GB2600397A - Antibody assay - Google Patents

Abstract

Test for the detection of antibodies to SARS-CoV-2 (COVID-19) in a biological test sample comprising a) a biological test sample; b) at least one protein, polypeptide or peptide derived from SARS-Cov-2; c) a negative control; d) a positive control; and e) at least two different dilutions of at least one antibody detection molecule. Also disclosed is a method for detecting antibodies to SARS-CoV-2 in a biological test sample comprising: a) adding a biological test sample, a positive control or a negative control to a well in an assay plate, the well being pre-coated with at least one protein derived from SARS-CoV-2; b) adding a first dilution of an antibody detection molecule to a number of wells in the assay plate and adding a second, different, dilution of the antibody detection molecule to the remaining number of wells; and c) detecting antigen-antibody binding.

Description

ANTIBODY ASSAY

The present invention relates to an antibody detection assay, in particular for the detection of SARS-CoV-2 antibodies in a biological sample.

Coronavirus disease 2019, or COVID-19, is an infectious disease caused by a newly discovered coronavirus, the severe acute respiratory syndrome coronavints 2 (SARS-00V-2 virus). Infection by the virus and the resulting disease have spread globally at an exponential rate, making it one of the worst pandemics in recent history. Although most people with COV1D19 have mild to moderate symptoms, the disease may cause severe medical complications and lead to death in some people. Older adults or people with existing chronic medical conditions are at greater risk of becoming seriously ill with COV1D-19 As a result, there is an urgent need for a test that identifies people who have previously been exposed to the virus. Not only does this provide an indication on the spread of the infection but it also provides an indication of the number of people who might be likely to be severely ill if infected and so health resources can be managed appropriately. Such information also provides a repository of potential therapies and a basis from which vaccines and treatments may be derived Antibodies are typically detected in blood using EL1SA and there are many commercial ELISA kits available for SARS-CoV-2 antibodies (Table 1).

Table 1: Accuracy figures for selected SARS-CoV-2 antibody tests (all accuracy claims made by companies) Company Test name Sensitivity (/o) Specificity (%) Cellex Cellex qSars-CoV-2 93.8 95.6 IgG.IgN4 cassette rapid test Ortho-Clinical Diagnostics Vitros anti-Sars-CoV-2 total reagent pack 83.3 100.0 Diasorin Liaison Sars-CoV-2 Si/S2 97.4 98.5 IgG test Ortho-Clinical Diagnostics Vitros anti-Sars-CoV-2 IgG reagent pack 87.5 100.0 Autobio Diagnostics Anti-Sars-CoV-2 rapid test (IgIST and IgG) 93.0 100.0 Abbott Laboratories Abbott Sars-CoV-2 IgG test 100.0 99.5 Bio-Rad Laboratories Platelia Sars-CoV-2 total Ab assay 98.0 99.0 Roche El ecsy s anti -Sars-CoV-2 100.0 99.8 antibody test Beckton Dickinson / Biomedics Biomedics Covid-19 88.7 90.6 IgNI/IgG Rapid Test Creative Diagnostics Creative Diagnostics Sars- 94.5 100.0 CoV-2 Antibody ELISA CTK Biotech OnSite COVED-19 IgG/IgNI Rapid Test 96.9 99.4 Epitope Diagnostics EDI Novel Corona% rus 100.0 100.0 Covid-19 IgG Elisa Kit Epitope Diagnostics EDI Novel Coronavirus 45.0 100.0 Covid-19 IgNI Elisa Kit lntec Products 1nTec Rapid Sars-CoV-2 95.2 98.0 Antibody (IgN1/1gG) Nirmidas Biotech Nirmidas Biotech Covid-19 93.8 99.5 (Sars-CoV-2) IgNI/IgG Antibody Detection Kit SD Biosensor Standard Q Covid-19 81.8 91.6 IgNI/IgG Duo Test Source: EvaluateMedTech and company websites However, as the figures in Table 1 show, the sensitivity and specificity of these tests are variable. The tests also tend to be expensive and do not give titre values. Titre values are a comparison of the optical densities between a test sample and a control and are an important measure because they provide an indication of the extent and strength of a host's immune response. So, a high titre value indicates that, if the subject were to be subjected to the virus again, the body is likely to have sufficient immune capacity to fight the infection and so render the subject less sick or present fewer or less severe symptoms, if any. Of particular concern is that there is still no definite proof that the presence of antibodies to SARS-CoV-2 does in fact confer immunity to subsequent infection by this virus in humans. Therefore, knowing the titre value in a subject provides valuable data to answer this question.

Accordingly, there remains a need for an antibody test that is sufficiently sensitive and specific, that is providable at a reasonable cost and that provides titre values. It is against this background that the present invention has been devised In one aspect the present invention resides in an antibody test or assay to detect antibodies to SARS-CoV-2. The test comprises: i) a biological test sample; ii) at least one protein, polypeptide or peptide derived from SARS-Cov-2; iii) a negative control; iv) a positive control; and v) at least two different dilutions of at least one antibody detection molecule.

Most of the antibody tests currently in commercial and research use are point of care tests which only give a positive or negative result and are not able to provide the titres or a comparison of values, such as optical density, between a sample and a control. Accordingly, the test of the present invention is preferably a plate-based assay test such as an ELISA. Such assays are designed for detecting and -importantly -quantifying peptides, proteins, antibodies and hormones. In particular, ELISAs rely on antibodies present in a test sample binding to a target antigen, and a detection system to indicate the presence and quantity of antigen binding.

In particular, an antigen must be immobilised to a solid surface and then complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.

ELISAs are typically performed in 96-well (or 384-well) polystyrene plates and passively bind antibodies and proteins. The binding and immobilisation of reagents makes ELISAs simple to design and perform. Having the reactants of the ELISA immobilised to the plate surface enables easy separation of bound from non-bound material during the assay. This ability to wash away non-specifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation.

ELISAs principles are very similar to other immunoassay techniques and so it will be understood that any similarly principled technology may be used to put the present invention into effect.

A clear advantage of using an ELISA-or immunoassay-based test is that multiple samples may be tested at the same time which reduces the test cost of each sample.

The biological test sample may be derived from any suitable biological tissue or fluid but, in the present application, a blood sample is preferred because it is easy and (relatively) painless to obtain, easy to store and simple to process for use in the test of the present invention. Ideally, the biological sample is plasma or serum While the COVID-19 pandemic is currently only seen in humans, it will be appreciated that the test is also suitable for non-human subjects, including mammals, birds and reptiles, particularly domestic pets, and animals and insects for human consumption.

In one embodiment, the at least one protein derived from SARS-CoV-2 is, or is derived from, a Spike protein or part thereof, such as a recombinant N-terminal S1 subunit. Recombinant Spike proteins are well described and defined in the scientific literature and are readily available commercially. While live virus could be included in the assay, alternatively or in addition to recombinant protein, the use of a recombinant antigen puts the assay into biosafety level 2, whereas use of live virus would require the assay to be carried out at biosafety level 3. A BSL-3 laboratory typically includes work on microbes that are either indigenous or exotic and can cause serious or potentially lethal disease through inhalation. The microbes are so serious that the work is often strictly controlled and registered with the appropriate government agencies. Laboratory personnel are also under medical surveillance and could receive immunisations for microbes they work with. Since immunisation against SARS-CoV-2 is not currently available, such a requirement for an antibody assay is clearly not practical.

An alternative or additional measure may be that the at least one protein derived from SARSCoV-2 is derived or part of live virusper se and the test sample is pre-processed before carrying out the assay of the invention. An example of suitable pre-processing is to heat the serum samples at a temperature above body temperature for a suitable period of time before use to reduce risk from any potential residual (live or viable) virus in the serum. A suitable temperature above body temperature might be 56°C and a heating time of 1 hour may be suitable.

In the test of the present invention, the target is a recombinant Spike 51 subunit because this part of the SARS-CoV-2 virus is a target for the development of a suitable vaccine. Its inclusion in an antibody assay should enable its detection and thus indication of a response to a vaccine.

Most known tests typically test for antibodies to the Spike protein. However, the test of the present invention has the optional ability to comprise a second or further protein, polypeptide or peptide derived from SARS-CoV-2, for example a polypeptide derived from the receptor binding domain (RBD). The RBD is part of the S1 subunit. In this way, antibodies to both Spike and the RBD may be checked simultaneously. The inclusion of at least a second or further antigen increases the specificity of the test for each sample and provides veracity for a positive result.

Tests and assays should always include a control and such controls are typically negative controls, i.e. proving the absence of the matter that is being tested, to flag any contamination or technical issue within the particular test A standard negative control would be the omission of the test sample. Alternatively, or in addition, a suitable negative control for the present invention is a biological sample that is known to be free of SARS-CoV-2 virus or proteins, polypeptide and peptides therefrom. For example, a historical blood sample obtained before the emergence of SARS-CoV-2 virus (a PCR-negative sample) or human serum which is sold commercially as a supplement for tissue cultures. As well as providing a quality check, the negative control also provides a measurement baseline for each assay which, in the preferred embodiment, is a base line optical density. From this baseline, values may be obtained and checked serially, for example, as titres or fold differences for investigating the antibody or immune response in an individual or particular test sample.

An important part of the assay of the present invention is the inclusion of a positive control. It will be appreciated that any suitable positive control may be used. An example is a biological sample known to contain SARS-CoV-2 or one or more peptide, polypepti de or protein derived therefrom, for example a sample derived from blood that is confirmed (for example, by PCR) to include SARS-CoV-2. The advantage of including a positive control, in addition to a negative control, is for quality control -the positive control confirms that the assay is able to detect virus is a sample. If the negative and or positive controls provide inaccurate results, then it is clear that data from the assay should not be used and the test repeated afresh.

Another important feature of the test is the inclusion of more than one dilution (or concentration) of at least one antibody detection molecule. It will be appreciated that, where the test is an ELISA, the antibody detection molecule will be a secondary antibody. Ideally the secondary antibody is an anti-IgM or an anti-IgG that is capable of identifying the presence of 181\4 or IgG in a test sample obtained from a subject. Again, it will be appreciated that, for an ELISA, the anti-IgM or anti-IgG will be conjugated to a suitable detection system such as horseradish peroxidase (HRP).

Currently available tests include just a single concentration of an antibody detection molecule because the aim of these tests is to provide simply yes/no result. The inclusion of two dilutions (or concentrations) enables the test of the present invention to provide a confirmed yes/no answer for the presence or absence of the virus. In addition, the two dilutions provide a value from which the amount of IgIVI or IgG in a sample may be quantified, thereby providing a titre for the sample. Any suitable dilutions may be used, for example 1:3000 and 1:5000. It will be appreciated that the terms "dilution" and "concentration" have the same meaning and so are used interchangeably herein.

It will be appreciated that a third or more dilutions may also be included to provide more detail on the quantity of antibody in the test sample.

While the test may include a single antibody detection molecule, it is advantageous if the test further includes at least a second antibody detection molecule which detects a different antibody to the first. Again, commercially available kits test for either 1gM or IgG but not both simultaneously. In the present invention it is conceived that the test includes IgM and IgG detection, or IgG and IgNI detection. Anti-spike IgNI antibodies appear 2-3 weeks after onset of the infection symptoms and persist for up to 6-8 weeks. Anti-spike IgG antibodies appear 56 weeks after onset of the infection symptoms and the duration of their presence in blood remains unknown. Therefore, the test of the present invention is able to provide information on the timeline of infection in a test sample.

It will be appreciated that more than two antibody detection molecules may be included in the test. While this increases the complexity, such addition may be used to provide additional data and information about the immune response to a SARS-CoV-2 infection.

While only a single dilution of the at least second antibody detection molecule is adequate, it is ideal if at least two dilutions of the at least second detection molecule are used. In this way, the specificity and veracity of the results is significantly increased. As with the first antibody detection molecule, any suitable dilutions may be used, such as 1:3000 and 1:5000.

In some embodiments, the assay may further include a correction factor. A correction factor may be typically calculated from a suitable number of negative samples. In the present instance, such negative samples are ideally collected from a pre-COV-19 era and known to be free of SARS-CoV-2. An optical density correction factor may be calculated as mean optical density + 3* the standard deviation (SD) of the samples. Other methods such as AUROC (area under receiver operating characteristic) curve, the Youdne's index and Euclidian index may be used alternatively or in addition.

Optionally, the optical density mean, standard deviation and correction factor calculations from the negative samples may be compared with the same calculations derived from a suitable number of samples collected from SARS-CoV-2-positive patients, such as patients who needed admission for COVID-1 9 infection. Where there is no overlap between the correction factor values for negative and positive control samples, this confirms use of the negative correction factor calculation for the assay.

In a particular embodiment, a cut off value for each individual assay may be additionally calculated based on a number of negative samples run in each assay (mean + 3*SD). If the optical density cut off is lower than the (negative) correction factor, the correction factor may be used for interpretation of the results.

In another aspect, the present invention resides in a method for detecting antibodies to SARS-CoV-2 in a biological test sample taken from a subject using the test described herein. The method comprises: a) adding a biological test sample, a positive control or a negative control to a well in an assay plate, the well being pre-coated with at least one protein derived from SARS-CoV-2; b) adding a first dilution of an antibody detection molecule to a number of wells in the assay plate and adding a second, different, dilution of the antibody detection molecule to the remaining number of wells; and c) detecting antigen-antibody binding.

In one embodiment, a second antibody detection molecule may be added to some or all of the wells of the assay plate. It will be appreciated that third, fourth etc antibody detection molecules may also be further added. As described above, the second and further antibody detection molecules detect a different antibody to the first antibody detection molecule.

While a single concentration of the second (or further) antibody detection molecule may be added to all the wells in the assay plate, the method encompasses the addition of a first dilution to a (discrete) number of wells in the assay plate and the addition of a second, different, dilution of the second (or further) antibody detection molecule to the remaining number of wells. The same option applies to any additional antibody detection molecules above the first and second. In addition, more than two different dilutions may be used In a particular example, the first dilution of first and second antibody detection molecules are the same. Alternatively, or in addition, the second dilution of the first and second antibody detection molecules are the same. The same examples apply to any further antibody detection molecules that may be added to the wells. It will be appreciated that the invention also encompasses adding different antibody detection molecules each having different dilutions.

In one embodiment, the first dilution of the second antibody detection molecule is added to the same wells as the first dilution of the first antibody detection molecule. Alternatively, the second dilution of the second antibody detection molecule is added to the same wells as the first dilution of the first antibody detection molecule. In another alternative, the wells with the first dilution of the first antibody detection molecule are not all the same as the wells to which the first dilution of the second antibody detection molecule are added. It will be appreciated that all combinations and permutations of dilutions and antibody detection molecules are encompassed.

A preferred method of detecting antigen-antibody binding is via a colorimetric or optical density change in each well of the assay plate compared to a negative control. However, it will be appreciated that the present invention nis not limited to such methods and that any suitable detection method is suitable.

In a further aspect, the present invention resides in an antibody detection kit for the test/assay described herein. The kit comprises: a) at least one protein, polypeptide or peptide derived from SARS-Cov-2; b) a negative control; c) a positive control; and d) at least two different dilutions of at least one antibody detection molecule.

It will be appreciated that the at least one protein, polypeptide or peptide derived from SARSCoV-2 in the kit may be or may be derived from a Spike protein or part thereof Optionally, the kit may further comprise at least a second protein, polypeptide or peptide derived from SARS-CoV-2. Such second or further protein, polypeptide or peptide may be or 20 may be derived from the receptor binding domain (RBD) of SARS-CoV-2.

In one embodiment, the negative control may a biological sample that is known to be free of SARS-CoV-2 virus or proteins, polypeptide and peptides derived therefrom.

In another embodiment, the positive control may a biological sample that contains SARSCoV-2 virus, or one or more proteins, polypeptide and peptides derived therefrom.

In a further embodiment, the at least one antibody detection molecule may be secondary antibody that is capable of identifying the presence of IgM or IgG in the test sample.

Thus, an assay has been developed to detect IgM and IgG anti-SARS-CoV-2 antibodies which is inexpensive, accurate and may be used in hospital laboratories to detect neutralising antibodies against the virus and also to confirm local populations who have achieved immunity.

The present invention will now be described by way of non-limiting examples and figures in which: Figure 1: A schematic retrieved from https://www.abcam. corn/novel -coronavi rus-i gm-antibody-detection-kit-sars-cov-2-ab272244.html#descriptionimages_l illustrating how the Abcam test results are to be read. Positive for Coronavirus: both the test line (T) and the quality control line (C) are coloured dark pink. Negative for Coronavirus: The test line (T) does not develop colour, or a faint grey band may be visible, but the quality control line (C) is coloured. Suspect: A light pink band is an inconclusive result. Invalid: There is no coloured control line (C) band.

Figure 2: A schematic grid providing details of the sample layout on a 96-well ELISA plate for use with an assay in accordance with the invention described herein. PC = positive control; S = sample; Ab = sample containing pairs of known recombinant antibodies used as a control; AB = human AB serum sample dating from pre-COVID to serve as a negative control; Blank = TPBS only.

Figure 3: ELISA plates from different assay runs in accordance with the invention, with samples laid out as illustrated in Figure 1. Plates A and C are assays using plasma samples. Plate B is as assay using serum samples from the same blood samples as the plasma samples used in Plate A. Figure 4: Validation against Abcam Coronavirus (SARS-CoV-2) IgM and IgG antibody detection kits (ab272244 and ab 272243 respectively). Figure 4A: three tests showing results for IgIVI Negative, IgM Positive and IgG Positive samples Figure 4B: five tests validating sample results using the IgM test kit ab272244 As used herein, the singular forms "a", "an", and "theinclude both singular and plural referents unless the context clearly dictates otherwise.

The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The term also encompasses "consisting of' and "consisting essentially of'.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The term 'about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of and from the specified value, in particular variations of +/-10% or less, preferably +/-5% or less, more preferably +/-1% or less, and still more preferably +1-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.

Whereas the term "one or more", such as one or more members of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alit( a reference to any one of said members, or to any two or more of said members, such as, e.g any or etc. of said members, and up to all said members.

REAGENTS Samples Plasma (including EDTA) or serum (in Serum Separator Tubes) samples obtained from whole human blood were stored at -80°C Target proteins: Cat No: 230-01102-500 Subunit Recombinant SARS-CoV-2 Spike Protein, S1 Subunit, Host Cell (500 Mg) -Cambridge Biosciences Cat No: 230-01101-100 Recombinant SARS-CoV-2 spike protein Si subunit host cell receptor binding domain (RBD) -Cambridge Biosciences.

Control antibodies: Recombinant Anti-SARS-CoV-2 Spike Glycoprotein Si antibody [CR3022] -Chimeric (ab273074) Abcam Development antibodies: Goat anti-Human IgG (Gamma chain) Cross-Adsorbed Secondary Antibody, HRP Catalog # 62-8420-Thermo Fischer Scientific Goat anti-Human IgNI (Heavy chain) Secondary Antibody, FIRP Catalog # A18835-Thermo Fischer Scientific.

Goat Anti-Rabbit IgG H&L (EIRP) (ab205718) Abcam Secondary anti-mouse HRP antibodies against control antibodies.

Solutions.

Tweenlm 20 Surfaet-Amps1M detergent solution -Thermo Fischer.

P ercerm 10 X TBS buffer.

Non-fat powdered milk.

Abcam TMB ELISA Substrate (High Sensitivity) (ab171523) 1000 ml Abeam 450 nm Stop Solution for TMB Substrate (ab171529) 1000m1.

Miscellaneous Thermo ScientificTM 96 Well Plate, Non-Treated Surface, No lid, Non-sterile, Pack of 5 (Thermo ScientificTM 269620) 3667 Sigma-Aldrich Human Serum -Heat Inactivated, from human male AB plasma,

METHODS

The ELISA protocol was established following the method by Amanat F et al ((2020) Nat lied 26, 1033-1036), the contents of which are incorporated herein by reference, and was optimised.

Solutions of 2 gg/mL recombinant Spike and RBD proteins were made up in sterile PBS (1x).

50 ittl of recombinant protein solution was then added to each well of a 96-well ELISA plate.

The plate was wrapped in aluminium foil to protect the contents from light and incubated overnight at 4°C.

On the day of each assay, the coating solution was removed Serum samples were heated at 56°C for 1 hour before use to reduce risk from any potential residual virus in the serum.

A blocking solution of 3% non-fat powered milk dissolved in 1xPBS with 0 1% Tween (TPBS) was prepared and 100 gl added to each well. Plates were then incubated at room temperature for 1 hour.

After an hour, the blocking solution was removed from each plate. Each plate was washed three times with TPBS and once with 1xPBS and was then ready for the transfer of samples.

Patient samples and control antibodies (anti-spike) were diluted 1:200 in 1% non-fat milk prepared in TPBS in the serum. Dilutions were prepared in a mixing plate.

One hundred and thirty negative serum samples diluted 1:200 from historical (prior to COVID- 19 pandemic) samples of human AB serum (Sigma) -a supplement for tissue cultures -were used as a negative serological control, together with fifty negative serum samples dating from 2014. The optical densities of all negative samples were collated, and the mean and standard deviation (SD) was calculated to establish a correction factor for the assay overall. This was calculated as mean + 3*SD of the negative control optical density. Other methods such as AUROC curve, the Youdne' s index and Euclidian index were also used. The negative controls were compared to TPBS only.

Positive serum samples diluted 1:200 from serum samples of PCR positive patients (diagnostic surplus from diagnostic repository in patients with abnormal LTFs as a part of the liver screening), as well as samples from twenty five SARS-CoV-2 positive patients who needed admission for COV1D-19 infection, were used as a positive serological control. Mean and 3* SD was again calculated. No overlap was seen between correction factors for negative and positive samples, thereby confirming setting the negative correction factor for the assay.

The cut off optical density calculated for each individual assay run was calculated using negative AB serum samples included in each assay (mean + 3*SD) If the optical density cut off calculation was lower than the negative correction factor, the correction factor was used for interpretation of the results.

pl of sample, positive control or negative control was added to each well and the plates incubated for 2 hours at room temperature. After incubation, the wells were washed four times 25 with 250 R1 per well of 0.1% TPBS and once with PBS.

A 1:3000 dilution of goat anti-human IgG-horseradish peroxidase (FIRP) and a 1:5000 dilution of goat anti-human IgG-horseradish peroxidase (HRP) or 1:3000 and 1:5000 goat-anti-human 181\4 FIRP conjugated secondary (detection) antibody were prepared in 0.1% TPBS. 100 pl of either IgG-or IgM secondary antibody was added to each well and incubated for 1 hour at room temperature. The samples were tested against two different dilutions of secondary antibodies to confirm the result of the assay.

Plates were then washed four times with 250 Ill per well, three times with 0.1% TBS and one time with PBS. Once completely dry, 100 ill per well of TMB solution was added and incubated for 10 minutes. The reaction was stopped by adding 50 il 1IV1B stop solution to each well.

The plates were then immediately read at an optical density of 450 nanometres on an ELISA plate reader. The background value was set at an optical density 490nm of 0.11 and area under the curve (AUC) was calculated. Data was analysed in Prism 7 (Graphpad).

Results were compared to tests carried out using Abcam Coronavirus (SARS-CoV-2) IgM and IgG antibody detection kits (ab272244 and ab 272243 respectively). These kits are currently for research use only and are suitable for the qualitative detection of SARS-CoV-2 IgNI or IgG antibodies in human serum, plasma, and whole blood The detection kit uses the principle of immunochromatography: the separation of components in a mixture through a medium using capillary force and the specific and rapid binding of an antibody to its antigen. Each cassette is a dry medium that has been coated separately with novel coronavirus N protein ("T" test line) and goat antichicken IgY antibody ("C' control line) (Figure 1). Two free colloidal gold-labelled antibodies, mouse anti-human IgM (mIgM) and chicken IgY, are in the release pad section (S). Once diluted serum, plasma, or whole blood is applied to the release pad section, the mIgM antibody binds to coronavirus IgNI antibodies if they are present, forming an IgNI-IgNI complex. The sample and antibodies then move across the cassette's medium via capillary action. If coronavirus IgM antibody is present in the sample, the test line (T) will be bound by the IgM-IgNI complex and develop colour. If there is no coronavirus IgNI antibody in the sample, free mIgM will not bind to the test line (T) and no colour will develop. The free chicken IgY antibody will bind to the control line (C); this control line should be visible after the detection step as this confirms that the kit is working properly.

Tests ab272243 and ab272244 have been tested by Abcam on confirmed positive Covid-19 samples and known negative samples. The following data set was the outcome of two different screening batches compiled together. Confirmed positive and negative control samples were tested for the presence of IgG and IgM novel coronavirus antibodies. The kit was used as instructed by the manual, and positive or negative indications were listed as such. The results of that testing indicate a sensitivity to viral antibodies of 87%, with a specificity of 84%.

Table 2: Data for Abcam Coronavirus (SARS-CoV-2) IgM and IgG antibody detection kits (ab272244 and ab 272243 respectively) Sample Sample 1gM - 1gG - Overall Overall Sensitivity Specificity Type Number Ab272244 Ab272243 Positive Negative Positive 30 24 24 26 4 87% Negative 90 12 3 14 76 84

RESULTS

Three hundred blood samples were provided by volunteers after consenting on a participation form for service development of an anti-Spike and anti-RBD antibodies IBM and IgG ELISA test. IgM was tested against Spike protein. IgG was tested for both against RBD (receptor binding domain) and Spike protein.

Figure 2 is an example of a grid template that has been used to record details of the sample layout on a 96-well ELISA plate for an assay as described herein ELISA plates from three different assay runs as described above are shown in Figure 3, with samples laid out as illustrated in Figure 1. Plates A and B corroborate results because both plates use the same blood sample, with Plate A using samples derived from plasma and Plate B using samples derived from serum. As can be seen, both plasma and serum samples provide the same results.

As can be seen from all three plates, the optical density can be used to quantify the antibody titre in each sample From the cohort of three hundred participants, thirty-three were PCR-positive for the SARSCoV-2 virus and, using the assay described herein, thirty-two of those participants were found to be positive for IgM or IgG (97%) and only one participant was negative Eighty-four participants had COVID-19 symptoms and seventy-five (89%) of these had IBM or IgG antibodies on our assay.

A comparison was made against the Abcam IgM and IgG kits. As shown in Figure 4A, samples from positive and negative controls provided expected results. Positive and negative controls and three samples tested using the IgM kit all showed readable and definitive results (Figure 4B).

Overall, a 100% concordance was seen in all tested positive and negative controls and samples.

CONCLUSION

The inventors set out to improve an existing serological assay to detect SARS-CoV-2 virus antibodies in blood samples from humans. The assay described by Amanat et al (supra) is an ELISA that uses a recombinant Spike or receptor binding domain (RBD) protein from SARSCoV-2 as the antigen. Plasma and serum samples were tested against one or other of these antigens and binding was identified by a single 1:3,000 dilution of goat anti-human IgGhorseradish peroxidase (HRP) conjugated secondary antibody. Samples confirmed with a human coronavirus NL63 infection were used as a negative control.

In the ELISA assay described herein, the target of the assay was a commercially available recombinant Spike S1 subunit polypeptide and the binding of IgNI if present in a test sample. An additional target of a recombinant RBD polypeptide was included, against which IgG antibodies present in blood samples was also tested, as well as to IgNI.

Positive control and negative controls were included in every assay run with the negative controls providing a baseline optical density for each assay. From this baseline, values were obtained which could be checked serially as titres or fold difference for investigating the antibody or immune response in a sample. Most of the kits currently in use are point-of-care test which only give positive or negative results, as the Abcam kits do, and are not able to provide the titres or optical densities values. However, as explained hereinabove, this information is useful to identify the strength of immune response that has been mounted by the individual which, in turn, may provide insight into how the individual may response if re-infected.

In a further addition, the samples were tested against two different dilutions of IgM antibodies and two different dilutions of IgG. Anti-spike IgNI antibodies appear 2-3 weeks after onset of the infection symptoms and persist for up to 6-8 weeks. Anti-spike IgG antibodies appears 5-6 weeks after onset of the infection symptoms and the duration of their presence in blood remains unknown. In assays currently available, samples are assay for either IgNI or IgG, but not both. In addition, the present assay exemplified identifies binding with Spike protein, as well as Spike and RBD binding.

The assay described herein has many advantages over the antibody tests currently available.

Although not able to provide an immediate result, as the point-of-care devices are able to provide, the assay may be performed in approximately 4-5 hours (excluding overnight plate preparation) and so results may be provided within 24 hours of a test sample being taken. In addition, the assay requires only a small amount of serum or plasma -500 pl. The assay is also safe and can be performed at biosafety level 2 because it does not involve live virus.

It is anticipated that the test will be used to assess past/current exposure to SARS-CoV-2 and enable the identification of individuals with neutralising antibodies to the virus. In this way, individuals may be deemed to be free of virus and having a low risk of re-infection. The test may also be used to identify specific neutralising antibodies that might be helpful in vaccine development and/or treatment.

Claims (25)

1. CLAIMS: 1 An antibody test to detect antibodies to SARS-CoV-2, the test comprising: a) a biological test sample; b) at least one protein, polypeptide or peptide derived from Sars-Co' c) a negative control d) a positive control; and e) at least two different dilutions of at least one antibody detection molecule.
2. 2. The antibody test of Claim 1, wherein the antibody test is a plate-based immunoassay, such as ELISA.
3. 3. The antibody test of Claim 1 or Claim 2, wherein the biological test sample is a blood sample, plasma or serum.
4. 4. The antibody test of any one of Claims Ito 3, wherein the biological test sample is derived from a human.
5. 5. The antibody test of any one of Claims Ito 4, wherein the at least one protein, polypeptide or peptide derived from SARS-CoV-2 is or is derived from a Spike protein or part thereof
6. 6. The antibody test of any one of Claims Ito 5, wherein the test further comprises at least a second protein, polypeptide or peptide derived from SARS-CoV-2.
7. 7. The antibody test of Claim 6, wherein the second or further protein, polypeptide or peptide is derived from the receptor binding domain (RED) of SARS-CoV-2.
8. 8. The antibody test of any one of Claims Ito 7, wherein the negative control is a biological sample that is known to be free of SARS-CoV-2 virus or proteins, polypeptide and peptides derived therefrom.
9. 9 The antibody test of any one of Claims Ito 8, wherein the positive control is a biological sample that contains SARS-CoV-2 virus, or one or more proteins, polypeptide and peptides derived therefrom.
10. 10. The antibody test of any one of Claims Ito 9, wherein the at least one antibody detection molecule is secondary antibody that is capable of identifying the presence of IgIVI or IgG in the test sample.
11. 11. A method for detecting antibodies to SARS-CoV-2 in a biological test sample taken from a subject, the method comprising: a) adding a biological test sample, a positive control or a negative control to a well in an assay plate, the well being pre-coated with at least one protein derived from SARS-CoV-2; b) adding a first dilution of an antibody detection molecule to a number of wells in the assay plate and adding a second, different, dilution of the antibody detection molecule to the remaining number of wells; and c) detecting antigen-antibody binding.
12. 12. The method of Claim 11, wherein a second antibody detection molecule is added to some or all of the wells.
13. 13. The method of Claim 12, wherein the second antibody detection molecule is added in a first dilution to some of wells in the assay plate and a second, different, dilution of the second antibody detection molecule is added to the remaining number of wells.
14. 14. The method of Claim 12 or Claim 13, wherein the first dilution of the first and second antibody detection molecules are the same.
15. 15. The method of any one of Claims 12 to 14, wherein the second dilution of the first and second antibody detection molecules are the same
16. 16. The method of any one of Claims 12 to 15, wherein the wells with the first dilution of the first antibody detection molecule are not the same as the wells in which the first dilution of the second antibody detection molecule are added.
17. 17. The method of any one of Claims 11 to 16, wherein the biological test sample is a blood sample, plasma or serum.
18. 18. The method of any one of Claims 11 to 17, wherein the biological test sample is derived 15 from a human.
19. 19. The method of any one of Claims 11 to 18, wherein the negative control is a biological sample that is known to be free of SARS-CoV-2 virus or proteins, polypeptide and peptides derived therefrom.
20. 20. The method of any one of Claims 11 to 19, wherein the positive control is a biological sample that contains SARS-CoV-2 virus, or one or more proteins, polypeptide and peptides derived therefrom.
21. 21. The method of any one of Claims 11 to 20, wherein the at least one protein, polypeptide or peptide derived from SARS-CoV-2 is or is derived from a Spike protein or part thereof
22. 22. The method of any one of Claims 11 to 21, wherein some or all of the wells of the assay plate are further pre-coated with at least a second protein, polypeptide or peptide derived from SARS-CoV-2.
23. 23 The method of Claim 22, wherein the second or further protein, polypeptide or peptide is derived from the receptor binding domain (RBD) of SARS-CoV-2.
24. 24 The method of any one of Claims 11 to 23, wherein the at least one antibody detection molecule is secondary antibody that is capable of identifying the presence of IgM or IgG in the test sample
25. 25. The method of any one of Claims 11 to 24, wherein detection of antigen-antibody binding is via a colorimetric or optical density change compared to the negative control.