2020). It has recently been shown that the current novel coronavirus SARS-CoV-2 uses ACE2 receptors to invade host cells just like SARS-CoV (Zhou et al, 2020).

Clinical and epidemiological data from the Chinese CDC on 72,314 case records (confirmed, suspected, diagnosed, and asymptomatic cases) shared in the Journal of the American Medical Association (JAMA) (February 24, 2020) indicate a three-level disease severity: Mild disease : non-pneumonia and mild pneumonia. This happened 81% of the time. Severe illness: dyspnea, respiratory rate > 30/min, blood oxygen saturation (SpO2) < 93%, PaO2/FiO2 or P/F ratio [the ratio between the arterial pressure of oxygen (partial pressure of oxygen, Pa02) and the percentage of of oxygen supplied (fraction of inspired oxygen, FiO2)] < 300 and/or lung filtrations > 50% within 24 to 48 hours? this occurred in 14% of cases.

In addition, it has recently been published that high mortality in patients with COVID-19 may be the result of virally driven hyperinflammation. Fatal coviD-19 cases showed pathological patterns of secondary hemophagocytic lymphohistiocytosis (sHLH), which is an underrecognized, hyperinflammatory syndrome characterized by a fatal hypercytokinemia with multiorgan failure. In adults, sHLH is most often caused by viral infections and occurs in 3-7-4-3% of sepsis cases. Features of sHLH include persistent fever, cytopenia, and hyperferritinemia; pulmonary involvement (including ARDS) occurs in approximately 50% of patients.

Currently, there are unrecognized disease patterns such as lymphopenia, hyperferritinemia possibly leading to coagulation abnormalities, and early hypoxemia with initially preserved lung function. In any case, the patterns of disease manifestation are variable and under investigation.

An "influenza virus-cytokine-trypsin" cycle has been proposed as one of the main underlying mechanisms of vascular hyperpermeability and multiorgan failure (MOF) in severe influenza. Severe influenza causes a significant increase in the levels of proinflammatory cytokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-6, and EL-1β. This potentially lethal immune response has been called a cytokine storm. Such hypercytokinemia alters the cellular redox state through different cytokine receptors and reduces the expression of four complex I subunits, oxygen consumption and ATP synthesis in mitochondria, as well as increasing mitochondrial O<2> production and intracellular calcium concentration [Ca <2+>]

Importantly, ATP depletion causes dissociation of zonula occludens-1, an intracellular tight junction component, from the actin cytoskeleton and thus increases septal permeability. These cytokines also negate trypsin through the activation of nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1), which mediates the post-translational proteolytic cleavage of the viral envelope hemagglutinin (HA) and is vital for viral entry and replication in various organs and endothelial cells. Trypsin also increases [Ca<2+>] and C1<->and K<+>secretion via protease-activated receptor (PAR)-2, resulting in loss of zonula occludens-1 in endothelial cells and severe swelling in the airways and large intestine. In addition, it has been reported that IAV infection induced glycogen synthase kinase 3β-mediated β-catenin degradation in vascular endothelial cell adherens junctions, which resulted in increased membrane permeability.

These observations led to the conclusion that serious viral infections, e.g. caused by influenza A are associated with an energy metabolic disorder.

Two sensitive mitochondrial enzymes, pyruvate dehydrogenase (PDH) in glucose oxidation and carnitine palmitoyltransferase (CPT) in long-chain fatty acid oxidation, have been reported to play key pathogenic roles in mitochondrial ATP crisis and MOF during of severe IAV infection. Severe IAV infection has been reported to be associated with a marked and specific up-regulation of pyruvate dehydrogenase kinase (PDK), but not the related PDKsl-3 kinases, in skeletal muscle, liver, lung and heart, but not in the brain .

PDK4 phosphorylates PDH, a mitochondrial gatekeeper enzyme involved in glucose oxidation, and suppresses its activity, resulting in a significant decrease in glucose regulation mediated by energy homeostasis, culminating in an ATP crisis. PDK4 expression is transcriptionally regulated by several nuclear receptor hormones that are specifically activated by various metabolites and hormones, such as peroxisome proliferator-activated receptors (PPARs: PPARa, PPARβ/δ and PPARγ), glucocorticoid receptors, receptors associated with estrogen and thyroid receptors.

These findings, combined with the growing evidence for the link between cytokines and metabolic disorders, between cytokines and PPARs, and between PPARs and metabolic disorders via PDK4, suggest a novel cellular host mechanism for MOF in severe influenza. This concept includes the host-metabolic-cytokine cycle, which links PPARs and PDK4, and is in turn closely related to the influenza virus-cytokine-trypsin cycle (H. Kido et al, 2016 ).

It has recently been reported that approximately 30 SARS-COV-2 proteins interact with proteins of human mitochondrial metabolism and over 25 with components of the electron transport chain (Gordon DE et al., 2020). Components of mitochondrial metabolism (eg, pyruvate dehydrogenase) and/or electron transport chain activity (ie, complex I, Complex IV) can be used as biomarkers to predict the severity and prognosis of Covidl9. Comorbidities associated with reduced mitochondrial function and loss of ΔΨm (eg, diabetes) render innate immunity against SARS-COV-2 insufficient for an effective antiviral response, thus making patients highly susceptible to severe forms of the disease.

Working with new models of neuroinflammation (NIE) we know that hypoxia is a key event in neuroinflammation as oxygen delivery can reverse the neurological deficit. In this model of neuroinflammation (NIE) the cascade of inflammatory events leads to the expression of HIF-la as expected, but also to the inhibition of protein translation that counteracts the possible consequences of energy deficiency resulting from hypoxia and inflammation.

(Unpublished data M. Kasti, Experimental Neuroinflammation, A study of hypoxia and protein translation - UCL Dissertation 2013).

It is known that one of the most important protective mechanisms to counteract cellular stress, including viral-induced stress, is the phosphorylation of eukaryotic protein synthesis initiation factor 2α (eIF-2α), by the endoplasmic reticulum kinase PERK, which is part of of a process known as the unfolded protein response (UPR).

SUMMARY OF THE INVENTION

The invention includes a diagnostic immunoassay for the detection and treatment of SARS-CoV-2 in a patient, and includes the steps of:

a) the in vitro examination of an already collected and isolated biological sample from a subject suspected of being infected with SARS-CoV-2 - and

b) detecting the concentration of eukaryotic protein synthesis initiation factor 2α (eIF-2α) in said sample by immunological means-

wherein the reduced concentration of eIF-2α in said sample, compared to the concentration of eIF-2α in a plasma sample from a healthy mammal is predictive of SARS-CoV-2.

In some embodiments, the subject is a mammal, preferably a human.

In some embodiments, the detection comprises binding of eIF-2α by an antibody that specifically binds eIF-2α.

In some embodiments, the immunological agent uses a monoclonal antibody.

In some embodiments, the antibody is labeled and such detections include detecting the amount of label associated with said eIF-2α/antibody complex.

In some embodiments, the label is selected from the group consisting of a radioactive label, an enzymatic label, a colorimetric label, and a fluorescent label.

In some embodiments, the detection includes:

contact of the antibody with a labeled second antibody that binds said antibody? and

detecting the amount of labeled second antibody bound to said antibody immobilized on a substrate.

In some embodiments, the detection comprises a competitive assay, comprising the steps of:

a) providing a first antibody that specifically binds eIF-2α;

b) contacting said eIF-2α labeled first antibody and said sample-

c) detection of the amount of labeled eIF-2α that forms an eIF-2α/antibody complex.

In some embodiments, the first antibody is immobilized on a solid support.

In some embodiments, the detection comprises detecting the labeled eIF-2α bound to said first antibody.

In some embodiments, the detection comprises locating the labeled eIF- 2<α>that remains unbound in solution.

In some embodiments, the detection comprises Western Blotting using an antibody that specifically binds eIF-2α.

In some embodiments, phosphorylation of eIF-2α causes immediate, but reversible inhibition of protein synthesis.

Without being bound by theory, it is thought to be part of an eIF-1α-independent response to hypoxia.

In some embodiments, the adaptive mechanisms to counteract hypoxia and energy deficiency are a drastic reduction in ATP consumption, leading to a severe downregulation of the energy cycle by turning off certain parts of cell metabolism, and also using the available ATP more efficiently than during normoxic conditions. In some embodiments, a similar mechanism is present in severe Sars-CoV-2 infection.

In some embodiments, the proposed model of an energy metabolic disorder mentioned above reinforces the conclusions that severe SARS-CoV-2 infection can lead to a previously unrecognized metabolic disorder sooner or later in the disease process.

Another enzyme that is phosphorylated and inactivated during torpor is pyruvate dehydrogenase (PDH). This enzyme complex converts pyruvate to acetyl CoA, thereby controlling aerobic carbohydrate oxidation in the mitochondrial tricarboxylic acid cycle. PDH activity is controlled by various allosteric factors and by covalently bound kinases and phosphatases that, respectively, phosphorylate (inactivation) and dephosphorylate (reactivate) the PDH complex. Dramatic reductions in aerobic carbohydrate oxidation during the torpedo have been explained by PDH kinase-mediated phosphorylation of the PDH complex.

In some representations, similar observations of PDH upregulation are suggested in influenza A metabolic disorder, which makes us suspect a common underlying mechanism of energy conservation.

In some embodiments, the results show that the current lethal viral disease causes for various reasons a low oxygen concentration that could compromise energy homeostasis because it will not only reduce mitochondrial electron transport and ATP synthesis, but also activate AMP- activated fragment protein kinases (AMPKs). This activation down-regulates oxygen-consuming processes, including Na+/K+-ATPase and global protein synthesis allowing a metabolic adaptation to reduced oxygen levels.

In some embodiments, the downstream effect of severe hypoxia can lead to a metabolic disorder that has not been recognized so far and causes a number of symptoms that have been noticed for the first time around the world.

Metabolic adaptation designed to limit energy expenditure includes inhibition of protein translation by phosphorylation of eukaryotic initiation factor 2α (eIF-2α) and the formation of stress granules (SGs) already known to be affected by Sars -CoV-2.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts an exemplary SDS gel (left) and western blot (right, anti-His-tag antibody, penta-His conjugated) for the HCV helicase protein.

Figure 2 depicts a graphical representation of the data listed in Table 2.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

"Synthetic oligonucleotide" refers to polymeric nucleic acid molecules of 2 or more nucleotide bases that are not derived directly from genomic DNA or living organisms. The term synthetic oligonucleotide is intended to include DNA, RNA, and DNA/RNA hybrids that have been chemically constructed, or enzymatically synthesized in vitro.

An "oligonucleotide" is a numerical polymer with two or more nucleotide subunits covalently linked together. Oligonucleotides are generally about 10 to 100 nucleotides. The sugar groups of the nucleotide subunits can be ribose, deoxyribose, or modified derivatives thereof, such as OMe. Nucleotide subunits can be joined by bonds such as phosphodiester bonds, modified bonds, or by non-nucleotides that do not prevent hybridization of the oligonucleotide to the complementary nucleotide sequence. Modified bonds include those in which the phosphodiester bonds are replaced with a different bond, such as a phosphorosulfate or methylphosphonate bond, or a neutral peptide bond. Nitrogenous base analogs can also be components of oligonucleotides according to the invention.

A "target nucleic acid" is a nucleic acid comprising a target nucleic acid sequence. A "target nucleic acid sequence", "target nucleotide sequence" or "target sequence" is a specific deoxyribonucleotide or ribonucleotide sequence that can hybridize to a complementary oligonucleotide.

A "probe oligonucleotide" is an oligonucleotide that has a nucleotide sequence sufficiently complementary to its target nucleic acid sequence to be able to form a hybrid probe:target bidirectional under high stringency hybridization conditions. An oligonucleotide probe is an isolated chemical species and may include additional nucleotides outside the target region as long as said nucleotides do not inhibit hybridization under high stringency hybridization conditions. Non-complementary sequences, such as promoter sequences, restriction endonuclease recognition sites, or sequences that impart desired secondary or tertiary structure, such as a catalytically active site, can be used to facilitate detection using the probes found. A probe oligonucleotide can optionally be labeled with a detectable submolecule such as a radioisotope, a fluorescent submolecule, a chemiluminescent, a nanoparticle, an enzyme, or a linker, which can be used to detect or confirm probe hybridization to its target sequence. Probe oligonucleotides are preferably in the size range of about 10 to about 100 nucleotides in length, although it is possible for probes to be as much as over about 500 nucleotides in length, or under 10 nucleotides in length.

A "hybrid" or "duplex" is a complex formed between two single stranded nucleic acid sequences by Watson-Crick base pairs or non-canonical base pairings between complementary bases. "Hybridization" is the process by which two complementary strands of nucleic acid combine to form a double-stranded ("hybrid" or "duplex") structure. A "fungus" or "yeast" means any organism of the kingdom Fungi, and preferably, is directed to any organism of the phylum

Ascomycota.

"Complementarity" is a property conferred by the base sequence of a single-stranded DNA or RNA that can form a hybrid or double-stranded DNA:DNA, RNA:RNA or DNA:RNA through hydrogen bonding between Watson-Crick base pairs in the respective branches. Adenine (A) usually complements thymine (T) or uracil (U), while guanine (G) usually complements cytosine (C).

The term "stringency" is used to describe the temperature, ionic strength, and composition of the solvent present during the hybridization and subsequent processing steps. Those skilled in the art will recognize that "stringency" conditions can be modified by varying these parameters either individually or together. Under conditions of high stringency, only highly complementary nucleic acid hybrids will form, hybrids without a sufficient degree of complementarity. Accordingly, the stringency of the assay conditions determines the amount of complementarity required between two nucleic acid strands that constitute a hybrid. Stringency conditions are chosen to maximize the difference in stability between the hybrid formed with the target and the non-target nucleic acid.

Under "high stringency" conditions, nucleic acid base pairing will only occur between nucleic acid fragments that have a high frequency of complementary base sequences (for example, hybridization under "high stringency" conditions can occur between homologues with about 85-100 % identity, preferably about 70-100% identity). Under medium stringency conditions, nucleic acid base pairing will occur between nucleic acids with an intermediate frequency of complementary base sequences (for example, hybridization under "medium stringency" conditions may occur between homologues with approximately 50-70% identity). Thus, conditions of "weak" or "low" stringency are often required with nucleic acids derived from organisms that are genetically diverse, since the frequency of complementary sequences is usually lower.

One skilled in the art will understand that the substantially corresponding probes of the invention can vary from the reported sequence and still hybridize to the same target nucleic acid sequence. This deviation from the nucleic acid can be expressed in terms of a percentage of identical bases within the sequence or the percentage of completely complementary bases between the probe and its target sequence. The probes of the present invention substantially match a nucleic acid sequence if these percentages are from about 100% to about 80% or from 0 base mismatches in about 10 nucleotide target sequences to about 2 base mismatches in a target sequence of about 10 sequences target nucleotides. In preferred embodiments, the percentage is from about 100% to about 85%. In more preferred embodiments, this percentage is from about 90% to about 100%. In other preferred embodiments, this percentage is from about 95% to about 100%

By "sufficiently complementary" or "substantially complementary" is meant nucleic acids having a sufficient amount of contiguous complementary nucleotides to form, under high stringency hybridization conditions, a hybrid that is stable for detection. "Substantially complementary" may also refer to sequences with at least 90% identity, e.g., 95, 96, 97, 98, 99, or 100% identity to, a given reference sequence.

The terms "identical" or percent "identity", in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or annotations that are the same or have a certain percentage of amino acid or nucleotide residues that are the same (ie, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity in a specified region when compared and aligned for maximum match in a comparison window or specified region), as measured using a BLAST or BLAST 2.0 sequence comparison with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI website at ncbi.nlm.nih.gov/BLAST/ or similar). Such sequences are then said to be "substantially identical." The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can cover gaps as well as substitutions. Preferably, the identity is present in a region that is at least about 25 amino acids or nucleotides in length, or preferably over a region that is 50-100 amino acids or nucleotides in length.

For sequence comparison, usually one sequence acts as a reference sequence, against which the test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, sub-auditory coordinates are defined if necessary, and sequence algorithm program parameters are defined. Preferably, default program parameters can be used or alternative parameters can be defined. The sequence comparison algorithm then calculates percent sequence identities for the test sequences relative to the reference sequence, based on program parameters.

A "comparison window," as used herein, includes reference to a portion of any number of contiguous positions selected from the group consisting of 20 to 600, typically about 50 to about 200, and more specifically about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of aligning sequences for comparison are known. Optimal alignment of sequences can be performed for comparison, e.g., by the local homology algorithm of Smith & Waterman, Adv. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.

48:443 (1970), by the search similarity method of Pearson & Lipman, Proc. Acad. Sci. USA 85:2444 (1988), by the computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computing Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1987-2005, Wiley Interscience)).

"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and their polymers, single- or double-stranded, and their complements. The term includes nucleic acids containing known nucleotide analogs or modified residues or linkages, which are synthetic, natural and non-natural, which have similar binding properties to the reference nucleic acid and have metabolism similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorosulfates, phosphotides, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNA).

By "hybrid nucleic acid" or "target duplex" is meant a structure that is double-stranded, hydrogen-bonded, about 10 to 100 nucleotides in length, preferably 14 to 50 nucleotides in length, although this will depend on of the total length of the oligonucleotide probe. The structure is stable enough to be detected by means such as chemiluminescent or fluorescent light detection, autoradiography, electrochemical analysis, or gel electrophoresis. Such hybrids include RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

"RNA and DNA equivalents" refer to RNA and DNA molecules that have the same complementary “base-pair” hybridization properties. RNA and DNA equivalents have different sugar groups (ie, ribose vs. deoxyribose) and may differ by the presence of uracil in RNA and thymine in DNA. The difference between RNA and DNA equivalents does not contribute to differences in substantially corresponding nucleic acid sequences because the equivalents have the same degree of complementarity to a particular sequence.

By "preferentially hybridizes" is meant that under high stringency hybridization conditions the probe oligonucleotides can hybridize to their target nucleic acids to form stable probes: target hybrids (thereby indicating the presence of the target nucleic acids) without forming stable probes: non-target hybrids (which will demonstrate the presence of non-target nucleic acids from other organisms). Thus, the probe hybridizes to target nucleic acid to a sufficiently greater extent than to non-target nucleic acid to allow one skilled in the art to accurately detect the presence (for example Candida) and distinguish these species from other organizations. Preferential hybridization can be measured using techniques known and described herein.

Examples

For the selection of complex datasets and high-throughput virtual viewing, a number of integrated complex databases were used. These databases are based on both computational and experimental criteria.

The experimental techniques that have been used revolve around analog assays in a previously specially designed enzyme assay for the rapid screening of antiviral helicase modulators. The techniques and methodologies used are described below:

Prepare all solutions using ultrapure water, prepared by deionized water purification, and store all reagents at room temperature unless otherwise indicated.

1.1 Preparation of Recombinant HCV Helicase

1. Escherichia coli induction: Prepare 4 conical flasks with 750 mL LB and add 34 µg/mL chloramphenicol and 25 pg/mL kanamycin to each.

2. Cell buffer: 20 mM sodium phosphate pH 7.5, 300 mM NaCl

3. Buffer S: 20 mM sodium phosphate pH 7.4, 500 mM NaCl

4. Exchange buffer: 25 mM Tris-HCl pH 7.5, 0.05% CHAPS (3-[(3-cholamidopropyl)-dimethylammonium]-1-propane sulfate), 20% glycerol, 5 mM DT (dithiothreitol)

1.2 HCV Helicase Chemical Test Procedure

1. Oligonucleotide Mix:

Oligonucleotides (5-biotinGCTGACCCTGCTCCCAA TCTAA TCTA TAGTGTCACCTA-3 5'-DIG

CGA TTGGGAGCAGGGTCAGC-3) 1 : 1 molar, HEPES 2mM, NaCl 0.05M, EDTA 0.1mM, SDS 0.01% w/v.

2. Neutravidin stock solution: add Neutravidin to a final concentration of 1 mg/ml in phosphate buffered saline (1 M PBS - pH 7.0).

3. BSA solution: 0.1% w/v BSA.

4 Substrate solution: mix 2.5 ng of partially annealed DNA duplex and 75 µL of 1 M PBS containing 1 M NaCl, for each well.

5. Substrate wash solution: 50 mM Tris HCl pH 7.5, 50 mM NaCl.

6. Helicase reaction mixture: 11 nM purified full-length HCV NS3 protein, 25 mM 4-morpholine-propanesulfonic acid (MOPS) pH 7.0, 2 mM DTT, 3 mM MnCl2, 100 µg/ml BSA and 5 mM ATP. The reaction mixture for the negative control lacks ATP.

7. Reaction wash solution: 150 mM NaCl

8. Detection wash buffer: 0.1 M maleic acid, 0.15 M NaCl, 0.3%, Tween 20, pH 7.5

9. Blocking solution: 10% BSA w/v, 0.1 M maleic acid, 0.15 M NaCl, pH 7.5

10. Antibody solution: 1:10,000 solution of anti-Dig antibody (75 mU/mL) in blocking solution

11. Detection buffer: 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5

12. Chemiluminescence substrate solution: CSPD - 0.25 mm

All procedures are performed at room temperature unless otherwise specified.

2.1 Preparation of HCV NS3 Recombinant Helicase

1. Insert the full-length HCV helicase coding region into a pET28a vector, with an N-terminal 6xHis-Tag region.

2. Verify that the gene is intact before inducing the production of the protein.

3. Transform Escherichia coli cells (strain C41, DE3) with the helicase plasmid and inoculate the prepared LB flasks with them.

4. Induce production of recombinant helicase by adding 0.5 mM IPTG to each flask, then allow the culture to grow for 3 h at 18 °C.

5. Resuspend the cell pellet from the 4 cultures in 30 mL lysis buffer and then add 100 µg/mL lysozyme and 0.1% Triton X-100.

6. Incubate on ice for 30 min and then sonicate four times for 20 sec at 15 sec intervals.

7. Centrifuge the suspension at 15,000 x g for 20 minutes.

8. Adjust the clarified homogenates to 10 mM imidazole and filter through a 0.45 pm membrane.

9. Place the homogenates twice on nickel affinity (Ni-NTA) columns.

10. Wash each column with five times the column volume of buffer S, containing 10 mM imidazole.

1 1. Elution of NS3 helicase with buffer S containing 300 mM imidazole.

12. Immediately after elution, exchange the buffer in the helicase-containing fractions with the Exchange buffer, by dilution. This step is critical in order to avoid sedimentation.

13. Assess protein concentration using Bradford assay with BSA as standard.

14. Make aliquots of NS3 helicase and store at -80°C.

This recombinant protein preparation was estimated to be more than 85% pure by SDS gel electrophoresis and Coomasie blue staining, yielding nearly 1.6 mg of HCV NS3 per 3 L of E. coli cultures (Figure 1).

Chemiluminesence measurements were taken using all the different control combinations (presence/absence of helicase, DNA substrate and ATP) of the experiment in order to ensure the reliability of the measurements (table 1).

Table 1. Each enzyme activity assay was performed in triplicate and the results were averaged. All reactions were allowed to proceed for 60 min (concentrations as described in the methods section).

We demonstrated that NS3-mediated unwinding is proportional to the amount of DNA substrate in the well, but also to the HCV helicase concentration in the reaction. The reactions were ATP dependent (Table 2 and Figure 2).

Table 2. Different concentrations of DNA substrate (60 min-run).

2.2 Annealing reaction

1. Prepare for annealing by heating the oligonucleotide mixture d; ioy at 100°C for 5 minutes.

2. Incubate at 65°C for 30 min and then at 22°C for 4 h to allow stepwise annealing.

3. Store the annealed NS3 helicase substrate at -20°C (see Note 1).

2.3 Neutravidin coating of 96-well plates

1. Plate 96-wells overnight at 4°C with 100 µl/well of 5 µg/ml Neutravidin solution in 0.5 M sodium carbonate buffer pH 9.3.

2. Wash the plates three times with 100 µl/well PBS and air dry at room temperature.

2.4 Exclusion by BSA

1. Add 100 µL of the 0.1% w/v BSA solution

2. Incubate at 22°C for 2 hours.

3. Wash the plate three times with PBS, 200 µl/well and air dry at room temperature.

4. Store the plate at 4°C with desiccant (see note 2))

2.5 Substrate application to the 96-well plate

1. Prewarm all solutions to 37°C (see Note 3J)

2. Mix 75 µl of the substrate solution with 2.5 ng of the partially annealed DNA duplex for each well.

2. Incubate at 22°C for 4 hours.

3. Wash each well twice with 200 µl PBS per well and once with substrate wash solution.

2.6 Helicase reaction

1. Add 90 µL of the reaction mixture per well.

2. Incubate the reactions at 37°C for 1 hour.

3. Wash the plate twice with 200 µL of reaction wash buffer per well and allow to dry for 15 min at room temperature.

2.7 Activity determination - chemiluminescence preparation

1. Wash all wells for 5 min with detection wash buffer.

2. Then fill each well with blocking solution for 30 min and then incubate for 30 min in 20 µl antibody solution.

3. Wash the plate twice with 100 µl detection buffer.

4. Apply 20 µL detection buffer for equilibration and 1 µL chemiluminescence substrate working solution per well and incubate for 5 min at 17 °C.

5. Drain the wells and incubate the plate at 37°C for 30 min to evaporate any remaining solution.

6. The brightness has a constant intensity for about 24 hours and continues for about 48 hours. The remaining DIG in each well is counted for 10 min against controls (one lacking protein and one lacking ATP) in a luminescence plate reader (see Note 4).

Note 7: The oligonucleotides used in this protocol are those described by Hicham Alaoui-Ismaili et al. (Hicham et al. , 2000), modified by DIG labeling of the release arm. Other sequences could work, but a single 3' latent region in the substrate is necessary to drive strand displacement (Tai et al., 1996).

Note 2: Regarding the blocking procedure, it is important to ensure that all potential binding sites are occupied to avoid direct binding of the detection antibody to the well at a later stage. Wells should be filled with blocking solution, completely coating the plate.

Note 3: Preheating solutions allows reactions to proceed at their optimum temperatures and avoids rate changes due to temperature equilibration.

Note 4: All reaction wells should be filled with blocking solution to ensure that the entire well is blocked, preventing non-specific binding of any detection system components.

3. GENERAL MOLECULAR TECHNIQUES ARE USED

BIOLOGY

3.1 BACTERIAL TRANSFORMATION:

1. 1 µL of plasmid DNA is added to the cells

2. Leave on ice for 45 minutes

3. Heat in a 42 °C water bath for 2 min

Add 0.5 µL of LB medium without antibiotic

Leave on ice for 1 h at 37 °C

Microcentrifuge at 8000 rpm for 1 minute

Discard 0.5 mL of supernatant

Gentle suspension of residual volume

Plate on LB agar AMP Petri dishes

3.2 ECOLI SURGERY

Picking a healthy (round, consistent) colony with a loop

Inoculate a universal tube with 10 mL of LB AMP

Incubate in gradient centrifugation at 37 °C for 13 to 14 h

3.3 PREPARATION for 3.4

1.Add 1.5 mL of the overnight culture of E.Coli to an eppendorf and spin at 13000 for 1 minute

2. Repeat 3 times for each tube → a total of 4.5 mL of cell suspension is passed through each tube

Each time discard the overhead

3.4 MINI - PREPARATION

using a microcentrifuge

This protocol is designed to purify up to 20 µg of high-copy plasmid DNA from 1-5 mL overnight cultures of E. coli in LB medium (Luria-Bertani).

Note: All protocol steps must be performed at room temperature.

Procedure

1. Suspend bacterial cell pellets in 250 µL of Buffer P1 and transfer to a microcentrifuge tube.

2. Ensure that RNase A has been added to the PI buffer. No clumps of cells should be visible after resuspending the pellet.

3. Add 250 µL of Buffer P2 and gently invert the tube 4-6 times to mix.

4. Mix gently by inverting the tube. No vortexing, as this will result in shearing of the genomic DNA. If necessary, continue to invert the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 minutes.

5. Add 350 µL N3 buffer and invert the tube immediately but gently 4-6 times.

6. Mix the solution gently but thoroughly, immediately after adding Buffer N3. The solution should become cloudy.

7. Centrifuge for 10 min at 13,000 rpm (-17,900 xg) in a benchtop microcentrifuge.

8. A solid white pellet will form.

9. Apply the supernatants from step 4 to the QIAprep spin column by decantation or pipetting.

10. Centrifuge for 30-60 s. Discard the stream.

11. (Optional): Wash the QIAprep spin column by adding 0.5 mL PB buffer and centrifugation for 30-60 s. Discard the stream.

12. This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB 101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content . Host strains such as XL-1 Blue and DH5a™ do not require this additional washing step.

13. Wash the QIAprep spin column by adding 0.75 mL of PE buffer and centrifugation for 30-60 s.

14. Press flow and centrifuge for an additional 1 minute to remove residual wash buffer

15. Note: Residual wash buffer will not be completely removed unless the flow-through is discarded prior to this additional centrifugation. Residual ethanol from the PE buffer can inhibit subsequent enzyme reactions.

16. Place the QIAprep column in a clean 1.5 mL microcentrifuge tube. To denature DNA, add 50 µL of EB buffer (10 mM Tris-Cl, pH 8.5) or water to the center of each QIAprep

17. Spin column, let stand for 1 minute, and centrifuge for 1 minute.

3.5 DNA COLLECTION PROTOCOL

Materials:

1. TE solution

10 mM Tris (pH up to 7.5)

1 mM EDTA (pH to 8.0 for dissolution)

2. DNA sample

3. SYBR Green I(R) nucleic acid gel stain (Molecular Probes)

4. Plastic film

5. Distilled water

6. DNA Marker Stock (10 mg/mL)

Supplies:

1. Pipes

2. Polaroid organization (with the appropriate filter - photographic filter SYBR Green/Gold gel stain) and UV light box

3. Micropipetter and tips

Procedures:

1. Prepare 6 DNA templates from the DNA marker stock:

standard I (5 ug/ul): 1 :2 dilution of DNA marker stock

standard II (2.5 ug/ul): 1 :2 dilution of standard I;

standard III (1.25 ug/ul): 1 :2 dilution of standard II

standard IV (0.625 ug/ul): 1:2 dilution of standard III;

standard V (0.313 ug/ul): 1 :2 dilution of standard IV

standard VI (0.156 ug/ul): 1:2 dilution of standard V;

1. Make a 1 : 5000 dilution of SYBR Green I(R) with TE solution.

2. Mix 5 ul of the DNA sample and each of the 6 standards with 5 ul of the diluted SYBR Green I(R) dye.

3. Place a sheet of plastic wrap smoothly over the UV light box.

4. Spot the mixtures separately on plastic wrap.

5. Locate all 6 patterns.

6. Turn on the UV light and take a photo (Polaroid 667 black and white print film).

7. Compare the brightness of the DNA sample to the DNA standards and calculate the concentration.

3.6 LIGATION

LIGATION - PCR

Add 1 µL of the vector

Add 1 µL of 10x buffer

Addition of PCR product (1 µL)

Add 8 µL of SIGMA water

Add 1 µL Ligase

LIGATION - OVERNIGHT

Add 0.5 µL of the vector

Add 5 µL 10x Buffer Add 3.5 µL Insert DNA

Add 1 µL Ligase (last)

Incubate overnight at 17 °C LIGATION - BENCHTOP

Add 0.5 µL of the vector

Add 5 µL 10x Buffer Add 3.5 µL Insert DNA

Add 1 µL Ligase (last)

Leave at room temperature for 2 hours 30 minutes.

3.7 Preparation of SDS-Page Gel

The Running buffer is made with the pr contains 288g Glycine and 80g Tris). 20 mL will give the SDS buffer.

prepare 200 mL of SDS Page Buffer (that of SDS 10% buffer in 2000 mL of dH2O

Claims (12)

DIAGNOSTIC FOR THE DETECTION OF SARS-COV-2 CLAIMS What is being requested is: 1. A diagnostic immunoassay for the detection of SARS-CoV-2 in a subject, the assay comprising the steps of: a) the in vitro examination of an already collected and isolated biological sample from a subject suspected of being infected with SARS-CoV-2- b) detecting the concentration of eukaryotic protein synthesis initiation factor 2α (eIF-2α) in said sample by immunological means; wherein a reduced concentration of eIF-2α in said sample, compared to the concentration of IGFBP-1 in a plasma sample from a healthy mammal is predictive of SARS-CoV-2. 2. The assay of claim 1, wherein the detection comprises the binding of eIF-2<α>by an antibody that specifically binds eIF-2a. 3. The assay of claim 1, wherein said immunological means utilizes a monoclonal antibody. 4. The assay of claim I, wherein said antibody is detected and the detection comprises detecting the amount of label bound to the eIF-2α/antibody complex. 5. The assay of claim 4, wherein said label is selected from the group consisting of a radioactive label, an enzymatic label, a colorimetric label, and a fluorescent label. 6. The assay of claim 5, wherein said detection comprises: contacting said antibody with a labeled second antibody that binds said antibody? and detecting the amount of labeled second antibody bound to said antibody immobilized on a substrate. 7. The assay of claim 1, wherein said detection comprises a competitive assay comprising the steps of: a) providing a first antibody that specifically binds eIF-2α; b) contacting said eIF-2a labeled first antibody and said sample- c) detection of the amount of labeled eIF-2α that forms an eIF-2α/antibody complex. 8. The assay of claim 7, wherein the first antibody is immobilized on a solid support. 9. The assay of claim 7, wherein detecting comprises detecting labeled eIF-2α bound to said first antibody. 10. The assay of claim 9, wherein detecting comprises detecting eIF-2<α>labeling that remains unbound in solution. 11. The assay of claim 1, wherein said detection comprises Western Blotting using an antibody that specifically binds eIF-2α. 12. The test of claim 1, wherein the subject is a human.