WO2013162456A1

WO2013162456A1 - Method for detecting concentration of antibody or fusion protein

Info

Publication number: WO2013162456A1
Application number: PCT/SE2013/050449
Authority: WO
Inventors: Lin Chen; Klaus HOCHLEITNER; Rong HOU; Lianghua LU; Jianjun Yang
Original assignee: General Electric Company; Ge Healthcare Bio-Sciences Ab
Priority date: 2012-04-28
Filing date: 2013-04-24
Publication date: 2013-10-31
Also published as: CN103376327A

Abstract

A method comprises: providing a test strip having a sample portion, a conjugation portion comprising a control protein bindable to a fragment crystallizable (Fc) portion of an antibody or a fusion protein and a label conjugated with the control protein, a detection portion comprising an antigen specific for the antibody or the fusion protein, and a control portion comprising a control antibody of the control protein; contacting a sample solution comprising the antibody or the fusion protein with the sample portion; allowing the sample solution to migrate from the sample portion through the control portion; detecting an intensity of a signal of the detection portion; and determining the concentration of the antibody or the fusion protein in the sample solution using a formula in which the concentration of the antibody or the fusion protein is a function of the intensity of the signal.

Description

METHOD FOR DETECTING CONCENTRATION OF ANTIBODY OR FUSION

PROTEIN

BACKGROUND

[0001] The invention relates generally to methods for detecting concentrations of antibodies or fusion proteins.

[0002] Antibodies and fusion proteins are used for treating diseases. For example, Trastuzumab (Herceptin), an anti-HER2/neu receptor monoclonal antibody that inhibits growth of ErbB2-overexpressing breast cancer, is used to treat such cancers. Infliximab, a monoclonal antibody against tumour necrosis factor alpha (TNF- a )_? is used to treat autoimmune diseases.

[0003] One method of producing antibodies or fusion proteins is using the cell culture technology. However, cell culture usually takes many days during which some changes may happen anytime to change the immunogenicity of the antibody or fusion protein, so the growing of the cell needs to be monitored closely to see whether changes happen. One way of monitoring the growing of the cell is to detect the concentration of the antibody or fusion protein in the cell culture.

[0004] Most of the current technologies can only detect the presence of antibodies or fusion proteins and cannot tell the quantity (concentration) of antibodies or fusion proteins. For instance, US patent No. 6,841, 159 discloses a method for detecting the presence of at least one antibody to mycobacterium antigens present in a sample selected from one or more patient bodily fluids by detecting the presence of a signal caused by the formation of an antigen-antibody complex.

[0005] US patent No. 6,394,952 discloses that the concentration of fetal fibronectin in a patient sample may be determined based upon the ratio of the integrated area beneath each of the two peaks, occurring at the detection and control stripes of a test strip, of the reflectance ratio graphed as a function of the steps of moving two light sources above the test strip. The reflectance ratio is calculated using the readings of a reflectance reader and signals of both the detection and the control stripes are read for determining the concentration. In addition, the reagent in the conjugate pad of the test strip of US patent No. 6,394,952 is an antibody specific for the analyte (fetal fibronectin), which means that once the analyte is changed, the conjugate pad needs to be changed accordingly, which is undesirable in certain application environments.

[0006] Eva Engvall and Peter Perlmann disclose in an article entitled as

"ENZYME-LINKED IMMUNOSORBENT ASSAY, ELISA III. Quantitation of Specific Antibodies by Enzyme-Labeled Anti-Immunoglobulin in Antigen-Coated Tubes", THE JOURNAL OF IMMUNOLOGY, Vol. 109, No. 1, July 1972 a method for the quantitative determination of antibodies. The method in the article takes at least 22 hours for incubation and is thus time consuming.

[0007] Therefore, there is a need to develop a new or improved method for detecting the concentration of an antibody or fusion protein.

BRIEF DESCRIPTION

[0008] In one aspect, a method is provided, comprising: providing a test strip having a sample portion, a conjugation portion, a detection portion, and a control portion, the conjugation portion comprising a control protein bindable to a fragment crystallizable (Fc) portion of an antibody or a fusion protein and a label conjugated with the control protein; the detection portion comprising an antigen specific for the antibody or the fusion protein, the control portion comprising a control antibody of the control protein; contacting a sample solution comprising the antibody or the fusion protein with the sample portion; allowing the sample solution to migrate from the sample portion through the control portion; detecting an intensity of a signal of the detection portion; and determining the concentration of the antibody or the fusion protein in the sample solution using a formula in which the concentration of the antibody or the fusion protein is a function of the intensity of the signal.

[0009] These and other advantages and features will be better understood from the following detailed description of preferred embodiments of the invention that is provided in connection with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 is a schematic exploded perspective view of a test strip provided in accordance with one embodiment of the invention;

[0011] FIG. 2 is a schematic top plan view of the test strip of FIG. 1;

[0012] FIG. 3 is a diagram obtained in example 5; and

[0013] FIG. 4 is a diagram obtained in example 6 and comparative example 1.

DETAILED DESCRIPTION

[0014] Approximating language, as used herein throughout the specification and claims, may be applied to modify any quantitative representation that could permissibly vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as "about", is not to be limited to the precise value specified. In some instances, the approximating language may correspond to the precision of an instrument for measuring the value.

[0015] In the following specification and claims, the singular forms "a", "an" and "the" include plural referents, unless the context clearly dictates otherwise. Moreover, the suffix "(s)" as used herein is usually intended to include both the singular and the plural of the term that it modifies, thereby including one or more of that term.

[0016] As used herein, the terms "may", "could", "could be" and "may be" indicate a possibility of an occurrence within a set of circumstances; a possession of a specified property, characteristic or function; and/or qualify another verb by expressing one or more of an ability, capability, or possibility associated with the qualified verb. Accordingly, usage of "may" and "may be" indicates that a modified term is apparently appropriate, capable, or suitable for an indicated capacity, function, or usage, while taking into account that in some circumstances, the modified term may sometimes not be appropriate, capable, or suitable. For example, in some circumstances, an event or capacity may be expected, while in other circumstances, the event or capacity maynot occur. This distinction is captured by the terms "may", "could", "could be" and "may be".

[0017] Preferred embodiments of the present disclosure will be described herein below with reference to the accompanying drawings. In the following description, well-known functions or constructions are not described in detail to avoid obscuring the disclosure in unnecessary detail.

[0018] Please refer to FIGS. 1 and 2, a test strip 1 provided in accordance with one embodiment of the invention comprises: a sample portion 2, a conjugation portion 3, a detection portion 4, and a control portion 5. The conjugation portion 3 comprises a control protein (not shown) bindable to a fragment crystallizable (Fc) portion of an antibody or a fusion protein (not shown) and a label (not shown) conjugated with the control protein. The detection portion 4 comprises an antigen (not shown) specific for the antibody or the fusion protein. The control portion 5 comprises a control antibody (not shown) of the control protein.

[0019] In some embodiments, the test strip 1 comprises an absorbing portion 6 located further from the detection portion 4 than from the control portion 5.

[0020] The test strip 1 may be in the form of an elongate strip including any bibulous material to which the sample solution will sorb and along which the sample solution may migrate. The sample portion 2, the conjugation portion 3, the detection portion 4, the control portion 5 and the absorbing portion 6 of the test strip 1 may be made of same materials or different materials. The sample portion 2, the conjugation portion 3, the detection portion, the control portion 5 and the absorbing portion 6 of the test strip 1 may be integral with each other or may be assembled together.

[0021] In some embodiments, the test strip 1 may comprise a backing pad made of such as a plastic material like polyester (Mylar^®) or PET to support the sample portion 2, the conjugation portion 3, the detection portion 4, the control portion 5 and the absorbing portion 6 thereon. [0022] The sample portion 2 may be in any shape or configuration that the sample solution could be sorbed thereto. The sample portion 2 may be either a section of the test strip 1 or a sample pad.

[0023] In some embodiments, the sample portion 2 may be a sample pad prepared by saturating a strip of glass fiber with a solution comprising, e.g., bovine serum albumin (BSA), Tween 20 and phosphate buffered saline (PBS) and drying the strip of glass fiber.

[0024] The conjugation portion 3 may be in any shape or configuration that the sample solution could migrate along. The conjugation portion 3 may be either a section of the test strip 1 or a conjugation pad.

[0025] In some embodiments, the conjugation portion 3 may be a conjugation pad prepared by sorbing a conjugation solution on a strip of glass fiber followed by drying the strip of glass fiber. The conjugation solution comprises a control protein bindable to a fragment crystallizable (Fc) portion of an antibody or a fusion protein and a label conjugated with the control protein.

[0026] The label may be any physical or chemical label capable of being detected on a solid support using an optical instrument and capable of being used to distinguish the antibody or the fusion protein to be detected from other compounds and materials in the detection portion 4 of the test strip 1. Examples of the label include, but are not limited to, enzyme- substrate combinations that produce color upon reaction, colored particles, such as latex particles, colloidal metal labels, metal or carbon sol labels, fluorescent labels, and liposome or polymer sacs. In some embodiments, the colloid gold is used as the label in the conjugation solution.

[0027] The control protein may be at least one of protein A and protein G. The control protein is bound to the label and, when the sample solution migrates through the conjugation portion 3, is bound to an Fc portion of an antibody or a fusion protein in the sample solution. In the detection of the concentration of an antibody or a fusion protein, changing the analyte, i.e., the antibody or the fusion protein, does not necessarily cause the changing of the control protein and the label, so the conjugation portion may be universal for test strips of detection of any antibody or any fusion protein, thereby saving the designing and producing cost of the test strips.

[0028] The detection portion 4 and the control portion 5 may be also called T- line and C-line, respectively. In some embodiments, the detection portion 4 and the control portion 5 may be formed by dispensing a T-line solution and a C-line solution respectively to two spaced areas of a membrane 8, respectively. The membrane 8 may be made of such as nitrocellulose.

[0029] The T-line solution comprises an antigen specific for the antibody or the fusion protein. For example, in some embodiments, the antibody or fusion protein is tumor necrosis factor-alpha (TNF-a) antibody, the antigen is tumor necrosis factor- alpha (T F-α) protein.

[0030] The C-line solution comprises a control antibody corresponding to the control protein. In some embodiments, the control protein is protein A and the control antibody is the protein A antibody (anti-protein A).

[0031] The absorbing portion 6 helps enhancing a wicking force directed away from the sample portion 2 to draw the sample solution from the sample portion 2 through the control portion 5. The absorbing portion 6 may be either a section of the test strip 1 or an absorbing pad. In some embodiments, the absorbing portion 6 may be an absorbing pad made of bibulous material such as cotton paper, cellulose, silica microfiber filter, glass fiber, and quartz fiber filter.

[0032] In some embodiments, the sample portion 2, the conjugation portion 3, the membrane 8 and the absorbing portion 6 are assembled together. To enhance the migration of the sample solution from the sample portion 2 through the control portion 5, the conjugation portion 3 overlaps with both the sample portion 2 and the membrane 8 and the membrane 8 also overlaps with the absorbing portion 6.

[0033] In some embodiments, a plastic housing (not shown) may be provided to enclose the test strip 1 therein. In some embodiments, the sample portion 2 may be located outside of the plastic housing. [0034] The sample solution may be a solution comprising antibodies or fusion proteins. The sample solution may be a solution prepared from the antibodies or fusion proteins. In some embodiments, the sample solution may be prepared by diluting the cell culture during the cell growth. Besides the antibody or the fusion protein, the sample solution may comprise such as BSA and PBST (Tween 20 in PB/ NaCl) buffer.

[0035] The sample solution may be contacted with the sample portion 2 by, e.g., applying the sample solution to the sample portion 2 or immersing the sample portion 2 into the sample solution.

[0036] The sample solution migrates from the sample portion 2 forwardly under the wicking force. When the sample solution migrates through the conjugation portion 3, the antibody or the fusion protein binds at the fragment crystallizable (Fc) portion thereof to some of the control protein conjugated with the label and migrates forwardly together with the control protein and the label.

[0037] When the sample solution migrates through the detection portion 4, the antibody or the fusion protein binds to the specific antigen and is bound to the detection portion 4 while the sample solution comprising the rest of the control protein conjugated with the label keeps migrating forwardly so that the control protein conjugated with the label binds to the control antibody of the control portion 5.

[0038] The signals produced by the labels in the detection portion 4 and the control portion 5 may be visible by naked eyes or detectable using optical instruments. The presence of the signal in the control portion 5 indicates the completion of the migration of the sample solution through the detection portion 4 to the control portion 5.

[0039] The intensity of the signal of the label in the detection portion 4 may be detected by any optical instruments. The intensity of the signal of the detection portion 4 may be the output of the optical instrument. In some embodiments, the intensity of the signal is the height of a peak of the signal outputted from a strip reader. [0040] Allowing solutions having known concentrations of the antibody or the fusion protein to migrate along test strips and detecting intensities of signals of the corresponding detection portions may establish a formula in which the concentration of the antibody or the fusion protein is a function of the intensity of the signal of the detection portion. The concentration of the antibody or the fusion protein in the sample solution is determined by calculating using the formula and the intensity of the signal of the detection portion 4.

[0041] The method of the present invention takes very short time to allow the sample solution contacts and migrates along the test strip 1 and to determine the concentration of the antibody or the fusion protein using a formula in which the concentration of the antibody or the fusion protein is a function of the intensity of the signal of the detection portion. Therefore, it is simple and fast. From the following examples, it can be seen that the method has compatible test results with ELISA test and is thus reliable. The control protein, such as at least one of protein A and protein G in the conjugation portion 3 is bindable to the Fc portion of the antibody or the fusion protein, so when the antibody or the fusion protein is changed, the conjugation portion does not need to be changed, thereby saving cost for designing and manufacturing the test strip.

EXAMPLES

[0042] The following examples are included to provide additional guidance to those of ordinary skill in the art in practicing the claimed invention. Accordingly, these examples do not limit the invention as defined in the appended claims.

Example 1

[0043] One milliliter of Au-Nano particle solution (Au- P, 40 nm) (from

Shanghai Quicking Biotech Co., Ltd., Shanghai, China) was added to a 1.5 mL microcentrifuge tube, followed by adding 5 ul of 0.2 M K₂C0₃ solution (from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and vortexing the micro-centrifuge tube to mix the two solutions. After the further addition of 4 ul of Protein A (5 mg/ml, from Hangzhou LongGene Biotech Co., Ltd., Hangzhou, China), the micro-centrifuge tube was vortexed immediately before waiting for 10 minutes. 100 ul of 10wt% Bovine serum albumin (BSA, obtained from Acros Organics, Belgium) in water were added subsequently to the micro-centrifuge. After vortex, the solution in microcentrifuge was stood still for 10 minutes. Then, the solution was centrifuged at 11,000 rpm for 10 minutes. After removal of the supernatant, 100 uL of re-suspension solution containing 1/1000 Bovine serum albumin (BSA, obtained from Acros Organics, Belgium) in 0.01 M of phosphate buffered saline (PBS, pH 7.4, prepared by dissolving Na₂HP0₄, NaH₂P0₄, and NaCl in water) was added into the microcentrifuge tube to re-dissolve the conjugated colloidal gold particles with Protein A (Protein A-AuNP) to obtain the Protein A-AuNP solution.

[0044] A strip of glass fiber (33 glass, 0.8 cm-width x 4 cm-length, from GE

Healthcare Bio-Sciences Corp., New Jersey, USA) was placed in a micro-centrifuge tube containing 20 uL of the Protein A-AuNP solution and 180 uL of dilution buffer (3% trehalose, 0.5% BSA, and 1% Tween 20 in 0.01 M PBS) for 1-2 minutes until all the solution was adsorbed evenly on the glass fiber. The glass fiber was then dried in an oven at 37 °C for 3 hours to obtain the conjugation pad.

Example 2

[0045] A strip of glass fiber (33 glass, 1.5 cm-width x 4 cm-length, from GE

Healthcare Bio-Sciences Corp., New Jersey, USA) was soaked in 350 uL of sample pad solution comprising 1% BSA and 0.5% Tween20 in 0.1 M PBS till saturated. The strip was dried in an oven at 37 °C for 3 hours to obtain the sample pad.

Example 3

[0046] A strip of FF85 nitrocellulose membrane from GE Healthcare Bio-

Sciences Corp., New Jersey, USA was stuck on one side of a DB-6 plastic adhesive backing pad (obtained from Shanghai Joey BioTech Co., Ltd., Shanghai, China).

[0047] C-Line solution (1 mg/ml Anti-Protein A solution) was prepared by mixing 20 ul of 3 mg/ml chicken Protein A antibody (Anti-Protein A) (obtained from Hangzhou LongGene BioTech, Hangzhou, China) with 40 ul of 0.01 M PBS (pH 7.2). [0048] Recombinant Human Tumor Necrosis Factor-alpha (TNF-a) protein (1 mg/ml) obtained from Beijing BioSynthesis BioTech, China was used as the T-line solution.

[0049] The T-line and C-line solutions were dispensed, respectively, onto the detection portion and the control portion of the FF85 membrane with a 0.5 ul/cm volume using a BioDot XYZ-3050 dispensing platform (BioDot Inc., Irvine, CA, USA).

Example 4

[0050] A strip of cotton paper (470, from GE Healthcare Bio-Sciences Corp.,

New Jersey, USA) was attached onto one end of the DB-6 plastic adhesive backing pad on the same side as the FF85 membrane to be used as the absorbing pad and covered 1-2 mm of the FF85 membrane. The absorbing pad was located further from the detection portion than from the control portion.

[0051] The conjugation pad prepared in example 1 was assembled on the opposite end of the backing pad and covered 1-2 mm of the opposite end of the FF85 membrane.

[0052] The sample pad prepared in example 2 was applied to the opposite end of the backing pad covering 1-2 mm of the conjugation pad to obtain an assembled strip. The assembled strip was cut into 4 mm-wide test strips by a cutter ZQ2000 (Shanghai Goldbio Tech. Co., Ltd., Shanghai, China).

Example 5

[0053] Eight sample solutions were prepared by diluting an Anti-TNF-a sample from an ELISA kit from Shanghai Medipharm Biotech pharmaceutical Co. Ltd, Shanghai, China with 0.5% BSA in PBST buffer (0.1% Tween 20 in 0.1 M PB/0.01 M NaCl, pH 7.4) to have concentrations of 0, 1.25 ng/mL, 2.5 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, 40 ng/mL, and 80 ng/mL, respectively.

[0054] The eight sample solutions (150 μΕ each) were added respectively to eight wells of a 96-well plate. The sample pad of one test strip was inserted into each of the wells and the test strip stood to allow the sample solution migrate upward therealong. After 10 minutes, the test strip was put into a strip reader (DT2030, from Shanghai GoldBio Tech Co., Ltd., Shanghai, China) for reading the intensity of the signal of T-line, i.e., the peak height of the signal of the detection portion. A two dimensional diagram was obtained as shown in Figure 3 using the concentration of the Anti-T F-α in the sample solutions as horizontal axis and the intensities of the signals of the T-lines as the vertical axis and a formula in which the concentration and the intensity are functions of each other was established as: y=16.8251n(x)-10.285, in which y is the intensity of the signal and x is the concentration of tumor necrosis factor-alpha (T F-α) antibody in the sample solution. The relevant degree (R²) was 0.9606.

Example 6

[0055] A sample of the TNF-a antibody cell culture was collected during cell growth each day from the second day (after 1 day of growing) to the seventh day (after 6 days of growing) and was diluted in different ratios (the dilution ratio of samples of the second day and the third day: 1 : 100, of the sample of the fourth day: 1 :200; of the sample of the fifth day: 1 :400; of the sample of the sixth day: 1 :600; of the sample of the seventh day: 1 :800) with 0.5% BSA in PBST buffer to prepare 6 sample solutions.

[0056] 150 uL of each of the sample solutions was loaded into a well of a 96- well plate. The sample pad of one test strip was inserted into each of the wells and the test strip stood for 10 minutes to allow the sample solution migrate upward therealong. The test strips were read by the GoldBio Strip reader to provide the intensities of the T-line signals, i.e., the peak heights of signals of the detection portions.

[0057] The concentrations of the TNF-a antibody in the sample solutions were obtained by calculating using the formula obtained in example 5 and the intensities read by the reader. The concentrations of the TNF-a antibody in the cell cultures with respect to the days of cell growth were obtained by dividing the concentrations of the T F-α antibody in the sample solution by the dilution ratios in preparing corresponding sample solutions and are shown in line 1 of FIG. 4.

Comparative example 1

[0058] Six sample solutions were prepared in the same way as in example 6.

The concentrations of T F-α antibody in the sample solutions were detected using an ELISA kit obtained from Medipharm Biotech pharmaceutical Co., Ltd. following the standard protocol of the ELISA kit. The concentrations of the TNF-a antibody in the cell cultures with respect to the days of cell growth were obtained by dividing the concentrations of the TNF-a antibody in the sample solutions by the dilution ratios in preparing the sample solutions and are shown in line 2 of FIG. 4.

[0059] As can be seen from FIG. 4, the concentrations obtained in example 6 are compatible with the ones obtained in comparative example 1.

[0060] While the disclosure has been illustrated and described in typical embodiments, it is not intended to be limited to the details shown, since various modifications and substitutions may be made without departing in any way from the spirit of the present disclosure. As such, further modifications and equivalents of the disclosure herein disclosed may occur to persons skilled in the art using no more than routine experimentation, and all such modifications and equivalents are believed to be within the spirit and scope of the disclosure as defined by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method comprising: providing a test strip having a sample portion, a conjugation portion, a detection portion, and a control portion, the conjugation portion comprising a control protein bindable to a fragment crystallizable (Fc) portion of an antibody or a fusion protein and a label conjugated with the control protein; the detection portion comprising an antigen specific for the antibody or the fusion protein, the control portion comprising a control antibody of the control protein; contacting a sample solution comprising the antibody or the fusion protein with the sample portion; allowing the sample solution to migrate from the sample portion through the control portion; detecting an intensity of a signal of the detection portion; and determining the concentration of the antibody or the fusion protein in the sample solution using a formula in which the concentration of the antibody or the fusion protein is a function of the intensity of the signal.

2. The method of claim 1, wherein the formula is established by allowing solutions having known concentrations of the antibody or the fusion protein to migrate along test strips and detecting corresponding intensities of signals of detection portions.

3. The method of claim 2, wherein the intensity of the signal is the peak height of the signal outputted from a strip reader.

4. The method of claim 1, 2 or 3, wherein the antibody or fusion protein is tumor necrosis factor-alpha (TNF-a) antibody.

5. The method of claim 4, wherein the antigen is tumor necrosis factor-alpha (TNF-a) protein.

6. The method of claim 1, further comprising: preparing the sample solution from cell cultures collected during cell growth.

7. The method of claim 1, wherein the test strip comprises an absorbing portion located further from the detection portion than from the control portion.

8. The method of claim 1, wherein the control protein is at least one of protein A and protein G.

9. The method of claim 8, wherein the control protein is protein A.

10. The method of claim 9, wherein the label is derived from colloidal gold.