DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to an immunoassay to determine the presence and concentration of antibodies capable of binding a protein antigen in a biological sample isolated from an individual.

STATE OF THE ART

The growing spread of infectious diseases, in particular, infectious respiratory tract diseases, is driving scientific research towards a quest for new diagnostic and therapeutic strategies. An epidemiological situation already abounding in new potential pathogenic microorganisms was aggravated by the fact that the COVID-19 pandemic caused by the SARS-CoV-2 coronavirus (CoV) led to an unprecedented demand for diagnostic tests to determine the presence of the virus. Sometimes this demand greatly exceeded the production capacities of manufacturers, with a consequent lengthening of delivery times and often a supply failure. In fact, the epidemic caused by the SARS-CoV-2 virus had an unexpectedly rapid spread; in just a few months nearly every country in the world reported infections. In very little time it went from an epidemic to a pandemic status. Therefore, identifying everyone who has contracted a coronavirus infection, whether asymptomatic or symptomatic, is of vital importance in efforts to isolate outbreaks of infection and limit the spread of COVID-19 as much as possible.

Furthermore, it has become increasingly important also to identify people who have been exposed to COVID-19 and have thus developed antibodies against the Sars- CoV-2 virus. In fact, the presence of antibodies in blood makes it possible to assess whether a person has protection against COVID-19.

In addition, the determination of the presence of antibodies against the Sars-CoV-2 virus makes it possible to assess vaccine effectiveness and the duration of protection over time. Several rapid qualitative tests and several quantitative serological tests are available today on the market.

Rapid qualitative serological tests make it possible to find out whether an individual has come into contact with the virus and whether his or her immune system has thus produced antibodies in response. Quantitative serological tests, on the other hand, allow a specific assay of the antibodies produced.

The antibodies involved are immunoglobulins, IgM (the first to be produced in case of infection) and IgG (they follow IgM, when the level of the former decreases). If IgG is detected in a blood sample, it means that the infection occurred in the past.

Therefore, a serological test reveals the presence of antibodies against the virus and indicates any previous exposure to SARS-CoV-2; the positivity is late, and it is thus not a suitable test for detecting an ongoing infection. This type of test can be useful in the field of epidemiology for estimating the spread of infection within a community.

Furthermore, serological tests entail a waiting time of several days before a result is obtained and have a rather high cost. Rapid tests, on the other hand, enable solely a result of a qualitative type, i.e. the presence or absence of antibodies, to be obtained rapidly and at a low cost.

Thus, there is a greatly felt need for a test that enables a quantitative or semi- quantitative result to be obtained rapidly and at a low cost.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to an immunoassay to determine the presence and concentration of antibodies capable of binding a protein antigen in a biological sample isolated from an individual. Said immunoassay preferably comprising:

- the protein antigen and

- a plurality of anti-human antibody concentrations.

Preferably, said protein antigen is the S1 subunit of the spike glycoprotein or at least a portion thereof, even more preferably it is the receptor-binding domain (RBD) of the S1 subunit (RBD-S1 ) of the spike glycoprotein of a virus belonging to the Coronaviridae family.

A second aspect of the present invention relates to the use of the immunoassay to determine the presence and concentration of antibodies capable of binding an antigen in a biological sample. Preferably, the immunoassay is used to detect and determine the relative concentration of antibodies capable of binding a protein, or a part thereof, of the Sars-CoV-2 virus, as described above in detail.

A third aspect of the present invention relates to a method for determining the presence and concentration of antibodies capable of binding an antigen in a biological sample. Preferably, the method comprises the steps of a) bringing the isolated biological sample into contact with the protein antigen in order to obtain a protein antigen-antibody complex, b) contacting the protein antigen-antibody complex obtained in step (a) with a plurality of concentrations of anti-human antibodies, and c) detecting the binding of the protein antigen-antibody complex to at least one of the different concentrations of anti-human antibodies.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a schematic representation of the immunoassay according to the present invention.

DEFINITIONS

In the context of the present invention, the term “spike” means a glycoprotein structure present as a protuberance on the outside of the viral envelope, the double lipid layer that encloses some viruses, present in viruses of the Coronaviridae family. These protuberances bind to some receptors of the host cell and are essential both for host specificity and for viral infectivity.

In the context of the present invention, the term coronavirus (abbreviated as “CoV”) means a broad family of respiratory viruses that can cause diseases from mild to moderate, which range from the common cold to respiratory syndromes such as MERS (Middle East respiratory syndrome) and SARS (severe acute respiratory syndrome).

In the context of the present invention, the term “anti-human antibodies” means affinity-purified antibodies with a well-characterised specificity for human immunoglobulins.

In the context of the present invention, the term “anti-mouse antibodies” means affinity-purified antibodies with a well-characterised specificity for mouse immunoglobulins.

In the context of the present invention, the term “individual” means a human or animal individual.

In the context of the present invention, the term “BAU” means “binding antibody units”, which is a unit of measurement established by the World Health Organization (WHO) as an international standard. DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to an immunoassay to determine the concentration of antibodies capable of binding a protein antigen in a biological sample isolated from an individual.

Said immunoassay preferably comprising:

- the protein antigen and

- a plurality of anti-human antibody concentrations.

In one embodiment, the antibodies capable of binding the protein antigen are immunoglobulins of the IgG and/or IgM class, preferably of the IgG class.

In one embodiment, the immunoassay further comprises anti-mouse antibodies and antibodies obtained in mice (mouse antibodies). Preferably, the antibodies obtained in mice (mouse antibodies) are conjugated with at least one marker, preferably with at least a gold nanoparticle, preferably a colloidal gold nanoparticle. Alternatively, the antibodies obtained in mice (mouse antibodies) are conjugated with an immunofluorescent probe, or with biotin or else with an enzymatic marker.

Preferably, the protein antigen is a protein, preferably a spike glycoprotein, or at least a portion thereof, of a virus belonging to the Coronaviridae family, selected from: SARS-CoV, MERS-CoV and SARS-CoV-2. In a preferred embodiment of the invention, the protein antigen is a protein, preferably a spike glycoprotein, of the SARS-CoV-2 virus or at least a portion thereof.

In one embodiment, the protein antigen comprises an amino acid sequence substantially identical to SEQ ID NO.1. For example, the protein antigen comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or more to SEQ ID NO. 1.

In a preferred embodiment, the protein antigen comprises an amino acid sequence at least 80%, more preferably at least 90% identical to SEQ ID NO. 1 .

In one embodiment, the protein antigen consists of an amino acid sequence substantially identical to SEQ ID NO. 1. For example, the protein antigen consists of an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or more to SEQ ID NO. 1. In a preferred embodiment, the protein antigen consists of an amino acid sequence at least 80%, more preferably at least 90% identical to SEQ ID NO. 1 .

Preferably, said protein antigen is the S1 subunit of the spike glycoprotein or at least a portion thereof; even more preferably, it is the receptor-binding domain (RBD) of the S1 subunit (RBD-S1 ) of the spike glycoprotein of a virus belonging to the Coronaviridae family.

Preferably, said protein antigen is the S1 subunit of the spike glycoprotein or at least a portion thereof, even more preferably, it is the RBD-SD1 of the spike glycoprotein of SARS-CoV-2.

In one embodiment of the invention, the protein antigen comprises modifications to the N-terminal and/or C-terminal region. Said modifications are preferably selected from deletions, additions, alterations of amino acids and combinations thereof. Alternatively, said protein antigen can be modified, preferably in the primary structure thereof, by acetylation, carboxylation, phosphorylation and combinations thereof. In one embodiment, the biological sample is selected from serum, plasma, venous blood or capillary blood.

In one embodiment, the protein antigen is conjugated with at least one marker, preferably with at least one gold nanoparticle, preferably a colloidal gold nanoparticle. Alternatively, the protein antigen is conjugated with an immunofluorescent probe, or with biotin or else with an enzymatic marker.

Table 1

In one embodiment of the invention, the immunoassay comprises at least two, preferably at least three different concentrations of said anti-human antibodies. In one embodiment, the anti-human antibodies are immunoglobulins of the IgG and/or IgM class, preferably of the IgG class. In a preferred embodiment of the invention, the immunoassay is capable of detecting a protein antigen concentration in the biological sample between 15 and 1500 BAU/ml.

Preferably, said immunoassay comprises a first concentration of said anti-human antibodies capable of detecting a concentration of antibodies binding the protein antigen in the biological sample, said concentration being between 15 and 200 BAU/ml (binding antibody units), preferably between 20 and 170 BAU/mL

Preferably, said immunoassay comprises a second concentration of said anti-human antibodies capable of detecting a concentration of antibodies binding the protein antigen in the biological sample, said concentration being between 100 and 1000 BAU/ml, preferably between 150 and 900 BAU/ml.

Preferably, said immunoassay comprises a third concentration of said anti-human antibodies capable of detecting a concentration of antibodies binding the protein antigen in the biological sample, said concentration being higher than 700 BAU/ml, preferably higher than 800 BAU/ml.

Preferably, said immunoassay comprises a first concentration of said anti-human antibodies between 0.03 and 0.3 mg/ml, a second concentration between 0.3 and 0.7 mg/ml, and a third concentration between 1 and 5 mg/ml.

In one embodiment of the invention, said immunoassay comprises a first concentration of said anti-human antibodies between 0.05 and 0.3 mg/ml, a second concentration between 0.4 and 0.6 mg/ml, and a third concentration between 2 and 4 mg/ml.

With reference to figure 1 , the anti-human antibodies are fixed on a support 100. Preferably, said support 100 is composed of a material selected from: cellulose, nitrocellulose or nylon, it is preferably composed of nitrocellulose. Said different antihuman antibody concentrations are preferably fixed on the support 100 separately from one another.

In one embodiment, the protein antigen is contained in an absorbent medium 10, preferably an absorbent medium made of paper or another absorbent material known to the person skilled in the art. The absorbent medium 10 preferably also comprises the antibodies obtained in mice (mouse antibodies

In one embodiment, said absorbent medium 10 is fixed or positioned on the support 100.

In a preferred embodiment of the invention, the different anti-human antibody concentrations are fixed, in order, at different positions of the support 100, i.e. the first concentration is fixed at a first position 1 on the support 100 which is proximal relative to the absorbent medium 10; the second concentration and the third concentration are fixed sequentially at a second position 2 and third position 3, distally relative to the absorbent medium 10.

In one embodiment, the anti-mouse antibodies are fixed on the support 100, they are preferably fixed at a fourth position 4.

Preferably, the biological sample isolated from the individual is brought into contact with the protein antigen and with the antibodies obtained in mice (mouse antibodies), or with the absorbent medium 10. In one embodiment, if the biological sample comprises at least one antibody capable of binding the protein antigen, a protein antigen-antibody complex is formed. The protein antigen-antibody complex that is formed binds to at least one of the different concentrations 1 , 2, 3 of anti-human antibodies fixed on the support 100.

As the first concentration of anti-human antibodies is at the first position 1 , i.e. the one closest to the absorbent medium 10 on the support 100, the protein antigenantibody complex binds at least to the first concentration of anti-human antibodies.

If the amount of protein antigen-antibody complexes is greater than the binding capacity of the first concentration of anti-human antibodies, the protein antigenantibody complex will migrate towards the second position 2 and also bind to the second concentration of anti-human antibodies.

If the amount of protein antigen-antibody complexes is also greater than the binding capacity of the second concentration of anti-human antibodies, the protein antigenantibody complex will migrate towards the third position 3 and also bind to the third concentration of anti-human antibodies.

The binding of the protein antigen-antibody complex to each of the different antihuman antibody concentrations on the support 100 is preferably detected with the methods known to the person skilled in the art, more preferably by direct observation. In one embodiment, the protein antigen is conjugated with at least one gold nanoparticle and the binding of the protein antigen-antibody complex to the fixed antihuman antibodies results in the appearance of at least one coloured band detectable by direct observation.

Preferably, the appearance of a coloured band at the first position 1 corresponds to a concentration of antibodies capable of binding the protein antigen in the isolated sample defined as “low”, preferably between 15 and 200 BAU/ml, more preferably between 20 and 170 BAU/ml.

Preferably, the appearance of a coloured band at the second position 2 corresponds to a concentration of antibodies capable of binding the protein antigen in the isolated sample defined as “medium”, preferably between 100 and 1000 BAU/ml, more preferably between 150 and 900 BAU/ml.

Preferably, the appearance of a coloured band at the third position 3 corresponds to a concentration of antibodies capable of binding the protein antigen in the isolated sample defined as “high”, preferably higher than 700 BAU/ml, more preferably higher than 800 BAU/ml.

In one embodiment, the antibodies obtained in mice (mouse antibodies) comprised in the absorbent medium 10 and brought into contact with the sample isolated from the individual also migrate towards the different anti-human antibody concentrations. The antibodies obtained in mice migrate and form an antigen-antibody complex with the anti-mouse antibodies at the fourth position 4 of the support 100. Said antigenantibody complex is detected with means known to the person skilled in the art, preferably by direct observation.

In one embodiment, the antibodies obtained in mice (mouse antibodies) are conjugated with at least one gold nanoparticle and the binding thereof to the antimouse antibodies results in the appearance of a coloured band detectable by direct observation. Preferably, the appearance of a coloured band at the fourth position 4 indicates that the isolated sample brought into contact both with the protein antigen and with the antibodies obtained in mice (anti-mouse antibodies) has migrated correctly in the support. In other words, the appearance of a coloured band at the fourth position 4 is used as an internal control.

The Applicant has in fact discovered that by fixing increasing concentrations of antihuman antibodies, preferably of the IgG type, on a support, for example nitrocellulose, it is possible to determine the relative concentration of antibodies in a biological sample. In fact, the immunoassay comprises a protein antigen, for example a domain of the spike protein of the Sars-CoV-2 virus which, in the presence of antibodies capable of binding the antigen in the biological sample to be tested, causes the creation of an antigen-antibody complex which is in turn bound by the anti-human antibodies fixed on the support. The presence of colloidal gold linked to the antigen enables a direct observation and detection of both the presence of antibodies in the biological sample and the concentration of said antibodies in the biological sample. In other words, the immunoassay makes it possible both to detect the presence of antibodies in the biological sample and to have a relative quantification of the antibodies.

A second aspect of the present invention relates to the in vitro use of the immunoassay described above in detail to determine the presence and concentration of antibodies capable of binding an antigen in a biological sample. Preferably, the immunoassay is used to detect and determine the relative concentration of antibodies capable of binding a protein, or a part thereof, of the Sars-CoV-2 virus, as described above in detail.

In one embodiment, the use of the immunoassay comprises at least a step of bringing the isolated biological sample into contact with the protein antigen.

A third aspect of the present invention relates to an in vitro method for determining the presence and concentration of antibodies capable of binding a protein antigen in a biological sample.

Said protein antigen is preferably the S1 subunit of the spike glycoprotein or at least a portion thereof, even more preferably it is the receptor-binding domain (RBD) of the S1 subunit (RBD-S1 ) of the spike glycoprotein of a virus belonging to the Coronaviridae family, as described above in detail.

In one embodiment, the method comprises the steps of a) bringing the isolated biological sample into contact with the protein antigen in order to obtain a protein antigen-antibody complex, b) contacting the protein antigen-antibody complex obtained in step a) with a plurality of increasing concentrations of anti-human antibodies, and c) detecting the binding of the protein antigen-antibody complex to at least one of the several increasing concentrations of anti-human antibodies.

In one embodiment of the invention, the anti-human antibody concentrations are at least two, preferably at least three different concentrations of said anti-human antibodies, as described above in detail.

In one embodiment, the antigen-antibody complex obtained in step a) is brought into contact with the first concentration of anti-human antibodies. Preferably, if the amount of protein antigen-antibody complexes is greater than the binding capacity of the first concentration of anti-human antibodies, the protein antigen-antibody complex will be brought into contact with the second concentration of anti-human antibodies.

Preferably, if the amount of protein antigen-antibody complexes is also greater than the binding capacity of the second concentration of anti-human antibodies, the antigen complex will be brought into contact with the third concentration of antihuman antibodies.

Preferably, the detection in step c) of the binding of the protein antigen-antibody complex to the first anti-human antibody concentration corresponds to a concentration of antibodies capable of binding the protein antigen in the isolated sample defined as “low”, preferably between 15 and 200 BAU/ml, more preferably between 20 and 170 BAU/ml.

Preferably, the detection in step c) of the binding of the protein antigen-antibody complex to the second anti-human antibody concentration corresponds to a concentration of antibodies capable of binding the protein antigen in the isolated sample defined as “medium”, preferably between 100 and 1000 BAU/ml, more preferably between 150 and 900 BAU/ml.

Preferably, the detection in step c) of the binding of the protein antigen-antibody complex to the third anti-human antibody concentration corresponds to a concentration of antibodies capable of binding the protein antigen in the isolated sample defined as “high”, preferably higher than 800 BAU/ml.

In a preferred embodiment of the invention, the method is implemented using the immunoassay as described above in detail.

EXAMPLE

1. Materials and Methods

The kit is an immunoassay with a lateral flow chromatography system.

The kit comprises a strip that includes:

1) a pad containing the RBD subunit of the spike protein of the Sars-CoV-2 virus (S- RBD) conjugated with colloidal gold, with mouse IgG antibodies.

2) a nitrocellulose membrane containing high (line H), medium (line M), and low (line L) concentrations of anti-human IgG antibodies and a control line (C). The lines H, M, and L are coated with a different concentration of anti-human IgG antibodies. When the sample is added, if anti-SARS-CoV-2 IgG antibodies are present in the sample, the anti-SARS-CoV-2 IgG antibodies and the S-RBD conjugated with colloidal gold will form an antigen-antibody complex.

These complexes will continue to migrate along the strip as far as the lines H, M and L, where they will be captured by the anti-human IgG, visible red lines will be generated at the lines H, M, and L, according to the concentration of anti-SARS-CoV- 2 IgG antibodies. If the sample does not contain anti-SARS-CoV-2 IgG antibodies or if the level of antibodies is too low, the lines H, M and L will not appear.

The line C is coated with anti-mouse IgG antibodies, obtained in goats, which bind to the antibodies obtained in mice conjugated with colloidal gold and form a red line irrespective of the presence of anti-SARS-CoV-2 IgG antibodies in the sample.

2. Preparation of SARS-CoV-2 S-RBD

SARS-CoV-2 S-RBD recombinant protein (Bio-Mapper, Fapon, Genscript)

Anti-Human IgG (Boson)

Colloidal gold solution

0.1 mol/L phosphate buffer

10% BSA solution

0.01 mol/L phosphate buffer-BSA

1 mL of 0.1 M phosphate buffer was added slowly to the colloidal gold solution under stirring. 0.08 mg of S-RBD were added and the solution was kept under stirring for 30 minutes.

1 ml of 10% BSA solution was added and the solution was kept under stirring for 30 minutes and it was then centrifuged at 12000 rpm, 4°C for 30 minutes; the supernatant was discarded.

The precipitate was resuspended with 2 mL of 0.01 mol/L phosphate buffer-BSA and stored at 2-8 °C.

The labelled colloidal gold was joined to the nitrocellulose membrane (Satorus CN1410) coated with anti-human IgG antibodies so as to produce a reagent strip for comparison.

Selection criteria

Sensitivity: sample L (low) detected; Linearity: identification of L/M/H and the colour difference is more than 1 +

White: not detected

Test result

Table 2

Conclusion

The SARS-CoV-2 S-RBD supplied by Genscript has the best performance.

3. Anti-human IgG screening

SARS-CoV-2 S-RBD recombinant protein (Genscript)

Anti-human IgG (Boson, Fapon, Bio-Mapper)

Colloidal gold solution

0.1 mol/L phosphate buffer 10% BSA solution

0.01 mol/L phosphate buffer-BSA

Experimental method

1 mL of 0.1 M buffer phosphate was added under stirring to 10 mL of colloidal gold solution, 0.08 mg of S-RBD, and 1 mL of 10% BSA solution.

The solution was centrifuged at 12000 rpm at 4°C for 30 minutes. The precipitate was resuspended in 2 ml of 0.01 mol/L phosphate buffer-BSA.

The nitrocellulose membrane (Satorus CN1410) was coated with anti-human IgG with a certain concentration of 0.2 mg/ML, 1 p L/cm.

The labelled colloidal gold was added onto the nitrocellulose membrane coated with anti-human IgG to create a reagent strip for comparison.

Eligibility criteria

Sensitivity: sample L identified

Linearity: L/M/H White: not identified

Test results

Table 3

Conclusions

SARS-CoV-2-RBD supplied by Bio-Mapper has the best performance.

4. Optimisation of the concentration of anti-human IgG in line L

Materials

SARS-CoV-2 S-RBD labelled with colloidal gold (Genscript)

Anti- Bio Mapper

Nitrocellulose (Satorius CN140)

Experimental method

The nitrocellulose membrane was coated with anti-human IgG with the following concentrations in position L, no coating in positions M and H.

Table 4

Eligibility criteria

Sensitivity: sample L identified

White: not identified

Line L test result

Table 5

Conclusions

The concentration of anti-human IgG should be 0.2 mg/L

5. Optimisation of the concentration of anti-human IgG in line M

Materials

SARS-CoV-2 S-RBD labelled with colloidal gold (Genscript)

Anti- Bio Mapper

Nitrocellulose (Satorius CN140)

Experimental method

The nitrocellulose membrane was coated with anti-human IgG with a concentration of 0.2 mg/ml in position L and with the following concentrations in position M, no coating in position H.

Table 6

Eligibility criteria

Sensitivity: sample L identified in line L; sample L not identified in line M; sample M identified in line M.

White: not identified

Line L test result

Table 7

Line M test result Table 8

Conclusion

The concentration of anti-human IgG should be 0.5 mg/L

6. Optimisation of the concentration of anti-human IgG in line H

Materials

SARS-CoV-2 S-RBD labelled with colloidal gold (Genscript)

Anti- Bio Mapper

Nitrocellulose (Satorius CN140)

Experimental method

The nitrocellulose membrane was coated with anti-human IgG with a concentration of 0.2 mg/ml in position L, with a concentration 0.5 mg/ml in position M and with the following concentrations in position H.

Table 9

Eligibility criteria

Sensitivity: sample L identified in position L; sample L not identified in position M; sample M identified in position M; sample M not identified in position H; sample H identified in position H.

White: not identified

Line L test result

Table 10

Line M test result

Table 11

Line H test result

Table 12

Conclusions

Concentration of anti-human IgG should be 3 mg/L

7. Optimisation of the amounts of SARS-CoV-2 RBD labelled with colloidal gold

Materials

SARS-CoV-2 S-RBD labelled with colloidal gold (Genscript)

Anti- Bio Mapper

Nitrocellulose (Satorius CN140)

Experimental method

The nitrocellulose membrane was coated with anti-human IgG with a concentration of 0.2 mg/ml in position L, with a concentration 0.5 mg/ml in position M and a concentration of 3 mg/ml in position H. The SARS-CoV-2 S-RBD recombinant protein labelled with colloidal gold was fixed on the membrane in the following amounts.

Table 13 Eligibility criteria

Sensitivity: sample L identified in position L; sample L not identified in position M; sample M identified in position M; sample M not identified in position H; sample H identified in position H.

White: not identified Line L test result

Table 14

Conclusions

The amount of SARS-CoV-2 S-RBD labelled with colloidal gold should be 3 pL/cm.