AU4553993A

AU4553993A - Monoclonal antibodies to egg yolk immunoglobulins (igy)

Info

Publication number: AU4553993A
Application number: AU45539/93A
Authority: AU
Inventors: George Jackowski; Mikoyo Takahashi
Original assignee: SPECTRAL DIAGNOSTICS Inc
Current assignee: SPECTRAL DIAGNOSTICS Inc
Priority date: 1993-03-03
Filing date: 1993-07-14
Publication date: 1995-02-13
Also published as: JPH09500271A; EP0710252A1; ZA944547B; WO1995002612A1

Description

Monoclonal antibodies to egg yolk immunoglobulins ( IGY) .

This is a continuation-in-part application of U.S. serial no. 08/026,453 filed March 3, 1993, which is in turn a continuation-in-part application of U.S. serial no. 07/695,381 filed May 3, 1991.

field of Invention

The present invention relates to monoclonal antibodies to chicken yolk immunoglobulin (referred to herein as IgY) and the use of the same in immunoassays. The present invention further relates to a diagnostic system using the monoclonal antibodies to chicken IgY as a reporter antibody.

Background and Description of the Related Art

Immunoassay is a technique for measuring the presence of a substance using a reaction between antibody and antigen in vitro. This includes simple precipitation of antibody/antigen complexes to enzyme immunoassays (EIA). Today, EIA is used as the technique of choice. Since the introduction of hybridoma technology, which was developed nearly two decades ago, monoclonal antibodies (MAb) have been widely used in EIA. The advantages of MAbs are: their defined antigen specificity, unlimited supply, constant activity (i.e. no variation) and their easy production once identified. The most important advantage MAb offer for EIA is the possibility to standardize the assay methods, specificities and sensitivities.

Despite the popularity of MAb, polyclonal antibodies from goats, rabbits and other mammals are still used in immunoassays. The limitations of polyclonal antibodies obtained from mammals are: lot-to-lot variations due to their complex immunoregulatory mechanisms, very costly maintenance and often difficult and prolonged immunization protocols.

Recently the use of avian antibodies are gaining increasing popularity for use in immunoassays. The reason for the popularity is due to several advantages the chicken offers over mammals. Mammalian proteins are usually more immunogenic in the phytogenetically distant chicken. As an example, high affinity antibody against the human insulin receptor has been obtained from hen's eggs after repeated unsuccessful attempts to produce the same in rabbits (C. A. Stuart et al., Anal. Biochem., 173, 142, 1988). Another advantage is the simplicity of production. The ability of chickens to produce high levels of specific antibodies following immunization with antigens, and the transfer of large amounts of these antibodies from the serum of the laying hen to the yolk of the unfertilized egg, enables investigators to obtain antibodies, without the need of invasive techniques, by instead the simple collection of eggs.

In addition to the advantages of easy and continuous sample collection, it has been shown that antibodies in egg yolk are stable for a long period of time when stored at 4°C (J. C. Jensenius et al., J. Immunol. Method. 46, 63, 1981) and that large quantities of antibody can be obtained from the egg yolk exceeding the potential production from serum (M. Gassman et al., FASEB J. 4, 2528, 1990).

The properties of avian immunoglobulins, IgY, have been shown to be different from those of mammalian IgG (G. A. Leslie and L. W. Clem, J. Exp. Med. 130, 1337, 1969). The IgY does not react with mammalian Fc receptors, mammalian complement, staphylococcal protein A or streptococcal protein G (J. C. Jensenius et. al., J. Immunol. Methods. 46, 63, 1981). A more recent important finding is that chicken IgY does not react with rheumatoid factor (RF) which is a major cause of false positives in many immunological assays (A. Larsson et. al., Clin. Chem. 37, 411, 1991).

Many analytes in clinical diagnosis are detected in blood by EIA.

The clinical samples (i.e. plasma, serum or whole blood) often contain materials such as immunoglobulins and RF which cause false positive results. The properties of IgY described above enable unique application of this immunoglobulin in immunoassays where false positive results can be prevented due to non-specific interaction between mammalian antibodies or RF reaction with mammalian antibody.

An example of IgY application in EIA is the two-site assay, often called sandwich assay. Capture antibody, of a mammalian species, is bound to a suitable support and after reacting with antigen in the sample enzyme-labelled detector IgY antibody is added, which first binds and subsequently identifies the . presence of the antigen. Although it is generally best to use enzyme-labelled detector antibody to simplify the assay, the use of a reporter antibody specific to the detector antibody cuts down on the number of enzyme labelled detector reagents that must be monitored. To date the titre or presence of chicken immunoglobulin (IgY) in yolk has been monitored using rabbit polyclonal antibodies to chicken IgY. All the experiments by authors mentioned earlier have used as a reporter antibody, polyclonal antibodies raised in rabbits against IgY and coupled to alkaline phosphatase or labelled with ^I25I.

Peroxidase conjugated rabbit polyclonal anti-IgY is commercially available. These rabbit polyclonal antibodies produce high background levels in immunoassays. This is particulary problematic in in vitro diagnostics where discrimination between reactive and non-reactive specimens is critical. The monoclonal antibodies of the present invention were developed against egg yolk IgG (IgY), which can be used as a continuous source of reagents for immunoassays utilizing avian IgY. These monoclonal antibodies overcome the disadvantages of the prior art rabbit polyclonal antibodies.

Summary of Invention

Chicken antibodies are used in immunoassays with increasing popularity. New monoclonal antibodies against chicken IgY can be used as either capture antibodies or as detector antibodies for IgYs in enzyme-linked immunosorbent assay (ELIS A). The monoclonal antibodies described herein are specific for the heavy chain of IgY and show little or no cross reactivity with antibodies commonly used in EIAs such as human IgG, goat IgG, rabbit IgG or sheep IgG. In sandwich assays, where chicken IgYs are used as detectors, the detector IgY can be labelled directly. As well, a single enzyme conjugated second antibody (recorder antibody) can be used for many different assays, which thereby avoid the extensive labour to conjugate each detector chicken IgY antibody with marker enzymes. Furthermore the second antibodies can be used to form an amplified detector system.

According to the present invention there is provided monoclonal antibodies developed against chicken egg yolk immunoglobulins (IgY).

In one embodiment of the present invention there is provided hybridoma cell lines 2Y-114, 1Y-161, 1Y-162 and 1Y-263. These correspond to ATCC deposit numbers: , , and

, respectfully. Further according to the present invention there is provided monoclonal antibodies produced from the cell lines noted above.

According to a further embodiment of the present invention there is provided a method of using monoclonal antibodies developed against chicken egg yolk immunoglobulins as a reporter antibody in an immunoassay, wherein the immunoassay comprises: a capture antibody; an antigen in a test sample corresponding to the capture antibody; a chicken egg yolk detector antibody corresponding to the antigen to be tested in the test sample; and a labelled monoclonal reporter antibody developed against chicken egg yolk immunoglobulin.

This invention is also directed to a diagnostic assay system comprising at least a first, second and third antibody wherein: said first antibody is a capture antibody corresponding to a test antigen in a test sample; said second antibody is a chicken egg yolk detector antibody corresponding to said test antigen in said test sample; and said third antibody is a labelled monoclonal antibody developed against chicken egg yolk immunoglobulins.

Brief Description of the Drawings

Figure 1 shows a schematic presentation of direct- (Fig. 1A) and indirect- (Fig. IB) sandwich ELISA.

Figure 2 shows the cross-reactivity of anti-IgY monoclonal antibodies with mammalian IgGs.

Figure 3 shows the cross-reactivity of peroxidase conjugated rabbit polyclonal anti-IgY with mammalian IgGs.

Figure 4 compares the sensitivity of MAb 1Y263-HRP, of the present invention, and rabbit polyclonal anti-IgY-HRP as reporter antibody, using MAb capture and chicken IgY detector in an immunoassay for myosin heavy chain.

Figure 5 depicts a comparison of antibodies as described in Figure 4 except the immunoassay is for troponin I.

Figure 6 shows the results of a comparison of antibodies as described for Figure 4 except the immunoassay is for myosin light chain.

Detailed Description of the Preferred Embodiment

Antigens and antibodies used in the invention may be prepared by standard techniques well known in the art. Antibodies, used in the examples as capture antibodies, may be prepared from the serum of mammals such as rabbits, horses, sheep or goats.

In the two-site assay (sandwich assay) the capture antibody is bound to a solid support. Solid supports that have been used for RIA

(radioimmunoassay) or ELISA (enzyme linked immunosorbent assay) include for example: plastic test tubes, microtiter plates, disks, filters or beads; glass fiber filters; paper disks or filters, Sepharose beads, polyacrylamide gel, and Staphylococcal protein A. The capture antibody is then reacted with a sample, to test for the presence of the corresponding antigen. The expression

"corresponding" is well known in the art, and when applied to an antigen or antibody means that one such substance forms with its corresponding substance an immunocomplex or antigen-antibody complex.

According to the one embodiment of the present invention, the immunocomplex or antigen-antibody pair is detected by means of a detector chicken IgY antibody corresponding to the test antigen. The use of avain antibodies in immunoassays are gaining increasing popularity and as discussed above offer several advantages over mammalian antibodies. The IgY can be used in a direct-sandwich ELISA (see Figure 1A), wherein the IgY is appropriately labelled in order to identify the presence of the antigen. Conventional labels include radioactive tags, enzymes, chromophores, fluorophores and enzyme cofactors and effectors. Although the use of enzyme-labelled detector antibodies will simplify the assay, the use of a reporter antibody, raised against the detector antibody, (see Figure IB) reduces the number of labelled detector reagents that must be prepared and monitored. Therefore the use of a labelled reporter antibody, of the present invention, provides a "universal" reagent which can be used in the detection of any antigen-antibody complex, when chicken IgY is used as a detector antibody.

The reported antibody of the prior art is a polyclonal antibody raised in rabbits against IgY and is either enzyme labelled, for example with alkaline phosphatase or labelled with ¹²⁵I. Peroxidase conjugated rabbit polyclonal anti-IgY is commercially available. The monoclonal antibodies of the present invention were developed against egg yolk IgG, which can be used as a continuous source of reagents for immunoassays utilizing avian IgY. The production of monoclonal antibodies, against chicken IgY, was as described by

G. Kohler and C. Milstein, in Nature (London) Vol. 256, 1975, p. 495 and Eur. J. Immunol. 1976, 6:511-519; each of which are incorporated herein by reference. The reporter antibodies of the present invention are labelled, as is well known in the art and as discussed briefly above, to identify the presence of the antigen being tested.

As is known in the art, in the production of monoclonal antibodies, a vast number of hybridomas are produced which must be screened for affinity and specificity to the antigen. In the present invention, the trial and error production and analysis of monoclonal antibodies has been greatly simplified by the use of surface plasmon resonance (SPR), which can yield information on association rates, disassociation rates, and equilibrium kinetics and stoichiometry. Therefore, a sensible decision in selecting clones for recloning and expansion can be made based on the affinity, specificity and stability of an antibody's reaction with an antigen.

Kinetic and affinity constants of the interaction between the monoclonal antibodies of the present invention and the chicken yolk IgG were determined using the BIAcore™ system. (Pharmacia Biosensor AB, Uppsala, Sweden). The system uses surface plasmon resonance, which detects changes in optical properties at the surface of a thin gold film on a glass support. Detailed theoretical background and procedures are described by R. Karlsson et. al., (J. Immunol. Methods, 145, 229, 1991, incorporated herein by reference).

The association rate constants of the monoclonal antibodies of the present invention can range from 3.0 x 10⁴ M'V¹ to 1.0 x 10° M^'V. These monoclonal antibodies with an association rate constant within the range noted above were found to be suitable monoclonal antibodies for the diagnostic test systems described herein and are considered within the scope of the present invention. In one embodiment of the present invention, monoclonal antibodies with an association rate constant ranging from 5.6 x 10⁴ M^'V¹ to 1.0 x 10^s M^'V¹ were found to be very useful.

The disassociation rate constants can range from 4 x 10⁴ s^*1 to 2 x 10"² s"¹. In one embodiment of the present invention, the disassociation rate constants of the monoclonals prepared herein ranged from 1.6 x 10⁴ s^*1 to 3.2 x 10"* s'¹. The affinity constants calculated as the association rate constant/disassociation rate constant can range from 1.5 x 10⁶ M^"1 to 2.5 x 10⁹ M"¹. In one embodiment of the present invention, monoclonal antibodies were prepared wherein the affinity constant ranged from 2 x 10⁸ to 4.1 x 10⁸ M^"1.

As noted above, calculations of the kinetic and affinity constants provide a person skilled in the art with a quick and efficient method of selecting suitable monoclonal antibodies without having to await test results following expensive and laborious cloning and recloning and expansion of the hybridoma cell lines.

It will be apparent to persons skilled in the art that any antigen can be detected using the monoclonal antibodies to chicken IgY, provided that chicken IgY has been used as a detector antibody in the assay system. In one embodiment of the present invention, the monoclonal antibodies to chicken IgY are used as reporter antibody in a diagnostic assay to detect the presence of cardiac proteins. In one aspect of the invention, there is provided a diagnostic assay for detecting a myocardial infarction at early onset of patient chest pain. The assay comprises reacting a sample of blood, serum or plasma of the patient, containing an analyte to be measured (a cardiac protein), with at least one antibody, which corresponds to the cardiac protein. In this aspect of the invention at least one of the antibodies will be a chicken IgY and thus the monoclonal chicken IgY of the present invention can be used as a reporter antibody.

In one aspect of this embodiment, the analyte to be tested is at least three different markers of cardiac damage. The assay further comprises at least one antibody specific to each of said markers of cardiac damage, recognizing cardiac specific sequences, wherein one of said antibodies is a chicken IgY. The test further comprises the monoclonal chicken IgY of the present invention as a reporter antibody.

According to another aspect of the invention, there is provide a method for rapidly diagnosing the cause and stage of chest pain of a patient in the early onset thereof by determining the presence or absence of at least three proteins (analytes), which are present in blood within 6 hours of the onset of chest pain. The proteins comprise: a first protein (analyte) that is present in blood from onset of a myocardial infarction and necrosis but not anoxic injury in absence -of necrosis and is diagnostic between 4 hours and 8 hours after onset of chest pain, a second protein (analyte) that is cardiac specific that is present in blood following ischemic injury and is diagnostic from at least 2 hours after onset of chest pain, and a third protein (analyte) that is present in blood 6 hours after onset of myocardial infarction and necrosis but not anoxic injury in absence of necrosis and is diagnostic 6 hours after onset of chest pain.

In another embodiment of the present invention, the diagnostic assay is in the form of a panel test which utilizes an immunoassay sandwich dry chemistry format. In this embodiment a capture antibody is immobilized to a solid support in the panel test. The capture antibody corresponds to one of the cardial protein markers described above. A detector chicken antibody, which corresponds to the cardiac protein markers, binds to the protein marker, if present. The monoclonal antibodies to chicken egg yolk immunoglobulins, of the present invention are then used as reporter antibodies. Aspects of these embodiments of the present invention have been described in co-pending applications 07/695,381 and 08/026,453, both incorporated herein by reference.

In this embodiment of the invention, the cardiac marker proteins are selected from creatine kinase (CK-MB), myoglobin, myosin light chains

(MLC), troponin-I, troponin-T, tropomyosin and myosin heavy chains. In one aspect of this embodiment at least three cardiac protein markers are detected in this diagnostic assay.

The immunoassays of the present invention can be conducted in solid or liquid phase. Immunochemical assays include competitive and non- competitive binding assays, which have been described in the scientific and patent literature, and a large number of such assays are commercially available. Exemplary immunoassays which are suitable for the present invention include these described in U.S. Patents: 3,791,932; 3,817,837; 3,839,153; 3,850,752;

3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074 and 4,098,876 (all incorporated herein by reference).

There are currently available several types of automated assay apparatus which can undertake an assay on a number of samples contemporaneously. These automated assay apparatus include continuous/random access assay apparatus. Examples of such systems include OPUS™ of PB

Diagnostic System, Inc. and the IMX™ Analyzer introduced by Abbott

Laboratories of North Chicago, Illinois in 1988. The automated assay instrument of PB Diagnostic Systems, Inc. is described in U.S. Patent 5,138,868, which is incorporated herein by reference. A description of the IMX Analyzer is included in the "Abbott IMX Automated Bench Top Immunochemistry Analyzer System" by Fiore, M. et al, Clinical Chemistry. Vol. 34, No. 9, 1988, which is incorporated herein by reference. The automated analyzers have been developed for use in conjunction with solid phase immunoassay procedures to preform a variety of assays such as sandwich assays and competitive assays, according to the embodiments of the invention. An example of such an analyzer has been described in U.S. Patent 4,956,148 entitled "Locking Rack and Disposable Sample Cartridge" issued to C.J. Grandone on September 1, 1990, and assigned to Abbott Laboratories (disclosure incorporated herein by reference), which describes a carousel for carrying a plurality of reaction cells for use in connection with the Abbott IMX™ system. A further development in the art has been described in Canadian Patent Application 2,069,531, ChadwickM. Dunn etal, assigned to Abbott Laboratories (incorporated herein by reference) wherein the immunochemistry analyzer system has the capability of testing for up to three or four analytes in a single batch during a single run using currently available instrumentation. The system described in the Canadian application referred to above enables the users to group three small batches of assays together rather than run three separate analysis.

The present invention is further illustrated by the following examples, which are not intended to be limiting.

Examples

Preparation of Monoclonal Antibodies Against Chicken IgY Immunization

Balb/c female mice were immunized with subcutaneous injection of purified chicken egg yolk IgG (IgY) in complete Freund's adjuvant.

Subsequent immunizations were carried out at intervals of 3 - 4 weeks by intraperitoneal inoculation of IgY in incomplete Freund's adjuvant. Immunized mice were sacrificed 3 days after the last immunization without adjuvant.

Production of the Hybridomas and Antibodies

Hybridomas 2Y-114, 1Y-261, 1Y-262 and IY-263 were produced by fusion of Sp2/0 mouse myeloma cells with immunocytes obtained from spleen of immunized Balb/c mice as described by Fuller, S.A., Takahashi, M., and Hurrell, J.G.R., (Preparation of Monoclonal Antibodies; In: Ausubel F, Brent B, Kingston R., et. al., eds. Current Protocols in Molecular Biology. New York: Greene Publishing Associates, 1987: Unit 11). Briefly, fused cells in selective medium containing hypoxanthine, aminopterin, and thymidine were added to 300 to 500 wells of tissue culture plates (Costar No. 3598, 96-wells per plate) which were pre-seeded with feeder cells. Hybridoma cultures were subcloned 2 - 3 times by the limiting dilution method on a feeder layer of 1 - 3 X 10⁴ mouse peritoneal macrophages. Culture supernatants were screened for antibody activity by solid phase ELISA using polystyrene plates coated with chicken IgY. Balb/c mice previously treated with 0.5 ml of pristane were injected intraperitoneally with 1 - 3 x 10⁶ cloned hybridoma cells in 0.5 ml phosphate buffered saline, pH 7.4. Approximately 2 weeks later, ascites were collected and the monoclonal antibodies were affinity purified on Protein A or Protein G. Purified monoclonal antibodies were used for immunochemical studies.

Some properties of the four hybridoma lines are:

(1) cell doubling time ranging from 10.4h to 12h (2) average ml ascites produced ranging from 2.5 ml to 5.0 ml

(3) average mg/ml purified MAb yield from ascites ranging from 2.0 mg to 4.2 mg.

(4) stability of the cell lines in terms of MAb production - stable as tested up to 100 days of continuous culture in flasks.

A. Physics-chemical Characterization of Anti-IgY Monoclonal Antibodies 1. Antibody Class and Subclass Antibody class and subclass were determined by ELISA with a commercial kit (Bio-Rad, no. 172-2055). As shown in Table 1, all four monoclonals are IgGl, k. Table 1

Monoclonal Antibody Class and Subclass

Hybridoma line Isotype

2Y-114 IgGl, k

1Y-261 IgGl, k

1Y-262 IgGl, k

1Y-263 IgGl, k

2. Isoelectric Point (pi)

Isoelectric focusing on the four monoclonal antibodies was performed using the Model 111 Mini IEF cell (Bio-Rad, no. 1702975) following the instructions provided by the manufacturer. Results are summarized in Table 2. All four monoclonals recognize the heavy chain (H-chain) of IgY but do not react with light chain (L-chain). The pi value of the four monoclonals varies from 6.6 to 7.1.

Table 2

Hybridoma line Immunoblot against IgY pi value

2Y-114 H-chain specific 6.6

IY-261 H-chain specific 6.6

1Y-262 H-chain specific 6.7

1Y-263 H-chain specific 7.1 3. Kinetic and Affinity Constants

Kinetic and affinity constants for the interaction between the present four monoclonals and IgY were determined using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden). The system uses surface plasmon resonance, which detects changes in optical properties at the surface of a thin gold film on a glass support. Detailed theoretical background and procedures are described by R. Karlsson et. al., (J. Immunol. Methods, 145, 229, 1991, incorporated herein by reference).

Kinetic runs were performed as follows: Monoclonal antibodies, i.e. anti-IgY, at a constant concentration of 30 μg/ml in 10 mM Hepes, 0.15 M NaCl, 3.4 mM ethylenediaminetetraacetic acid disodium salt, 0.05% surfactant 20 (HBS, pH 7.4) were allowed to interact with sensor surfaces on which rabbit anti-mouse IgG_Fc had been immobilized. The antigen, chicken IgY, at concentrations ranging from 1.25 μg/ml to 20 μg/ml, was allowed to interact with the bound monoclonal antibodies. The runs were performed at 25 °C, at a flow rate of 5 μl/min during 6 min. (30 μl injection), taking a total of 24 report points. After injection of the antigen was complete, dissociation of the antigen from the antibody was monitored by taking a total of 18 report points. After the run, the surface was regenerated by injecting a 1M formic acid solution during 1 min. (5 μl injection). The instrument software produces a table of dR_A/dt and R_A values that can be directly used in a plotting program (Microsoft Excel).

Table 3 shows the results obtained. All 4 monoclonals have a good affinity for the antigen, IgY. Table 3

Mouse Monoclonal Anti-chicken IgY Antibodies

(Kinetic and Affinity Constants measured using the BIAcore)

ka - association rate constant kd - dissociation rate constant

Ka - affinity constants calculated as ka/kd

Kd - affinity constants determined from steady state binding

B. Immunological Properties 1. Antigenic Specificity

The antigenic specificity of the four monoclonal antibodies was determined by immunoblotting assay. Purified chicken IgY was electrophoresed on polyacrylamide gel in sodium dodecyl sulphate (SDS-PAGE) and transferred onto nitrocellulose paper. The nonspecific binding sites on blots were blocked with 5% skim milk solution in Tris-buffered saline with Tween 20 (TTBS buffer) followed by incubation with TTBS buffer containing purified monoclonal anti-IgY for lh. Blots were washed with TTBS buffer, pH 7.5, and incubated further with goat anti-mouse IgG labelled with horseradish peroxidase (HRP) (Bio-Rad, no. 170-6516). Colour was developed using 4-chloro-l-naphthol. The colour development was stopped by washing in distilled water. The results obtained as summarized in Table 2 indicated that all four monoclonals react specifically with the H-chain (heavy chain) of IgY.

2. Species Specificity

Cross-reactivity of anti-IgY monoclonals was assessed by solid- phase ELISA against IgGs of various species. Microtiter plates (Immulon 4 plate, Dynatech Co.) were coated with 100 μl of IgG solution (sheep-, rabbit-, goat-, human- and chicken-IgG) at 2 μg/ml in 50 mM carbonate buffer, pH 9.6 and allowed to stand overnight at 4° C. Plates were then washed once with 0J

M phosphate buffered saline, pH 7.4 (PBS) containing 0.05% Tween 20 (WB) and blocked with 0.1 M PBS containing 2 mg/ml bovine serum albumin (BSA) for 60 minutes at 37°C. After washing with WB, 2-fold dilutions of each monoclonal antibodies, starting at 4 μg/ml were added to the wells in duplicate. After washing the plates, goat anti-mouse IgG conjugated to peroxidase (Jackson

ImmunoResearch Laboratory Inc. no. 115-035-008) was added and incubated for 60 minutes at 37°C. The plates were washed and OPD substrate solution was added. Reaction was stopped with 2 M H₂SO₄ after 30 minutes in dark at room temperature. Fig. 2 shows that the four murine monoclonal, anti-IgY antibodies, do not cross react with rabbit IgG, goat IgG, sheep IgG or human IgG.

The two-site assay, sometimes referred to as a sandwich assay, which exemplifies some of the best features of ELISA uses a pair of antibodies, one bound to the plate (capture antibody) and the other labelled (detector antibody), to first bind and subsequently identify the presence of an analyte.

Often an enzyme-labelled second antibody (reporter antibody) to the detector is used. The present anti-IgY monoclonals can be most effectively used as enzyme labelled conjugate to the chicken IgY detector to various antigens. In two-site assays where two to three antibodies from different species are used, often one antibody reacts with another which leads to a false-positive reaction. The lack of cross reaction of the anti-IgY monoclonal antibodies of the present invention with IgGs from various mammalian species is clearly an advantage as a reagent in immunoassays. Fig. 3 shows an example which demonstrates this cross reaction between rabbit polyclonal anti-IgY with IgGs of different mammalian species. Microtiter plates were coated with IgGs from different species including chicken IgY. Then commercially obtained rabbit polyclonal anti-IgY labelled with a marker enzyme was added followed by addition of substrate solution. The results shown in Figure 3, when compared to Figure 2, clearly shows the improvement in specificity of the monoclonal antibodies of the present invention.

3. Performance of Coηjugated Anti-IgY Monoclonal Antibodies as "Reporter" Antibody

The four anti-IgY MAbs of the present invention were labelled with horse-radish peroxidase (HRP) using the Nakane method (P.K. Na ane and A. Kawaoi, J. Histochem. Cytochem., 22, 1084, 1974). Quality of HRP-labelled MAbs were assessed on the detectability of antigen (chicken IgY) with serial dilutions of the conjugates in solid-phase ELISA. The dilution of conjugates which gave an OD_490nm reading of 1.500 varied from 1/8,000 to 1/100,000.

The performance of HRP-labelled anti-IgY monoclonal antibody 1Y-263 was studied in the two-site ELISA assay using monoclonal antibody capture, chicken IgY detector and 1 Y-263-HRP as the reporter antibody (see Fig. IB for schematic presentation) for the detection of myosin heavy chain (MHC), myosin light chain 1 (MLC 1) as well as cardiac troponin I (cTnl). In the identical assays, HRP-labelled rabbit polyclonal anti-IgY (commercially obtained) was also evaluated as reporter antibody.

Immulon 4 plates (Dynatech) were coated with 2 μg/ml anti-MHC MAb 3 μg/ml anti-MLC I MAb, or 3 μg/ml anti-cTnl MAb in 0.05M carbonate buffer, pH 9.6 (coating buffer). Plates were sealed and stored at 4°C at least over night prior to use. The standard curve was made for each analyte with series of dilutions of purified either MHC, cTnl or MLC 1 in 0.15% BSA, 0.05% Tween 20 in PBS, pH 7.4 (diluent buffer). The standards were run in triplicate, while the zero (diluent buffer only) was run in quadruplicate. After 30 minutes incubation at 37°C, plates were washed and detector IgY antibody (double affinity purified anti-MHC, anti-Tnl or anti-MLC 1) was added at 2 μg/ml in diluent buffer. Following the incubation with the detector antibody, conjugate which was either rabbit polyclonal anti-chicken IgG-HRP (Jackson, no

303-035-003) at 1/50,000 or anti-IgY MAb 1 Y-263-HRP at 1/12,500 was added. After the incubation with the conjugates, OPD substrate solution was added. Reaction was stopped with 2M H₂SO₄ after 30 minutes in dark at room temperature. Fig. 4 shows the titration curve of MHC and Fig. 5 of cTn-I and Fig. 6 of MLC 1 using either rabbit conjugate or MAb conjugate. Analytical sensitivity, i.e. antigen detection level of assays using rabbit polyclonal reporter antibody is similar to that of assays using MAb reporter. Although the rabbit detector generates higher signal at equivalent antigen concentrations, it also produces significantly greater "background" noise (1.2- to 7.7-fold in MHC assay, 1.2- to 6.7-fold in Tnl assay and approximately 3-fold in MLC 1 assay at clinically relevant lower level). This dramatically reduces the OD range between positive and negative results. This is particularly problematic in vitro diagnostics where discrimination between reactive and nonreactive specimens is critical.

C. Examples of Use of the Monoclonal Antibodies of the Invention

1. Sandwich Assay

Mammalian cardiac proteins are highly conserved and it is difficult to raise high affinity antibodies to these proteins. Using the chicken, we were able to develop unique high affinity antibodies against myosin heavy chain, cardiac myosin light chain 1, myoglobin, cardiac troponin 1 and other cardiac proteins. As described in the section B.3., using these chicken antibodies as detector and the present mouse monoclonals against chicken antibodies as reporter, highly sensitive and specific assays can be developed. 2. Dotblot

Different amounts of samples are bound to the nitrocellulose membrane. Detector IgY binds to the sample. Enzyme-labelled MAbs of the present invention binds to the detector antibody. An insoluble substrate is used to visualize the bound enzyme.

3. Western blot

Protein samples are separated by electrophoresis and transferred to nitrocellulose membrane. The bands containing the sample binds detector IgY.

Enzyme-labelled MAb of the present invention binds to the detector. An insoluble substrate is used to visualize the bound enzyme.

4. Screening of chicken Monoclonal Antibody by Hybridoma Technology

There are numbers of chicken B-cell lines established as demonstrated by Nishinaka, S., et. al., (J. Immunol. Methods, 139, 217, 1991). Some of these lines may be used for fusion with specific immunoglobulin-secreting splenic lymphocytes from chicken. Antigen-specific antibody (IgY) secreting chicken hybridomas can be screened by ELISA using the antigen-coated plates. Antigen-specific monoclonal IgY in the culture supernatants is bound to the antigen. The bound monoclonal IgY is detected by labelled-MAb of the present invention. A substrate solution is added to visualize the bound enzyme.

5. Monoclonal anti-IgY as Capture Antibody on BIAcore Analysis

Chicken IgY can be analyzed for specificity/identity and kinetic analysis using the monoclonals of the present invention covalently coupled onto the sensor chip of BIAcore as described in the section A. 3. The present monoclonals are used as a capture antibody for the various characterization of chicken IgY. Although the disclosure describes and illustrates preferred embodiments of the invention, it is to be understood that the invention is not limited to these particular embodiments. Many variations and modifications will now occur to those skilled in the art. For a definition of the invention, reference is to be made to the appended claims.

Claims

We Claim:

1. A monoclonal antibody developed against chicken egg yolk immunoglobulin.

2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is specific for the heavy chain of the chicken egg yolk immunoglobulin.

3. The monoclonal antibody according to claim 2, wherein the monoclonal antibody is labelled.

4. The monoclonal antibody according to claim 2, wherein the affinity constant of the monoclonal antibody against the chicken egg yolk immunoglobulin ranges from 1.5 x 10⁶ M'¹ to 2.5 x 10⁹ M^*1.

5. The monoclonal antibody according to claim 4, wherein the affinity constant of the monoclonal antibody against the chicken egg yolk immunoglobulin ranges from 2.0 x 10⁸ M^-1 to 4.1 x 10⁸ M^-1.

6. The monoclonal antibody according to claim 2, wherein the monoclonal antibody is produced from hybridoma cell lines selected from the group consisting of: 2Y-114, 1Y-261, 1Y-262 and 1Y-263.

7. A method of using the monoclonal antibody of claim 1 as a reporter antibody in an immunoassay, wherein the immunoassay comprises:

a capture antibody; an antigen in a test sample corresponding to the capture antibody; a chicken egg yolk detector antibody corresponding to the antigen to be tested in the test sample; and a labelled monoclonal reporter antibody developed against chicken egg yolk immunoglobulin.

8. A diagnostic assay system comprising at least a first, second and third antibody wherein:

said first antibody is a capture antibody corresponding to a test antigen in a test sample; said second antibody is a chicken egg yolk detector antibody corresponding to said test antigen in said test sample; and said third antibody is a labelled monoclonal antibody developed against chicken egg yolk immunoglobulins.

9. The system according to claim 8, wherein the monoclonal antibody is specific for the heavy chain of the chicken egg yolk immunoglobulin.

10. The system according to claim 9, wherein the monoclonal antibody is enzyme labelled.

11. The system according to claim 10, wherein the monoclonal antibody is produced from hybridoma cell lines selected from the group consisting of 2Y-114, 1Y-261, 1Y-262 and 1Y-263.

12. The system according to claim 11 wherein the test antigen is a cardiac protein.

13. The system according to claim 12 wherein the test is selected from the group consisting of myosin heavy chain, cardiac myosin light chain, myoglobin, cardiac troponin-I, troponin-T, tropomyosin, myoglobin and creatine kinase (CK-MB).

14. A hybridoma cell line producing monoclonal antibodies developed against chicken egg yolk immunoglobulins.

15. The hybridoma cell line according to claim 14, wherein the hybridoma cell line is specific for the heavy chain of the chicken egg yolk immunoglobulin.

16. The hybridoma cell line of claim 15, wherein said hybridoma cell line produces a monoclonal antibody against chicken egg yolk immunoglobulin which has an affinity constant ranging from 1.5 x 10⁶ M^"1 to 2.5 x 10⁹ M"¹ when tested against chicken egg yolk immunoglobulin.

17. The hybridoma cell line of claim 15, wherein said hybridoma cell line produces a monoclonal antibody against chicken egg yolk immunoglobulin which has an affinity constant ranging from 2.0 x 10⁸ M^"1 to 4.1 x 10⁸ M^'1 when tested against chicken egg yolk immunoglobulin.

18. The hybridoma cell line of claim 17, selected from the group consisting of: 2Y-114, 1Y-261, 1Y-262 and 1Y-263.