• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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    • 专利摘要:
    The present disclosure relates to a process for detecting and optionally quantifying an analyte in a sample in the presence of a soluble target of the analyte. The soluble target forms a complex with the analyte and thus may interfere in determining the total analyte concentration. The process of the present invention utilizes a unique modified citrate buffer for diluting the sample containing the analyte and the soluble target which in turn helps in dissociating the analyte-soluble target complex, thereby enabling the process of the disclosure to detect and optionally quantify measure the analyte accurately.
    公开号:AU2013219968A1
    申请号:U2013219968
    申请日:2013-02-14
    公开日:2014-10-02
    发明作者:Vishika HEGDE；Ramakrishnan Melarkode；Vivek Gopal Nayak；Ranjit Ravindran Pillai；Shilpa Govinda Ramaswamy；Nilanjan Sengupta；Senthil THANGAMUTHU；Anita Rao Udiavar；Laxmikant VASHISHTA
    申请人:Mylan GmbH；Biocon Ltd；
    IPC主号:G01N33-53
    专利说明:
    WO 2013/121368 PCT/IB2013/051193 A PROCESS FOR DETECTION AND OPTIONAL QUANTIFICATION OF AN ANALYTE TECHNICAL FIELD OF THE DISCLOSURE 5 The present disclosure relates to a process of utilizing a unique acidic dissociation buffer in a single step ligand binding assay to quantify total amount of an analyte present in a biological matrix containing a soluble target of the analyte. More specifically, the disclosure relates to a process for the development of immunoassays using an ELISA format to reliably quantify the total analyte using this unique buffer. The disclosure can be 10 extended to immunogenicity assays for the detection of anti-analyte antibodies in the presence of excess free analyte. BACKGROUND OF THE DISCLOSURE Immunoassays are primarily used for the detection and quantitation of a wide variety of 15 analytes such as hormones, tumor antigens, bacterial or viral antigens, protein therapeutics etc. in biological samples. Quantitative immunoassays are critical for understanding the Pharmacokinetic profile of the therapeutic proteins in support of efficacy and safety studies. In addition, immunoassays are also used to quantitate the soluble targets of the therapeutic proteins and anti-therapeutic protein antibodies. For soluble protein targets or 20 shed receptor targets of the analyte, two different forms of the analyte exist in the biological matrix: the free analyte and the analyte complexed with the soluble target. In order to get an accurate pharmacokinetic profile of the analyte, it is ideal to develop ligand binding assay methods aimed at quantitating the total analyte concentrations instead of only the free analyte concentrations. 25 A frequent problem often encountered in quantitative immunoassays is accurate estimation of the total analyte concentrations in the presence of an interfering substance (soluble target) which competes with the immunoassay reagents for binding to the analyte. This leads to an underestimation of the analyte and compromises the true assessment of the 30 pharmacokinetic profile of the analyte. The interfering substance can be removed by pre treatment of the samples. However this step leads to loss of sample and introduces additional variability.
    WO 2013/121368 2 PCT/IB2013/051193 STATEMENT OF THE DISCLOSURE Accordingly, the present disclosure relates to a process for detection and optional quantification of analyte in biological sample, said process comprising providing modified citrate buffer to the sample prior to carrying out assay for said detection and optional 5 quantification of the analyte within the biological sample; a method of disassociating complex formed by interaction of analyte and at least one interfering substance as target for the analyte in a biological sample, said method comprising act of providing modified citrate buffer to the biological sample for disassociating the complex; an enzyme linked immunosorbent assay (ELISA) kit for detecting and optionally quantifying analyte in 10 biological sample, said kit comprising components selected from group comprising modified citrate buffer, capture antibody, detection antibody, reagent for reacting with the detection antibody, microtitre plates, coating buffer, washing solution, and stopping solution or any combination thereof; and a modified citrate buffer for detecting and optionally quantifying presence of an analyte in a biological sample. 15 BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES In order that the disclosure may be readily understood and put into practical effect, reference is made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with a detailed description below, are 20 incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where: Figure 1 shows graphs depicting interference of recombinant HER2-ECD on a Trastuzumab calibration curve. 25 Figure 1A shows a graph depicting that spiking of HER2-ECD results in a dose-dependent locational shift of Trastuzumab calibration curves when diluted in phosphate buffered saline Tween20 (PBST). 30 Figure 1B shows a graph depicting that modified citrate buffer diluent nullifies the dose dependent interference of HER2-ECD on a Trastuzumab calibration curve. Figure 2 shows the comparison of different acidic buffers at pH 3.5, to nullify the interference of recombinant HER2-ECD on a Trastuzumab calibration curve. Particularly, WO 2013/121368 3 PCT/IB2013/051193 Figure 2 is a graph showing that use of modified citrate buffer diluent results in a minimal locational shift induced by 1 gg/mL HER2-ECD spiking in serum. DETAILED DESCRIPTION OF THE DISCLOSURE 5 The present disclosure relates to a process for detection and optional quantification of an analyte in a biological sample, said process comprising providing modified citrate buffer to the sample prior to carrying out assay for said detection and optional quantification of the analyte within the biological sample. 10 In an embodiment of the present disclosure, the process comprises: (a) providing modified citrate buffer to the sample for obtaining diluted biological sample; and (b) carrying out a conventional assay for detecting and optionally quantifying the analyte in the sample. 15 In another embodiment of the present disclosure, the biological sample comprises at least one interfering substance as a target for the analyte, and wherein said interfering substance and said analyte combine to form a complex. 20 In yet another embodiment of the present disclosure, the formation of the complex reduces amount of free analyte in the biological sample, thereby hindering the accurate detection and quantification of said analyte. In still another embodiment of the present disclosure, the modified citrate buffer dissociates 25 the complex and restores the free analyte within the sample to enable the accurate detection and quantification of said analyte. In still another embodiment of the present disclosure, the modified citrate buffer comprises Citrate-HCl at concentration of about 125mM and Tween-20 at concentration of about 30 0.l1% w/v, and wherein pH of the buffer ranges from 2.5 to 3.8, preferably 3.5. In still another embodiment of the present disclosure, the biological sample is selected from group comprising blood, plasma, and serum or any combinations thereof.
    WO 2013/121368 4 PCT/IB2013/051193 In still another embodiment of the present disclosure, the analyte is selected from group comprising a hormone, a tumor antigen, a bacterial antigen, a viral antigen, a protein therapeutic and an antibody or any combinations thereof. 5 In still another embodiment of the present disclosure, the analyte is an antibody. In still another embodiment of the present disclosure, the antibody is against human epidermal growth factor receptor 2 (HER-2). 10 In still another embodiment of the present disclosure, the interfering substance is an extracellular domain of HER-2 (HER-2-ECD). In still another embodiment of the present disclosure, the assay is enzyme linked immunosorbent assay (ELISA). 15 In still another embodiment of the present disclosure, the enzyme linked immunosorbent assay is sandwich enzyme linked immunosorbent assay and is carried out in presence of components selected from group comprising a capture antibody, a detection antibody, a reagent for reacting with the detection antibody, a microtitre plate, a coating buffer, a 20 washing solution and a stopping solution or any combinations thereof. In still another embodiment of the present disclosure, the capture antibody is an antibody against the analyte within the biological sample. 25 In still another embodiment of the present disclosure, the detection antibody is labelled with an enzyme selected from group comprising horseradish peroxidase and alkaline phosphatase and ruthenium. In still another embodiment of the present disclosure, the reagent for reacting with the 30 labelled antibody is chromogenic substrate selected from ABTS (2,2'-Azinobis[3 ethylbenzothiazoline-6-sulfonic acid] -diammonium salt, TMB (3,3',5,5' tetramethylbenzidine) and electro chemiluminscent compatible substrate.
    WO 2013/121368 5 PCT/IB2013/051193 In still another embodiment of the present disclosure, the coating buffer is phosphate buffered saline. In still another embodiment of the present disclosure, the washing solution comprises 5 phosphate buffered saline and Tween-20. In still another embodiment of the present disclosure, the stopping solution is 0. IN sulphuric acid. 10 In still another embodiment of the present disclosure, the enzyme linked immunosorbent assay (ELISA) comprises: a. coating a solid surface with the capture antibody against the analyte; providing a modified citrate buffer to the biological sample for obtaining a diluted biological sample; 15 b. adding the diluted sample to the antibody coated solid surface or support of step (a); c. incubating and washing the support to remove any unbound analyte; d. adding a labelled detection antibody against the analyte and washing the support to remove any unbound detection antibody; and 20 e. adding a reagent for reacting with the labelled antibody for detecting and optionally quantifying the analyte in the sample. The present disclosure further relates to a method of disassociating complex formed by interaction of analyte and at least one interfering substance as target for the analyte in a 25 biological sample, said method comprising act of providing modified citrate buffer to the biological sample for dissociating the complex. The present disclosure further relates to an enzyme linked immunosorbent assay (ELISA) kit for detecting and optionally quantifying an analyte in a biological sample, said kit 30 comprising components selected from group comprising modified citrate buffer, a capture antibody, a detection antibody, reagent for reacting with the detection antibody, microtitre plates, coating buffer, washing solution, and stopping solution or any combination thereof.
    WO 2013/121368 6 PCT/IB2013/051193 The present disclosure further relates to a modified citrate buffer for detecting and optionally quantifying presence of an analyte in a biological sample. In an embodiment of the present disclosure, the modified citrate buffer comprises citrate 5 HCl at concentration of about 125 mM and polysorbate-20 at concentration of about 0.l1% w/v, and wherein pH of the buffer ranges from 2.5 to 3.8, preferably 3.5. In another embodiment of the present disclosure, the biological sample comprises at least one interfering substance as a target for the analyte, and wherein said interfering substance 10 and said analyte interact to form complex. In yet another embodiment of the present disclosure, the analyte is a human epidermal growth factor receptor 2 (HER-2) antibody. 15 In still another embodiment of the present disclosure, the interfering substance is an extracellular domain of HER-2 (HER-2-ECD). DEFINITION OF TERMS 20 Modified Citrate Buffer- A Modified Citrate Buffer according to the present disclosure is a citrate buffer comprising sodium citrate and citric acid also comprising polysorbate 20 and HCl. The HCl is added in order to bring down the pH of the buffer to a desired range of 2.5-3.8. 25 The present disclosure relates to a process of utilizing a unique acidic dissociation buffer in a single step ligand binding assay/analytical assay to accurately quantitate the total amount of an analyte present in the biological matrix containing a soluble target of the analyte (also referred to herein as interfering substance). Using this buffer resolves the problem of underestimation of the analyte due to the presence of the interfering substance, without 30 introducing any pre-treatment of samples, thus preserving the simplicity and ease of immunoassays. The process comprises the following steps: a) Coating a solid surface such as a microtiter plate with an antibody specific to the analyte.
    WO 2013/121368 7 PCT/IB2013/051193 b) Diluting the biological samples containing the analyte and the interfering substance in modified Citrate buffer and adding the diluted samples to the coated solid surface from the previous step. c) Detecting the capture antibody-analyte complex by adding a labeled antibody against the 5 analyte. d) Adding a reagent capable of reacting with the labeled antibody and quantitating the signal. In an embodiment of the present disclosure Modified Citrate buffer is prepared by 10 modifying a citrate buffer by addition of a strong acid, i.e., the modified citrate buffer of the present disclosure is a combination of the salt of a weak acid (Sodium citrate) and a weak acid (citric acid) with the addition of a strong acid to obtain the required pH of between 2.5 to 3.8. 15 In one aspect, the modified citrate buffer contains polysorbate. In another aspect, the modified citrate buffer contains polysorbate-20. In yet another embodiment of the present disclosure, the capture and the detection antibodies may be of monoclonal or polyclonal origin. The epitope on the analyte 20 recognized by the detection antibody is different from the epitope recognized by the capture antibody. The label conjugated to the detection antibody may be an enzyme such as horse radish peroxidase, alkaline phosphatase or other enzymes. A substrate for the enzyme conjugated with the detection antibody is used as an indicator. Common substrates used are selected from ABTS (2,2'-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium 25 salt and TMB (3,3',5,5'-tetramethylbenzidine). In still another embodiment, the present disclosure relates to a process of utilizing a unique acidic dissociation buffer in a single step ligand binding assay to quantitate total amount of analyte present in the serum matrix containing the interfering substance. 30 In still another embodiment, the disclosure discloses a method of accurately determining the levels of an anti-HER2 antibody in the presence of an interfering substance, an extracellular domain (ECD) of HER2 receptors. The biological sample in which the WO 2013/121368 8 PCT/IB2013/051193 accurate determination of the level of anti-HER2 antibody is sought may be serum or plasma derived from a cancer patient undergoing anti-HER2 therapy. In still another embodiment of the present disclosure, accuracy (analytical recovery) of trastuzumab QCs made in metastatic breast cancer sera containing endogenous HER2-ECD 5 is maximum when modified citrate buffer is used as diluent. The present disclosure is illustrated by its application to the accurate analytical recovery of trastuzumab in serum spiked with recombinant HER2-ECD, the soluble target of trastuzumab. Over-expression of the human epidermal growth factor receptor 2 (HER 10 2/neu) occurs in 25% of human breast cancers and is associated with poor prognosis. Trastuzumab is a humanized monoclonal antibody against the extracellular domain (ECD) of HER-2/neu receptor and binding results in inhibition of cancer cell proliferation. Trastuzumab is approved for the treatment of HER-2/neu over-expressing Metastatic Breast Cancer. The concentration of serum trastuzumab should be maintained above 15 lOgg/mL during therapy, in order to effectively inhibit the growth of HER-2/neu over expressing breast cancer cells. Accurate quantitation of serum trastuzumab concentrations is hence critical during therapy. In an embodiment of the present disclosure, the full length HER-2/neu protein is composed 20 of a cytoplasmic domain, a transmembrane domain and an extracellular domain (ECD) that is shed from the surface of cells. ECD is shed into the blood of normal individuals and is often elevated in metastatic breast cancer patients. Trastuzumab administered for treatment of these patients exists in two different forms in the matrix, the free drug and the drug complexed with the ECD. Elevated levels of circulating endogenous ECD result in 25 increased levels of complexed trastuzumab. Standard immunoassays for quantitating trastuzumab measure only free trastuzumab leading to the underestimation of the circulating total trastuzumab levels. The assay disclosed herein eliminates the problem of ECD interference and thus underestimation by utilizing a unique acid-dissociation buffer which dissociates the trastuzumab-ECD complex in a single step. 30 The present disclosure is further elaborated with the help of following examples and associated figures. However, these examples should not be construed to limit the scope of the present disclosure.
    WO 2013/121368 9 PCT/IB2013/051193 EXAMPLES EXAMPLE: Preparation of Modified Citrate Buffer 5 In the present disclosure, 13.04 g of Citric Acid Monohydrate and 18.48g of Sodium Citrate Dihydrate are added to 950 mL of Milli Q water and dissolved. The pH of the solution is adjusted to 3.5 by adding Concentrated HCl (~ 4 mL). The volume of the solution is made up to 1000ml and the solution is filtered using 0.22 Micron filter unit to 10 obtain the modified citrate buffer. EXAMPLE 2: Single step ELISA for accurate quantitation of trastuzumab in the presence of interfering HER2- ECD. 15 In the present disclosure, 100 gL of a monoclonal antibody against trastuzumab Complementary Determining region (CDR) is coated on 96 well microtiter plates in PBS buffer at a final concentration of about lgg/mL and incubated overnight at about 4 0 C. Calibrators and Quality Controls (QC) are prepared using trastuzumab in serum samples 20 spiked with or without recombinant HER2-ECD. Samples are diluted about 50 fold (minimum required dilution, MRD) in modified citrate buffer containing 125 mM citrate HCl and 0.1% Tween-20 (polysorbate 20) at pH between 2.8 to 3.5, and added to the blocked antibody coated microtiter plates. The plates are incubated at 22 0 C for one hour and washed five times with PBST. After washing, about 100 gL of Peroxidase conjugated 25 polyclonal detection antibody against trastuzumab CDR (1:2500 in PBST) is added to each well and incubated at 22 0 C for one hour. The plates are washed seven times with PBST and about 100 gL of chromogenic substrate TMB is added to each well. After an incubation of 15 minutes, the reaction is stopped by the addition of 0.1N Sulphuric Acid and the plate is read at 450nm with a background subtraction at 630nm. 30 EXAMPLE 3: Investigation of interference of HER2-ECD on serum calibration curve of Trastuzumab and studying the Effect of Modified Citrate Buffer on the Same WO 2013/121368 10 PCT/IB2013/051193 In the present disclosure, in order to investigate the interference of HER2-ECD on the serum calibration curve of trastuzumab, trastuzumab calibration curves are made in pooled healthy female serum containing either 0 ng/mL, 250ng/mL and 500ng/mL of spiked recombinant HER2-ECD. Samples are made to undergo 50 fold MRD in appropriate 5 diluents - Figure 1. The Trastuzumab calibration curve was done using the ELISA assay as described above. Figure (1A) shows that increasing the amount of spiked recombinant HER2-ECD resulted in a locational shift of the Trastuzumab Calibration curves in PBST diluent. Figure (1B) 10 shows that the interference of spiked HER2-ECD is overcome when the serum samples underwent the MRD in modified citrate buffer (pH 3.5) diluent containing polysorbate 20. Dilution of samples containing spiked HER2-ECD and trastuzumab in modified citrate buffer containing polysorbate 20 resulted in the dissociation of immune complex formed between the analyte (trastuzumab) and target (HER2-ECD). 15 EXAMPLE 4: Determining the Optimal Acidic pH of Buffer for Disassociating Trastuzumab-HER2-ECD complex 20 In the present disclosure, to determine the optimal acidic pH required to dissociate the trastuzumab-HER2-ECD complex, an acidic buffer, Citrate is tested at three different pH values as assay diluents for MRD. PBST diluent is also used as a control. Trastuzumab calibration curves are made in serum without spiked HER2-ECD and trastuzumab QC samples (500 ng/mL in neat serum) are prepared in 500 ng/mL recombinant HER2 25 ECD spiked serum. Calibration standards and QCs are made to undergo MRD in each of the four diluents. Analytical recovery of QC samples in each diluent is back calculated from the calibration curve prepared in the corresponding diluent. The lower the percentage relative error of recovery of trastuzumab QC, the better the buffer. As shown in table 1 above, a pH of 2.5 to 3.8 is required to obtain maximum recovery of the trastuzumab QC 30 samples made in HER2-ECD spiked serum, with a pH of 3.5 showing the best result. The Trastuzumab calibration curve was done using the ELISA assay as described above. TABLE 1- Optimum pH of Assay Diluent for Maximum Analytical Recovery of Trastuzumab LLOQ-QC (Lower Limit of Quantitation Quality Control) WO 2013/121368 11 PCT/IB2013/051193 S. No Buffer pH % Analytical Recovery of % Relative error of trastuzumab QC (500ng/ml) in recovery of trastuzumab 500ng/ml HER2 ECD spiked QC (500 ng/mL) in 500 serum ng/mL HER2 ECD spiked serum 1 PBST 7.4 32.4 67.6 2 Citrate buffer+ 0.1% 4.5 46.5 53.5 polysorbate-20 3 Modified Citrate + 4.0 74.78 25.22 0.1% polysorbate-20 4 Modified Citrate + 3.8 90.29 9.71 0.1% polysorbate-20 5 Modified Citrate + 3.5 104.67 4.67 0.1% polysorbate-20 6 Modified Citrate + 3.0 108.41 8.41 0.1% polysorbate-20 7 Modified Citrate + 2.5 88.77 11.23 0.1% polysorbate-20 8 Modified Citrate + 2.0 ND ND 0.1% polysorbate-20 EXAMPLE 5: Comparison of Buffers for Obtaining Maximum Analytical Recovery of Trastuzumab QC 5 In the present disclosure, various acidic buffers at pH 3.5 are compared for MRD of samples to assess the most suitable combination of salt and weak acid in order to nullify the interference by HER2-ECD and maximize the analytical recovery of QC samples. Trastuzumab calibration curve, trastuzumab QCs (500 ng/mL and 1500 ng/mL in neat serum) are made inl gg/mL HER2-ECD spiked serum and made to undergo 50 fold MRD 10 in four different diluents. The ELISA assay also includes a control trastuzumab calibration curve made in serum without spiked HER2-ECD and diluted 50 fold in PBST. Analytical WO 2013/121368 12 PCT/IB2013/051193 recovery of QC samples in each diluent is back calculated from the control trastuzumab calibration curve made in serum without spiked HER2-ECD and diluted 50 fold in PBST. Figure 2 shows that usage of Modified citrate buffer diluent results in minimal calibration 5 curve shift induced by 1 gg/mL HER2 -ECD spike, as compared to the control un-spiked serum calibration curve diluted in PBST. In the present disclosure, dilution of samples in other acidic buffers of same pH (conventional citrate buffer without the addition of HCl and glycine-HCl) failed to nullify 10 the HER2-ECD induced calibration curve shift as shown in Table 2 below. Maximum recovery of the Trastuzumab QC samples (500ng/mL and 1500 ng/mL in neat serum) is observed when modified citrate buffer diluent is used. TABLE 2- Comparison of Various Buffers for Obtaining Maximum Analytical 15 Recovery of Trastuzumab QC S. No. Buffer % Analytical Recovery of Trastuzumab QCs in 1 pg/ml HER2-ECD spiked serum LLOQ QC LOW QC (500ng/ml) (1500ng/ml) 1 PBST 10.28 40.97 2 Glycine HCl, pH 3.5 Out of Range 9.82 3 Citrate buffer+ 0.1% 72.56 75.16 polysorbate 20 pH 3.5 4 Modified Citrate buffer+ 86.06 86.82 0.1% polysorbate 20 pH 3.5 This emphasizes the criticality of the modified citrate buffer composition on trastuzumab QC recovery in the presence of HER2-ECD. 20 EXAMPLE 6: HER2-ECD Tolerance Determination of the Modified Immunoassay using Modified Citrate Buffer Diluent WO 2013/121368 13 PCT/IB2013/051193 In the present disclosure, to determine the maximum concentration of shed HER2-ECD that can be tolerated by the improved immunoassay using modified citrate buffer diluent containing polysorbate 20, trastuzumab QCs (375 ng/mL, 1125 ng/mL and 7500 ng/mL) are made in serum samples containing increasing amounts of recombinant HER2-ECD. 5 The calibration standards and QC samples are made to undergo a 50 fold MRD in modified citrate buffer containing polysorbate 20. The analytical recoveries of QCs are back calculated from a trastuzumab calibration curve made in un-spiked serum. It is observed that trastuzumab QCs can be reliably recovered from samples containing up to 2gg/mL of HER2- ECD as shown in Table 3 below. The assay used in this experiment is ELISA. 10 TABLE 3- HER2-ECD Tolerance Determination of the Modified Immunoassay using Modified Citrate Buffer Diluent S. No. Spiked HER2-ECD level % Analytical Recovery of Trastuzumab QCs (ng/ml) in recombinant HER2-ECD spiked samples 375ng/ml 1125ng/ml 7500ng/ml 1 50 106.11 100.37 93.11 2 500 97.11 98.27 95.04 3 1000 96.45 94.49 94.64 4 2000 98.01 92.09 95.30 15 EXAMPLE 7: Analytical Recovery of Trastuzumab QC in Metastatic Breast Cancer Patient Samples with Varying Levels of shed HER2-ECD 20 In the present disclosure, analytical Recovery of trastuzumab QCs made in Metastatic Breast Cancer (MBC) serum samples containing varying levels of endogenous shed HER2 ECD is shown in Table 4 below. A trastuzumab calibration curve made in un-spiked pooled healthy female sera and trastuzumab QCs made in MBC serum samples are diluted 50 fold using either PBST or modified citrate buffer containing polysorbate 20. The 25 analytical recoveries of QCs are back-calculated from the corresponding diluent calibration curve. As seen in table 4 below, usage of modified Citrate buffer diluent results in optimal Trastuzumab QC recoveries compared to PBST diluent. The assay performed is ELISA.
    WO 2013/121368 14 PCT/IB2013/051193 The concentrations of endogenous shed HER2-ECD are evaluated in MBC patient samples by a commercially available ELISA kit. TABLE 4: Analytical recovery of Trastuzumab QC in metastatic breast cancer 5 patient samples with varying levels of shed HER2-ECD. Usage of modified Citrate buffer diluent resulted in optimal Trastuzumab QC recoveries compared to PBST diluent. *ND: Not done; BQL: Below Quantitation Limit. % Analytical Recovery of Trastuzumab QCs in Shed HER2- metastatic breast cancer patients at LLOQ QC Patient No ECD level (500ng/mL) (ng/mL) Modified Citrate buffer diluent PBST diluent containing polysorbate 20 1 33.7 95.02 99.93 2 56.8 87.37 97.02 3 77.5 86.23 100 4 170 83.02 103.32 5 211.6 74.22 95.3 6 580 67.06 106.17 7 716.9 ND 104.48 8 833.6 ND 95.09 9 4761.8 BQL 87.29 10 Therefore, from the above examples, it is clear that the process of the present disclosure utilizing the modified citrate buffer is capable of accurately determining and quantifying analytes, such as HER-2 antibody in a biological sample which contains a soluble target for the analyte such as HER-2 ECD. It is observed that Trastuzumab QCs can be reliably recovered from samples containing up to 4.7 pg/mL of HER2- ECD as shown in Table 4 15 above. OTHER EMBODIMENTS While a number of embodiments and examples of this invention are described herein, it is apparent that these embodiments and examples may be altered to provide additional WO 2013/121368 15 PCT/IB2013/051193 embodiments and examples which use the processes of this invention. Therefore it will be appreciated that the scope of this invention is to be defined by the appended claims, rather than by the specific embodiments that have been represented by way of example above.
    权利要求:
    Claims (27)
    [1] 1. A process for detection and optional quantification of analyte in biological sample, said process comprising providing modified citrate buffer to the sample prior to 5 carrying out assay for said detection and optional quantification of the analyte within the biological sample.
    [2] 2. The process as claimed in claim 1, wherein the process comprises: (a) providing modified citrate buffer to the biological sample for obtaining diluted 10 biological sample; and (b) carrying out conventional assay for detecting and optionally quantifying the analyte in the sample.
    [3] 3. The process as claimed in any of the claims I to 2, wherein the biological sample 15 comprises at least one interfering substance as a target for the analyte, and wherein said interfering substance and said analyte combine to form a complex.
    [4] 4. The process as claimed in claim 3, wherein the formation of the complex reduces amount of free analyte in the biological sample, thereby hindering the accurate 20 detection and quantification of said analyte.
    [5] 5. The process as claimed in claim 1, wherein the modified citrate buffer dissociates the complex formed in claim 3 and restores the free analyte within the sample to enable the accurate detection and quantification of said analyte. 25
    [6] 6. The process as claimed in claim 1, wherein the modified citrate buffer comprises Citrate-HCl at concentration of about 125mM and polysorbate-20 at concentration of about 0.l1% w/v, and wherein pH of the buffer ranges from 2.5 to 3.8. 30
    [7] 7. The process as claimed in claim 1, wherein the biological sample is selected from group comprising blood, plasma, and serum or any combinations thereof. WO 2013/121368 17 PCT/IB2013/051193
    [8] 8. The process as claimed in claim 1, wherein the analyte is selected from group comprising hormone, tumor antigen, bacterial antigen, viral antigen, protein therapeutic and antibody or any combinations thereof. 5
    [9] 9. The process as claimed in claim 8, wherein the analyte is an antibody.
    [10] 10. The process as claimed in claim 9, wherein the antibody is against human epidermal growth factor receptor 2 (HER-2). 10
    [11] 11. The process as claimed in claim 3, wherein the interfering substance is an extracellular domain of HER-2 (HER-2-ECD).
    [12] 12. The process as claimed in any of the claims 1 to 2, wherein the assay is enzyme linked immunosorbent assay (ELISA). 15
    [13] 13. The process as claimed in claim 12, wherein the enzyme linked immunosorbent assay is sandwich enzyme linked immunosorbent assay and is carried out in presence of components selected from group comprising capture antibody, detection antibody, reagent for reacting with the detection antibody, microtitre 20 plate, coating buffer, washing solution and stopping solution or any combinations thereof.
    [14] 14. The process as claimed in claim 13, wherein the capture antibody is an antibody against the analyte within the biological sample. 25
    [15] 15. The process as claimed in claim 13, wherein the detection antibody is labelled with an enzyme selected from group comprising horseradish peroxidase and alkaline phosphatase and ruthenium. 30
    [16] 16. The process as claimed in claim 13, wherein the reagent for reacting with the labelled antibody is chromogenic substrate selected from ABTS (2,2'-Azinobis[3 ethylbenzothiazoline-6-sulfonic acid]-diammonium salt, TMB (3,3',5,5' tetramethylbenzidine) and electro chemiluminescent substrate. WO 2013/121368 18 PCT/IB2013/051193
    [17] 17. The process as claimed in claim 13, wherein the coating buffer is phosphate buffered saline.
    [18] 18. The process as claimed in claim 13, wherein the washing solution comprises 5 phosphate buffered saline and Tween-20.
    [19] 19. The process as claimed in claim 13, wherein the stopping solution is 0. IN sulphuric acid. 10
    [20] 20. The process as claimed in any of the claims 12 to 19, wherein the enzyme linked immunosorbent assay (ELISA) comprises: a. coating a solid surface with the capture antibody against the analyte; b. providing modified citrate buffer to the biological sample for obtaining 15 diluted biological sample; c. adding the diluted sample to the antibody coated support of step (a); d. incubating and washing the support to remove any unbound analyte; e. adding a labelled detection antibody against the analyte and washing the support to remove any unbound detection antibody; and 20 f. adding reagent for reacting with the labelled antibody for detecting and optionally quantifying the analyte in the sample.
    [21] 21. A method of disassociating complex formed by interaction of analyte and at least one interfering substance as target for the analyte in a biological sample, said 25 method comprising providing modified citrate buffer to the biological sample for dissociating the complex.
    [22] 22. An enzyme linked immunosorbent assay (ELISA) kit for detecting and optionally quantifying analyte in biological sample, said kit comprising components selected 30 from group comprising modified citrate buffer, capture antibody, detection antibody, reagent for reacting with the detection antibody, microtitre plate, coating buffer, washing solution, and stopping solution or any combination thereof. WO 2013/121368 19 PCT/IB2013/051193
    [23] 23. A modified citrate buffer for detecting and optionally quantifying presence of an analyte in a biological sample.
    [24] 24. The modified citrate buffer as claimed in claim 23, wherein the modified citrate 5 buffer comprises Citrate-HCl at concentration of about 125mM and Tween-20 at concentration of about 0.1% w/v, and wherein pH of the buffer ranges from 2.5 to 3.8.
    [25] 25. The modified citrate buffer as claimed in claim 23, wherein the biological sample 10 comprises atleast one interfering substance as target for the analyte, and wherein said interfering substance and said analyte interact to form complex.
    [26] 26. The modified citrate buffer as claimed in claim 23, wherein the analyte is a human epidermal growth factor receptor 2 (HER-2) antibody. 15
    [27] 27. The modified citrate buffer as claimed in claim 25, wherein the interfering substance is an extracellular domain of HER-2 (HER-2-ECD).
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    同族专利:
    公开号 | 公开日
    WO2013121368A2|2013-08-22|
    KR102076354B1|2020-02-10|
    ES2796253T3|2020-11-26|
    AU2013219968B2|2018-11-08|
    US20160003826A1|2016-01-07|
    NZ700025A|2018-07-27|
    WO2013121368A3|2016-07-07|
    KR20150034676A|2015-04-03|
    US9638696B2|2017-05-02|
    EP2815238A2|2014-12-24|
    EP2815238B1|2020-04-08|
    EP2815238A4|2017-05-03|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US4604348A|1981-11-19|1986-08-05|New York Blood Center, Inc.|Composition for use in immunoassays|
    US4657851A|1984-01-03|1987-04-14|Georgetown University|Breast cancer diagnostic blood test|
    US5256541A|1991-11-06|1993-10-26|Sangstat Medical Corporation|Detection of soluble alloantigen immune complexes|
    SE9201984D0|1992-06-29|1992-06-29|Pharmacia Biosensor Ab|IMPROVEMENT IN OPTICAL ASSAYS|
    DE19538641C2|1995-10-05|2000-09-21|Privates Inst Bioserv Gmbh|Patient-specific immunoadsorbents for extracorporeal apheresis and processes for their production|
    US20030125536A1|1996-01-11|2003-07-03|Corixa Corporation|Compositions and methods for the therapy and diagnosis of breast cancer|
    DE19963859A1|1999-12-30|2001-07-12|Apotech Res & Dev Ltd|Bi- or oligomer of a di-, tri-, quattro- or pentamer of recombinant fusion proteins|
    US6984494B2|2000-08-15|2006-01-10|Genentech, Inc.|Analytical method|
    US20080182796A1|2001-08-31|2008-07-31|Syngenta Participations Ag|Modified Cry3A toxins and nucleic acid sequences coding therefor|
    ES2594893T3|2009-12-16|2016-12-23|Abbvie Biotherapeutics Inc.|Anti HER2 antibodies and their uses|EP3117211A4|2014-03-13|2017-10-11|Pharmacophotonics, Inc. D/B/A Fast Biomedical|Improved measurement of body fluid volumes|
    EP2904393B1|2012-10-03|2018-11-28|Gyros Patent Ab|Method and kit for analyte determination at acidic conditions|
    US10027165B2|2016-06-20|2018-07-17|Fairchild Semiconductor Corporation|Power supply circuit with secondary side to primary side communication|
    法律状态:
    2019-03-07| FGA| Letters patent sealed or granted (standard patent)|
    优先权:
    申请号 | 申请日 | 专利标题
    IN566/CHE/2012||2012-02-15||
    IN566CH2012||2012-02-15||
    PCT/IB2013/051193|WO2013121368A2|2012-02-15|2013-02-14|A process for detection and optional quantification of an analyte|
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