WO2001044499A9

WO2001044499A9 - Modified labeled complement components for immunoassays

Info

Publication number: WO2001044499A9
Application number: PCT/US2000/041081
Authority: WO
Inventors: Stanley Paul Racis; Steve Sinka
Original assignee: Diagen Corp
Priority date: 1999-12-16
Filing date: 2000-10-05
Publication date: 2002-06-06
Also published as: WO2001044499B1; AU4301001A; WO2001044499A1

Abstract

The invention is in the field of immunologic serological in vitro diagnostics. One aspect of the invention is the reagent itself. The reagent is a polypeptide modified complement componentthat is conjugated to one or more signal emitting molecules, such that it facilitates reactions in assays targeting the presence of analytes in samples. A second aspect of the invention is a method for making the reagent. A third aspect of the invention involves the use of the reagent to eliminate materials, time and costs in many types of immunoserological assays. The invention further includes a kit for detecting the presence of analytes in samples.

Description

TITLE MODIFIED LABELED COMPLEMENT COMPONENTS FOR IMMUNOASSAYS

Inventors Stanley Paul Racis Steve Sinka

BACKGROUND OF THE INVENTION It is important to be able to quickly and accurately quantify various analytes in order to aid in the diagnosis, monitoring and prognosis of various disease conditions. This is true for a host of diseases including many cancers, infectious diseases, and autoimmune conditions, such as systemic lupus erythematosus and rheumatoid arthritis. While it is often preferable to assay for antigens directly for infectious diseases, in autoimmune situations, the antibodies against a host of different self-antigens are themselves the marker of interest. Numerous techniques, such as electrophoresis and immunoassays. are known for detecting analytes in a sample, including cell culture and biological fluids, such as blood, urine and cerebrospinal fluid. The problem with electrophoresis is that it requires complicated, time-consuming and costly instruments that are not always readily available. However, many of the more simple common detection techniques and assays are immunoassays.

The basis for many immunoassays is similar, the binding of an antigen or hapten to a specific antibody. This binding is specific, sensitive and generally easily reproducible. To facilitate measurement, the analyte, the substance to be measured, usually antigen (Ag) or antibody (Ab), is displayed in solution or on some form of solid support, such as cell membranes, microscope slides, micro titer wells, or diffusion or electrophoretically separated bands that are stabilized in gels. The fluid to be tested, usually blood, urine, cerebrospinal fluid or cell cultures, is then placed in contact with the solid support that is displaying the analyte. If ligand. usually antibody or antigen, is present in the test fluid, it will bind to the solid support via the analyte display. Unbound materials are washed free of the solid support. Most often, but not necessarily, a secondary antibody with an attached signal emitting molecule, such as fluorochromes, enzymes, radioisotopes, magnetic substances, or chemiluminescent compounds, is incubated with the solid support to facilitate measurement of binding to the ligand. After incubation, unbound labeled secondary antibody is washed free of the solid support. Finally, quantitative detection of the signal is performed to determine the concentration of analyte in the fluid sample.

Such assays can be heterogeneous or homogeneous. Heterogeneous assays require the separation of bound labeled-reagents from free or unbound labeled reagents. On the other hand, homogeneous assays do not require a separation step so they are usually faster, more convenient and easier to automate than heterogeneous assays. Several examples of immunoassays are radioimmunoassays, immunofluorescent assays, and enzymatic immunoassays.

A radioimmunoassay (RIA) is a type of immunoassay that employs radioisotopes as labels to detect antigens or antibodies. RIAs are generally the most sensitive, the most reproducible, and the most amenable to automation. However, RIAs require a separation step so they are also time consuming. In addition, RIAs are unable to be performed in many academic and clinical settings because the isotopes are relatively expensive and the RIAs require capitol intensive and complex equipment. Furthermore, other types of assays are favored over RIAs because RIAs require specialized safe working facilities, specially trained and certified technicians, and a means for nuclear waste disposal.

Immunofluorescent assays (IF A) are another type of immunoassay. IF As allow for the detection and localization of antigens in which specific antibody is conjugated with fluorescent compounds to provide direct detection of the label by fluorescent microscopy. In general, IF As using organic fluorochromes, such as fluorescein or rhodamine derivatives, cannot match the sensitivity of RIAs. In addition, the non-specific background effects of IF As can be particularly vexing. However, their ease of use and relative rapidity has rendered them amenable to many medical and scientific applications. Furthermore, IFAs are readily adaptable to homogeneous assay procedures. Another type of immunoassay is the enzymatic-immunoassay (EIA). EIAs use enzymes to covalently label antibody or antigen in order to detect substances. Several variations of EIAs exist, including sandwich assays and enzyme linked immunosorbent assays, commonly referred to as ELISA. Examples of some commonly used enzymes are horseradish peroxidase. alkaline phosphatase, and β-galactosidase. A problem with EIAs is that they involve an additional step whereby modification of the enzyme's substrate is measured, usually by a colorimetric reaction. A problem with the above procedures is that they require a number of incubations and washing steps, which add significant time and manipulations to the assays. Even though many of the assays can be purchased in kits, these kits still involve multiple sequential incubations and washes prior to development and recording, which results in increased time and costs.

Another aspect of the immune response involves complement. Complement is a collective term used to designate a group of plasma and cell membrane proteins that interact in a precise sequential manner, termed the complement cascade, and play a key role in host defense processes, particularly in regulating features of the inflammatory and immune responses. The complement cascade consists of two pathways: the classic pathway and the alternative pathway. The classic complement pathway is activated when complement component Cl binds to antigen-antibody complexes. On the other hand, the alternative complement pathway does not require antibody binding for activation. Despite the two different activation pathways, the proteins of the complement system are termed components. Two of the complement components are Cl and C3.

Cl is a macromolecular complex composed of three different proteins, Clq, Clr and Cls. Each Cl complex is composed of one Clq, two Clr and two Cls chains. Antibody binding is mediated by the Clq portion of the complex, which binds the Fc portion of immunoglobulins (Ig). Clq can bind IgM, IgGl, IgG2 or IgG3. On the other hand, Clq does not bind IgG4, IgE, IgA or IgD. Clq is composed of three Y-shaped subunits that are joined together. Each Y-shaped subunit consists of six polypeptide chains. At the end of each Y- shaped branch is a globular head that is capable of binding the Fc region of immunoglobulin. Thus, Clq has six individual binding sites for the Fc region of immunoglobulin. However, the affinity and avidity of these individual globular regions are not sufficient for stable binding to an immunoglobulin because the six globular heads have significant freedom of movement due to the flexibility of the Y-shaped subunits. However, if a stabilized array of Fc regions, as formed by an antigen-antibody complex, is present, Clq can form a stable bond with it.

C3 is a glycoprotein that consists of an alpha and beta disulphide-linked chain. An inactive peptide C3a can be cleaved from the alpha chain, resulting in the formation of C3b. C3b has a strong tendency to interact with IgG molecules. Some types of assays take advantage of complement's ability to bind antigen- antibody complexes in order to amplify the detection reaction. After the antigen-antibody complex is formed, complement is added to the system. The bound complement can then be detected through the use of labeled antibody that is specific for complement. When the antigen-antibody reaction is amplified by the addition of complement, the number of incubations and washing steps required is increased resulting in a significant increase in time required to complete the assay.

There is a need for a detection method that is more rapid, reliable and cost effective than the presently available detection methods and test kits. BRIEF SUMMARY OF THE INVENTION

The invention is in the field of immunologic serological in vitro diagnostics. The invention provides reagents that may be used in many types of immunoserological formats, obviating the need for discrete sequential incubated reactions. The invention facilitates a savings in time, materials and cost in the measurement of analytes, particularly antibodies, without sacrificing specificity.

One aspect of the invention is the reagent itself. The reagent is comprised of a complement component, one or more polypeptide linkers bound to the complement component, and one or more signal emitting molecules bound to one or both of the linker(s) and the complement component. The complement component is preferably Clq or C3b. The one or more polypeptide linkers may be selected from the group consisting of poly-1-lysine, poly-1-alanine, poly-1-arginine, poly-1-cysteine, poly-1-glutamate, poly-1-glycine, poly-1- asparagine, poly-1-serine, poly-1-glutamine, poly-1-tyrosine, poly-1-histidine, poly-1-leucine, poly-1-proline, poly-1-threonine, poly-1-tryptophan, poly-1-phenylalanine, poly-1-ornithine, poly-1-hydroxyproline. poly-1-glutamic acid and poly-1-isoleucine. The one or more signal- emitting molecules may be selected from the group consisting of fluorochromes, enzymes, radioisotopes, chemiluminescent molecules, latex particles, and magnetic compounds. The reagent of the present invention facilitates reactions in assays targeting the presence of analytes, preferably antibodies, in samples. The polypeptide linkers are bound to the complement component at sites that are not involved in antigen-antibody binding. The polypeptide linker has a plurality of conjugation sites for binding signal emitting molecules. The invention further includes a kit comprised of the reagent described above, antigen specific to an antibody of interest bound to a support, and preferably positive and negative controls. The kit is particularly useful for detecting the presence of antinuclear antibodies in a patient sample.

A second aspect of the invention is a method of making the reagent. The method of the present invention includes two phases comprising: binding one or more first signal emitting molecules to one or more polypeptide linkers to form one or more labeled polypeptide linkers, conjugating the labeled polypeptide linkers to a complement component to form a reaction product. In a preferred embodiment, the method of the present invention further includes binding at least one second signal emitting molecule to the reaction product to form a reagent. A third aspect of the invention involves use of the reagent, or the kit, in any of a number of different immunoassays. The present invention includes a method for detecting antigen specific antibodies comprising: incubating together a mixture that includes a reagent, a sample suspected of containing an antibody of biological interest and a bound antigen specific to an antibody of interest in amounts and for a period of time sufficient to allow any antibody present in the sample having the capacity to bind with the bound antigen, to bind thereto to form a bound antigen-antibody complex, and to allow the reagent to bind to any bound antigen-antibody complex present to form a bound reagent; removing any unbound reagent; and detecting signal emitted from the bound reagent.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings are for illustrative purposes to facilitate better understanding of the invention and not for limiting the same.

Figure 1 is a diagram of an embodiment of a reagent.

Figure 2 is a flowchart showing an embodiment of the method for making a reagent. Figure 3 is a diagram illustrating the differences between a standard immunoassay and an immunoassay using a reagent according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a reagent for use in immunoassays, a novel immunoassay and a method for making the reagent. The reagent of the present invention is shown schematically in Figure 1. The reagent is a complement component that is conjugated to one or more signal emitting molecules and one or more polypeptide linkers. The reagent facilitates reactions in assays targeting the presence of antibodies in samples. The reagent comprises a complement component. The complement component preferentially binds to complexed antibody over unbound antibody. The term complexed antibody as used herein means antibody bound to antigen that is displayed on some form of solid support or in some similar manner stabilizing immunoglobulins (Ig) with respect to each other in close proximity. Complement components Clq and C3b are preferred. Complement is species dependent so complement components derived from mammals, preferably humans and guinea pigs, and synthetic complement components are preferred for detecting human antibodies while complement components derived from mice and rabbits are less preferred for detecting human antibodies. Human Clq and human C3b are the most preferred complement components for detecting human antibodies.

Complement component Clq may be derived using any of the methods described by Kolb et al., "Clq: Isolation from human serum in high yield by affinity column chromatography and development of a highly sensitive hemolytic assay", J. Immunology, vol. 122, p. 2103 (1979); Paul et al., "Isolation and characterization of highly stable rabbit Clq", J. Immunological Methods, vol. 21, p. 341 (1978); Agneilo et al., "Precipitin reactions of the Clq component of complement with aggregated gamma globulins and immune complexes in gel diffusion", Immunology, vol. 19, p. 909 (1970); Yonemasu et al, "Clq: Rapid purification method for preparation of monospecific antisera and for biochemical studies". J. Immunology, vol. 106, p. 304 (1971); Calcott et al., "Clq protein of human complement", Biochemistiγ, vol. 11, p. 3442 (1972); Sledge et al., "Purification of the human complement protein Clq by affinity chromatograpy", J. Immunology, vol. 1 1 1, p. 661 (1973); Pohl et al., "Isolation and purification of human Clq from plasma", J. Immunological Methods, vol. 36, p. 13 (1980); and Tenner et al., "Purification and radiolabeling of human Clq", J. Immunology, vol. 127(2), p. 648 (1981), the disclosures of each of which are incorporated herein by reference. Complement components are also commercially available and are expected to work well in the reagent, provided there is sufficient quality control in the manufacturing process, as unstable, labile or agglutinated materials do not work well.

The reagent also comprises one or more polypeptide linkers. The polypeptide linkers have the capacity to bind the complement component at sites and the capacity to bind one or more signal-emitting molecules. The polypeptide linkers are bound to the complement component at sites. The sites can be any location on the complement component as long as they do not interfere with the complement component's ability to bind to antigen-antibody complexes. Interference is determined by any significant inhibition of the conjugate's ability to agglutinate IgG coated latex particles compared to Clq and/or by its performance characteristics in the antinuclear antibody immunofluorescent assay application. In addition, the binding of the polypeptide linkers to the complement component can occur by any known method in the art provided that the complement component's binding sites for antigen- antibody complexes are not directly or sterically inhibited and as long as the method does not interfere with the complement component's ability to bind antigen-antibody complexes. Of course, the method of binding will vary, depending upon the particular polypeptide linker used. The polypeptide linkers may be derived from any source, including mammals, preferably humans, synthetic polypeptide linkers and commercially available polypeptide linkers. Although any size polypeptide linker may be used, it should be of sufficient size to provide numerous conjugation sites for binding signal emitting molecules. Polypeptide linkers between 15-30 kilodaltons (KD) of molecular weight (MW) are preferred. In addition, polypeptide linkers comprising the 1-enantiomer are preferred. Some examples of polypeptide linkers include: poly-1-lysine, poly-1-alanine, poly-1-arginine, poly-1-cysteine, poly-1-glutamate, poly-1-glycine, poly-1-asparagine, poly-1-serine, poly-1-glutamine, poly-1- tyrosine. poly-1-histidine, poly-1-leucine, poly-1-proline, poly-1-threonine, poly-1-tryptophan, poly-1-phenylalanine, poly-1-ornithine, poly-1-hydroxyproline, poly-1-glutamic acid and poly- 1-isoleucine. However, poly-1-lysine is the most preferred.

The reagent also comprises one or more signal -emitting molecules. The signal- emitting molecules are bound to the polypeptide linkers and/or the complement component. Only one signal-emitting molecule needs to be bound to the reagent. However, detection is easier with multiple signal-emitting molecules bound to the reagent. The signal-emitting molecule may be any type of label used in serology, including fluorochromes, enzymes, radioisotopes, chemiluminescence and magnetic labels. The signal-emitting molecules may be either synthetically made or commercially available. Latex particles may also be used. Although latex particles may not be typically thought of as emitting signals, the latex will suffice as a visually detectable tag for the bound reagent and, therefore, as used herein, signal emitting molecules shall include latex particles.

In addition, binding of the signal-emitting molecules to the reagent can be by any means known in the art, including various types of electrostatic, hydrophilic and/or hydrophobic bonds. Some ionic or covalent bonds may also be acceptable, however, this varies depending upon the polypeptide, the specific signal emitting molecules and individual application constraints. In addition, the use of active cross-linking agents, such as gluteraldehyde and cyanagen bromide, should be avoided as these agents usually lead to interference and background problems. Of course, the method for binding will depend upon the particular type of signal-emitting molecules used.

A second aspect of the invention is a method for making the reagent. The signal- emitting molecule or molecules are not all added at one time. Rather, the method is a two phase procedure, as shown in Figure 2. The first step of the method is to obtain a purified complement component.

Purification of the complement component is not a necessary step of the method, but if performed, will enhance results. As stated previously, complement may be derived from mammals, synthetically made, or bought from a commercial source. Therefore, the complement component may already exist in purified form or may need to be subsequently purified. If performed, purification may be by any method or methods known in the art, including: chromatography, based upon size and/or charge; gradient centrifugation; affinity chromatography, immunoprecipitation or electrophoresis. The purification procedure(s) should preferably result in a complement component recovery of about 90% or more of the binding units, as measured by latex agglutination titer, and preferably about a 99% or more decrease in free label, although some variation may be tolerated. The percent purity of the complement component is not as important as the percent recovery of active binding units normalized to complement component mass. The goal of purification is to avoid as much background noise in the detection step of the immunoassay as possible.

The next step of the invention involves binding one or more first signal-emitting molecules to a polypeptide linker to form a labeled polypeptide linker. As stated previously, the polypeptide linker can be any one of a number of linkers. In addition, the first signal- emitting molecule may be any type of label used in serology, including fluorochromes, enzymes, radioisotopes, chemiluminescence and magnetic and latex labels.

One or more first signal-emitting molecules is bound to the polypeptide linker at this time at any position on the linker that does not lead to significant steric hindrance or direct blockage of binding. It is most preferable to use an equimass ratio or near equimass ratio of signal- emitting molecules and polypeptide linker. An equimass ratio is determined by titering to the point where interference with binding occurs, i.e., loading the linker with signal-emitting molecules until steric hindrance or direct blocking of binding occurs. In addition, the binding of signal-emitting molecules to the polypeptide linker can be by any means currently known in the art that leads to various types of electrostatic, hydrophilic and/or hydrophobic bonds. Some ionic or covalent bonds may also be acceptable, however, this varies depending upon the polypeptide, the specific signal-emitting molecules and individual application constraints. Of course, the method for binding will depend upon the particular type of signal-emitting molecules used. However, general methods such as those in the Sigma catalog and those in "Immunochemistry in Practice" by Johnstone & Thorpe have proven workable.

Thereafter, the labeled polypeptide linker is preferably isolated and concentrated. Isolation and concentration of the labeled polypeptide linker can be by any method or methods known in the art. including: chromatography, based upon size and/or charge; gradient centrifugation; affinity chromatography, immunoprecipitation or electrophoresis. Although isolating and concentrating the labeled polypeptide linker is not required, the increased purity obtained is advantageous. A labeled polypeptide linker with a purity level of around 99% or better is believed to be sufficient. Obtaining a purified labeled polypeptide linker is more important than obtaining purified complement component or reagent.

Following optional isolation and concentration of labeled polypeptide linker, it is conjugated to the complement component to form a reaction product. Preferably, an equimolar concentration of complement and polypeptide linker is used. Thereafter, additional signal-emitting molecules may be bound to the reaction product. As stated previously, labeled polypeptide linker is conjugated to the complement component at sites. The sites can be any location on the complement component as long as they do not interfere with the complement component's ability to bind to antigen-antibody complexes. Therefore, sites within the complement component's binding site for antigen-antibody complexes and sites which sterically inhibit the complement component's binding properties to antigen- antibody complexes will not work. In addition, sites that restrict the flexibility of the complement component's arms are probably also problematic. Although not necessary for practicing the invention, those skilled in the art can determine in advance the sites on the complement component that won't interfere with antigen-antibody binding. The determination can be carried out to a limited degree by doing a detailed structural analysis of the complement component. In addition, the determination may be made by binding antigen-antibody complexes to the complement component to block the binding sites, carrying out the labeling and attachment steps, and then releasing the antigen-antibody complexes and isolating the complement component. The conjugation of labeled polypeptide linker to the complement component can occur by many known methods in the art, including most electrostatic, hydrophobic and/or hydrophilic methods, plus some ionic or covalent methods. The method of conjugation will vary depending upon the polypeptide linker originally used and the complement component. The reaction product is isolated and optionally concentrated by any suitable known method or methods, including: chromatography, based upon size and/or charge; gradient centrifugation: affinity chromatography, immunoprecipitation or electrophoresis. The step of isolating and concentrating is not required, but if performed, should achieve a reaction product with a complement component recovery of about 90% or more of the binding units, as measured by latex agglutination titer, and preferably about a 99% or more decrease in free label, although some variation may be tolerated.

The method of the invention further includes binding one or more second signal- emitting molecules to the reaction product to form a reagent. The second signal-emitting molecules may bind to either the complement component or to one or more of the polypeptide linkers. The addition of second signal-emitting molecules aids in detection of the antibody and also aids in decreasing the level of background noise in the detection step of the immunoassay. In addition, the binding of second signal-emitting molecule(s) causes a change in the reagent that gives it better detection properties than it had before the step of binding second signal-emitting molecules.

The second signal-emitting molecules may be any type of label used in serology, including fluorochromes, enzymes, radioisotopes, chemiluminescence and magnetic labels. In addition, the binding of the second signal-emitting molecules to the reaction product can be by any means known in the art that leads to various types of electrostatic, hydrophilic and/or hydrophobic bonds. Some ionic or covalent bonds may also be acceptable, however, this varies depending upon the polypeptide, the specific signal-emitting molecules and individual application constraints. As stated previously, the method for binding will depend upon the particular type of signal-emitting molecules used. This additional binding procedure may utilize the same or different method than was used to bind the initial signal- emitting molecules to the original polypeptide linker.

The additional binding of signal-emitting molecules preferably adds more of the same signal-emitting molecule that was originally added to the polypeptide linker in the initial binding step. However, in an alternate embodiment, different labels may be added to the reaction product to form a multi-purpose reagent. The reagent may be isolated and optionally concentrated as described hereinabove.

A third aspect of the invention involves using the reagent in many types of assays to detect the presence of antibodies in a sample. The conjugate allows for a one-step reaction, rather than multiple serial incubations, resulting in a substantial savings in materials, time and cost. Figure 3 is a diagram illustrating the differences between a standard immunoassay and an immunoassay using a reagent according to the present invention.

The present invention includes a method for detecting antigen specific antibodies comprising: incubating together a mixture of a reagent, a sample suspected of containing an antibody of biological interest and a bound antigen specific to an antibody of interest, in amounts and for a period of time sufficient to allow any antibody present in the sample having the capacity to bind with an antigen, to bind with the antigen to form a bound antigen- antibody complex, and also to allow the reagent to bind to any bound antigen-antibody complex present to form a bound reagent; removing any unbound reagent; and detecting one or more signal-emitting molecules. In an alternative embodiment, the detection step can include detecting one or more different types of signal-emitting molecules.

Before detecting antigen specific antibodies according to the method of the present invention, an optional step may be performed that includes diluting the samples and preparing the reagents. The procedures employed in this step are the same as for an analogous type of standard assay.

The method of the invention involves incubating the reagent with a sample suspected of containing an antibody of interest and a substrate bound display of the target antigen, and using radioimmunoassays, fluorescent immunoassays, enzymatic immunoassays, or chemiluminescent assays, to detect the presence of the antibody. The reagent will be synthesized and have the characteristics as described hereinabove. In addition, tests indicate that, upon binding polypeptide linker to Clq, the reagent's stability and binding kinetics to antigen-antibody complexes are improved. The incubating can occur by any one of several means that employ a solid phase antigen capture substrate, i.e., an antigen that is displayed on some form of solid support, such that the reagent will bind to a bound antigen-antibody complex. Many different types of solid phase antigen substrates may be employed in the present invention, including immunofluorescent assay slides, well plates, membranes, filters and beads. In addition, the sample can be derived from any source, such as biological fluids or cell culture. Preferred biological fluids are blood, urine and cerebrospinal fluid, with blood being the most preferred.

Another step of the invention involves washing the incubation mixture to isolate the reagent bound to antigen-antibody complexes on the solid phase antigen substrate from the other materials in the sample. Any solution that will facilitate the separation of the reagent bound to antigen-antibody complexes on the solid phase antigen substrate from the other materials in the sample will work. However, preferred washing solutions are phosphate buffered saline (PBS), PBS with less than 1% Tween or another emulsification agent, or PBS with less than 1% bovine serum albumin (BSA).

Another step of the invention involves detecting the signal emitted from the signal- emitting molecules of the reagent following binding to antigen-antibody complexes in a patient sample. Detection can occur by any of the standard means employed in the art. This will, of course, depend upon the type or types of signal-emitting molecules bound to the reagent. Therefore, for example, if the type of signal-emitting molecule were a radiolabel, then one would employ standard detection methods for radioimmunoassays to detect the signal emitted. Likewise, if the type of signal-emitting molecule used were an enzyme, then one would employ standard enzymatic detection methods for enzymatic immunoassays to detect the signal. A series of tests and procedures were conducted to make the reagent of the present invention. Further tests and procedures were conducted to utilize the reagent of the invention in immunoassays.

Materials

Various separation methods have yielded Clq useable in this invention, including the methods described by Kolb et al., "Clq: Isolation from human serum in high yield by affinity column chromatography and development of a highly sensitive hemolytic assay", J. Immunology, vol. 122, p. 2103 (1979); Paul et al., "Isolation and characterization of highly stable rabbit Clq", J. Immunological Methods, vol. 21, p. 341 (1978); Agneilo et al., "Precipitin reactions of the Clq component of complement with aggregated gamma globulins and immune complexes in gel diffusion", Immunology, vol. 19, p. 909 (1970); Yonemasu et al., "Clq: Rapid purification method for preparation of monospecifϊc antisera and for biochemical studies", J. Immunology, vol. 106, p. 304 (1971); Calcott et al., "Clq protein of human complement", Biochemistry, vol. 11, p. 3442 (1972): Sledge et al., "Purification of the human complement protein Clq by affinity chromatograpy", J. Immunology, vol. 111, p. 661 (1973); Pohl et al., "Isolation and purification of human Clq from plasma", J. Immunological Methods, vol. 36, p. 13 (1980); and Tenner et al., "Purification and radiolabeling of human Clq", J. Immunology, vol. 127(2), p. 648 (1981).

All of the polypeptide linkers used are commercially available from Sigma Chemical. Example 1 Labeling of Clq or reaction product with fluorescein isothiocyanate (FITC).

Dialyzed fresh Clq, not frozen, is brought to a concentration of approximately 2 mg/ml in carbonate buffer. The carbonate buffer is a solution containing 0.25M sodium carbonate at a pH of 9.0 and 0.1M sodium chloride. Next, 0.05 mg of FITC per mg of Clq is added. The solution is incubated overnight at 4° C. In order to separate the unbound fluorochrome, the solution is run on a Sephadex column and eluted with PBS. All bands are tested for latex agglutination capacity and the presence of label by visual inspection using an ultraviolet (UV) light. In order to be useable, at least 80% of the latex agglutinating units per unit volume must be recovered.

In labeling the reaction product, the label appears to occupy any appropriate site on the complement component and/or the polypeptide linker. Example 2 Labeling of C 1 q or reaction product with tetramethyrhodamine isothiocyanate.

The same procedure as in Example 1 above was followed, except that 0.125 mg of rhodamine was substituted for 0.05 mg of FITC. As in Example 1, all bands are tested for latex agglutination capacity and the presence of label by visual inspection using an ultraviolet (UV) light. In order to be useable, at least 80% of the latex agglutinating units per unit volume must be recovered.

In labeling the reaction product, the label appears to occupy any appropriate site on the complement component and/or the polypeptide linker. Example 3

Tests comparing the cross-linked versions of reagents.

Equal reaction volume of a 1% gluteraldehyde solution was employed and an additional dialysis step against the carbonate buffer was added. Then, the gluteraldehyde preparation(s) are compared with the starting Clq and other reagents for their latex agglutinating titers and ability to specifically stain a homogenous positive antinuclear antibody (ANA) slide. In all cases where gluteraldehyde was utilized, it either led to substantial decreases in latex bead agglutination titers, and thus diminished Clq's ability to discriminate and or bind immune complexes, and/or severe non-resolvable, non-specific background problems. Example 4 Labeling of polypeptide linkers.

Commercially available labeled polypeptides from Sigma Chemical were used, such as FITC labeled polypeptides or Alkaline phosphatase/peroxidase labeled polypeptides. However, if one wishes to label the polypeptide rather than using commercially available labeled polypeptides, the same procedure as used in Examples 1, 2, 5 and 6 were followed, except that the ratio of signal-emitting molecule to protein was doubled. The ratio is determined in a standard titration experiment by varying this ratio and examining the product for its ability to bind to Clq at the established ratio of 2 parts polypeptide to 1 part Clq.

In order to separate the unbound signal-emitting molecules, the solution is run on a Sephadex column and eluted with PBS. All bands are optically monitored at an OD of 280nm for the presence of protein, and visually with UV light for the presence of label. Only the band showing protein and label is collected. It is then dialyzed overnight against PBS and normalized to 2 mg/ml.

In all cases, the commercially available labeled polypeptides were more stable and resulted in less background readings and caused less of an inhibiting effect on Clq's ability to bind immune complexes. Example 5 Labeling of C 1 q or reaction product with peroxidase.

The Clq or reaction product is dialyzed in phosphate buffer and the final concentration is adjusted to approximately 2.0 mg/ml. 5.0 mg of peroxidase is dissolved in 0.5 ml of phosphate buffer and is added to 0.5 ml of conjugate or polypeptide linker. 1.0 ml of gluteraldehyde is then added to the mixture. The solution is mixed gently either at room temperature for one hour or for 4 to 8 hours at 4° C. The mixture is then run through a Sephadex column and eluted with PBS. The two fractions that are eluted from the exclusion volume, which shows absorbance at 280 nm, are collected and tested for their ability to agglutinate IgG coated latex beads. Only the conjugate fraction has agglutinating ability. The unbound enzyme does not. The fraction can then be stored at 4°C until use.

Neither enzyme significantly inhibited the agglutinating ability of the starting Clq at the ratio indicated above. If the ratio of enzyme to Clq is more than doubled, the agglutinating ability of the conjugate is significantly inhibited.

Table I

In this experiment, a typical ELISA for circulating immune complexes was employed where an enzyme labeled antibody directed against human immunoglobulin was part of the kit (Diagen CIC ELISA). The peroxidase or alkaline phosphatatase labeled Clq was substituted for the CIC kits Enzyme labeled Antibody (E-Ab). The enzyme labeled conjugates showed equivalent positive and negative control responses in the typical ELISA format. Example 6 Labeling of C 1 q or reaction product with alkaline phosphatase.

The Clq or reaction product was dialyzed in phosphate buffer and the final concentration was adjusted to approximately 2.0 mg/ml. 0.85 ml of this solution was added to the alkaline phosphatase pellet and dialyzed overnight against PBS to remove the sulphate precipitin from the pellet. The volume of the solution was then brought up to 1.0 ml with PBS and 8 μl of gluteraldehyde was added. The solution was incubated at room temperature for 2 hours. Next, the solution was dialyzed against PBS overnight. This dialysis step was followed by dialysis against Tris buffer for 24 hours. The mixture was then eluted with PBS through a Sephadex column and the two fractions eluted from the exclusion volume (which show absorbance at 280 nm) were collected and tested for their ability to agglutinate IgG coated latex beads. Only the reagent fraction demonstrated agglutinating ability. The unbound enzyme did not. The fraction can then be stored at 4° C until use. The data from Examples 6 & 7 are presented in Table II.

Table II Enzyme Labeled Conjugates

This experimental series involves a typical Human Clq-based enzyme-linked immunosorbent assay (ELISA) for circulating immune complexes (CICs). A standard was employed such that maximum binding and thus OD (280) absorption (an OD of approximately 1.200) was achieved. All results were normalized to the kit control at 100%. Unlabeled Clq was set to zero with an actual OD reading of approximately 0.100 recorded. Each of the test groups reported here are the optimal readings achieved for a range of ratios of Clq to enzyme. While it seems clear that the two stage labeling procedure gave the best binding results, it is worth noting that the C 1 q-(poly-l-lysine-Enzyme) groups all registered significant backgrounds of approximately 0.250 where no standard was present, whereas all other test groups recorded backgrounds of less than 0.050. It seems clear that the linking of the polypeptide is at least allowing for increased signal to binding unit ratios and increased non-specific attachments, while the polypeptide and two phase labeling methodology further increases the signal to binding unit ratio without the non-specific activity. Example 7 Latex agglutination procedure.

10 lambda of Sigma Human IgG coated Latex beads were placed onto a microscope slide and an equal volume of test solution, or dilutions thereof, were added. The mixture was gently swirled on the slide for 10-30 seconds at room temperature and visually observed agglutination was recorded. By doing serial dilutions of the test solution, a titer was obtained at which agglutination ceases which reflects the binding activity per unit mass or volume for that sample. Example 8

Conjugation of labeled polypeptide linker to Clq.

Clq is used at 2 mg/ml in PBS (1L H₂O, 1.2gm Na₂HP0₄, 0.192 gm NaH₂PO₄, and 8.5 gm NaCl) and Poly-L-lysine - FITC in carbonate buffer is added. Generally equal volumes of each are mixed in the dark at room temperature for 2 hrs. Two different carbonate buffers were used. Solution 1 contained 5.3 gm Na₂CO₃ in 100 ml 0.85% saline. Solution 2 contained 4.2 gm NaHCO₃ in 100 ml 0.85% saline. 17.4 mis of solution 1 are added to 30 mis of solution 2 and the pH is adjusted to 9.5 with HC1 or NaOH. A typical labeling procedure employs 0.5 mis of each component and the resulting 1 ml is purified by gel filtration with Sephadex G-25 using PBS to elute. The first visible eluted band has been shown to contain protein (OD280) , with an orange color indicating the presence of FITC. The unattached labeled poly-L-lysine appears as a later band that is bright yellow in color. Latex agglutination is used to confirm that only the first orange band has any agglutinating ability and that the agglutinating titer is within 1 dilution of the starting preparation (normalized per unit mass). Concentrations of C 1 q and labeled poly-L-lysine have been varied between 1-5 mg/ml for Clq and 1-5 mg labeled poly-L-lysine. The volume of labeled poly-L-lysine has been varied. Optimal results in terms of latex agglutinating ability and in positive antinuclear antibody staining are achieved with 2 mg/ml for each agent.

The time and temperature of interaction were also varied. The time was varied from 30 minutes to 12 hours with temperatures of 4°C, room temperature (23°C) and 37°C. Room temperature and 2 hours were optimal. Example 9 Alterations to typical immunoassay serology.

The reagent described above was added directly to a volume of a patient sample. The solution was then agitated gently and applied directly to the substrate displaying the assay's target antigens, for example, a glass slide with fixed cells or proteins, plastic wells, membranes or beads, in a manner similar to that in the typical application procedures for IF As, RIAs and EIAs. It has been determined that the order of addition of the Clq or the patient sample to a test vessel does not matter. The temperature for the mixing step does not seem to be critical, providing that all liquids are below about 40° C and the period of incubation is maintained for approximately 10-30 minutes, with approximately 85% of the reaction occurring within the first 15 minutes as a function of temperature. As a result, the method of the present invention is able to remove the labeled antigen specific monoclonal antibody solutions, the counter stain solutions, and the additional wash solutions from the procedure.

Example 10

Effect of different labeling procedures and different polypeptides on Clq binding ability. Numerous polypeptides were attached, both with and without signal emitting molecules, to Clq. All the solutions were then titrated and latex agglutination activity was measured to determine the effect on Clq's binding ability for antigen-antibody complexes.

The latex beads were coated with IgG (Sigma Chemical).

This experiment was also performed on standardized microscope slides coated with heat aggregated human IgG, which is the circulating immune complex (CIC) standard, to measure fluorescent ability per unit binding ability.

The results shown in Table III indicate that poly-1-lysine was clearly superior in terms of minimal effect to latex agglutination and maximum signal to Clq ratio.

Table III

Example 11

Effect of various labeling regimens on agglutination titer (Agg. Titer) and fluorescent intensity (FI. Int.) in antinuclear antibody (ANA) immunofluorescent assay (IF A).

This experiment utilizes conventional ANA IFA test kits and procedures (i.e., Zeus®, Sigma, etc.). The positive control for the assay involves using the kits secondary antibody (labeled with FITC) and the positive homogeneous control sample. The secondary antibody

19

SUBSTTTUTE SHEET (RULE 26) is replaced in each run by the various test groups described below (all volumes were 40 lambda- same as the kits antibody and all Clq concentrations in each sample set were normalized to 1 mg/ml):

Clq - unlabeled Clq

Clq* - FITC labeled Clq

Clq-PIL - Clq linked to unlabeled Poly-L-lysine

Clq-PIL* - Clq linked to FITC labeled poly-L-lysine

(Clq-PIL)* - Clq linked to poly-L-lysine and the reagent labeled with FITC

(Clq-PIL*)* - Clq linked to FITC labeled poly-L-lysine and the reagent relabeled with FITC

P1L* - Labeled poly-L-lysine

Table IV

Agglutination titers were performed immediately prior to initiating the ANA IFA tests. All FI. Intensity reports with a (!) also displayed nucleolar staining. Thus, it appears that the use of both the poly-L-lysine and the double staining result in increased signal, and possibly sensitivity, and elimination of the poly-L-lysine affinity for nucleolus. Example 12 Effect of polypeptide length and size.

Essentially polypeptides having MW 15-30KD were better than 1-4KD, 4-15KD, 30- 70KD in terms of both agglutination titers and fluorescent intensity. Example 13

Effect of different labeling regimens on the stability of the reagent. Clq* - FITC labeled Clq

Clq-PIL* - Clq linked to FITC labeled poly-L-lysine (Clq-PIL)* - Clq linked to poly-L-lysine and the reagent labeled with FITC (Clq-PIL*)* - Clq linked to FITC labeled poly-L-lysine and the reagent relabeled with FITC

Table V

Reagents were employed in antinuclear antibody (ANA) kits with the new modified procedure and time of stability is the time point prior to a less than maximum fluorescent intensity reading in terms of the standard typical kit positive controls.

Example 14

Effect of Clq conjugate concentration on antinuclear antibody (ANA) readings.

Concentration of C 1 q**(micrograms/ml). Table VI

The typical antinuclear antibody (ANA) immunofluorescent assay (IFA) kit was employed with samples A-E:

A - negative control sample B,C - Borderline positive samples D - Low positive sample E - High positive sample

Subsequent experimentation showed that 40 lambda of a 1 to 2 mg/ml concentration of (Clq- PIL*)* was optimal for application in the typical ANA format.

A-E were run with the protocol of the present invention using the (Clq-PIL*)* reagent of the present invention.

A-E were run with standard Zeus & Sigma ANA IFA kits with the following results:

Table VII

Example 15

This example illustrates the differences between a typical antinuclear antibody (ANA) procedure and an antinuclear antibody procedure using a reagent according to the present invention.

The typical antinuclear antibody procedure is as follows: remove the specimen and controls from storage and allow them to warm to room temperature; label 12 X 75 mm glass test tubes, one for each patient, and place the tubes in a rack; using a pipette, dispense 1.9 ml of buffer into each tube; mix the specimen by vortexing and add 0.1 ml of each specimen to the appropriate tube and mix again; remove the appropriate number of slides from the freezer and allow them to warm to room temperature; add 0.5 ml of controls and patient samples to the appropriate areas on the slides; place the slides in a moist chamber and incubate in the dark for 30 minutes; remove the slides from the chamber, rinse the slides in running buffer and place them into a slide washing dish with sufficient buffer for 10 minutes; remove the slides from the dish and blot dry; add 0.05 ml FITC labeled anti-human conjugate to each test well on the slides; incubate for 30 minutes at room temperature in a moist chamber; remove the slides from the chamber, rinse the slides in running buffer and place them into a slide washing dish with sufficient buffer for 10 minutes; add one drop of glycerol buffer and cover with a cover slip; and read the test wells for the results.

The total incubation time for the above procedure is approximately 80 minutes. In addition, the total technician time for the above procedure is approximately 2.5 hours.

In contrast, a new ANA procedure using the reagent of the present invention involves the following steps: remove the specimen and controls from storage and allow them to warm to room temperamre; label 12 X 75 mm glass test tubes, one for each patient, and place the tubes in a rack: using a pipette, dispense 0.19 ml of buffer into each tube; mix the specimen by vortexing and add 0.01 ml of each specimen to the appropriate tube and mix again; add 0.20 ml of the reagent of the present invention to each tube and mix; remove the appropriate number of slides from the freezer and allow them to warm to room temperature; add 0.05 ml of controls and patient samples to the appropriate test wells on the slides; incubate the slides in a moist chamber at room temperature for 20 minutes; rinse the wells in running buffer for 5 minutes: add one drop of glycerol buffer and cover with a cover slip; and read the test wells for results.

The total incubation time for the above procedure is approximately 25 minutes. In addition, the total technician time for the above procedure is approximately 1 hour. Example 16

The effect of different labeling regimens on stability of the reagent.

Table VIII

Reagents were employed in ANA kits with the procedure of the present invention. Time of stability is the time point prior to a less than maximum fluorescent intensity reading in terms of the standard typical kit positive controls, i.e., the time of storage at the indicated temperature before and observable decrease in scoring intensity. It seems clear that the addition of the polypeptide linker to the Cl q helps to stabilize it and the secondary labeling further enhances this effect. While this may appear attributable to just an increased signal to molecule ratio, all reagents were titrated such that even a 1 :2 dilution would have reduced the scoring. Thus, it appears more likely that the reagent's structure has been stabilized at least in addition to an increased signal to molecule ratio. Although the foregoing invention has been described in detail using illustrations and specific commercial components for clarity, it will be apparent to persons having ordinary skill in the art that certain changes and modifications may be made without departing from the spirit and scope of the invention.

Claims

CLAIMS We claim:

1. A reagent for use in immunoassays comprising: a complement component; one or more polypeptide linkers bound to said component to form a reaction product; and one or more signal-emitting molecule bound to said reaction product to form a reagent.

2. The reagent of claim 1 wherein said complement component is selected from the group consisting of C 1 q and C3b.

3. The reagent of claim 1 wherein said complement component is selected from the group consisting of human complement and synthetic complement

4. The reagent of claim 1 wherein said one or more polypeptide linkers is selected from the group consisting of poly-1-lysine, poly-1-alanine, poly-1-arginine, poly-1-cysteine, poly-1- glutamate, poly-1-glycine, poly-1-asparagine, poly-l-serine, poly-1-glutamine, poly-1-tyrosine, poly-1-histidine, poly-1-leucine, poly-1-proline, poly-1-threonine, poly-1-tryptophan, poly-1- phenylalanine, poly-1-ornithine, poly-1-hydroxyproline, poly-1-glutamic acid and poly-1- isoleucine.

5. The reagent of claim 1 wherein said one or more polypeptide linkers is selected from the group consisting of human complement, synthetic complement, and commercially available complement.

6. The reagent of claim 1 wherein said one or more signal-emitting molecules is selected from the group consisting of fluorochromes, enzymes, radioisotopes, chemiluminescent compounds and magnetic compounds.

7. The reagent of claim 1 wherein said one or more signal -emitting molecules is either synthetically made or commercially available.

8. A method for making a reagent comprising: binding one or more first signal emitting molecules to one or more polypeptide linkers to form one or more labeled polypeptide linkers; and conjugating said one or more labeled polypeptide linkers to a complement component o form a reaction product.

9. The method of claim 8 further comprising further binding one or more second signal- emitting molecules to said reaction product to form a reagent.

10. The method of claim 8 wherein said complement component is selected from the group consisting of Clq and C3b.

11. The method of claim 8 wherein said one or more first signal emitting molecules is selected from the group consisting of fluorochromes, enzymes, radioisotopes. chemiluminescent compounds and magnetic compounds.

12. The method of claim 8 wherein said one or more polypeptide linkers is selected from the group consisting of poly-1-lysine, poly-1-alanine, poly-1-arginine, poly-1-cysteine, poly-1- glutamate, poly-1-glycine, poly-1-asparagine, poly-l-serine, poly-1-glutamine, poly-1-tyrosine, poly-1-histidine, poly-1-leucine, poly-1-proline, poly-1-threonine, poly-1-tryptophan, poly-1- phenylalanine, poly-1-ornithine, poly-1-hydroxyproline, poly-1-glutamic acid and poly-1- isoleucine.

13. The method of claim 8 wherein said one or more second signal-emitting molecules is selected from the group consisting of fluorochromes, enzymes, radioisotopes, chemiluminescent compounds and magnetic compounds.

14. The method of claim 8 wherein said binding further comprises using an equimass concentration of polypeptide linker and one or more first signal-emitting molecules.

15. The method of claim 8 wherein said conjugating occurs at one or more sites on said complement component that are not involved in binding an antigen-antibody complex.

16. The method of claim 8 wherein said conjugating further comprises using an equimolar concentration of polypeptide linker and complement.

17. The method of claim 9 wherein said one or more first signal-emitting molecules is different than said one or more second signal-emitting molecules.

18. The method of claim 9 wherein more than one type of signal-emitting molecule is bound to the reagent.

19. A method for making a reagent comprising: binding labeled polypeptide linker to a complement component selected from Clq or C3b to form a reaction product; isolating and concentrating said reaction product; binding one or more second signal-emitting molecules to said reaction product to form a reagent; and isolating and concentrating said reagent.

20. An immunoassay comprising: incubating together a mixture comprising a reagent, a sample suspected of containing an antibody of interest and a bound antigen specific to the antibody of interest, in amounts and for a period of time sufficient to allow any antibody present in the sample having the capacity to bind with the bound antigen, to bind thereto to form an antigen-antibody complex and to allow said reagent to bind to any antigen-antibody complex present to form bound reagent, wherein said reagent comprises a complement component, one or more polypeptide linkers, and one or more signal-emitting molecules; and, removing unbound reagent; detecting signal emitted from said bound reagent.

21. The immunoassay of claim 20 wherein said one or more signal-emitting molecule is selected from the group consisting of fluorochromes. enzymes, radioisotopes, chemiluminescent molecules, latex particles, and magnetic compounds.

22. The immunoassay of claim 21 wherein said reagent comprises more than one signal-emitting molecule and at least two of said signal emitting molecules are different.

23. The immunoassay of claim 20 wherein said complement component is selected from the group consisting of Clq and C3b.

24. The immunoassay of claim 20 wherein said one or more polypeptide linkers is selected from the group consisting of poly-1-lysine, poly-1-alanine, poly-1-arginine, poly-1-cysteine, poly-1-glutamate. poly-1-glycine. poly-1-asparagine. poly-l-serine, poly-1-glutamine. poly-1- tyrosine, poly-1-histidine, poly-1-leucine, poly-1-proline, poly-1-threonine, poly-1-tryptophan, poly-1-phenylalanine, poly-1-ornithine, poly-1-hydroxyproline, poly-1-glutamic acid and poly- 1-isoleucine.

25. The immunoassay of claim 20 wherein said one or more polypeptide linkers are different.

26. The immunoassay of claim 20 wherein said sample is selected from the group consisting of biological fluids.

27. The immunoassay of claim 26 wherein said biological fluids are selected from the group consisting of blood, urine and cerebrospinal fluid.

28. The immunoassay of claim 20 wherein said sample is from a cell culture.

29. A kit for use in an immunoassay comprising: a reagent comprised of a complement component, one or more polypeptide linkers bound to said component, and one or more signal-emitting molecules bound to at least one of said linker and said component; and, an antigen specific to an antibody of interest bound to a support.

30. The kit of claim 29 further comprising positive and negative control samples.

31. The kit of claim 29 wherein said complement component is Clq or C3b.

32. The kit of claim 29 wherein said complement component is human.

33. The kit of claim 29 wherein said one or more polypeptide linkers is selected from the group consisting of poly-1-lysine, poly-1-alanine, poly-1-arginine, poly-1-cysteine, poly-1- glutamate, poly-1-glycine, poly-1-asparagine, poly-l-serine, poly-1-glutamine, poly-1-tyrosine, poly-1-histidine, poly-1-Ieucine, poly-1-proline, poly-1-threonine, poly-1-tryptophan, poly-1- phenylalanine, poly-1-ornithine, poly-1-hydroxyproline, poly-1-glutamic acid and poly-1- isoleucine.

34. The kit of claim 29 wherein said one or more signal-emitting molecules is selected from the group consisting of fluorochromes, enzymes, radioisotopes, chemiluminescent molecules, latex particles, and magnetic compounds.

35. The kit of claim 29 wherein said one or more signal-emitting molecules is an enzyme, further comprising a substrate and a substrate buffer.

36. The kit of claim 29 wherein the antibody of interest is an antinuclear antibody.

28

SUBSTTTUTE SHEET (RULE 26) AMENDED CLAIMS

[received by the International Bureau on 12 July 2001 (12.07.01 ); original claims 3 and 5 amended; remaining claims unchanged (1 page)]

2. The reagent of claim 1 wherein said complement component is selected from the group consisting of Clq and C3b.

3. The reagent of claim 1 wherein said complement component is selected from the group consisting of human complement and synthetic complement.

5. The reagent of claim 1 wherein said one or more polypeptide linkers is selected from the group consisting of human polypeptide linkers, synthetic polypeptide linkers, and commercially available polypeptide linkers.

6. The reagent of claim 1 wherein said one or more signal-emitting molecules is selected from the group consisting of fluorochromes, enzymes, radioisotopes, chemiluminescent compounds and magnetic compounds. 7. The reagent of claim 1 wherein said one or more signal-emitting molecules is either synthetically made or commercially available.

8. A method for making a reagent comprising: binding one or more first signal emitting molecules to one or more polypeptide linkers to form one or more labeled polypeptide linkers; and conjugating said one or more labeled polypeptide linkers to a complement component to form a reaction product.