Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

• the present invention relates to improvements in immunoassays and analogous assays for other specific binding 5 pairs, especially assays to be carried out on samples of body fluids and other samples obtained from humans and non-human animals.
• immunoassay format There are essentially two types of immunoassay format, one in which an antibody or antigen is captured selectively C onto a capture zone of a solid phase from a sample under investigation (heterogeneous phase or "capture” assays), and another, a homogeneous phase assay format, in which the antibody or antigen under investigation is detected in solution.
• heterogeneous phase or “capture” assays a homogeneous phase assay format
• Assays for specific binding pairs other than antibodies and antigens for example, for receptors and their ligands, may be carried out analogously to immunoassays.
• the present invention is concerned with capture assays.
• the solid phase on which the capture occurs should be capable of being washed in some way and is, for example, of a plastics or polymeric material, for example, of nitrocellulose, polyvinyl chloride, polystyrene, polyamide, polyvinylidene fluoride or another synthetic polymer.
• the solid phase may be formed into microtitre wells, beads, dipsticks, aspiration tips, electrodes and optical devices.
• the solid phase may be particles, for example, so-called "latex" particles, stabilised blood or red blood cells, bacterial or fungal cells, spores, gold or other metallic sols, and proteinaceous colloids.
• the size of particles is generally from about 0.02 to 5 microns, and beads generally larger, for example, from about 2 mm to 10 mm.
• the solid phase may be a slide or "tile" having coated hollows. Slides,- cards and tiles may be used as immunoassay vessels for agglutination assays.
• Microtitre pi ⁇ tes and beads of polymeric material are particularly convenient, especially for large-scale use, since automated systems are available commercially for handling such test formats.
• Other solid phases that may be used include laminar solid phases, for example, membranes, sheets and strips, for example, of a porous, fibrous or bibulous material, for example, of nylon, polyvinyl chloride or another synthetic polymer, of a natural polymer, for example, cellulose, of a derivatized natural polymer, for example, cellulose acetate or nitrocellulose, or of glass fibres.
• Paper for example, diazotized paper may be used.
• Films and coatings, for example, of fibrous or bibulous material, for example, as described above, may be used as the solid phase.
• a solid phase for example, a membrane, sheet, strip, film or coating, may be incorporated in a device for the determination of multiple or, more generally, single samples.
• An assay device may incorporate the solid phase within a rigid support or a housing, which may also comprise some or all of the reagents required for carrying out an assay.
• a laminar solid phase may be used for a so-called “membrane” assay, for example, a dot-blot or Western biot assay.
• the capture component is immobilized in a predetermined "capture zone".
• the capture zone may be substantially the entire surface of the solid phase, for example, the inner surface of a microwell, or the outer surface of a bead or other particle.
• the capture zone is generally a defined region that is less than the total surface of the solid phase for example, one or more dots, stripes or other defined areas of predetermined size and shape.
• the capture component is generally a polyclonal or monoclonal antibody that forms a complex with that antigen.
• the capture component may be an antigen that binds that antibody species selectively.
• the capture component may be any substance that is capable of capturing antibodies non-selectively (an "im unoglobulin capture” assay). A representative proportion of all antibodies is captured, and then the antibody species of interest is detected by means of a labelled component that interacts specifically with that antibody species.
• the labelled component is generally a labelled antigen that binds specifically with the antibody species of interest.
• Suitable capture components for an immunoglobulin capture assay include, for example, protein A, protein G, anti-Fc antibodies, anti-IgG, anti-IgM and anti-IgA antibodies.
• immunoassay techniques described herein in connection with antibodies and antigens can be applied analogously to the detection and/or determination of members of specific binding pairs other than antigens and antibodies, especially receptors and their ligands, and references herein to immunoassays also include other specific binding pair assays, unless specified otherwise.
• ligands are hormones, for example, insulin and glucagon, a ino acid transport proteins, viral coat proteins, molecules involved in neurotransmission, for example, adrenergic and cholinergic transmitters, serotonin and dopamine, adrenalin, hista ine and cytokines.
• an antibody to a ligand or a receptor it may it may preferable to determine a ligand or a receptor by a conventional immunoassay. If it is difficult or impossible to produce an antibody, or if it is desirable for any other reason, a ligand or receptor may be determined in a capture assay analogous to a capture immunoassay. In such an assay, either the ligand or the receptor may be immobilised on the capture zone of a solid phase. Agonists and antagonists of ligands may also be determined in such a system.
• capture component accordingly denotes any substance that is immobilized on a capture zone of a solid phase for use in a heterogeneous phase (capture) assay for an antigen or hapten, antibody, or member of another specific binding pair, for example, a ligand or receptor, or an agonist or antagonist of a ligand.
• Testing of samples of body fluids and of solid samples, for example, of cells or tissue, obtained from a body for is carried out routinely. Testing is carried out in two main contexts. One is often called “clinical” testing and involves testing samples of body fluids and solid samples in a clinical context, for example, for the purpose of diagnosing disease and disorders, monitoring the course of disease and/or monitoring treatment in an individual.
• Another major type of testing is the screening of donated blood in order to maintain a supply of blood and blood products free from contamination by pathogenic organisms.
• Controls also enable timings, temperatures and functionality of nearly all components of an immunoassay system to be validated. Thus operator error is minimised and controllable. There still remains the problem that during manufacture it is possible that the capture zone of the solid phase on which an assay is to be conducted failed to be coated appropriately with the capture component of the system.
• manufacturers check that each well in a microtitre plate is filled with reagent at each stage of manufacture, either colorimetrically, gravimetrically or by some physical measurement of liquid level in the well. Similar checks are carried out in the coating of beads and other particles.
• Binding failure could be, for example, because an individual well became dirty during plate moulding, contaminated in store or during pre-manufacture stages, exposed to contamination during the binding phase, or even replaced with an unauthorised strip of wells during the coating or quality assurance procedures. Similar considerations apply to beads and other particles and also to other solid phases, for example, laminar solid phases for use in blot assays or in assay devices.
• the present invention provides methods and means to ensure that the coating component is actually present on the capture zone of the solid phase, and also that it is functional.
• the present invention provides a method for detecting the presence of a capture component immobilized on a capture zone of a solid phase for use in an immunoassay or other specific binding pair assay for an analyte of interest, wherein
• the capture component is labelled with a labe' capable of producing a detectable signal directly or indirectly, and the signal is detected before or after the assay is carried out;
• a further test which comprises contacting the solid phase with an agent that is capable of interacting with the capture component or the captured analyte of the immunoassay or other specific binding pair assay and is also capable of producing a detectable signal directly or indirectly, and detecting the signal.
• the signal produced may be detected qualitatively, quantitatively or semi-quantitatively (i.e. the term "detect” as used herein includes “determine”). Absence of the signal indicates that the capture component is not present on the capture zone of a solid phase, or is not present in an adequate amount. A signal lower than expected indicates incomplete or inadequate immobilization of capture component, that is to say, the amount of immobilized capture component is less than expected.
• the method according to the invention therefore provides a valuable method for eliminating from immunoassays or other specific binding pair assays false negative results due to manufacturing defects.
• the capture component itself is labelled with a label that is capable of producing a detectable signal, either indirectly or especially directly, it is a simple matter for the manufacturer to detect the signal on completion of manufacture to ensure that the labelled capture molecules are located as expected on the solid phase, for example, in a microwell, on a bead or on a capture zone of a laminar solid phase.
• the test to check for the presence of capture component on the capture zone of a solid phase may be carried out by the user of the solid phase, either before or after performing the immunoassay (or other specific binding pair assay) itself. If no signal is detected, or the signal is lower than expected for a particular solid phase, that solid phase should not be used for the intended assay. If the test is carried out after the assay itself has been performed on a sample, the result of that assay should be ignored and the assay should be repeated.
• the capture component may be labelled before it is applied to the capture zone of a solid phase, or labelling may be carried out after the capture zone of a solid phase has been coated with unlabelled component. It is generally preferable to label the capture component before it is immobilized, since labelling in situ may result in non ⁇ specific binding of the label to the solid phase.
• the present invention accordingly provides a solid phase for use in an immunoassay or another specific binding pair assay, which solid phase comprises a capture component immobilized on a capture zone, characterised in that the capture component is labelled with a label capable of producing a detectable signal directly or indirectly.
• the present invention further provides a method for producing a solid phase for use in an immunoassay or another specific binding pair assay, which comprises
• detectable signal (b) immobilizing a capture component on a capture zone of the solid phase and labelling the immobilized capture component with a label capable of producing a detectable signal directly or indirectly.
• Small molecules that produce (or emit) a predetermined detectable signal without changing the biological properties of the labelled molecule are well known and are widely used in immunoassays and other specific binding pair assays.
• the detectable signal may be optical, radioactive or physico- chemical and may be produced when the label is stimulated or excited, as in the case with fluorescent labels. (The term “produce” used herein includes “emits".)
• the signal may result from agglutination, a diffraction effect or a birefringence effect.
• directly detectable labels include, for example, radioactive markers; fluorescent markers, for example, fluoresca ine, rhodamine or fluorescein; and coloured markers, for example, dyes and coloured particles, for example, gold sol.
• fluorescent markers for example, fluoresca ine, rhodamine or fluorescein
• coloured markers for example, dyes and coloured particles, for example, gold sol.
• directly detectable labels for example, magnetically resonant and electroactive species, and methods for labelling molecules that can be used as capture components according to the present invention are described in the literature of the art, including standard text books, for example, Kemeny &
• Coloured and fluorescent labels are generally preferable to and more commercially acceptable than radiolabels.
• the present invention relates to both immunoassays (RIAs) and the more commercially important enzyme immunoassays (EIAs).
• EIAs enzyme immunoassays
• radiolabels are generally unsuitable for labelling the capture component.
• a coloured or fluorescent label is preferred for labelling the capture component. If a radiolabel is used for the capture component in an RIA, it should emit a different type of radiation from that of the radiolabel for the assay itself.
• Coloured labels and fluorescent labels may be read either by eye or by machine. Fluorescence may be determined by eye using an ultra-violet lamp and scanning the solid phase while looking for fluorescent and non-fluorescing units or regions. Determining the label using a machine has the advantage that a permanent record of the reading may be obtained.
• Fluorescent labels have an advantage over coloured labels of less potential interference with later steps in the immunoassay, which may involve coloured reagents and/or colour changes. They also have the further advantage that fluorescence is a much more sensitive technique than colirimetry. Fluorescent labels are therefore generally the preferred choice.
• the signal produced by the label may be detected (or determined) qualitatively, quantitatively or semi- quantitatively. Fluorescent labels are particularly suitable for semi-quantitative and quantitative determinations, as shown in Figure l of the accompany drawing.
• Figure 1 is a graph of fluorescence readings of microwells against coating volumes for fluorescein-labelled antibodies used to coat the microwells. The fluorescence readings were made on dry microwells after the coating process had been completed.
• the manufacturer of a coated solid phase can easily incorporate a detection step after completion of the conventional coating process, for example, after the product has been dried and before it is packaged, in order to check that the coating component is present as required.
• the user of the solid phase may check for the detectable marker and hence the capture component on the solid phase before use i.e. while it is still in the dry state, in addition to or as an alternative to a manufacturer's quality control detection step.
• the capture component may carry a label that is capable of producing a signal indirectly.
• a particularly preferred example of an indirect label is an enzyme that is capable of giving rise to a measurable or otherwise detectable change of any sort, particularly one in which the enzyme catalyses a detectable colour or fluorescence change in the presence of a suitable substrate.
• enzyme systems are widely used in enzyme-linked immunoassays (EIAs) and are well known per se, see for example, Kemeny & Challacombe loc. cit. (which also describes other direct and indirect labels).
• EIAs enzyme-linked immunoassays
• Examples of typical enzyme systems are those using alkaline phosphatase, ⁇ -galactosidase, urease or peroxidase, for example, horse-radish peroxidase.
• the same enzyme system may be used in the capture component detection step, but it may be preferable to use an enzyme that catalyses a different reaction.
• the capture component is labelled with biotin, which is capable of producing a detectable signal on reaction with avidin or streptavidin that is itself labelled with a direct or indirect label.
• hepatitis B surface antigen for example hepatitis B surface antigen (HBsAg)
• the manufacturer would normally coat the capture zone of a solid phase, for example, microwells or beads, with antibodies which react with the antigen Labelling the antibody with a label that produces (emits) a detectable signal, especially a direct label, for example, a fluorescent label, enables measurement of the label as a last stage during manufacture.
• a detectable signal especially a direct label, for example, a fluorescent label, enables measurement of the label as a last stage during manufacture.
• the signal may be read by the user prior to using the wells or beads for the intended assay or after carrying out the intended assay.
• the anti-human antibody may be labelled.
• the plate-coating antigen may be labelled.
• the solid phase may be contacted (after the immunoassay or other specific binding pair assay) with an agent that is capable of interacting with the capture component or the captured analyte and is also capable of producing a detectable signal, directly or indirectly.
• the user of the assay will carry out the confirmatory test.
• the agent that is capable of interacting with a capture component may be a binding partner for the capture component.
• the binding partner- is generally an antibody (polyclonal or monoclonal) that binds selectively to that antigen.
• the binding partner may be an antigen or may be a second antibody that will bind to the capture antibody, for example, the second antibody may be an anti-species antibody e.g. if the capture antibody is a sheep antibody, the second antibody may be an anti-sheep antibody. Similar considerations apply to other types of binding partners, for example, ligands and their receptors.
• the binding partner is labelled with a label capable of producing a signal directly or indirectly.
• the solid phase is contacted with an agent that is capable of interacting with the captured analyte and is also labelled with a label capable of producing a detectable signal directly or indirectly.
• an agent that is capable of interacting with the captured analyte and is also labelled with a label capable of producing a detectable signal directly or indirectly.
• the agent capable of interacting with the captured analyte is preferably a directly or indirectly labelled binding partner for the capture analyte and is especially a directly or indirectly labelled anti-human antibody.
• the agent that interacts with the capture component or the captured analyte is labelled with a label capable of producing a detectable signal directly or indirectly.
• the signal may be an optical, radio-active or physico-chemical signal.
• the agent may be labelled with a label that is detectable as such, for example, a dye, coloured particle, radiolabel, electroactive species, magnetically resonant species or fluorophore.
• the agent may be provided with a label that produces a signal indirectly, for example, by labelling the agent with biotin or with an enzyme that is itself capable of giving rise to a measurable change of any sort.
• the signal may result from agglutination, a diffraction effect or a birefringence effect involving the agent.
• the present invention provides a kit that comprises component(s) for the detection of a capture com ⁇ ponent immobilized on the capture zone of a solid phase by a method according to the present invention and optionally also comprises component(s) required for the immunoassay or other specific binding pair assay for the analyte of interest.
• a substrate for the enzyme may be provided; when the capture component is labelled with biotin, labelled avidin or streptavidin may be provided. If the avidin or streptavidin is labelled with an indirect label, for example an enzyme, the appropriate substrate may also be provided.
• the capture component or captured analyte is to be detected after the immunoassay or other specific binding pair assay, for example, by interaction with an agent capable of producing a detectable signal, that agent may be provided, together with any further reagents required.
• the agent may be a directly labelled specific binding partner, in which case no further reagents are required. If the specific binding partner is labelled with an enzyme or with biotin, then further reagents may be provided as described above.
• a kit may also comprises further reagents, selected for example, from diluents, buffers, wash solutions, stop solutions, standard and control solutions.
• an EIA involves the use of a component that carries an enzyme capable of catalysing a detectable reaction, especially a reaction with a substrate that results in a colour or fluorescence change.
• a capture (heterogeneous phase) assay the enzyme-labelled component forms a complex with another component that is bound (directly or indirectly) to the capture zone of a solid phase. Addition of the substrate results in a detectable, for example, colour change. It is conventional to add a so-called "stop solution” (often a strong acid) to stop the colour reaction. The signal, for example, colour or fluorescence, is then observed by eye or read by mach.ine. After completion of the assay, the various liquid and soiid components are generally thrown away.
• a capture (heterogeneous phase) RIA the solid phase is generally disposed of after measurement of any bound radiolabel.
• the confirmatory assay is carried out before the assay by the manufacturer and/or by the user of the assay or, alternatively, after the assay itself.
• the solid phase on which an assay (either EIA or RIA) for the analyte of interest has been carried out is not discarded but further reactions or tests are carried out to determine whether capture component is present, if it is present in an adequate amount, and/or if it is functional. It is generally (although not always) necessary to wash the solid phase (after discarding any liquid, for example, the coloured contents of a microwell, or a tube in which a bead has been assayed).
• the solid phase is then contacted with the agent that is capable of interacting with the capture component or the captured analyte and also of producing a detectable signal and is also contacted, simultaneously or sequentially, with any further reagents that are required to produce the detectable signal.
• the agent capable of producing the signal may be a binding partner for the capture component or captured analyte and may be labelled with a label that itself produces (emits) a detectable signal, for example, a coloured particle or a fluorescent label.
• a detectable signal for example, a coloured particle or a fluorescent label.
• the agent may be simply incubated with the solid phase, any agent remaining unbound after the incubation is washed off, and the solid phase is then examined for the presence of the signal.
• a coloured or fluorescent label may be determined by eye or, at the appropriate wavelength, by machine.
• a binding partner for the capture component may carry an indirect label, that is to say, a label that requires a further reagent for the production of a detectable signal.
• an indirect label is generally an enzyme capable of catalysing a reaction involving production of a detectable signal, generally a colour change or a change in fluorescence.
• the solid phase is generally washed, and is then incubated with the enzyme-labelled binding partner for the capture component and with the appropriate substrate for the enzyme in the same manner as a conventional EIA is carried out.
• the capture component may be determined indirectly via the captured analyte, using a directly or indirectly labelled binding partner for the captured analyte, such an assay may be carried out analogous to the method described above for the determination of the capture component itself. If the agent that is capable of interacting with the capture component or the captured analyte is labelled with biotin, labelled avidin or streptavidin is used to provide a detectable signal. In a further embodiment, the capture component itself is labelled with a label capable of producing a signal indirectly. That label may be an enzyme capable of catalysing a reaction that gives rise to a detectable signal.
• the solid phase is incubated with an appropriate substrate in the conventional manner for an EIA.
• the solid phase is then washed and the assay itself is carried out. If the test for capture component is carried out after the immunoassay or other specific binding pair assay itself, the solid phase is washed after the assay and the solid phase incubated with the substrate for the enzyme.
• An indirect label for the capture component may be biotin, in which case the agent capable of producing a detectable signal may be avidin or streptavidin, labelled with a direct or indirect label, as described above for direct and indirect labels for binding partners for a capture component.
• the solid phase is incubated with the labelled avidin or streptavidin.
• the direct or indirect label may then be determined as described above for binding partners for a capture component.
• the solid phase is generally washed before carrying out the main assay.
• the biotin assay is carried out after the main assay, generally after the solid phase has been washed.
• Coloured labels and fluorescent labels used as direct labels for any labelled component in any of the methods described above may be read either by eye or by machine, as may enzyme-catalysed colour or fluorescence changes. Fluorescence may be determined by eye using an ultra-violet lamp and scanning the solid phase while looking for fluorescent and non-fluorescing units, for example, individual microwells, or regions, for example, capture zones or laminar solid phases. Determining a coloured label or the product of a colour reaction using a machine has the advantage that a permanent record of the reading may be obtained.
• Capture component may be present on the capture zone of a solid phase but not functional.
• the capture component may have become contaminated such that it is present in the correct amount on the solid phase but it is unable to form a complex with the analyte of interest.
• a negative result will be obtained if an attempt is made to determine the capture component via the captured analyte, because none will be present. Analogously, if the method of the present invention is carried out using a binding partner for the coating component, a negative result will be obtained because none will be bound.
• the present invention accordingly provides a method of determining the presence of functional capture component immobilized on a capture zone of a solid phase used for an immunoassay or other specific binding pair assay for an analyte of interest wherein, after the immunoassay or other specific binding pair assay for the analyte has been carried out, there is performed a further test wherein the solid phase is contacted with a binding partner for the capture component or for the captured analyte, the binding partner being capable, directly or indirectly, of producing a detectable signal.
• the absence of signal in this method of the present invention indicates that the capture component is not present on the capture zone of a solid phase, is not present in an adequate amount, or is not functional.
• a signal lower than expected indicates incomplete or inadequate immobilization of capture component or inadequate functionality.
• a negative result in the assay for the analyte should therefore be ignored and the assay repeated.
• the capture component is immobilized on a predetermined capture zone of the solid phase.
• the capture component may be coated onto the entire surface of the solid phase, for example, over the inner surface of a microwell or over the outer surface of a bead or other particle.
• the capture component may be immobilized on defined regions of the solid phase only, for example, in dots, stripes or other defined areas of predetermined size and shape, for example, in the case of laminar solid phases to be used for blot assays.
• an antibody or antigen is dissolved at an experimentally determined optimum concentration, usually between about 0.5 and 5 micrograms/ml, in an optimum pH buffering medium, for example, a sodium carbonate/sodium bicarbonate buffer, for example, at about pH 9 to 10, for example, pH 9.6; sometimes a pH around 5 is better, and allowed to adsorb passively to the solid phase.
• an optimum pH buffering medium for example, a sodium carbonate/sodium bicarbonate buffer, for example, at about pH 9 to 10, for example, pH 9.6; sometimes a pH around 5 is better, and allowed to adsorb passively to the solid phase.
• chemical bonding is used to bind the reagent more firmly.
• the capture component is generally allowed to bind to the solid phase for at least 30 minutes, and usually overnight, then the solid phase is washed, generally with a pH buffering solution. After the binding (or bonding) step, a solution which will block any unfilled binding sites is added.
• the blocking agents is generally a protein, for example, albumin or casein, but other materials, including non-proteinaceous materials can be used. After a further incubation, generally more than about 30 minutes, not usually overnight, the solid phase is finally washed and allowed to dry. It is then ready for use.
• the capture component is to be immobilized on a capture zone that is less than the total surface of the solid phase, for example, if the capture zone is dots, stripes or predetermined shapes on a laminar solid phase, the capture component may be painted or printed onto the capture zone.
• a further method of applying capture components in the case where a laminar solid phase comprises a number of capture zones each with a different capture component, for example, as in Western blots, is to apply a mixture of capture components to the solid phase and to separate the capture components by electrophoresis.
• the capture component carries a label, either a direct label, for example, a coloured or fluorescent label, or an indirect label, for example, an enzyme or biotin, it may be labelled before it is applied to the solid phase, or labelling may be carried out after coating with unlabelled component. It is preferable to label the capture component before it is immobilized, since labelling in situ may result in non-specific binding of the label to the solid phase.
• the signal produced by the label may be determined qualitatively, quantitatively or semi-quantitatively. Fluorescent labels are particularly suitable for semi- quantitative and quantitative determinations, as shown in Figure 1 of the accompany drawing.
• Figure 1 is a graph of fluorescence readings against coating volumes for fluorescein-labelled antibodies used to coat microwells.
• the absence of signal in a method of the present invention indicates that the capture zone of a solid phase has not been coated with the capture component or that an inadequate amount of capture component is present or that the capture component is not functional.
• a signal lower than that expected indicates incomplete or inadequate coating of the capture zone or inadequate functionality of the capture component.
• a negative result in the previous assay for the analyte of interest should therefore be ignored and the assay replaced.
• the present invention provides a valuable method of eliminating false negative results due to manufacturing defects. The following Examples illustrate the invention:
• Sheep anti-human IgG was mixed with fluorescein iso hiocyanate at an experimentally determined optimum concentration ratio (which was about 80:1, at a protein concentration of about 5 mg/ml), allowed to react overnight, and then the excess fluorescent reagent was removed by gel filtration.
• Antibody labelled with fluorescein isothiocyanate as described above was brought to concentration of 5 microgram/ml in a carbonate/bicarbonate buffer pH 9.6.
• F7, G1-G7 and H1-H7 were used.
• Wells A8-A12, B8-B12, C8-C12, D8-D12, E8-E12, F8-F12, G8-G12 and H8-H12 of the plate were incubated with 100 ⁇ l of the antibody-containing solution, with the exception of wells
• the plate was incubated overnight at room temperature, and then free sites on the wells were blocked. The plates were then drained and dried at room temperature.
• Fluorescence of the dry microwells was measured using a Dynatech plate reading filter fluorimeter.
• the wavelengths for fluorescein are activation at 490nm with fluorescence at 535nm.
• Table 1 sets out the fluorescence readings obtained for the plate (wells Al- H12) and indicates the volume of antibody-containing solution used in the wells.
• Figure 1 is a graph showing fluorescence of the dry wells vs. coating volume.
• the method of the present invention may be used for qualitative, semi-quantitative or quantitative determination of a capture component on a capture zone of a solid phase for use in an immunoassay or another specific binding pair assay.
• microwells of a 96-well microtitre plate (Nunc, Maxisorb) were coated with a mixture of polyclonal rabbit antihuman IgG (DAKO) and polyclonal mouse antihuman IgM
• DAKO DAKO
• HIV-l recombinant protein comprising core and envelope antigens from the CBL-1 isolate of HIV-l (Sattentau Q.J. et al. (1986) Science 234 1120), HIV-2 peptide comprising gp36 envelope antigen
• the mixture of antibodies or of antigens was brought to a concentration of 5 microgram/ml in a carbonate/-bicarbonate buffer pH9.6.
• the solution was incubated in the microwells overnight at room temperature, and then free sites on the wells were blocked. The microtitre plates were then drained and dried at room temperature.
• Immunoassay for HIV-1+2 lOO ⁇ l of diluted samples (10 ⁇ l sample and 90 ⁇ l sample diluent) were added to wells of the microtitre plate and incubated under humid conditions at 37°C for 60 minutes. Some of the samples were HIV-l positive samples, some were HIV-2 positive, and some were HIV-1+2 negative.
• the wells were then washed thoroughly five times with a wash solution, each wash step involving removal of the contents of each well by aspiration, filling the well with wash solution (glycine borate buffer containing Tween) , and soaking for 30 seconds. After the final wash step the contents of the well were removed and the wells were inverted and tapped dry on a paper towel or tissue.
• HIV-l and HIV-2 conjugates 50 ⁇ l of a working strength solution of the HIV-l and HIV-2 conjugates (HIV-l recombinant protein comprising core and envelope antigens from the CBL-l isolate of HIV-l (Sattentau Q.J. et al. (1986) Science 234 1120) conjugated to HRP (horse-radish peroxidase and HIV-2 peptide comprising gp36 envelope antigen, ' conjugated to HRP) in HEPES buffer containing bovine serum albumin and detergents were added to the wells either singly or in combination as described below and the plate was incubated at 37°C for 30 minutes under humid conditions.
• HRP horse-radish peroxidase and HIV-2 peptide comprising gp36 envelope antigen, ' conjugated to HRP
• results obtained for the capture component test in those wells that were coated with the antibody capture component are greater by a factor of about 10 than the results for those wells that were coated with HIV antigens. Surprisingly, this result was observed regardless of whether the wells had contained HIV-positive or HIV-negative samples.

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