WO2001027621A2 - Competitive inhibition elisa for antibody detection - Google Patents

Abstract

The present invention is a specific and sensitive method of detecting antibodies in biological samples. This method of inhibition enzyme linked immunosorbent assay (iELISA) is generally applicable for the detection of antibodies found in biological fluids. The inhibition ELISA immunoassay (iELISA) of the present invention greatly increases the sensitivity, specificity, and convenience of obtaining serologic data. The present invention also relates to a test kit for use in an immunoassay to detect an antibody to an antigen in a test sample.

Description

INHIBITION ENZYME LINKED IMMUNOSORBENT ASSAY (/ELISA) FOR

THE DETECTION AND QUANTIFICATION OF ANTIBODIES

IN BIOLOGICAL SAMPLES

The present application claims priority under Title 35, United States Code, § 1 19 of

United States Provisional application Serial No. 60/158090 filed October 07, 1999

FIELD OF THE INVENTION

The present invention relates to an inhibition enzyme linked immunosorbent assay

(/ELISA) for detecting and quantifying antibodies in biological samples.

BACKGROUND OF THE INVENTION

The present invention is a specific and sensitive method of detecting antibodies in biological samples. This method of inhibition enzyme linked immunosorbent assay (/ELISA) was developed in particular, for the detection of antibodies to recombinant polypeptides in serum. This method, however, is generally applicable to the other antigens and their corresponding antibodies found in biological fluids and tissues such as serum, tears, saliva, urine, milk, semen, lymph, spinal, and vaginal secretions.

Many immunoassay techniques exist for the immuno-surveillance of antigens and antibodies (Kemeny D.M. and Challacombe S.J. ELISA and Other Solid Phase Immunoassays Theoretical and Practical Aspects, John Wiley & Sons, 1988).

Direct ELISA assay and protein Western electroblotting assays are among the most sensitive. However, one of the drawbacks of these procedures is the background interference caused by non-immune immunoglobulin and other serum factors present in all normal sera. This background, by its tendency to overlap and obscure low levels of specific reactive immunoglobulin, decreases the sensitivity of the assay and creates the need for supplementary confirmation tests in order to demonstrate true positivity. One such confirming test used extensively is the protein

Western blotting procedure which, although sensitive, more specific, and informative for distinct proteins, is highly labor intensive, difficult to interpret, and is qualitative in nature, rather than quantitative.

Competition ELISA immunoassays exist for detection and quantification of antigens and antibodies in serum, which have increased the sensitivity, specificity, and convenience of obtaining serologic data.

EP 202 890 describes a competition ELISA immunoassay for detecting human T-cell leukemia-lymphoma virus type III (HTLV-IJJ).

US 5,798,219 describes a competition ELISA immunoassay for detecting antibodies to rickettsial diseases of cattle, sheep, and goats.

EP 353 822 B l discloses a competition ELISA immunoassay for detecting malaria infections caused by a protozoon of the Plasmodium genus. However, these methods can also have considerable background, require large quantities of purified antigen for coating the support surface, require a labeled secondary detection antibody, and have numerous steps which are time consuming and labor intensive.

The inhibition ELISA immunoassay (/ELISA) of the present invention greatly increases the sensitivity, specificity, and convenience of obtaining serologic data. The method increases sensitivity of specific antibody measurements by eliminating the background associated with binding of non-specific immunoglobulin. The method increases specificity by incubating the affinity-purified antibody and labeled antigen simultaneously with the test serum. The present method reduces assay time and labor requirements as a consequence of the elimination of the secondary detection step. SUMMARY OF THE INVENTION

Enzyme linked immunosorbent assays (ELISA), in general, are performed by binding either antigen or antibody as a reference reagent to a solid phase support. Test sample is then reacted with the bound reference reagent. The reagents undergo a series of dilution, incubation, and washing steps in order to separate bound and free reagents. The process concludes with a detection step compatible with the type of label used, designed to indirectly measure the amount of antibody (or antigen) in the test sera.

A novel method is described for the detection and measurement of antibodies in biological fluids such as serum. The advantage of this assay method is that it reduces or eliminates false positives and decreases assay incubation time by eliminating the need for a separate detection step. This invention is an inhibition assay, which differs from direct binding assay formats in that it allows for the inhibition of bound specific affinity purified antibody and test sera antibody for labeled antigen. This avoids elevated backgrounds caused by non-specific binding of secondary detection antibodies to antigen bound to a solid phase, as in the sandwich assay. A second advantage of this assay is that it allows for the determination of concentration in serum in addition to titer dilution of antibodies. A third advantage is a reduction in assay time as a consequence of the elimination of the secondary detection step.

One aspect of this invention is to provide a method for the detection of endogenous antibodies against recombinant polypeptides in serum. This is an important because increasing concentrations of specific antibody to a recombinant protein could have a neutralizing effect on both drug and the drug target. Typically, the levels of circulating antibody to drugs in the serum are very low. The inhibition enzyme linked immunosorbent assay (/ELISA) method of the present invention allows for sensitive, accurate, and rapid detection of antibodies to recombinant polypeptides. This method is, however, generally applicable for detecting other antigens and their corresponding antibodies in biological fluids such as serum. There are many important potential uses for a simple method for the detection of antibody concentrations in sera. Normal plasma from normal donors can be screened for higher than normal concentrations of endogenous antibodies to known pathogens.

One could easily test sera from humans or animals that have been vaccinated with a particular virus, for example the rabies virus, and quickly determine if titers were sufficiently elevated to give protection against infection. One could also detect the levels of a class of antibodies such as immunoglobulin E (IgE) when the body is responding to an allergen.

Another embodiment of the present invention is a method to determine exogenous antibody levels in biological fluids such as serum. The /ELISA of the present invention could be used to determine the pharmacokinetics (PK) and pharmacodynamics (PD) parameters of exogenous antibodies to evaluate efficacy.

Another embodiment of the present invention is to provide a method for diagnosis of a disease by detecting an antigen or its corresponding antibody. The /ELISA can be used to detect an antigen or its corresponding antibody.

Another aspect of the invention is the use of the assay in epidemiological studies in regions where a particular pathogen infects one or more species endemic to that region. A field /ELISA test kit for detecting serum antibody levels would be amenable for accurately determining particular species involved and rates and spread of infection and could be an important tool in control efforts.

Another aspect of the invention are multitudinous in vitro applications for an inhibition ELISA that could detect antibody levels from experimental tissue culture media or purification samples or the detection of antibody level in biological fluids other than serum such as urine, saliva, lymph, tears, milk, semen, vaginal secretions, or spinal fluid. Another application of this method is for the detection of virus, including but not limited to, human T-cel 1 leukemia-lymphoma virus type III (HTLV-III), the causative agent of acquired immune deficiency syndrome (AIDS). This method is also applicable to the detection of other pathogenic organisms including but not limited to bacteria, yeast, fungus, and parasites. In addition, this method can be used for detecting antibodies raised in response to vaccines, including but not limited to, OspA vaccine for the prevention of Lyme disease.

Another aspect of the present invention is to provide a kit for the rapid screening and detection of antigens and their corresponding antibodies in biological fluids and tissues such as serum.

Definitions

The following is a list of abbreviations and the corresponding meanings as used interchangeably herein:

Mab monoclonal antibody

C constant C_H heavy-chain C-region

C_L light-chain C-region

ScFv single-chain Fv molecule

V variable

V_H heavy-chain V-region L light-chain V-region

Fab fragment antigen binding (V_H, C_H, V_L, C_L)

Fv fragment variable (V_H, V_L,) g gram(s) mg milligram(s) ml or mL milliliter(s)

RT room temperature PEG poly (ethylene glycol)

The following is a list of definitions of various terms used herein:

The term "antibody", as used herein, includes various forms of modified or altered

(engineered) antibodies such as; an intact immunoglobulin; an Fv fragment containing only the light and heavy chain variable regions (V_L and V_H); an Fv fragment linked by a disulfide bond (Brinkmann, et al. Proc. Natl. Acad. Sci. USA, 90: 547-551 (1993)); an Fab fragment containing the variable regions and parts of the constant regions, (Fab)'2, dimeric Fabs or trimeric Fabs, which can be multivalent and/or multispecific; a single-chain antibody (ScFv) (Bird et al., Science 242: 424-426 (1988); Huston et al., Proc. Nat. Acad. Sci. USA 85: 5879-5883 (1988)), single-chain multimers (diabodies, triabodies, tetrabodies, etc.), which can be multivalent and/or multispecific). The antibody may be of animal (especially mouse or rat) or human origin or may be chimeric (Morrison et al., Proc Nat. Acad.

Sci. USA 81 : 6851-6855 (1984)) or humanized (Jones et al., Nature 321 : 522-525 (1986), and published UK patent application #8707252). These various antibody forms are schematically illustrated in Figure 9. As used herein the term "antibody" also includes whole antibodies or fragments, which have been fused to radioisotopes, drugs, toxins, enzymes and biosensor surfaces. The term "antibody", as used herein, moreover includes whole antibodies or fragments, which have been modified by the addition of a PEG molecule.

The term "chimeric antibodies" refers to hybrid immunoglobulins in which the original murine variable regions are preserved and the constant regions are switched for those of a human antibody.

The term "humanized antibodies", refers to hybrid immunoglobulins in which the murine residues that conform to specific complementarity determining regions and others of possible structural relevance are transplanted to a human antibody framework. The term "transgenic plants" refers to plants modified genetically to constitutively produce functional antibodies or their fragments.

The term "plantibodies", as used herein, refers to Mabs produced in plants.

As used herein the term "MPO" or "Myelopoietin" is defined as a family of multifunctional receptor agonists comprising an IL-3 variant and a G-CSF receptor agonist as taught in U.S. patent Nos. 6,057,133; 6,030,812; and 5,738,849

As used herein the term "PMP" or "Promegapoietin" is defined as a family of multifunctional receptor agonists comprising a circularly permuted c-mpl ligand and a G-CSF receptor agonist as taught in U.S. Patent No. 6,066,318, and WO97/12985.

As used herein the term "ProGP" or "Progenipoietin" is defined as a family of multifunctional receptor agonists comprising a circularly permuted flt3 ligand as taught in WO98/17810.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 schematically shows a standard direct binding sandwich ELISA compared to one form of the inhibition ELISA of the present invention.

Figure 2 shows antibody titers from serum samples from rhesus monkeys treated with a recombinant protein over a six-month period. Rhesus monkeys were immunized with 100 μg/kg of myelopoietin (MPO), a member of a family of recombinant proteins which are multifunctional agonists of human interleukin-3 (IL- 3) and human granulocyte colony stimulating factor (G-CSF) receptors (WO 95/21197 and WO 95/21256), with 0.5 mg/kg Titermax (adjuvant from Hyclone Company) subcutaneous administration on day 1 and boosted on days 21 , 42, 63,

84, 105, 126, and 147. Spike controls consisted of normal rhesus serum with addition of 4 μg/ml affinity purified goat anti-MPO, which is then serial diluted 1:2 to generate a standard curve.

Figures 3 and 4 show serum antibody titers from breast cancer patients (chemotherapeutic agent was docetaxel, 100 mg/m² on day 1) treated with 5.0 μg/kg/day of MPO. The control is normal human serum with the addition of 4 μg/ml affinity purified goat anti-MPO diluted as above.

Figure 5 shows serum antibody titers from lymphoma cancer patients (Chemotherapeutic regimen was ESHAP-etoposide, methylprednisolone, cisplatin, cytosin arabinoside, Journal of Clinical Oncology, 13: 1734-1741, 1995) treated with 7.5 μg/kg/day of MPO. Control standard is normal human serum with the addition of 4 μg/ml affinity purified goat anti-MPO.

Figure 6 shows serum antibody titers from normal female rhesus monkey immunized with Promegapoietin (PMP), a member of a family of recombinant proteins, which are multifunctional agonists of human IL-3 and human c-mpl receptors (WO 97/12985), 50 μg/kg/day with 0.5 mg/kg subcutaneous injections for 10 days.

Figure 7 shows serum antibody titers from a human male sarcoma cancer patient treated with 0.5 μg/kg/day for five days for 3 cycles with PMP. Control is normal human serum with the addition of 4 μg/ml affinity purified goat anti-PMP.

Figure 8 demonstrates the antibody is being detected by the removal of IgG by

Protein G from the serum. Anti-PMP titers were measured in a patient in Cycle 1 of the chemotherapy at Day 1 with no titer as a negative control and at Day 22 with positive titer before and after Protein G treatment for 1 hour at room temperature. Various concentrations of affinity purified goat anti-PMP antibody were added to normal serum as a positive control. Figure 9 Schematic representations of the engineered Mab fragment formats described herein, (a) Intact IgG (bivalent), (b) Monovalent immunoglobulin fragments (Fab, Fv, and scFv), (c) multibodies (bivalent, bispecific, trivalent, and trispecific). Variable domains (V) and Constant domains (C) are represented by ovals (V_H-domains are shaded gray, V_L-domains are black and C-domains are white) and linkers are represented as black lines, (d) Chimeric, human sequences are dotted and mouse sequences are crosshatched, (e) Dimeric Fab (Fab). Only one V- domain arrangement is shown for each structure, there are obviously alternative orientations of V-domains and linkers.

DETAILED DESCRIPTION OF THE INVENTION

The invention described is an inhibition ELISA (/ELISA). In the preferred embodiment, as shown in Figure 1 , the affinity purified specific antibody (primary antibody) is bound to a 96-well microtiter plate, which is then exposed to a predetermined concentration of labeled antigen and a solution containing an unknown concentration of test sample antibody (secondary antibody). The microtiter plate containing the antigen-antibody mixture is incubated to allow the primary and secondary antibodies to compete for binding of labeled antigen. After washing thoroughly to remove unbound antigen and secondary antibody-antigen complex, measurement of bound labeled antigen that was not removed by competition with the secondary antibody, is determined by the addition of a substrate, which yields a chromogenic product. The amount of chromogen produced is directly related to the amount of labeled antigen bound to the primary antibody and the percent inhibition of labeled antigen by the unknown concentration of secondary antibody. The percent inhibition is determined by a graphic transformation of the absorbance readings and the 50% inhibition concentration (IC50) of this curve is used to determine titer of the secondary antibody in the unknown solution. One could also envision a similar assay in which antigen is coated to a microtiter plate and labeled primary antibody, either monoclonal or affinity purified, compete with unknown test solution antibody for binding to the precoated antigen. It is also envisioned that the assay could be performed in solution by separating the primary antibody and the unknown secondary antibody. This may be accomplished by labeling the antigen or primary antigen with a magnetic tag or attaching them to agarose and then using a magnetic field and centrifugation, respectively, to separate them.

The assay may also be performed by binding antibody to binding surfaces other than microtiter plates. For example, antibodies could bind to small Biacore sensor chips (BR- 1000- 14) coated with affinity molecules such as Protein A or G or carboxymethyl dextrans. Additionally, other binding surfaces such as small agarose beads or magnetic beads could also be anticipated as being employed in the assay.

There are many important potential uses for the simple detection of antibody titers in sera. Normal plasma from normal donors can be screened for higher than normal titers of naturally occurring antibodies to known pathogens. One could easily test sera from humans or animals that had been vaccinated with a particular virus, for example the rabies virus, and quickly determine if titers were sufficiently elevated to give protection against infection. The assay may lend itself to epidemiological studies in regions were a particular pathogen infects one or more species endemic to that region. A field ELISA test kit for detecting serum antibody levels would be amenable for accurately determining particular species involved and rates and spread of infection and could be an important tool in control efforts.

One could also imagine many other in vitro applications for an inhibition ELISA that could detect antibody levels from experimental tissue culture media or purification samples or the detection of antibody level in biological fluids other than serum such as urine or lymph or spinal fluid. One could envision the use of engineered antibodies for the capture step in an ELISA or for the detection of therapeutic engineered exogenous antibodies from biological fluids, purification samples or tissue culture media. This could include; mouse Mabs such as OKT3; scFvs such as D2E7 (adalimumab) and CAT 152, chimeric antibodies such as Rituxan (Rituximab), ReoPro (Abciximab), Remicade

(Infliximab), and Simulect (Basiliximab); humanized antibodies such as Herceptin (Trastuzumab), Zenapax (Dacliximab), and Synagis (Palivizumab); Fab fragments; and pegylated antibodies such as CDP-870.

The use of exogenous antibodies for "antibody therapy" began with the approval by the FDA in 1986 of Orthoclone OKT3 (Muromomab) anti-CD3 mouse Mab for acute renal therapy (Shield, C.F. et al. Am. J. Kidney Dis. 27:855-864 (1996). However, when rodent Mabs were used therapeutically, a human antimurine antibody response developed that resulted in a rapid and strong sensitization that completely neutralized the therapeutic effectiveness of the monoclonal antibody.

Since that time a number of approaches to engineering antibodies have been developed to overcome this problem (for a review see Gavilondo, JN. et al., Biotechniques 29: 128-145, 2000). One approach to engineering antibodies is "chimeric antibodies". A number of chimeric antibodies have been approved in the U.S. or Europe for the treatment of diseases such as; (Rituximab), which is targeted against CD20 for the treatment of Νon- Hodgkin's lymphoma (for review see Grillo-Lopez, A.J., et al., Semin. Oncol. 26(5, Suppl. 14):66-73, 1999; Maloney, D.G., Semin. Oncol. 26(5 suppl 14):74-78, 1999); ReoPro (Abciximab), which binds lib/ Ilia on platelets for the treatment of Cardiac ischemic complications in high risk PTCA patients; Remicade (Infliximab) which binds TΝF- α for treatment of

Crohn's disease and Rheumatoid arthritis (Markman, A., et al., Drugs 59(6): 1341- 1359, 2000); and Simulect (Basiliximab), which binds IL- 2 α subunit for kidney transplant rejection. Another approach to engineering antibodies is "humanized antibodies". A number of humanized antibodies have been approved in the U.S. or Europe for the treatment of diseases including; Herceptin (Trastuzumab), which binds the extracellular domain of Her2/ Νeu receptor for the treatment of metastatic breast cancer (for review see Albanell, J., et al., Drugs Today 35(12) 931-946, 1999); Zenapax (Dacliximab), which binds IL- 2 subunit for kidney transplant rejection (90% human 10% murine); Synagis (Palivizumab), which binds the A antigenic site of the respiratory syncytial virus (RSV) F- protein for treatment of RSV infections (95% human, 5% murine). A different approach is to produce the

Mab in a "humanized" mouse in which the expression of mouse Abs is suppressed and replaced with human antibody genes. Several fully human antibodies produced in "humanized" are in development including; ABX-IL8, which binds IL-8 and is being developed for the treatment of Rheumatoid arthritis and psoriasis; and MDX CD4, which is targeted to CD4 for the treatment of Rheumatoid arthritis. There are several products in development using single chain antibodies including; D2E7 (adalimumab), which binds TNF α for the treatment of Rheumatoid arthritis (for review see Chantry, D., Curr. Opin. Anti-inflammatory Immunomodulatory Invest. Drugs 2(l):31-34, 2000); and CAT 152, which binds TGFb2 for the prevention of scarring following glaucoma surgery. Mabs have also been "pegylated" to increase half-life. On such example of a "pegylated" Mab is CDP-870, which is a PEGylated single Fab against TNF α with two 20K PEG molecules attached at a cysteine in the hinge region for the treatment of Rheumatoid arthritis.

In addition to the use of Mabs for the treatment of diseases numerous Mabs have been approved in the U. S. or Europe for diagnostic purposes including: Indimacis 125 (Igovomab), which is a murine (Fab)'2 for diagnosis of ovarian adencarcinoma; CEA-Scan (Arcitumomab), which is a murine Fab for the detection of recurrent/metastatic colorectal cancer; ProstaScint (Capromab-Pentetate), which is a murine Mab for detecting prostate adenocarcinoma; Tecnemab KI, which is a

Fab/(Fab)'2 mix for the diagnosis of cutaneous melanoma lesions; Veluma (Nofetumonab) which is a murine Fab for detecting small-cell lung cancer; and Humaspect (Votumumab), which is a human Mab for the detection colon and rectum carcinoma. An embodiment of the present invention is the use of the /ELISA method for diagnostic purposes to detect antigens or antibodies associated with variety of diseases. One such example is for the detection of antibodies associated with B cell lymphomas. About 80% of adult lymphoproliferative malignancies involve Ig- containing or Ig-producing tumors. These tumors, called B cell tumors, include those associated with leukemias and lymphomas such as chronic lymphocytic leukemia, lymphosarcoma cell leukemia, nodular lymphoma, large-cell lymphoma, Burkitt's lymphoma, hairy-cell leukemia, "undifferentiated" lymphoma, and acute lymphocytic leukemia of pre-B cell type. The tumor cell populations of these cancers are clonal in nature and appear to contain cells arrested in various stages of differentiation with respect to Ig synthesis and secretion. Because the tumor population is monoclonal, the Ig produced by the population is likewise monoclonal. That is, the Ig that is expressed or secreted is restricted to a single V_H and V_L region and to a single light chain of either the K or λ type. It is usually of the IgM class (μ heavy chain). Another example is for the detection of IgE levels to determine if a patient is responding to an allergen. When a patient is challenged by an allergen the symptoms, runny nose, sneezing, congestion, coughing, itchy eyes, headache, or skin rash, are often difficult to distinguish from those of a cold, flu, a bacterial infection. By being able to rapidly measure IgE levels, which increase with an allergen challenge, a physician would easily be able to determine if the patient was suffering from an allergy and be able to treat the patient accordingly. In this form of the /ELISA the 'antigen' is an anti-IgE antibody.

Additional Mabs that are currently in development are summarized in Table 1 (Gavilondo, JN. et al., Biotechniques 29: 128-145). The method of the current invention could also be used to detect the antibodies listed in Table 1 but is not limited to such antibodies. Table l¹

Representative Antibodies in Clinical Evaluation

Institution/Company Antibodies Characteristics/Application (in order)

CANCER

Ixsys/Scπpps Vitaxin ™ MAb against αvβ3 integπn Cancers Coulter Pharm Inc Anti-BI Mouse MAb anti-B l antigen, coupled to ¹³¹I B cell lymphoma NCI/NIH, Bethesda Anti-Tac Fv- 1 scFv fragment anti-Tac receptor, genetically coupled to PE38, LMB-7, Pseudomonas toxin domains (PE38) Leukemia/lymphoma Anti-transferπn 2 scFv fragment against a tumor associated glycoprotein, receptor, Ch 14 1 J genetically coupled to Pseudomonas toxin domains (PE38) Colon and breast cancers, other advanced stage solid tumors

3 Advanced refractory solid tumors 4 Chimeric anti-melanoma antibody Melanoma and neuroblastoma

NeoRx/Dupont Verluma^{I M} MAb for imaging in small-cell and non-small-cell lung tumors Janssen Pharmaceutica/ Avicidin MAb conjugate Colorectal, lung, prostate cancers NeoRx NeoRx NR-LU-10 In situ radiotherapy with ⁰Y indirectly labeled pan carcinoma antibody Colon metastases, ovarian cancer, prostate cancer

Perlmmune Anti-colon cancer Human MAb Colorectal cancer imaging Genentech Anti-VEGF Humanized antibody Cancers Chiron Bispecific antibody Cancer

Wyeth-Ayerst Labs CMB-401. CMA-676 1 Ovarian cancer 2 Relapsed acute myelogenous leukemia Memorial Sloan- hB72 3 Humanized B72 3 Ab against the TAG72 tumor associated Ketteπng, NY glycoprotein Breast cancer Centocor/Glaxo Panorex^{"1 M} Chimeric 17- 1 A anti Lew is yAb Adjuvant therapy tor metastatic edrecolomab colorectal cancer

Cytel/Upjohn CY 1748 Humanized anti-P selectin Ab Inflammation and cancer

Royal Free Hospital MFE-23 scFv fragment against the B3 CEA domain obtained

Sch Medicine, London from a phage library Radioimaging in colon carcinoma

Contra Costa Cancer Center, HuBrE 3 ^9I,Y indirectly labeled humanized anti mucin Ab In situ

California radiotherapy in breast cancer

ImClone Systems C225, BEC-2 1 Chimeric anti EGF-receptor Ab Many EGF receptor-positive cancers 2 Anti-idiotypic MAb that mimics the GD3 ganglioside found in some tumor cells Small cell lung cancer

CIMAB SA, Havana/York ιor-R3 lor r3hum, 1 Anti EGF receptor MAb EGF receptor-positive cancers in Medical ιor-c5 combination with radiotherapy I Humanized anti EGF-receptor antibody EGF receptor-positive cancers 99Tc-labelcd mouse

MAb against carcinoma-associated glycoprotein Recurrent and metastatic colorectal carcinoma imaging

ImmunoGen Oncolysin B ^{I M}, 1 lmmunotoxin composed by a MAb against lymphoma cells C242-DM 1 coupled to a very potent proprietary drug 2 MAb against a colon cancer antigen, coupled to a very potent proprietary drug

Bristol-Myers Squibb BR96 Conjugate between a genetically engineered improved MAb against the Lewis \ antigen and doxorubicin Carcinomas

Techniclone Int and ChTNT- 1/b, 1 Chimeric Ab Non-Hodgkin s B cell lymphoma, solid tumors 2 Techmclone InVAlpha I- ChTNT- l/b, Radiolabeled chimeric Ab Malignant glioma 3 Chimeric anti Therapeutic Oncolym¹ " HLA-DR Ab radiolabeled with ^lI Non-Hodgkin s lymphoma

Novartis Anti-idiotype MAb Cancers

IDEC Pharm IDEC-Y2B8 1 Non Hodgkin s B cell lymphoma Combination with Rituximab®

IDEC-InB8 2 Non-Hodgkin s B-cell lymphoma

¹ Gavilondo, JN. et al., Biotechniques 29: 128-145 Table 1 (cont)

Representative Antibodies in Clinical Evaluation (continued)

Institution/Company Antibodies Characteristics/Application (in order)

Neoprobe RlGScan® CR49 MAb for colorectal cancer imaging. Application submitted. Cytogen OncoRad® PR CYT- 1. In situ radiotherapy with ^I)Y labeled pan carcinoma antibody. 356-Y-90, OncoScint Prostate cancer.2. Detection, staging and follow-up of breast cancer.

Medarex MDX-447, 1. Bispecific Ab. Head and neck, renal cancers.2. Bispecific Ab. MDX-H210, MDX-220 Breast, colorectal, kidney, ovarian, prostate.3. Bispecific Ab.

Cancers.

Immunomedics CEA-Cide^{I M}, hMN14, 1. Humanized anti-CEA Ab. CEA-positive cancers.2. ⁹ mTc

CEA-Scan^{I M} , labeled anti-CEA MAb. Breast and lung cancer. 3. Staging and

LymphoScan^1M, residual disease in non-Hodgkin's B-cell Iymphoma.4 ⁹⁹mTc

Lymphocιde^{1 M}, labeled antι-CD22 humanized Ab. Non-Hodgkin's B

AFP-Scan^1M, Lymphoma.5. mTc labeled Fab fragment against germ cells.

ImmuRAIT-CEA, Liver and germ cell cancer stagιng.6. Anti-CEA ^πlI labeled lmmuRAIT-LL2 MAb. Colorectal cancer. 7. 'I labeled MAb. Non-Hodgkin's B lymphoma.

Millenium Ca path 1 LDP-03 Antι-CD52 humanized Ab. Chronic lymphocytic leukemia

Protein Design Labs/ SMART'^M M 195, 1. Antι-CD33 humanized Ab. Acute myeloid leukemia Acute

Kanebo, and Protein SMART^{1 M} l D10, promyelocytic leukemia .2. Anti-HLA humanized Ab Non-

Design Labs / Zenapax® Hodgkin's Iymphoma.3. Anti-CD25 humanized Ab.

Hoffmann-La Roche Advanced myeloid leukemia. Certain blood cancers.

Altarex OvaRex Antι-CA125 MAb. Ovarian cancer.

ICOS Anti-ICAM 3 Humanized Ab. Cancer treatment.

AUTOIMMUNE DISORDERS

Celltech/Bayer CDP 571 Humanized anti-TNF alpha Ab Crohn's disease and

Ulcerative colitis. Rheumatoid arthritis.

Protein Design Labs SMART¹" antι-CD3 Humanized antι-CD3 Ab. Autoimmune diseases.

IDEC/Smith Kline, Clenoliximab, 1. Humanized Ab. Rheumatoid arthritis.2. Humanized

IDEC, IDEC/ Glaxo/ IDEC- 131 , antι-CD40 L Ab. Systemic lupus erythematosus. Multiple

Smith Kline IDEC 151 Sclerosis 3. Antι-CD4 pπmatized Ab Rheumatoid arthritis.

Biogen Antι-CD40L Humanized Ab. Lupus, immune thrombocytopenic purpura

Alexion h5G 1 1 Humanized antι-C5 Ab. Treatment ot collagen-induced

Pharmaceuticals arthritis, glomerulonephπtis. Rheumatoid arthritis Systemic lupus erythematosus

Ortho Biotech Orthoclone OKT4 Antι-CD4 mouse MAb CD4 mediated autoimmune diseases

Millenium LDP-02 Antι-α4β7 humanized Ab. Ulcerative colitis.

Cambridge Antibody J695 Antι-IL- 12 human Ab trom phage library. Rheumatoid arthritis

Technology/BASF Crohn's disease.

Cambridge Antibody D2E7 Anti-TNF alpha human Ab trom phage library. Rheumatoid

Technology/BASF arthritis.

Medarex/Centeon, MDX 33, 1. Antι-sCD64 (FcR) human MAb trom transgemc mice

Medarex/Eisai/ MDX CD4 Autoimmune hematological disorders.2. Antι-CD4 human MAb

Genmab trom transgemc mice. Rheumatoid arthritis

HEART/VASCULAR DISEASE AND BLOOD DISORDERS

Rephgen h60.1 Humanized anti CD1 1 B Ab. Ischemia /reperlussion in thoracoabdominal aneurysm

Alexion Pharm h5Gl . l Humanized anti C5 scFv. Stroke, myocardial infarction and surgical trauma

Genentech Antι-CD18 Antι-CD 18 humanized Fab'2. Acute myocardial infarction

Boehπnger Ingelheim BIRR- 1 Anti-ICAM 1 MAb. Prevention of repertussion damage. Table 1 (cont)

Representative Antibodies in Clinical Evaluation (cont)

Institution/Company Antibodies Characteristics/Application (in order)

Centocor Capiscint, ReoPro® 1 Atherosclerotic plaque imaging agent 2 Chimeπc anti-gpllbllla

Ab Unstable angina Acute myocardial infarction

American Biogenetic Sci MH l -Fab Fab In vivo imaging cardiovascular thrombosis ICOS nHu23F2G Humanized Ab anti-adhesion molecules Myocardial infarction

Hemorragic shock Ischemic stroke

Biogen VL4-A integπn Humanized Ab Artheπopathy Centocor Corsevιn ^{I M} M Anti-Factor VH -Fab or imaging Disseminated intravascular coagulation

NEUROLOGIC DISORDERS

Boehπnger Ingelheim Enlimomab Anti- ICAM 1 Ab Stroke

ICOS nHu23F2G, Rovelizumab Humanized Ab anti-adhesion molecules Multiple sclerosis

Athena Neurosciences Antegren ^,M Anti-LMF humanized antibody Multiple sclerosis flares

Millenium LDP-01 Humanized beta 2 lntegπn Stroke

Protein Design Labs/ Zenapax® Antι-CD25 humanized Ab Tropical spastic paraparesis

Hoffmann-La Roche dacli7umab

Centocor ReoPro® abciximab Chimeric anti gpllbllla Ab Stroke

IDEC Pharm IDEC 131 Humanized antι-CD40L Ab Multiple sclerosis

Cambridge Antibody J695 Antι-IL- 12 human Ab trom phage library Multiple sclerosis

Technology/BASF

T Cell Sciences ATM027 Humanized antibody Multiple sclerosis

INFLAMMATION

Cell Genesys Anti-selectins Humanized Abs Inflammatory disease

ICOS nHu23F2G Humanized Ab anti-adhesion molecules Inflammatory disease

Protein Design Labs Anti-E/P and L Humanized antibodies Trauma, stroke, digestive tract selectins inflammatory diseases, and repertussion

Boehπnger Ingelheim Anlι-ICAM- 1 Murine anti-lCAM 1 MAb Burns

TRANSPLANTATION

Bochπnger Ingelheim Antι-ICAM- 1 Muπne anti-ICAM 1 MAb Phase II/II1 for kidney transplant rejection

BioTransplant Medimmune BTI-322 1 Antι-CD2 rat MAb Allogratt rejection 2 Ann T cell receptor

T10B9 Allogratt rejection

Medimmune MEDI-507 1 Antι-CD2 humanized Ab GVHD Acule kidney transplant

MEDI-500 rejection 2 Anti-TCR αβ humanized Ab GVHD

Sandoz Antι-CD25 Humanized Ab Allograft rejection

Abgenix ABX-CBL Antι-CD147(CBL) human MAb trom transgemc mice

GVHD Allogratt rejection

Millenium LDP-01 Humanized anti-beta 2 lntegπn Kidney transplantation

Bristol-Myers CTLA-4 Immunoligand with antι-CD28 activity Allogratt rejection

Protein Design Labs, SMART^{1 M} antι-CD3 1 Humanized antι-CD3 Ab Transplantation 2 Humanized anti

Protein Design Labs/ Zenapax ¹" CD25 Ab Liver transplant Pediatπc kidney transplant

Hotfmann-La Roche dachzumab Kidney transplant rejection with cyclospoπne elimination

Ortho Biotec and OKT4A, hOKT-4A 1 Antι-CD4 mouse MAb Prophylaxis of allogratt rejection 2

Ortho/Novum Humanized version of the OKT4 antι-CD4 MAb Allogratt rejection Table 1 (cont)

Representative Antibodies in Clinical Evaluation (continued)

Institution/Company Antibodies Characteristics/Application (in order)

Pasteur Meπeux/lmmunotech Anti-LFAl Anti-CDl 8 murine MAb Allograft rejection Biogen Antova Humanized antι-CD40L Ab Allograft rejection

SKIN DISORDERS

Genentech hul l 24 Anti-CDl l a humanized Ab Moderate to severe psoriasis

ICOS ICM3 Anti-ICAM 3 Ab Psoriasis

CIMAB SA Havana lor-tl Antι-CD6 MAb Psoriasis

Medimmune MEDI-507 Antι-CD2 humanized Ab Psoπasis

Abgenix ABX-IL8 Anti IL8 human MAb trom transgemc mice Psoriasis

Celltech CDP-850 E-selectin humanized Ab Psoriasis

IDEC/Mitsubishi IDEC-1 14 Antι-CD80 pπmatized Ab Psoπasis

Cambridge Antibody CAT 192 Anti-TGFB l tully human Ab derived from phage library

Technology Fibrotic disease Scarring

Protein Design Labs SMART'" antι-CD3 I Humanized antι-CD3 Ab Psoπasis 2 Humanized

Protein Design Labs/ Zenapax® Antι-CD25 Ab Psoπasis

Hoffmann-La Roche daclizumab

INFECTIOUS DISEASES

Knoll Pharm /BASF MAK 195F Anti TNF alpha murine Fab 2 Treatment of sepsis Immunomedics LeukoScan® Imaging of osteomyelitis infected prosthesis, appendicitis Protein Design Labs Protovιr^{I M}, 1 Anti-CMV humanized Ab Infection in liver transplantation

Protein Design Labs/ SDZ OST 577, 2 Anti-HBsAg human MAb Infection in liver transplantation

Novartis SDZ SL 109 3 Ann HBsAg human MAb Infection

Progenies/ PRO 542 Anti- gpl 20 HIV humanized Ab Neutralizing

Gen/ymeTransgemcs Peptide Therap HNK20 Ann RSV Fgp murine IgA MAb Respiratory infection Planet Biotechnology CaroRx ¹ Chimeric slgA anti Stiepocotcm mutans produced in transgemc tobacco Complementary treatment for the prevention of canes

RESPIRATORY AND ALLERGIC DISORDERS

Biogen Anti VL4-A lntegπn Humanized antibody Asthma

Genentech Tannox/Novartis rhuMabE25 Humanized anti IgE Ab hay tever Allergic asthma Allergic rhinitis

IDEC Pharm IDEC 152 Antι-CD23 pπmatized Ab Allergy Asthma

Glaxo/Smith Kline SB-240563 1 Antι-IL5 humanized Ab Allergy Asthma

2 Ann IL4 humanized SB-240683 Ab Allergy Asthma

Celltech/Scheπng SCH 55700 Antι-1L5 humanized Ab Allergv Asthma

MISCELLANEOUS

Cambridge Antibody CAT 152 Antι-TGFB2 fullv human Ab derived trom phage

Technology library Glaucoma surgery

Med are x MDX-RA Immunotoxin Prevention of secondary cataracts

ICOS IC 14 Anti-CD 14 Ab Toxic shock One could envision the use of the /ELISA in kit form. Antibodies against specific antigens could be precoated on plates followed by blocking of non-coated solid surface. These plates would then be stable, and could be stored at 4°C for several months. Peroxidase labeled antigen is also stable for several months at 4°C. Test samples could be added anytime within this window of reagent stability in a research or clinical setting.

MATERIALS AND METHODS:

Production of polyclonal antisera

Purified soluble antigens are generally mixed with an adjuvant to help enhance the immune response by stimulating various cellular components of the immune system. Typically, Freund's adjuvant (FA, Difco Laboratories) is used. Freund's adjuvant is a mineral oil with a stabilizer to yield stabile water-in-oil emulsions. Complete Freund's Adjuvant (CFA, Difco Laboratories) contains a dispersion of dried, heat killed Mycobacterium tuberculosis and causes formation of local granulomas, which are rich in macrophages and immunocompetent cells. Immunizations are routinely performed using CFA with subsequent boosts in FA at intervals of one month.

Intramuscular inoculations for goats are generally performed in the thigh muscle of the hind leg followed by subcutaneous boosts to decrease the chances of an anaphylactic reaction. Test bleeds should be drawn approximately 10 days after each boost until desired titer is obtained. Screening of test bleeds could be performed by ELISA, Westerns, or Ouchterlony agarose double diffusion precipitin assays.

Affinity Purification

Antibodies are routinely purified from tissue culture media for monoclonals or, from serum for polyclonals, using affinity chromatography methods. One such method employs the use of Protein A isolated from culture media of Staphylococcus aureus immobilized on sepharose beads. Protein A has an extremely high affinity for IgG and IgG sub-classes from a variety of mammalian species and will also bind to some IgA and IgM antibodies (Kronvall, G., et al. J. Immunol. 104: 140-147, 1970). To purify antigen specific antibodies, protein antigen ligands are coupled to activated immunoaffinity supports via primary amines. The gel containing approximately 5- 10 mg antigen per ml resin, is then packed into a column and the solution containing the desired antibody is slowly passed over the column, followed by washing, to remove weakly bound non-specific proteins. The specific antibody is then eluted. Acid elution is the most commonly employed desorption method. Eluants such as glycine-HCl pH 2.5, 20 mM HC1, sodium citrate pH 2.5, 0.5 M acetic acid pH 2.5 are routinely used to dissociate the antigen-antibody complexes. Base elution such as 50 mM diethylamine pH 1 1.5, and elution with chaotrophic agents, are less frequently used than acid elution, but in some cases, they are more effective.

Enzyme Labeling of antibodies and antigens

Widely used methods for detecting immunoassay antigen-antibody complexes involve the labeling of either the antigen or the antibody with a radioisotope, fluorescent tag, or an enzyme. Obvious safety and disposal concerns have increased the popularity of enzyme-linked immunoassays. There are three enzymes generally used with ELISA' s. The earliest was alkaline phosphatase and the most commonly used now is horseradish peroxidase. The third is beta-galactosidase. Phosphatase or galactosidase is used in situations where there may be endogenous peroxidase activity in the antigen. Conjugation is by a variety of methods. Two of the more commonly used cross-linking methods involve the use of glutaraldehyde (GA) (O'Sullivan, M.J. and Marks, V., Meth. Enzymol. 73: 147, 1979) and periodate (NaIO₄) (Nakane, P.K., and Kawaoi, A., J. Histochem. Cytochem. 22: 1084, 1974) the latter being very efficient for glycoproteins. This method has been most significant for the development of ELISA' s due to its high efficiency compared to the GA methods. With the NaIO₄ method, yields are at least 3 to 4 times higher than the conjugates prepared with GA. The NaIO₄ method was originally used for the conjugation of horseradish peroxidase to antibodies but may also be applied to other glycoproteins such as various cytokines. Another more recent method of labeling is with biotin and avidin. Commonly, proteins are labeled with biotin and avidin is tagged with either alkaline phosphatase or horseradish peroxidase. Biotin- avidin complexes have a high binding affinity and can be used as a tool to greatly enhance the detection signal.

Antibody generation

Anti-MPO and anti-PMP antiserum were developed in goats. Goats were chosen as animal of choice for antibody production because large volumes of antisera could be generated in a relatively short period of time. The goats were immunized with MPO and PMP antigens as follows. Antigen was diluted to a concentration of 1.0 mg/ml phosphate buffered saline (PBS) pH 7.4, and was then mixed with an equal volume of CFA. The animals were given intramuscular injections in the rear hindquarter. After four weeks, the goats were boosted with 0.5 mg of antigen mixed one to one with FA and then bled on day ten to check for antibody titer. Antibody titers were assessed using agarose double diffusion technique of Ouchterlony (Arkiv. Kemi Mineral Geoi, 263 (14): 1-9, 1948) with plates obtained from ICN Biomedicals,

Inc., (642751). Goats were given booster injections and bled every 4 weeks.

Affinity purification of antisera

Antibody was affinity purified using immobilized antigen at 15 mg/ml AffiGel 10 resin. Purified antigen was dialyzed into 3 ml 0.05 M sodium phosphate buffer pH 7.5, containing 150 mM sodium chloride. After dialysis against a 5000-fold excess of phosphate buffer, washed AffiGel (1 ml/ 15 mg protein) was added to the antigen solution and allowed to incubate overnight at 4°C with slow rotation. Blocking of remaining active esters was achieved by the addition of 0.1 ml 1 M glycine ethyl ester pH 8.0 (Sigma G-8001) per ml of gel for one more hour at 4°C. The gel was then transferred to a column and washed extensively with coupling buffer. The goat anti-sera was diluted 1 : 1 with PBS and pumped onto an affinity column at a flow rate of 0.5 ml/min. Affinity purified antibody was eluted from the column with 0.5 M acetic acid pH 2.5 elution buffer, collecting into 1: 10 volume 10 X phosphate buffered saline buffer to neutralize the pH. A final pH adjustment to 7.0 was made with NaOH. Approximately one mg of specific antibody was obtained per ml of serum.

Preparation of enzyme labeled antigen conjugate

The following conjugation reagents were purchased from Sigma Chemical Company, St. Louis, Missouri: sodium m-periodate (S-1878), sodium borohydride (S-9125), and horseradish peroxidase type XII (P-8415). AffiGel- 10, an active ester agarose used for affinity chromatography, was purchased from Bio Rad (153-6099). MPO and PMP were labeled with horseradish peroxidase (HRP) according to the method described by Nakane, et al. (J. Histochem. Cytochem. 22: 1084-1091, 1974). Briefly, 5.0 mg/ml of purified MPO was dialyzed into 10 mM carbonate/bicarbonate buffer pH 9.5. HRP was dissolved in 2 ml distilled water at 10 mg/ml and 0.1 ml of a 0.2 M sodium periodate solution was added and allowed to stir for 20 minutes, room temperature (on addition of the sodium periodate, there is a color change from brown to green). Next, the HRP-aldehyde solution was dialyzed against 1 mM sodium acetate buffer, pH 4.5 at 4°C, for four hours with 5-6 buffer changes (color changes back to green). The HRP-aldehyde was adjusted between pH 9-9.5 by the addition of 20ul of 0.2 M carbonate buffer pH 9.5, and 5 mg of MPO was immediately added to this solution. The pH of the mixture was rechecked and adjusted to pH 9-9.5, if necessary, and then incubated for two hours at room temperature. The mixture was then reduced by the addition of 0.05 ml sodium borohydride, 4 mg/ml, to give a final concentration of about 0.18 mg NaBH /ml and allowed to stand for two hours at 4°C with occasional shaking. The reaction mixture was then dialyzed overnight against 2000-5000 times its volume of PBS at

4°C. For storage, the antigen/HRP conjugate was mixed with bovine serum albumin (BSA final concentration 5 mg/ml) and 0.01% thimerosal (Sigma, T-5125) as a preservative.

Performance parameters

To determine the appropriate assay conditions, checkerboard ELISA's were performed by spiking various concentrations of affinity purified goat antibody into human serum from 5 normal donors and allowing it to compete with a constant amount of solid matrix absorbed affinity purified goat antibody for the binding sites of HRP labeled MPO or PMP. In a checkerboard ELISA, antigen concentrations are diluted in one direction on a microtiter plate, and antibody is diluted in the other direction. Optimum concentrations are selected based on predetermined criteria for time of development, maximum desired optical density (OD) and minimum noise to signal ratio. Results were plotted as either the concentration of competing antibody vs. OD₂₈o or as dilution vs. OD₂₈₀- It was determined that a coating antibody concentration of 0.25 μg/ml was limiting enough to allow for adequate competition for 10 ng/ml of HRP-MPO. Starting dilution of human patient serum was diluted 1 : 100 to eliminate any serum matrix effect and then serially diluted to 1 : 102,400. Optimally, the desired combination of coating antibody, serum antibody, and HRP conjugated MPO or PMP, would produce an optical density of 0.5-1.0 at 650nm in approximately 15 minutes at room temperature for the upper limit of the standard curve.

Inhibition enzyme linked immunosorbent assay (iELISA)

Bovine serum albumin (BSA RIA grade, fraction V) used in assay buffers, was purchased from Sigma (A-7888). For the ELISA, Immulon II, polystyrene 96-well microtiter plates (Dynatech), and TMB peroxidase substrate (Kirkegaard & Perry Laboratories, Inc. (50-76-04) were used. Tween 20 was purchased from Sigma (P- 1379). Polystyrene 96-well microtiter plates were precoated with 150 μl/well of a

0.25 mg/ml affinity purified goat anti-MPO or PMP antibody solution diluted in 0.1 M sodium carbonate buffer pH 8.2, and allowed to bind overnight at room temperature, 100% humidity. The plates were then emptied to remove unbound antibody and blocked with the addition of 200 μl/well 3% BSA-10 mM phosphate buffered saline-0.05% Tween 20 pH 7.4, for one hour at 37°C, and 100% humidity. The unbound blocking agent was then removed and the plates were washed three times with 0.05% Tween -isotonic saline (TS). Next, 75 μl/well of human serum diluted 1:50 (1: 100 final) was added to the appropriate wells diluted in 0.1% BSA- lOmM PBS-0.05% Tween 20. Then 75 μl of HRP/Antigen at a 1 :50,000 (1 : 100,000 final) was added to each well and the coated antibody and antibody in the serum were allowed to compete for HRP/MPO for 2 hours at 37°C and 100% humidity.

The plates are then emptied to remove unbound conjugate and washed three times with TS. In the next step, a colorimetric reaction was performed to determine the bound peroxidase activity by addition of 150 μl/well of TMB peroxidase substrate solution to each well and allowing the reaction to proceed for approximately 15 minutes. Finally, the absorbance of the resultant blue color was measured by an

ELISA autoreader (Dynatech, U.S.A.) at 650 nm.

EXAMPLES

Example 1 Measurement of MPO antibodies in Rhesus monkeys

The myelopoietin (MPO) family of proteins are multifunctional agonists of the human IL-3 and G-CSF receptor complexes and were developed to produce a significant attenuation in neutropenia and thrombocytopenia in patients receiving myelosuppressive cancer therapy. It is critical, when administering protein therapeutics, to determine if antibodies are being generated that may have important clinical significance such as neutralization. The /ELISA assay was to determine the serum antibody titers of a rhesus monkey immunized and boosted over a 6.5 month period with MPO. Immunization consisted of subcutaneous injections of 100 μg/kg MPO with 0.5 mg/kg Titermax (adjuvant) with subsequent 100 μg/kg boost on days 21, 42, 63, 84, 105, 126, and 147. Serum was obtained and then added to the assay at a 1 : 100 dilution and then serially diluted 1:2 for 10 dilutions to determine titers. Results suggest that titers first appear around day 14 and reach a peak between 2.5 and 5 months. For a positive control, affinity purified goat anti-MPO was added to normal rhesus serum at 4 μg/ml and then serially diluted 1:2 (Figure 2).

Example 2 Measurement of MPO antibodies in cancer patients

Serum from MPO-treated cancer patients were analyzed to determine if they were producing antibodies to MPO. Breast cancer patients were given 5.0 μg/kg/day

MPO after various cycles of docetaxel chemotherapeutic treatment and lymphoma patients were given 7.5 μg/kg/day MPO after receiving ESHAP chemotherapeutic regimen. Results of cancer and lymphoma patients treated with MPO showed either no anti-MPO antibodies or slight titers of less than 1 :500 using an IC50 calculation (Figures 3-5).

Example 3 Measurement of PMP antibodies in rhesus monkeys

The /ELISA was used to determine antibody titers in another class of hematopoietic growth factors. Promegapoietin (PMP), a dual receptor agonist for the IL-3 and thrombopoietin (TPO) receptors, has demonstrated enhanced recovery from chemotherapy induced thrombocytopenia. Serum was examined from normal rhesus monkeys immunized with PMP subcutaneously for 10 days with 50 μg/kg/day. A titer was first detectable at day 21 (Figure 6).

Example 4 Measurement of PMP antibodies in cancer patients

Serum from a male sarcoma patient treated with 3 cycles of 0.5 μg/kg/day PMP for 5 days was analyzed for antibodies. With the /ELISA, an increased antibody titer to PMP with subsequent treatment cycles was detected (Figure 7). With increasing concentrations of specific PMP antibody a neutralizing effect on both drug and endogenous growth factor could result inducing thrombocytopenia.

Example 5 Demonstration that the iELISA was measuring antibody in the serum

To demonstrate that the /ELISA was indeed measuring antibody in the patient serum, positive titer serum was pre-treated with 50 μg/ml Protein G for 1 hour to remove any IgG from the serum and then samples were assayed before and after Protein G treatment. Results clearly show that after the addition of Protein G, serum antibody levels return to baseline levels of pretreatment serum (Figure 8).

Example 6 Measurement of an exogenous TNF a antibody

A support surface, such as a 96 well microtiter plate, is precoated with a purified antibody (either affinity purified, monoclonal or synthetic) to TNF α. Labeled

TNFα and a test sample, such as serum from a patient that has been treated with an anti-TNFα Mab, such as Remicade (Infliximab), CDP-870 or D2E7 (adalimumab), are co-incubated in the antibody-coated microtiter well. The anti-TNFα Mab, Remicade (Infliximab), CDP-870 or D2E7 (adalimumab), should bind the labeled TNFα and thus inhibit the binding of labeled TNFα to the antibody bound to the plate. Concentration of antibody in the biological fluid could be determined by percent inhibition relative to a standard anti-TNFα antibody.

Example 7 Measurement of an exogenous CD20 antibody

A support surface, such as a 96 well microtiter plate, is precoated with a purified antibody (either affinity purified, monoclonal or synthetic) to CD20 ligand. Labeled CD20 ligand and a test sample, such as serum from a patient that has been treated with an anti-CD20 Mab, such as Rituxan (Rituximab), are co-incubated in the antibody-coated microtiter well. The anti-CD20 ligand Mab, Rituxan (Rituximab), should bind the labeled CD20 ligand and thus inhibit the binding of labeled CD20 ligand to the antibody bound to the plate. Concentration of antibody in the biological fluid could be determined by percent inhibition relative to a standard anti-CD20 antibody.

Example 8 Measurement of an exogenous HER 2 antibody

A support surface, such as a 96 well microtiter plate, is precoated with a purified antibody (either affinity purified, monoclonal or synthetic) to HER 2. Labeled HER 2 and a test sample, such as serum from a patient that has been treated with a anti-

HER 2 Mab, such as Herceptin (Trastuzumab), are co-incubated in the antibody coated microtiter well. The anti-HER 2 Mab, Herceptin (Trastuzumab), should bind the labeled TNFα and thus inhibit the binding of labeled Her 2 to the antibody bound to the plate. Concentration of antibody in the biological fluid could be determined by percent inhibition relative to a standard anti-HER 2 antibody.

Example 9 Measurement of an endogenous IgE antibody levels

IN this form of the /ELISA the 'antigen' is an anti-IgE antibody. A support surface, such as a 96 well microtiter plate, is precoated with a purified antibody (either affinity purified, monoclonal or synthetic) to IgE class antibodies. A labeled anti- IgE antibody and a test sample, such as serum from a patient that has been challenged by an allergen, are co-incubated in the antibody-coated microtiter well. The IgE antibody, should bind the labeled anti-IgE and thus inhibit the binding of labeled anti-IgE to the anti-IgE antibody bound to the plate. Concentration of antibody in the biological fluid could be determined by percent inhibition relative to a standard anti-HER 2 antibody.

Additional information on ELISA and other solid phase immunoassays can be found in Kemeny D.M. and Challacombe S.J. ELISA and Other Solid Phase

Immunoassays Theoretical and Practical Aspects, John Wiley & Sons, (1988). Methods of producing antibodies suitable for use in the present invention are well known to those skilled in the art and can be found described in such publications as Harlow & Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988), and Asai, Methods in Cell Biology Vol. 37: Antibodies in Cell Biology, Academic Press, Inc. New York (1993). Methods of producing engineered antibodies are also well known to those skilled in the art and can be found in such publications as Gavilondo, J.V. et al., Biotechniques 29: 128-145 (2000) and Hudson P.J. Current Opinion in Immunology 11:548-557 (1999). Methods of producing antibodies in plants suitable for use in the present invention are well known to those skilled in the art and can be found described in such publications as; Hein,

Biotechnology Process 7:455 (1991); Zeitlin, Nature Biotech 16: 1361 (1998); WO 98/10062, Ma, Eur J Immunol 24: 131 (1994), Schouten, Plant Mol Biol 30:781 (1996), Ravishanker Gα, J Biochem., Mol. Biol. Biophysics 4(2):73-102 (2000); Fisher, R., Biol. Chem. 380:825-839 (1999); Russel, D.A., Curr. Top. Microbiol. Immunol. 240: 1 19-138 (1999); and Smith M.D., Food Technol. Biotechnol.

35(3): 183- 191 (1997). Additional details about single chain antibodies and methods for producing such can be found in U.S. Patent NOs. 6,121 ,424; 6,103,889, 6,027,725, 6,025,165, 5,990,275; 5,917,021, 5869,620; 5,856,456; 5,757,260; 5,763,733; 5,656,730; 5,534,621, 5,518,889; 5,455,030; and 5,260,203.

All references, patents or applications cited herein are incorporated by reference in their entirety as if written herein.

Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention.

It is intended that all such other examples be included within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for the detection of a first antibody in a test sample, comprising the steps of: coating a binding surface or support with a purified second antibody to form an antibody-coated surface; combining a predetermined amount of a purified labeled antigen and said test sample containing said first antibody; adding the incubated mixture of antigen and first antibody to said antibody-coated surface; incubating said antibody-coated surface with said combination of antigen and first antibody; and measuring the amount of antigen-binding inhibition.

2. The method of claim 1 wherein said first antibody is an endogenous antibody.

3. The method of claim 2 wherein said endogenous antibody is against a recombinant polypeptide.

4. The method of claim 3 wherein said recombinant polypeptide is selected from the group consisting of; MPO, PMP, and ProGP.

5. The method of claim 2 wherein said endogenous antibody is against a vaccine.

6. The method of claim 1 wherein said first antibody is an exogenous antibody.

7. The method of claim 6 wherein said exogenous antibody is selected from the group consisting of; an intact immunoglobulin, a FV fragment, a Fab fragment, (Fab)2 fragment, a single chain antibody, a chimeric antibody, or a humanized antibody.

8. The method of claim 7 wherein said exogenous antibody is selected from the group consisting of; Vitaxin™, PE38, LMB-7, Ch 14.18, Verluma™,

Avicidin, NR-LU-10, CMB-401, CMA-676, hB72.3, Panorex™, CY1748, MFE-23, HuBrE.3, C225, BEC-2, ior-R3, ior-r3hum, ior-c5, Oncolysin B™, BR96, ChTNT-1/b, Oncolym™, IDEC-Y2B8, IDEC-InB8, RlGScan® CR49, OncoRad® PR CYT-356-Y-90, OncoScint®, MDX-447, MDX-H210, MDX-220, CEA-Cide™, hMN14, CEA-Scan™ , LymphoScan™, Lymphocide™, AFP-Scan™,

ImmuRAIT-CEA, ImmuRAιT-LL2, Campath 1 LDP-03, SMART™ M195, SMART™1 D10, Zenapax®, OvaRex, Anti-ICAM 3, CDP-571 , SMART™ anti-CD3, Clenoliximab, IDEC-131, IDEC 151, h5G 1.1, Orthoclone OKT4, Orthoclone OKT3 (Muromomab), LDP-02, J695, D2E7, MDX 33, MDX CD4, h60.1, h5Gl. l, BIRR-1, MHl-Fab, nHu23F2G, VL4-A integrin, Corsevin™ M,

Enlimomab, Rovelizumab (nHu23F2G), Antegren™, LDP-01, Zenapax®, daclizumab, ReoPro® Abciximab, IDEC 131, J695, ATM027, nHu23F2G, BTI-322, MEDI-507, ABX-CBL, LDP-01 , CTLA-4, SMART™ anti-CD3, Zenapax™, daclizumab, OKT4A, hOKT-4A, Anti-LFAl, Antova, hul 124, ICM3, ior-tl , MEDI-507, ABXTL8, CDP-850, IDEC-1 14, CAT 192, SMART™ anti-CD3,

Zenapax®, daclizumab, MAK 195F, LeukoScan®, Protovir™, SDZ OST 577, SDZ MSL 109, PRO 542, HNK20, CaroRx™, rhuMabE25, IDEC-152, SB-240563, SCH 55700, CAT 152, MDX-RA, IC14, Rituxan (Rituximab), Remicade (Infliximab), Simulect (Basiliximab), Herceptin (Trastuzumab), Zenapax (Dacliximab), Synagis (Palivizumab), and CDP-870.

9. The method of claim 8 wherein said exogenous antibody is selected from the group consisting of; Orthoclone OKT3 (Muromomab), Rituxan (Rituximab), ReoPro (Abciximab), Remicade (Infliximab), Simulect (Basiliximab), Herceptin (Trastuzumab), Zenapax (Dacliximab), Synagis (Palivizumab), D2E7

(adalimumab), CAT 152, CAT 192, J695, and CDP-870.

10. The method of claim 9 wherein said exogenous antibody is selected from the group consisting of; CDP 571, D2E7 (adalimumab), CAT 152, CAT 192, J695, and CDP-870.

11. The method of Claim 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 wherein said second antibody is a purified polyclonal antibody.

12. The method of claim 11 , wherein said purified polyclonal antibody is purified by a method selected from the group consisting of: antigen affinity purification, protein A affinity purification, and protein G affinity purification.

13. The method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 wherein said second antibody is a monoclonal antibody.

14. The method of claim 1,2,3,4,5,6,7,8,9, 10, 11, 12, or 13 wherein said antigen is labeled with an agent selected from the group consisting of: horseradish peroxidase, alkaline phosphatase, biotin and phosphorescently.

15. The method of claim 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 wherein said binding surface or support is a microtiter plate well.

16. The method of claim 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, or 15 wherein said test sample is serum from a mammal.

17. The method of claim 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, or 15 wherein said test sample is serum from a human.

18. A test kit for use in an immunoassay to detect a first antibody to an antigen in a test sample, comprising: an insoluble binding surface or support;

a purified second antibody bound to said binding surface or support; and

a purified and labeled antigen that specifically binds to and saturates said second antibody.

19. A test kit for use in an immunoassay to detect a first antibody to an antigen in a test sample further comprising washing reagents, incubating reagents, and label substrate.

20. The test kit of claim 18 or 19 wherein said first antibody is an endogenous antibody.

21. The test kit of claim 21 wherein said endogenous antibody is against a recombinant polypeptide.

22. The test kit of claim 21 wherein said recombinant polypeptide is selected from the group consisting of; MPO, PMP, and ProGP.

23. The test kit of claim 20 wherein said endogenous antibody is against a vaccine.

24. The test kit of claim 18 or 19 wherein said first antibody is an exogenous antibody.

25. The test kit of claim 18 or 19 wherein said exogenous antibody is selected from the group consisting of; an intact immunoglobulin, a FV fragment, a Fab fragment, (Fab)2 fragment, a single chain antibody, a chimeric antibody, or a humanized antibody.

26. The test kit of claim 25 wherein said exogenous antibody is selected from the group consisting of; Vitaxin™, PE38, LMB-7, Chl4.18, Verluma™, Avicidin, NR-LU-10, CMB-401, CMA-676, hB72.3, Panorex™, CY1748, MFE-23, HuBrE.3, C225, BEC-2, ior-R3, ior-r3hum, ior-c5, Oncolysin B™, BR96,

ChTNT-1/b, Oncolym™, IDEC-Y2B8, IDEC-InB8, RlGScan® CR49, OncoRad® PR CYT-356-Y-90, OncoScint®, MDX-447, MDX-H210, MDX-220, CEA-Cide™, hMN14, CEA-Scan™ , LymphoScan™, Lymphocide™, AFP-Scan™, ImmuRAIT-CEA, ImmuRAIT-LL2, Campath 1 LDP-03, SMART™ M195, SMART™ 1 D10, Zenapax®, OvaRex, Anti-ICAM 3, CDP-571, SMART™ anti-CD3, Clenoliximab, IDEC-131, IDEC 151, h5G 1.1, Orthoclone OKT4, Orthoclone OKT3 (Muromomab), LDP-02, J695, D2E7, MDX 33, MDX CD4, h60.1, h5Gl. l, BIRR- 1 , MH 1 -Fab, nHu23F2G, VL4-A integrin, Corsevin™ M, Enlimomab, Rovelizumab (nHu23F2G), Antegren™, LDP-01, Zenapax®, daclizumab, ReoPro® Abciximab, IDEC 131, J695, ATM027, nHu23F2G,

BTI-322, MEDI-507, ABX-CBL, LDP-01, CTLA-4, SMART™ anti-CD3, Zenapax™, daclizumab, OKT4A, hOKT-4A, Anti-LFAl, Antova, hul 124, ICM3, ior-tl , MEDI-507, ABX-IL8, CDP-850, IDEC-1 14, CAT 192, SMART™ anti-CD3, Zenapax®, daclizumab, MAK 195F, LeukoScan®, Protovir™, SDZ OST 577, SDZ MSL 109, PRO 542, HNK20, CaroRx™, rhuMabE25, IDEC-152, SB-240563, SCH

55700, CAT 152, MDX-RA, IC14, Rituxan (Rituximab), Remicade (Infliximab), Simulect (Basiliximab), Herceptin (Trastuzumab), Zenapax (Dacliximab), Synagis (Palivizumab), and CDP-870.

27. The test kit of claim 24 wherein said exogenous antibody is selected from the group consisting of; Orthoclone OKT3 (Muromomab), Rituxan (Rituximab), ReoPro (Abciximab), Remicade (Infliximab), Simulect (Basiliximab), Herceptin (Trastuzumab), Zenapax (Dacliximab), Synagis (Palivizumab), D2E7 (adalimumab), CAT 152, CAT 192, J695, and CDP-870.

28. The test kit of claim 24 wherein said exogenous antibody is selected from the group consisting of; CDP 571, D2E7 (adalimumab), CAT 152, CAT 192, J695, and CDP-870.

29. The test kit of claim 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 wherein said second antibody is a purified polyclonal antibody.

30. The test kit of claim 29, wherein said purified polyclonal antibody is purified by a method selected from the group consisting of: antigen affinity purification, protein A affinity purification, and protein G affinity purification.

31. The test kit of claim 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 wherein said second antibody is a monoclonal antibody.

32. The test kit of claim 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,

31 wherein said antigen is labeled with an agent selected from the group consisting of: horseradish peroxidase, alkaline phosphatase, biotin and phosphorescently.

33. The test kit of claim 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , or 32 wherein said binding surface or support is a microtiter plate well.

34. The test kit of claim 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, or 33 wherein said test sample is serum from a mammal.

35. The test kit of claim 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,

31 , 32, or 33 wherein said test sample is serum from a human.