Patent Grant
12145980
This Application is a 371 of PCT/CL2019/050155 filed on Dec. 27, 2019, which, in turn, claimed the priority of Chilean Patent Application No. 3871-2018 filed on Dec. 28, 2018, both applications are incorporated herein by reference.
Monoclonal antibodies, or fragments thereof are disclosed, that recognize the PB2 protein of the human influenza virus (Flu), where said monoclonal antibodies or fragments thereof comprise an antibody that comprises a light chain variable region where its CDR1 (CDRLC1) is defined according to SEQ ID NO: 1, its CDR2 (CDRLC2) is defined by SEQ ID NO: 2 and its CDR3 (CDRLC3) corresponds to SEQ ID NO: 3, and a heavy chain variable region where its CDR1 (CDRHC1) is defined according to SEQ ID NO: 4, its CDR2 (CDRHC2) is defined by SEQ ID NO: 5 and its CDR3 (CDRHC3) corresponds to SEQ ID NO: 6, or an antibody comprising a light chain variable region where its CDR1 (CDRLC1) is defined according to SEQ ID NO: 7, its CDR2 (CDRLC2) is defined by SEQ ID NO: 8 and its CDR3 (CDRLC3) corresponds to SEQ ID NO: 9, and a heavy chain variable region where its CDR1 (CDRHC1) is defined according to SEQ ID NO: 10, its CDR2 (CDRHC2) corresponds to SEQ ID NO: 11 and its CDR3 (CDRHC3) corresponds to SEQ ID NO: 12, where said antibody can be used as detection or capture antibody. Additionally, a method for diagnosing Flu infection in a biological sample is provided that uses monoclonal antibodies in diagnostic kit format to detect Flu, where said kit comprises at least one monoclonal antibody against Flu as previously described.
The present invention relates to monoclonal antibodies, or fragments thereof, that recognize the PB2 protein of the human influenza virus, useful for the development of diagnostic methods of influenza infection in humans.
Influenza is an infectious disease of the respiratory tract caused by the human influenza virus. This virus is responsible for producing severe or mild respiratory symptoms, mainly affecting the areas of the nose, throat, bronchial tubes and occasionally the lungs. In general, clinical symptoms of influenza are similar to those of seasonal flu, however, symptoms can be variable and range from an asymptomatic infection to severe pneumonia that can lead to death.1. The virus is easily transmitted from person to person or through drops or small particles that have been expelled through the cough or sneeze of a sick person, which makes it spread quickly and be part of seasonal epidemics. 11. www.who.int/csr/disease/swineflu/faq/es/#doesit
Influenza virus can be detected throughout the year, but its detection increases in the autumn-winter season, although time and duration can be variable.2. According to epidemiological statistics, in the United States in 2016, 310,000 people were hospitalized for complications related to influenza. In the same country, statistics indicate that this infection causes around 89,000 deaths annually. From the point of view of economic cost, losses due to human influenza viruses in the United States are estimated to reach an annual cost that ranges from 71 to 150 billion dollars3. 2 espanol.cdc.gov/enes/flu/about/season/flu-season.htm3 espanol.cdc.gov/enes/flu/about/disease/us_flu-related_deaths.htm
The most common diagnostic methods for the detection of Flu are called “Rapid Influenza Diagnostic Tests” (RIDT), these tests are based on the detection of Flu antigens (immunoassay) in swab or nasopharyngeal aspirate samples. These tests can give a result in a period of 15 to 20 minutes, however, they lack sensitivity and only confer a qualitative result (positive or negative), which can potentially be a false negative due to its low specificity4. 4 espanol.cdc.gov/enes/flu/professionals/diagnosis/rapidlab.htm
Until now, the standard technique for confirming an influenza virus infection corresponds to molecular analysis of reverse transcriptase polymerase chain reaction (RT-PCR). For example, Human Influenza Virus Real-Time RT-PCR Diagnostic Panel developed by the Center for Disease Control and Prevention (CDC) enables in vitro detection of influenza virus in respiratory tract samples from human patients exhibiting signs and symptoms of respiratory infection. This method detects influenza A and B viruses through the reaction of primers against genes encoding highly conserved proteins, such as matrix protein (M protein), nucleoprotein (NP protein) and non-structural protein (NS protein). This type of technique has disadvantages in terms of cost and optimization, since its implementation and start-up requires the acquisition of high-cost specialized equipment and reagents, as well as highly trained personnel.
Another common method for diagnosis is viral isolation in cell cultures. The problem with this type of technique is that it requires highly specialized equipment and personnel. On the other hand, it is a slow method that can deliver diagnostic results within 5 to 14 days after its initiation.
In the practice of clinical diagnosis, one of the main difficulties or problems is the sample itself, since a limited amount of it can be accessed, which also has a low antigen concentration and includes the presence of other proteins and cellular components that can interfere with the detection reaction.
Monoclonal antibodies have been previously described for the detection of human influenza virus antigens. In WO2012045001 (A2), for example, a human monoclonal antibody is disclosed that binds to the surface protein hemagglutinin. In U.S. Pat. No. 9,650,434B2a monoclonal antibody or antigen-binding fragment thereof is provided, which can specifically bind to HA1 domain of the hemagglutinin protein of influenza viruses H1 subtype and H5 subtype. In both documents, the proposed solution aims at a different antigen from the PB2 protein detection, and the efficiency, specificity and sensitivity of the antigen-antibody binding in clinical samples is not demonstrated.
Regarding detection of the Flu PB2 protein, JP2015189715 (A) provides a monoclonal antibody or an antigen-binding fragment thereof that binds to the PB2 subunit of RNA-dependent RNA polymerase. Sequences of the heavy chain variable regions and light chain variable regions of the antibody described therein differ substantially from the monoclonal antibodies' sequences part of the invention. On the other hand, JP2015189715 (A) does not perform antigen detection assays in human clinical samples, nor the specificity and sensitivity features of the antibodies is determined, in the context of clinical diagnosis. Let us remember that in clinical diagnostic conditions, biological samples that include very low concentrations of antigen are used, which hinders the specificity and sensitivity of the antigen-antibody reaction.
Therefore, a new alternative for the diagnosis of the human influenza virus is required that, unlike molecular diagnostic tests and cell culture tests that entail longer response times and a high cost for their implementation and maintenance, allows the detection of a wide variety of influenza types and subtypes quickly, sensitively, specifically and at a lower cost. Furthermore, even though until now monoclonal antibodies have been proposed for detection of other Flu proteins and even against PB2, these antibodies have only been evaluated in murine models and do not correspond in any case to a solution to the posed technical problem.
According to information provided, monoclonal antibodies that detect PB2 protein are proposed to be used in detection and rapid, efficient and accurate diagnosis in patients infected with Flu, where said antibodies specifically detect the protein in clinical samples at very low concentrations of the specific antigen (high sensitivity), even distinguishing the specific viral antigen in clinical samples that even include antigens from other respiratory viruses. Additionally, provided antibodies can form part of a diagnostic method and kit for the Flu diagnosis, where each antibody can be used in a versatile way as a detection antibody as well as a capture antibody.
The present invention relates to specific monoclonal antibodies against PB2 protein fragments thereof, of the human influenza virus. In particular, the invention corresponds to monoclonal antibodies or fragments thereof secreted by hybridoma cell lines called 1A3E2 and 2F11B1, that recognize the PB2 protein of the human influenza virus (Flu), where said monoclonal antibodies or fragments thereof comprise an antibody that comprises a light chain variable region where its CDR1 (CDRLC1) is defined according to SEQ ID NO: 1, its CDR2 (CDRLC2) is defined by SEQ ID NO: 2 and its CDR3 (CDRLC3) corresponds to SEQ ID NO: 3, and a heavy chain variable region where its CDR1 (CDRHC1) is defined according to SEQ ID NO: 4, its CDR2 (CDRHC2) is defined by SEQ ID NO: 5 and its CDR3 (CDRHC3) corresponds to SEQ ID NO: 6, or an antibody comprising a light chain variable region where its CDR1 (CDRLC1) is defined according to SEQ ID NO: 7, its CDR2 (CDRLC2) is defined by SEQ ID NO: 8 and its CDR3 (CDRLC3) corresponds to SEQ ID NO: 9, and a heavy chain variable region where its CDR1 (CDRHC1) is defined according to SEQ ID NO: 10, its CDR2 (CDRHC2) corresponds to SEQ ID NO: 11 and its CDR3 (CDRHC3) corresponds to SEQ ID NO: 12, where said antibody can be used as detection or capture antibody. Additionally, a method for diagnosing Flu infection in a biological sample is provided that uses monoclonal antibodies in diagnostic kit format to detect Flu, where said kit comprises at least one monoclonal antibody against Flu as previously described.
Antibodies described in the invention have important advantageous and remarkable technical features with respect to other antibodies and methods for detecting viral antigens already existing.
First, each virus has specific surface proteins, therefore, other diagnostic techniques based on monoclonal antibodies for other types of respiratory viruses are not comparable to the proposed invention. Detection specificity of antibodies against Flu PB2 protein respect to other viral antigens, for example against adenovirus pill protein, is demonstrated in the results provided in
Second, antibodies that are part of the scope of the invention allow specific detection of PB2 protein fragments thereof, in such a way that they do not compete with each other for the antigen-binding site, nor do they exert an impediment to simultaneously bind to it.
Third, they allow the detection of PB2 protein or fragments thereof with high sensitivity in samples containing a small antigen amount, such as nasopharyngeal swab samples, for example.
The proposed monoclonal antibodies are capable of detecting PB2 protein, a highly conserved protein. Strategy for detecting a conserved viral protein allows antibodies that are part of the scope of the invention to detect different types of human influenza, including influenza A, B and C.
When reference is made to CDR sequences in the present invention, these correspond to short sequences found in the variable domains of proteins that have antigen detection function. CDR sequences for the heavy chain (CDRHC) and light chain (CDRLC) of the antibodies secreted by hybridomas 1A3E2 and 2F11B1.
Monoclonal antibodies described can be used for Flu infection determination, diagnosis and/or detection assays. These antibodies can be used simultaneously to increase detection sensitivity of clinical samples where there is little quantity and availability of antigen. In this regard, it is also provided a Flu infection diagnosis method in a biological sample, which comprises contacting the biological sample with the monoclonal antibody against Flu PB2 protein or a fragment thereof according to the claim, and detecting the binding of the antibody with the antigen. Biological sample can be, but is not limited to, infected in vitro cells with Flu, nasal secretions, nasal washes, cerebrospinal fluid, pharyngeal secretions and/or bronchial washes or secretions. As part of the method, the assay used for the detection of antigen-antibody binding is selected from ELISA, luminex, immunofluorescence, immunohistochemistry, immunochromatography, flow cytometry, cell sorter, immunoprecipitation and/or Western blot.
Present invention also includes a diagnostic kit to detect the human influenza virus, which comprises: a monoclonal antibody against Flu PB2 protein or a fragment thereof, where said antibody can act as a capture or detection antibody, where particularly, the detection antibody is conjugated to a marker for its detection; a solid support to which the antibody is attached; and reagents for detecting the marker included in the detection antibody, such as fluorophores, biotin, radioisotopes, metals, and enzymes.
In the present invention when it refers to capture antibody, this corresponds to the antibody that specifically binds to the antigen. In the case of the detection antibody, this corresponds to the antibody to which a marker is conjugated to be detected by different tests such as immunochromatographic test, luminex, flow cytometry, immunofluorescence, radioimmunoassay, Western blot, Dot plot, ELISA, luminex, immunodiffusion. or immunoprecipitation.
Antibodies that are part of the present invention can act dually as a capture antibody or as a detection antibody when coupled to a detection marker. Detection marker will be conjugated to the detection antibody, and this can correspond, without limitation, to fluorophores, biotin, radioisotopes, metals and enzymes. Preferably, the detection antibody is conjugated to reporter system based on the detection of horseradish peroxidase (HRP) enzyme activity.
1A3E2 hybridoma was grown in DMEM-high glucose culture medium supplemented with 3.7 g/L of Sodium Bicarbonate and 10% fetal bovine serum, at 37° C. (98.6° F.) with 10% CO2, up to a cell density of 700,000 cells/mL. Total RNA of 3.5×106 cells was obtained, performing a treatment with Trizol compound (Invitrogen). 0.5 μg of RNA was used to generate the cDNA by reverse transcription reaction with the PrimeScript™ 1st Strand cDNA Synthesis kit, which uses isotype-specific universal primers. The antibody heavy and light chain were amplified according to the GenScript rapid amplification of cDNA ends (RACE) standard operating procedure (SOP). Amplified antibody fragments were separately cloned into a standard cloning vector. PCR colony was performed to identify clones which have the correct size inserts. At least five colonies with inserts of the correct size were sequenced for each fragment. Sequences of different clones were aligned and the consensus sequence of these clones was provided. Nucleotide sequences of heavy and light chains of antibodies secreted by 1A3E2 hybridoma were identified, being identified as SEQ ID NO. 1 and SEQ ID NO.3 for the case of heavy chains and SEQ ID NO. 2 and SEQ ID NO.4 for the case of light chains.
2F11B1 hybridoma was grown in DMEM-high glucose culture medium supplemented with 3.7 g/L of Sodium Bicarbonate and 10% fetal bovine serum, at 37° C. (98.6° F.) with 10% CO2, up to a cell density of 700,000 cells/mL. Total RNA of 3.5×106 cells was obtained, performing a treatment with Trizol compound (Invitrogen). 0.5 μg of RNA was used to generate the cDNA by reverse transcription reaction with the PrimeScript™ 1st Strand cDNA Synthesis kit, which uses isotype-specific universal primers. The antibody heavy and light chain were amplified according to the GenScript rapid amplification of cDNA ends (RACE) standard operating procedure (SOP). Amplified antibody fragments were separately cloned into a standard cloning vector. PCR colony was performed to identify clones which have the correct size inserts. At least five colonies with inserts of the correct size were sequenced for each fragment. Sequences of different clones were aligned and the consensus sequence of these clones was provided. From this, nucleotide sequences of heavy and light chains of antibodies secreted by 2F11B1 hybridoma were determined, corresponding to those identified as SEQ ID NO. 1 and SEQ ID NO.3 to the light chains and sequences identified as SEQ ID NO. 1 and SEQ ID NO.3 to heavy chains.
This assay aims to demonstrate the specificity for Flu PB2 protein antibodies produced by 1A3E2 and 2F11B1 hybridomas. Antigen detection was carried out using the indirect ELISA technique, where ELISA plate was activated with 50 ng of purified antigen for 1 hour at 37° C. (98.6° F.). Similarly, the plate was activated with 20 μg of uninfected MDCK cell lysate (as a negative control) and infected with Flu serotype A virus. Another negative control included was 50 ng of ADV pIII protein in a separate well. Subsequently, the plate was washed twice with 1×/Tween20 0.05% phosphate buffered saline (PBS). The plate was then blocked for 2 hours at 37° C. (98.6° F.) with 1×PBS/10% Fetal Bovine Serum (FBS). Subsequently, the washes were repeated and then each antibody (1A3E2 and 2F11B1) were incubated at a final concentration of 3.4 μg/mL (170 ng per well), diluted in 1×PBS/10% FBS, for 1 hour at 37° C. (98.6° F.) (each antibody on a separate plate). Under the same conditions, on a different plate, a control assay was performed using a commercial monoclonal antibody that recognizes the PB2 protein of Flu (Anti-Influenza A virus PB2 protein antibody, catalog number GTX125926, GeneTex) at a concentration of 3.4 μg/mL. After incubation time, the washes were repeated and a secondary anti-mouse IgG antibody labeled with horseradish peroxidase (HRP) in dilution 1 in 2000 (1.8 ng/μl per well) was added to each well in 1×PBS/10% FBS, for 1 hour at room temperature (≈25° C. (77° F.)), in the dark. Finally, washes were carried out and it was developed with 50 μL of citrate/tetramethyl-benzidine buffer (TMB, 3,3′,5,5′-tetramethylbenzidine, 1 mg/mL, Becton Dickinson). To stop the reaction, 50 μL of H2SO4 2 N were added and the result was read on an ELISA reader, at 450 nm. To determine that the reaction of the secondary antibody was specific in recognizing the primary antibody and also that the obtained signal was not caused by nonspecific binding of the secondary antibody to the viral antigen, controls were carried out in which only the secondary antibody was used with no primary antibody or sample (well not activated). Another control to determine that the primary antibody reaction is specific for the antigen, consisted of using the antibodies on an ELISA plate that has not been activated with the antigen (with no antigen) or using the antibodies on an ELISA plate that possessed 50 ng of ADV pIII protein or uninfected cells. Results show that monoclonal antibodies of the invention are capable of recognizing 50 ng of purified antigen, specifically, since they do not recognize ADV pIII protein, nor proteins of uninfected cells (
Assay was performed to determine the maximum protein dilution that Flu anti-PB2 monoclonal antibodies from 1A3E2 and 2F11B1 hybridomas are able to detect by indirect ELISA. For this, the same technique described in example 3 was used. The plate was activated with 11 serial dilutions of Flu PB2 protein 1:2, starting with 50 ng of purified antigen. Anti-PB2 1A3E2 and 2F11B1 antibodies were used in a concentration of 3.4 μg/mL (170 ng/well), and were diluted in 1×PBS/10% FBS. Subsequently, anti-mouse IgG detection antibody was added in a dilution of 1:2,000 (1.8 ng/μL per well) and incubated for 1 hour at room temperature (≈25° C. (77° F.)), in the dark. Finally, the washes were carried out and it was developed with 50 μL of citrate/Tetramethylbenzidine (TMB, 3-3′-5-5′-tetramethylbenzidine, 1 mg/mL, Becton Dickinson) buffer. To stop the reaction, 50 μL of H2SO4 2 N were added and the result was read on an ELISA reader, at 450 nm. Results showed that anti-PB2 1A3E2 antibody is capable of detecting up to 780 picograms (pg) of the Flu PB2 protein (
Assay was performed to determine the maximum dilution of Flu anti-PB2 monoclonal antibodies from 1A3E2 and 2F11B1 hybridomas which allow the detection of the viral antigen. For this, a plate was activated with 50 ng of purified antigen (protein PB2) and then the plate was blocked for 2 hours at 37° C. (98.6° F.) with 1×PBS/10% Fetal Bovine Serum (FBS). Anti-PB2 1A3E2 and 2F11B1 antibodies were used in 1:2 dilutions, starting from the working concentration (170 ng) up to dilution 11 (0.15 ng) in 1×PBS/10% FBS. Subsequently, anti-mouse IgG detection antibody was added in a dilution of 1:2000 (1.8 ng/μL per well) incubated for 1 hour at room temperature (≈25° C. (77° F.)), in the dark. Finally, the washes were carried out and it was developed with 50 μL of citrate/Tetramethylbenzidine (TMB, 3-3′-5-5′-tetramethylbenzidine, 1 mg/mL, Becton Dickinson) buffer. To stop the reaction, 50 μL of H2SO4 2 N were added and the result was read on an ELISA reader, at 450 nm. In
Availability and concentration of viral proteins is generally very low in clinical samples of nasopharyngeal swabs, so it was necessary to modify the ELISA assay that was previously performed. For this assay, a Sandwich ELISA was performed, using anti-PB2 antibody from the Flu 1A3E2 hybridoma as capture antibody and Flu anti-PB2 2F11B1 clone as detection antibody. Flu anti-PB2 2F11B1 detection antibody was conjugated to the HRP. Wells of an ELISA plate were activated with 3.4 μg/mL (170 ng/well) of anti-PB2 antibody from Flu 1A3E2 hybridoma, diluted in 1×PBS, for 1 hour at 37° C. (98.6° F.). 2 washes were carried out with 1×-Tween20 PBS 0.05% and later the plate was blocked with 200 μL of 1×PBS/10% FBS for 1 hour at 37° C. (98.6° F.). Washed again and incubated for 1 hour at 37° C. (98.6° F.) each well with 50 μL of nasopharyngeal swabs (previously treated) from patients positive for Flu according to the diagnostic method “D3 Ultra DFA Respiratory Virus Screening and ID ( ) Kit de DHI (Diagnostics Hibryds) USA”, routinely referred to as “viral panel”, and which were treated as described later. As controls were included: 1) specificity control: 50 μL of sample of patients diagnosed with Flu were used by the viral panel for anti-Flu antibodies; 2) positive control: 50 ng of recombinant PB2-Flu protein; 3) Negative control: corresponding to healthy control samples. Subsequently, the 2 corresponding washes were carried out with 1×-Tween20 PBS 0.05% and each well was incubated for 1 hour at room temperature with 50 μl of anti-PB2 antibody from 2F11B1 hybridoma, conjugated with HRP (1.8 ng/μL of final concentration). Detection antibodies were incubated for 1 hour at room temperature (≈25° C. (≈77° F.)), in the dark. The plate was then washed 2 more times, developed with 50 μL of TMB solution and incubated for 15 minutes in the dark. The reaction stopped with 50 μL of H2SO4 2N and the plate was read at 450 nm in an ELISA reader (Epoch model), certified for clinical diagnosis.
Obtained results for this test are shown in
This assay demonstrates the versatility of the antibodies from 1A3E2 and 2F11B1 hybridomas Flu anti-PB2, since they are capable of simultaneously binding to the antigen without competing for the binding site or interfering with each other. The above allows the capture and subsequent detection of PB2 protein in patient samples.
Treatment of clinical samples. The samples used for the tests were obtained from nasopharyngeal swabs contained in universal transport medium (UTM). The samples were centrifuged at 14,000 rpm for 4 minutes at room temperature. Subsequently, the supernatant (SN1) was separated from the pellet; the latter was incubated with 100 μL of RIPA Buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1%, SDS and a 1× protease inhibitor cocktail) for 15 minutes at 4° C. (39.2° F.), vortexing every 5 minutes. It was then centrifuged at 14,000 rpm for 4 minutes at room temperature. At the end, the supernatant obtained (SN2) was taken and mixed with SN1, vortexing was performed.
It is extremely important to use both antibodies for the detection of PB2 protein, due to the low availability of antigen in the sample. Using an ELISA Sandwich increases the specificity and sensitivity in the diagnosis of Flu. Assays were performed where the plate was activated directly with clinical samples of nasopharyngeal swabs, then anti-PB2 1A3E2 and anti-PB2 2F11B1 antibodies were incubated, separately. Then a secondary anti-mouse IgG antibody conjugated with HRP was incubated and absorbance generated by incubating the antibody complex plus sample with the TMB substrate was evaluated, and a positive diagnosis was not observed since the signal delivered was very low (data not shown).
Carrying out a diagnostic kit using the ELISA's Sandwich technique, where the plate can be activated and blocked, would reduce the time and cost of performing a diagnosis, since this technique is easy to perform and analyze compared to the standard technique (PCR). The kit does not need highly trained personnel to perform or analyze it.
As in ELISA technique, the availability and concentration of viral proteins is generally very low in clinical samples of nasopharyngeal swabs, so it was wanted to evaluate the obtained results by ELISA technique to another more sensitive technique (
Obtained results for this test are shown in
This assay, as in ELISA assay with patient samples, demonstrates the versatility of antibodies from 1A3E2 and 2F11B1 hybridomas of FLU, since they are capable of simultaneously binding to antigen without competing for the binding site or interfere with each other and detect poor antigen availability in nasopharyngeal swab sample.
Previously, ELISA tests were carried out in Sandwich where the previous diagnosis of the samples to be evaluated was known. After these tests, a blind study was carried out, where about 160 nasopharyngeal swab samples were evaluated, without knowing the microbiological diagnosis. For all assays in the blinded study, ELISA's Sandwich were performed where anti-L 1A3E2 antibody was used as capture antibody and anti-L 2F11B1 antibody was used as HRP-conjugated detection antibody. For all assays, wells of an ELISA plate were activated with 3.4 μg/mL (170 ng/well) of anti-L antibody from FLU 1A3E2 hybridoma, diluted in 1×PBS, for 30 minutes at 37° C. (98.6° F.). 2 washes were carried out with 1×-Tween20 PBS 0.05% and later the plate was blocked with 200 μL of 1×PBS/10% FBS for 30 minutes at 37° C. (98.6° F.). Each well with 50 μL of nasopharyngeal swabs from patients was washed again and incubated for 1 hour at 37° C. (98.6° F.), which were evaluated in parallel by the standard diagnostic method (PCR), routinely referred to as “viral panel”, and which were treated as previously described in example 6. As controls were included: 1) specificity control: 50 μL of BSA protein (50 ng) were used; 2) positive control: 50 ng of PB2-FLU recombinant protein; 3) Negative controls: wells with no sample and wells blocked and incubated with detection antibody. Subsequently, the 2 corresponding washes were carried out with 1×-Tween20 PBS 0.05% and each well was incubated for 30 minutes at room temperature (≈25° C. (≈77° F.), in the dark) with 50 μl of anti-PB2 antibody from 2F11B1 hybridoma, conjugated with HRP (1.8 ng/μL of final concentration). The plate was then washed 2 more times, developed with 50 μL of TMB solution and incubated for 15 minutes in the dark. The reaction stopped with 50 μL of H2SO4 2 N and the plate was read at 450 nm in an ELISA reader (Epoch model), certified for clinical diagnosis.
Results are shown in
In this application example, is demonstrated that both the specific monoclonal antibody against the PB2 protein can be detected by indirect ELISA. For detection of protein L, ELISA plates were activated with 50 μL of protein PB2 y BSA 50 ng. Nonspecific sites were blocked with 10% FBS diluted in 1×PBS. 170 ng (3.4 μg/mL) of Fab fragments of antibodies secreted by 1A3E2 (anti-Flu) and 2F11B1 (anti-Flu) hybridomas, both previously biotin conjugated. Incubation of biotin-binding molecules (Streptavidin), which is HRP-conjugated (1:2,000 dilution, 75 ng per well) (
The objective of this assay is to demonstrate the ability to detect fragments of anti-Flu antibodies, produced by 1A3E2 and 2F11B1 hybridomas, by PB2 protein. Prior to the indirect ELISA assay, IgG molecule of each anti-Flu antibody was fragmented. Fragmentation was performed using the “Thermo Scientific™ Pierce™ F(ab′)2 Fragment Preparation Kits” kit (#10381214, Thermo Scientific), which separates F(ab′)2 fragment and Fc from the antibody of interest, by using the enzyme pepsin that digests the Fc fragment and subsequently purification steps are carried out to separate the F(ab′)2 fragment from the digested Fc fragment. After antibody fragmentation, purified F(ab′)2 fraction was verified by the Western bolt technique. F(ab′)2 fractions were conjugated to biotin molecules using the rapid conjugation kit, Lightning-Link rapid biotin type A (#370-0010, Expedeon). Having ready all reagents, antigen detection was carried out by indirect ELISA technique, where ELISA plate was activated with 50 ng of purified PB2 antigen for 1 hour at 37° C. (98.6° F.). Two negative controls were included, one with no sample and the other incubating the well with 50 ng of BSA protein. Subsequently, the plate was washed twice with 1×/Tween20 0.05% phosphate buffered saline (PBS). The plate was then blocked for 2 hours at 37° C. (98.6° F.) with 1×PBS/10% Fetal Bovine Serum (FBS). Subsequently, the washes were repeated and then each antibody conjugated with biotin, unfractionated and F(ab′)2 fractions (1A3E2 and 2F11B1) was incubated at a final concentration of 3.4 μg/mL (170 ng per well), diluted in 1×PBS/10% FBS, for 1 hour at 37° C. (98.6° F.) (each antibody on a separate plate). After incubation time, washings were repeated and a biotin-binding protein (Streptavidin) labeled with horseradish peroxidase (HRP) enzyme was added to each well in dilution 1 in 2000 (25 ng/μL per well) in 1×PBS/10% FBS, for 1 hour at room temperature (□25° C. (□77° F.), in the dark. Finally, the washes were carried out and it was developed with 50 μL of citrate/Tetramethylbenzidine (TMB, 3-3′-5-5′-tetramethylbenzidine, 1 mg/mL, Becton Dickinson) buffer. To stop the reaction, 50 μL of H2SO4 2 N were added and the result was read on an ELISA reader, at 450 nm. To determine that the reaction of the secondary antibody was specific in recognizing the primary antibody and also that the obtained signal was not caused by nonspecific binding of the secondary antibody to the antigen, controls were carried out in which only the secondary antibody was used with no primary antibody or sample (well not activated). Another control to determine that the primary antibody reaction is specific for the antigen, consisted of using the antibodies on an ELISA plate that has not been activated with the antigen (with no sample) or using the antibodies on an ELISA plate that possessed 50 ng of PB2. Results show that monoclonal antibodies of the invention are capable of recognizing 50 ng of purified antigen, specifically, regardless of whether the complete antibody or a fragment thereof is used (
| Number | Date | Country | Kind |
|---|---|---|---|
| 3871-2018 | Dec 2018 | CL | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CL2019/050155 | 12/27/2019 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2020/132772 | 7/2/2020 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 9650434 | Chen et al. | May 2017 | B2 |
| Number | Date | Country |
|---|---|---|
| 2008309939 | Nov 2013 | AU |
| 2013540701 | Nov 2013 | JP |
| 2015189715 | Nov 2015 | JP |
| 2006051069 | May 2006 | WO |
| 2009046983 | Apr 2009 | WO |
| 2012045001 | Apr 2012 | WO |
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| Number | Date | Country | |
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| 20220119503 A1 | Apr 2022 | US |
