Patent Application
20100297778
1. Field of the Invention
This invention relates to reagents, more particularly, reagents for carrying out immunoassays.
2. Discussion of the Art
The KingFisher™ magnetic particle processor, commercially available from Thermo Fisher Scientific, Inc., Waltham, Mass., is designed to transfer coated magnetic particles between wells containing reagents in order to perform various biochemical processes. Movable magnetic rods are used to capture, transfer, and release the magnetic particles. The KingFisher™ magnetic particle processor has many programmable options, such as, for example, parameters of the magnetic particles, parameters relating to the release of the magnetic particles, incubation times, agitation of liquids in the wells, and sequence of usage of the wells.
The KingFisher™ magnetic particle processor can be used to perform immunoassays. In one embodiment of an immunoassay, a plastic strip holding five adjacent 1 mL wells can be used. The wells contain reagents used in the immunoassay. Magnetic particles are transferred between the wells by means of a movable magnet.
The KingFisher™ magnetic particle processor, used in conjunction with an immunoassay procedure, can increase the sensitivity of the immunoassay, as compared with currently available instruments and analyzers for carrying out immunoassays.
The primary mechanism underlying the improvement in detection of analytes is the use of a larger volume of sample that is used in commercially available immunoassay analyzers. A larger volume of sample contains a larger quantity of analyte. The KingFisher™ magnetic particle processor permits capture of this larger amount of analyte and subsequent quantification thereof.
The process carried out by the KingFisher™ magnetic particle processor improves detection of the analyte through reduction in the background signal resulting from non-specific binding of materials, other than the analyte, to the interior surface of a well. For example, certain conjugates having certain labels, e.g., an acridinium label, will bind to the interior surface of the well. The higher the concentration of conjugate having acridinium label in the reaction mixture, or the higher the concentration of acridinium on the conjugate, the more non-specifically bound conjugate will bind to the interior surface of the well.
Because the KingFisher™ magnetic particle processor moves the complex comprising the magnetic microparticle and the labeled conjugate to another well before the trigger reagent is introduced, and the reading carried out, the non-specifically bound conjugate is left behind, i.e., bound to the interior surface of the well, and, accordingly, will not contribute to the signal read. Non-specifically bound label increases the signal from the sample that is not associated with the analyte, i.e., the background signal. Elimination of the signal resulting from non-specific binding that is not associated with the analyte improves the sensitivity of the immunoassay.
In addition to the type of non-specific binding previously mentioned, there is a second type of non-specific binding. In this second type of non-specific binding, the conjugate containing the label, e.g., an acridinium label, non-specifically binds to the magnetic microparticle. This second type of non-specific binding results in an increase in noise and a reduction in the signal-to-noise ratio.
As shown in
The invention described herein involves a method and conjugate that can be used to eliminate the signal caused by non-specific binding of the conjugate, e.g., a specific binding member attached to a label, to a solid phase, e.g., a magnetic microparticle. The method and the conjugate involve the use of a cleavable linking agent for linking the label to the specific binding member that specifically binds to the analyte. The use of a cleavable linking agent allows the release of the label from the specific binding member from the complex comprising the magnetic microparticle, the analyte, and the conjugate into solution. After the release of the label, the magnetic microparticles having any label non-specifically bound thereto are removed from the reaction mixture. Only the label, e.g., acridinium, from the conjugate would remain in the elution well.
Any conjugate that is non-specifically bound through interaction between the label and the solid phase, e.g., a magnetic particle, would remain bound to the solid phase, and would subsequently be removed from the elution well when the solid phase is removed from the elution well and transferred to another well before the introduction of additional reagent(s), e.g., a trigger reagent.
If the conjugate is non-specifically bound through interaction of the label and the solid phase, the cleavage of the link between the label and the specific binding member (e.g., antibody) would only release the specific binding member (e.g., antibody). The label that is released from the specific binding member would remain bound to the solid phase.
In the immunoassay described herein, after the complex comprising the magnetic microparticles, the analyte, and the conjugate is formed, the cleavable linking agent is cleaved, the label from the complex is released, the magnetic microparticles are removed from the reaction mixture, the label is triggered, and then the signal is measured. In a sandwich immunoassay format, the immunoassay comprises the steps of:
In a competitive immunoassay format, the immunoassay comprises the steps of:
The invention also provides a kit for carrying out a competitive immunoassay and a kit for carrying out a sandwich immunoassay.
As used herein, the term “container” is intended to include both tubes and wells. The term “well” includes micro-wells and wells having greater volume than a micro-well. The KingFisher™ magnetic particle processor uses micro-wells. The KingFisher™ mL magnetic particle processor uses tubes. The principle of the method and conjugate described herein is the same regardless of whether micro-wells, wells, or tubes are used to perform the immunoassays described herein.
As used herein, the expressions “label”, “label group”, and the like mean a group attached to a specific binding member, e.g., an antibody or an antigen, to render the reaction between the specific binding member and its complementary binding member detectable. Representative examples of labels include enzymes, radioactive labels, fluorescein, and chemicals that produce light. A label is any substance that can be attached to an immunoreactant and that is capable of producing a signal that is detectable by visual or instrumental means. Various labels suitable for use in this invention include catalysts, enzymes, liposomes, and other vesicles containing signal producing substances such as chromogens, catalysts, fluorescent compounds, chemiluminescent compounds, enzymes, and the like. A number of enzymes suitable for use as labels are disclosed in U.S. Pat. No. 4,275,149, incorporated herein by reference. Such enzymes include glucosidases, galactosidases, phosphatases and peroxidases, such as alkaline phosphatase and horseradish peroxidase, which are used in conjunction with enzyme substrates, such as fluorescein di(galactopyranoside), nitro blue tetrazolium, 3,5′,5,5′-tetranitrobenzidine, 4-methoxy-1-naphthol, 4-chloro-1-naphthol, 4-methylumbelliferyl phosphate, 5-bromo-4-chloro-3-indolyl phosphate, chemiluminescent enzyme substrates, such as the dioxetanes described in WO 88100694 and EP 0-254-051-A2, and derivatives and analogues thereof. Preferably, the label is an enzyme and most preferably the enzyme is alkaline phosphatase.
As used herein, the expression “test sample”, the expression “biological sample”, and the term “sample” refer to a material suspected of containing an analyte. The test sample can be used directly as obtained from the source or following a pretreatment to modify the character of the sample. The test sample can be derived from any biological source, such as a physiological fluid, such as, for example, blood, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, amniotic fluid, and the like. The test sample can be pretreated prior to use, such as preparing plasma from blood, diluting viscous fluids, and the like. Methods of treatment can involve filtration, distillation, extraction, concentration, inactivation of interfering components, the addition of reagents, and the like. Other liquid samples besides physiological fluids can be used, such as water, food products, and the like, for the performance of environmental or food production assays. In addition, a solid material suspected of containing the analyte can be used as the test simple. In some instances it may be beneficial to modify a solid test sample to form a liquid medium or to release the analyte.
As used herein, the expression “specific binding member” means a member of a specific binding pair, i.e., two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. An example of such specific binding members of a specific binding pair is an antigen and an antibody that specifically binds to that antigen. Another example of such binding members of a specific binding pair is a first antibody and a second antibody that specifically binds to the first antibody.
As used herein, the term “conjugate” means a specific binding member, e.g., an antigen or an antibody, coupled to a detectable moiety, e.g., a chemiluminescent moiety. The term “conjugate” also means a specific binding member, e.g., an antigen or an antibody, coupled to a solid phase, e.g., a magnetic microparticle.
As used herein, the expression “cleavable linking agent” means an entity that covalently couples a specific binding member to a label, which entity can be cleaved by means of a change in the pH level of less than about 6 or greater than about 8 or by means of a chemical reaction with a chemical entity, such as, for example, a thiol, a periodate, a hydroxylamine.
As used herein, the expressions “solid phase”, “solid phase material”, and the like, mean any material that is insoluble, or can be made insoluble by a subsequent reaction. Representative examples of solid phase material include polymeric or glass beads, microparticles, tubes, sheets, plates, slides, wells, tapes, test tubes, or the like.
As used herein, the term “analyte” means the compound to be detected or measured. The analyte has at least one epitope or binding site.
As used herein, the expression “monoclonal antibodies” means antibodies that are identical because they were produced by one type of immune cell and are all clones of a single parent cell.
As used herein, the term “binding affinity of an antibody” means the strength of the interaction between a single antigen-binding site on an antibody and its specific antigen epitope. The higher the affinity, the tighter the association between antigen and antibody, and the more likely the antigen is to remain in the binding site. The affinity constant is the ratio between the rate constants for binding and dissociation of antibody and antigen. Typical affinities for IgG antibodies are 105 to 109 L/mole.
As used herein, the expression “normal human plasma” means human plasma that is free of the analyte of interest or other known abnormality or pathology.
As used herein, the term “pre-activated” means reacting 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide hydrochloride (hereinafter “EDAC”) and N-hydroxysulfosuccinimide (hereinafter “sulfo-NHS”) with the carboxyl groups on microparticles to provide semi-stable NHS esters that will react with NH2 groups on monoclonal antibodies to form stable amide bonds that couple the antibodies to the microparticles.
As used herein, the term “magnetic microparticles” means paramagnetic microparticles. Paramagnetic microparticles are attracted to magnetic fields, hence have a relative magnetic permeability greater than one. However, unlike ferromagnets, which are also attracted to magnetic fields, paramagnetic materials do not retain any magnetization in the absence of an externally applied magnetic field.
As used herein, the symbol “(s)” following the name of an item indicates that one or more of the subject items is intended, depending upon the context. As used herein, the symbol “S/N” means signal to noise ratio.
As used herein, the term “immunoassay” means a special class of assay or test that is performed in a container, e.g., a test tube, a well, a micro-well, which assay or test uses a reaction between and antibody and an antigen to determine whether a patient has been exposed to the antigen or has an antibody to the antigen. An immunoassay can be a heterogeneous immunoassay or a homogeneous immunoassay. The method described herein is primarily concerned with the heterogeneous immunoassay.
Heterogeneous immunoassays can be performed in a competitive immunoassay format or in a sandwich immunoassay format. In the competitive immunoassay format, a solid phase material is attached to a specific binding member specific for the analyte. The sample, which is suspected of containing the analyte, e.g., an antigen, is mixed with (a) the solid phase material attached to the specific binding member specific for the analyte and (b) a conjugate comprising the analyte attached to a detectable moiety. The amount of detectable moiety that binds to the solid phase material can be detected, measured, and correlated to the amount of analyte, e.g., antigen, present in the test sample. The analyte can also be an antibody, rather than an antigen. Examples of solid phase materials include beads, particles, microparticles, and the like.
The present invention is concerned primarily with the sandwich immunoassay format. However, other immunoassay formats, such as, for example, competitive assay formats, can be used. In the sandwich assay immunoassay format, a solid phase, e.g., a microparticle, is coated with antibodies. The antibody on the solid phase is known as the capture antibody. The assay is intended to detect and measure antigens in the sample. A second antibody is labeled with an appropriate label, e.g., acridinium. The second antibody is not attached to a solid phase. The second antibody is known as the detection antibody. The antibody and antigen attach in the following order to form a complex: antibody on solid phase-antigen-antibody having a label. Then the solid phase is removed from the complex. The antibody-antigen-antibody sandwich enables measurement of the antigen by activating the label, which can be used to determine the concentration of analyte in the sample. As used herein, the expression “sandwich complex” means an antibody-antigen-antibody sandwich.
In one example of the sandwich immunoassay format, a test sample containing an antibody is contacted with an antigen, e.g., a protein that has been immobilized on a solid phase material thereby forming an antigen-antibody complex. Examples of solid phase materials include beads, particles, microparticles, and the like. The solid phase material containing the antigen-antibody complex is typically treated, for example, with a second antibody that has been labeled with a detectable moiety. The second antibody then becomes bound to the antibody of the sample that is bound to the antigen immobilized on the solid phase material. Then, after one or more washing steps to remove any unbound material, an indicator material, such as a chromogenic substance, is introduced to react with the detectable moiety to produce a detectable signal, e.g. a color change, generation of light. The detectable signal change is then detected, measured, and correlated to the amount of antibody present in the test sample. It should also be noted that various diluents and buffers are also required to optimize the operation of the microparticles, antigens, conjugates, and other components of the assay that participate in chemical reactions. It should be further noted that other types of sandwich assays can be utilized, such as, for example, where the first antibody is immobilized on the solid phase material.
A heterogeneous immunoassay to determine the concentration of an analyte present at a low concentration in a biological sample can be performed with the apparatus described in U.S. Pat. Nos. 5,795,784 and 5,856,194, in a sandwich immunoassay format, which employs microparticles as the solid phase material. These patents are incorporated herein by reference.
In the case of HIV antigens, such as, for example, HIV-1 p24 antigen, it is preferred that monoclonal antibodies be used to carry out the immunoassay described herein. For example, monoclonal antibodies 120A-270 and 115B-151 can be used as a component of a solid phase capture antibody and as a detection antibody conjugate, respectively, to develop an ultra-sensitive immunoassay for HIV-1 p24 antigen for use in commercially available automated immunoassay analyzers. These monoclonal antibodies are described in greater detail in U.S. Pat. No. 6,818,392, incorporated herein by reference. Monoclonal antibodies are typically selected on the basis of their high binding affinities (e.g., greater than 5×109 liters/mole), compatibility between components for sandwich assays, and detection of all subtypes of the antigen tested. The monoclonal antibodies for the HIV-1 p24 antigen mentioned previously can be used to determine all subtypes of HIV-1 p24 antigen and HIV-2 p26 antigen.
Determination of the presence and amount of an analyte in a biological sample can be determined by a competitive diagnostic assay. Small molecule, competitive diagnostic assays usually require a labeled component that can compete with the analyte for available antibody sites. The labeled component is typically referred to as a tracer. Examples of the labeled component include radioactive tracers, fluorescent tracers, chemiluminescent tracers, and enzyme tracers. Typically, the labeled component consists of the analyte or an analogue of the analyte coupled to a label.
The probability that a particular reagent comprising a specific binding member for a given analyte and a labeled component will be useful in a sensitive assay for the given analyte can be assessed by knowledge of the dose response curve. The dose response curve for an immunoassay is a plot of the ratio of the response in the presence of the subject analyte to the response in the absence of the subject analyte as a function of the concentration of the subject analyte. The dose response curve for a given immunoassay is unique for each reagent comprising a specific binding member for a given analyte and a tracer and is modulated by the competition between the tracer and the analyte for sites on the specific binding member for the analyte.
Prior to carrying out an immunoassay for the subject antigens, the method described herein utilizes a processing technique to prepare biological samples for use in a commercially available automated immunoassay analyzer. Such a processing technique can be carried out with a KingFisher™ mL magnetic particle processor or a KingFisher™ magnetic particle processor, both of which are commercially available from Thermo Fisher Scientific, Inc., Waltham, Mass.
Referring now to
Before starting the magnetic particle processing via a keypad (not shown) and a display (not shown), the samples and reagents are dispensed into the tubes 116a, 116b, 116c, 116d, and 116e and the tip comb(s) 114 is (are) loaded into its (their) slot(s). The tube strip(s) 116 is (are) placed into the removable tray in the correct position and the tray is pushed into the end position. During the operation, the front and top lids can be closed or open. Closed lids protect the processing against environmental contamination. The KingFisher™ mL magnetic particle processor is described in detail in KingFisher™ mL User manual, Revision No. 1.0, February 2002, Catalog No. 1508260, incorporated herein by reference.
The KingFisher™ magnetic particle processor is designed for the automated transfer and processing of magnetic particles in volumes of liquids suitable for micro-wells. This is in contrast to the KingFisher™ mL magnetic particle processor, which employs greater volumes of liquids. The KingFisher™ magnetic particle processor is described in detail in KingFisher™ Micro-well User Manual, Revision No. 1.0, 1999-04-09, Catalog No. 1507730, incorporated herein by reference.
Referring now to
Before starting the magnetic particle processing via a keypad (not shown) and a display (not shown), the samples and reagents are dispensed into the micro-wells 216a, 216b, 216c, 216d, 216e, 216f, 216g, and 216h and the tip comb(s) 214 is (are) loaded into its (their) slot(s). The well strip(s) 216 is (are) placed into the removable tray in the correct position and the tray is pushed into the end position. During the operation, the front and top lids can be closed or open. Closed lids protect the processing against environmental contamination.
Regardless of which of the aforementioned KingFisher™ instrument is being used, the operating principle employed is inverse magnetic particle processing technology, commonly referred to as MPP. Rather than moving the liquids from one well to another, the magnetic particles are moved from the tube 116a (or from the micro-well 216a) to the tube 116b (or to the micro-well 216b), at least one tubes (micro-wells) containing specific reagent(s). This principle stands in contrast to the external magnet method, i.e., the type of separation used in the apparatus shown in U.S. Pat. Nos. 5,795,784 and 5,856,194. According to inverse magnetic particle processing technology, magnetic particles are transferred with the aid of magnetic rods covered with disposable, specially designed plastic tip combs.
Working with magnetic particles can be divided into five separate process steps:
As shown in
With respect to non-specific binding, the label can non-specifically bind to magnetic microparticles; furthermore, the specific binding member can non-specifically bind to magnetic microparticles.
The removal of a chemiluminescent label, e.g., acridinium, that is non-specifically bound to magnetic microparticles has not been an option for chemiluminescent instrument platforms, e.g., ARCHITECT®, PRISM®, IMX®, AXSYM® instruments. Prior to the development of the conjugate and the method described herein, there has not been a mechanism available to separate the solid phase, with any non-specifically bound chemiluminescent label, e.g., acridinium, from the reaction mixture prior to the sequence of the trigger step, the read step, and the quantitation step, in these instruments.
The use of a conjugate having a cleavable linking agent, with a Kingfisher™ magnetic particle processor or a Kingfisher™ mL magnetic particle processor, allows only the label specifically bound to the captured analyte to contribute to a signal. The Non-specifically bound label attached to the solid phase, e.g., magnetic microparticles, would be removed from the elution well and eliminated as a source of a non-specific signal, thereby improving the sensitivity of the assay.
The method described herein provides an opportunity to evaluate the use of increased concentrations of labeled conjugates, or to evaluate the incorporation of higher ratios of label into conjugates when preparing conjugates comprising a specific binding member attached to a label.
Increased concentrations of labeled conjugates would accelerate reaction kinetics, and the incorporation of higher ratios of labels into conjugates would increase the amount of label present for the detections system. Either of these actions could improve sensitivity of the assay by increasing the signal based on the specifically bound analyte. Higher concentrations of the labeled conjugates or higher incorporation ratios of the label into the labeled conjugates would likely result in more non-specific binding of the labeled conjugates to the solid phase, e.g., magnetic microparticles. However, the non-specifically bound label would not increase the signal resulting from non-specifically bound conjugates, because such non-specifically bound label would be removed from the elution reaction mixture along with the solid phase, e.g., magnetic microparticles.
If acridinium is used as the label, the cleavable linking agent would need to be cleavable under conditions that do not trigger the acridinium prematurely, or modify the acridinium in such a way that would result in reduction, or even elimination, of its chemiluminescent properties.
Cleavable linking agents suitable for use with the reagents and immunoasssays described herein are set forth in TABLE 1.
The cleavable linking agents suitable for use herein are insensitive to pH of the reaction mixture.
Techniques for preparing the first conjugate and the second conjugate are well-known to those having ordinary skill in the art. In order to prepare the first conjugate, the magnetic microparticle, which has a polymeric coating thereon, can be bound to the specific binding member in one of two ways. If the polymeric coating has reactive groups, the magnetic microparticle can be covalently bonded to the specific binding member. If the polymeric coating does not have reactive groups or if it has reactive groups that will not react with the specific binding member, the magnetic microparticle can be attached to the specific binding member by van der Waals force. The first conjugate suitable for use herein can be manufactured by Invitrogen Corporation, under the trademark Dynal®.
In order to prepare the second conjugate, one reactive group of the cleavable linking agent forms a bond with a functional group of the specific binding member, and the other reactive group of the cleavable linking agent forms a bond with a functional group of the label. See Pierce Catalog 2005/2006, incorporated herein by reference, for references that teach one of ordinary skill in the art how to attach the cleavable linking agent to functional groups of chemical entities, such as, for example, specific binding members and labels.
Conjugates suitable for use herein can be manufactured by Invitrogen Corporation.
The conjugate described herein has several advantages relative to those of the prior art. By removal of the non-specifically bound label, noise is reduced and the signal-to-noise ratio is increased. A higher concentration of the conjugate, e.g., acridinium attached to a specific binding member, can be added to the reaction mixture. A higher concentration of label, e.g., acridinium, can be incorporated into the conjugate.
Care must be taken in selection of the linking agent that the cleavage process must not adversely affect the label, e.g., acridinium. Care must be taken so that the range of pH in the presence of hydrogen peroxide does not exceed 8.0.
Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention, and it should be understood that this invention is not to be unduly limited to the illustrative embodiments set forth herein.
