WO2006031216A1

WO2006031216A1 - Quantitative immunoassays using tag/anti-tag chemistry

Info

Publication number: WO2006031216A1
Application number: PCT/US2004/029469
Authority: WO
Inventors: Jan Pawlak; Victor A. Manneh; John E. Bartz; Catherine Pawlak; Patrick M. Sexton; Charles P. Zahl; Tilden L. Capen-Frederick; Diem Q. Le; Danae L. Monroe; Karen C. Haber; Michael P. Allen; Joel M. Blatt
Original assignee: Metrika, Inc.
Priority date: 2004-09-10
Filing date: 2004-09-10
Publication date: 2006-03-23

Abstract

A one step lateral immunochromatographic immunoassay configured to compare at least one of Troponin, mitochondrial enzymes or low molecular weight polypeptide ischemic markers to an indirect Streptavidin/biotinylated antibody tertiary complex to assess ischemic heart events at early onset of patient chest pain.

Description

QUANTITATIVE IMMUNOASSAYS USING TAG/ANTI-TAG CHEMISTRY

RELATED APPLICATION

The present application repeats a substantial portion of prior application no. 08/455,236 entitled "Disposable Electronic Assay Device" filed May 31, 1995 by Michael P. Allen, now U.S. Patent 5,580,794, which is a continuation of application no. 08/111,347 entitled "Disposable Electronic Assay Device" filed August 24, 1993 by Michael P. Allen, now abandoned and prior application no. 08/657,894 entitled "Electronic Assay Device and Method" filed June 6, 1996 by Michael P. Allen, Joel M. Blatt, and Joseph T. Windunas which is a continuation-in-part of application no. 08/455,236 entitled "Disposable Electronic Assay Device" filed May 31, 1995 by Michael P. Allen, now U.S. Patent 5,580,794, which is a continuation of application no. 08/111 ,347 entitled "Disposable Electronic Assay Device" filed August 24, 1993 by Michael P. Allen and now abandoned. The present application adds and claims additional disclosure not presented in the prior applications. Since the present application names an inventor named in the prior applications, it constitutes a continuation-in-part of the prior applications.

The present application also repeats a substantial portion of prior application no. 08/512,844 entitled "Dry Reagent Particle Assay and Device Having Multiple Test Zones and Method Therefor" filed August 9, 1995 by Joel M. Blatt and Michael P. Allen, and prior application no. 08/703,479 entitled "Device and Method for Preventing Assay Interference" filed August 27, 1996 by Joel M. Blatt, Wilma M. Mangan, Paul J. Patel and Victor A. Manneh.

The subject jmatter of this application is related to a disposable single-use digital electronic instrument that is entirely self-contained, including all chemistry reagents, as disclosed in U.S. Application Serial No. 08/642,228 entitled "Method and Device for Measuring Reflected Optical Radiation" filed April 30, 1996 by Raymond T. Hebert, Joel M. Blatt, and Joseph T. Widunas. The above applications have the same assignee as the present invention and is incorporated herein by reference in its entirety. DESCRIPTION OF THE PREFERRED EMBODIMENTS

Example 1

This invention relates to a diagnostic tests and devices for conducting such tests at the point of care or in a diagnostic laboratory for accurate, simple, and rapid assessment of chest pain. In particular, the invention relates to differential diagnosis of the origin of chest pain, e.g., whether the pain is cardiac in origin, and for differentiating between unstable angina ("UA"), myocardial infarction ("MI"), congestive heart failure ("CHF"), and other ischemic events affecting the heart, at early onset of patient chest pain. The invention further relates to diagnosis of the stage of the MI in a patient suffering from MI, and to prognosis of such a patient.

MLCs are highly sensitive ischemic markers for UA and MI. MLCs appear in the serum rapidly, and their levels remain elevated for up to 10 days following myocardial necrosis. There are two principal types of MLC known as MLCl and MLC2, which exist as a soluble pool in the myocardial cell cytoplasm and also integral with the myosin myofibril. In the ventricular muscle, MLC2, and perhaps MLCl, is identical with the isotype expressed in slow skeletal muscle. MLCl is elevated in 80-85% of the patients with cardiac pain. Thus, MLCl is a very sensitive ischemic marker and is quite tissue specific.

Low molecular weight cardiac proteins (LMWCP) have been used as cardiac markers. Examples of such cardiac markers include components of the contractile apparatus, namely, troponin, including troponin T, troponin-I, and troponin-C; mitochondrial enzymes, such as triose P isomerase; low molecular weight polypeptides which are readily released from the heart; and sarcolemmal membrane proteins or protein fragments which may be released early after the onset of ischemia, in particular, a 15 kd sarcolemma protein and a 100 kd complex gylcoprotein that are cardiac specific. However, to date diagnostic tests and methods that make use of these markers have not been developed. They may be employed in the practice of this invention because they are ischemic markers which are cardiac specific. The cardiac isotype troponin-I inhibits the interaction between actin and myosin molecules during rest periods between contractions of the heart muscle. Troponin-I appears in serum of patient within 4-6 hours after MI and remains elevated for 7-8 days.

Troponin-T is part of the tropnin-tropomyosin complex of the thin filament, that is part of the muscle contractile apparatus, and that contains actin and tropomyosin regulatory elements. Troponin-T serves as a link between the tropolyosin backbone and the troponin-I/troponin-C complex. Troponin-T is a basic protein and has isotypes in cardiac and fast and slow skeletal muscles. It appears in serum within 3 hours of the onset of chest pain and remains elevated for at least 10 days following ML. Myosin heavy chains (MHC), and tropomyosin, are heavy molecular weight proteins which are useful as cardiac markers. MHC is part of the major contractile protein of muscle. Fragments of MHC can re released from the ventricle into serum after myocardial cell necrosis and subsequent irreversible membrane injury MI. MHC fragments do not appear quickly in the serum. MHCs remain elevated for at least 10 days following MI and peak levels of MHC are observed 4 days after MI.

Tropomyosin is a dimer formed from two polypeptide which are part of the regulatory system in muscle contraction. Tropomyosin is detectable

For the immunoassay for Troponin I, a "direct sandwich" format for Troponin I is compared to an "indirect Streptavidin/biotinylated antibody tertiary complex" approach in a one-step lateral (i.e. immunochromatographic) immunoassay. Figs. 1 and 2 depict an overview of a "direct sandwich" or an "indirect tertiary complex" immunoassay formats, respectively, for Troponin I, an important marker for myocardial infarction. ^•

To activate the labeling beads, one ml of 0.403 μm Dark Blue latex particles (MIPs; P/N 10012; Metrika, Inc., Sunnyvale, CA) at 10% (w/v) solids are combined with 1 ml of 0.5 M MES buffer (pH 6.0), 5.5 ml of deionized H₂0, 2.3 ml of 50 mg of N-hydroxysuccinimide (ISIHS; Product # 24500; Pierce Chemical Company, Rockford, IL) per ml deionized H₂O and 0.2 ml of 5 mg of 1- ethyl-3-(3-dimethylamino- propyl) carbodiimide hydrochloride (EDC; Product # 22980; Pierce Chemical Company) in deionized H₂O. The resultant mixture is sonicated on ice for 40 seconds and then allowed to react on a shaker at RT for 30 minutes. The activated MPs are then centrifuged at 10⁰C at 10,000xg and washed three times with cold 50 mM MES buffer (pH 6.0) by resuspension and centrifugation cycles. In a typical procedure, the final pellet of MPs is suspended in 3.666 ml of 50 mM MES buffer (pH 6.0), a 5 ml mixture of 0.6-1.2 mg of goat anti-Troponin I antibody (Product code # GT 149; Scripps Laboratories, Inc., San Diego, CA) and 2.5 mg of mouse IgG (cat. 1-8765; Sigma Chemical Company, St. Louis, MO) in the same buffer are added with mixing, followed by an addition of 5 ml of 0.1 M borate buffer (pH 8.5). The mixture is allowed to incubate at RT for 2 hr and then centrifuged as described above. Subsequently, 10 ml of 50 mM borate buffer (pH 8.5) containing 5 mM ethanolamine (cat. # E- 9508; Sigma Chemical Company) is added to the pellet, MPs are suspended, incubated at RT for 30 min, and the suspension is centrifuged as described above. The remaining hydrophobic sites on MPs are then blocked with FSG blocking solution composed of 0.1 % (w/v) of fish skin gelatin (FSG; cat. # G-7765; Sigma Chemical Company) in 50 mM borate buffer (pH 8.5) at RT for 30 min. The MPs blocked with FSG are centrifuged as described above and suspended in 0.2 M EPPS buffer (pH 8.0) containing 0.5 % (w/v) of FSG, 0.5 % (w/v) of Hammarsten casein

(Product # 440203H; BDH Laboratory Supplies, Poole, England), 0.5 % (v/v) of Tween 20 (cat. # P-1379; Sigma Chemical Company) and 0.01 % (w/v) OfNaN₃.

For the "direct sandwich" immunoassay, to prepare the capture zone membrane, nitrocellulose having a pore size of >5 um (Product # AE98; Schleicher and Schuell, Keene, NH) is affixed to an XY-plotter table. A mouse monoclonal antibody against Troponin I (Product # TRI-7F83;

Dako Corporation, Carpinteria, CA) prepared at 1.0 mg/ml in phosphate-buffered saline solution w/NaN_j (PBS; L/N RDP 033; Metrika, Inc.) is dispensed in a 2.0 mm zone at the distal end of the nitrocellulose membrane using an IVEK Digispense (IVEK Corporation, Springfield, VT) dispensing system. After air drying at 45°C, the membrane is placed into a tray containing the membrane blocking solution for 20 minutes at RT. The membrane blocking solution comprises of 0.2-0.35 % (w/v) of polyvinyl alcohol) (PVA; 80 % hydrolyzed; Av. MoI. Wt. 9,000-10,000; Cat. # 36,062-7; Aldrich Chemical Company, Inc., Milwaukee, WI) prepared in deionized water. ^' The membrane is removed and blotted for 5 minutes. The membrane is air dried at 45°C for 5 ~ minutes, and then placed at less than 5.0 % relative humidity (RH). For the "indirect Streptavidin/biotinylated antibody tertiary complex" immunoassay, a conjugate of Streptavidin and bovine serum albumin (BSA) is prepared as follows. Ten mg of Streptavidin (Catalog # SA 10; Prozyme, Inc., San Leandro, CA) are dissolved in 1 ml of 0.1 M sodium phosphate (NaP) buffer (pH 8.0) containing ImM dithiothreitol (DTT; Cat. # D 0632; Sigma Chemical Company) and 50 μl of 46 mg of 2-iminothiolane (2-IT; Product # 26101; Pierce Chemical Company) dissolved in the same buffer is added at RT with stirring. After allowing the reaction to proceed at RT for 45 min, the mixture is buffer exchanged into 0.1 M NaP buffer (pH 7.4) using Sephadex G-25F resin (PD 10 columns; Cat. # G-25-80; Sigma Chemical Company, St. Louis, MO). Simultaneously, 10 mg of BSA is dissolved in 1 ml of 0.1 M NaP buffer (pH 7.4) and 50 μl of 8.5 mg of SMCC (Product # 22360; Pierce Chemical Company) dissolved in 1 ml of anhydrous dimethylformamide (DMF; cat. # 27,054-7; Sigma Aldrich Chemical Company) is added with stirring at RT. After allowing the reaction to proceed at RT for 1 hr, the mixture is buffer exchanged into 0.1 M NaP buffer (pH 7.4) using PD column. The resultant products from the two reactions are combined with stirring and allowed to react for 2 hrs at RT. Subsequently, the reaction mixture is buffer exchanged into 0.1 M NaP buffer (pH 7.4) using PD columns, yielding the final Streptavidin-BSA conjugate product. To prepare the capture zone membrane, the nitrocellulose membrane is affixed to an XY-plotter table. A Streptavidin-BSA capture band is dispensed in a 2.0 mm zone at the distal end of the nitrocellulose membrane using Streptavidin-BSA conjugate at 0.85mg/ml. The solution is dispensed with an IVEK Digispense dispensing system. After air drying at 45⁰C, the membrane is placed into a tray containing the membrane blocking solution previously described for 20 minutes at RT. The membrane is removed and blotted for 5 minutes. The membrane is air dried at 45°C for 5 minutes, and then placed at less than 5.0 % RH overnight. Processed capture membranes remain at less than 5.0 % RH until assembly.

To prepare the biotinylated binding member antibody for an "indirect Streptavidin/biotinylated antibody tertiary complex" immunoassay, two hundred μl of the previously mentioned mouse monoclonal antibody against Troponin I are dialyzed overnight at 2-8⁰C against PBS using Slide Dialyzer system (Product # ...., Pierce Chemical Company) and 10 μl of 3.5 mg of sulfo-NHS- LC-biotin (EZ Link Sulfo-NHS-LC-biotin; Product # 21335; Pierce Chemical Company) dissolved in 1 ml of anhydrous DMF is added at RT with stirring. After allowing the reaction to proceed at RT for 2 hrs, the process is stopped by adding 20 μl of 30 mM glycine prepared in 0.1 M NaP buffer (pH 8.0). Finally, the reaction mixture is dialyzed overnight at 2-8⁰C against PBS using SlideDialyzer system. To carry out immunoassays in "direct sandwich" or indirect tertiary complex" format, a commercial set of Opus^® TnI calibrators (cat. #703-006; Behring Diagnostics Inc., Westwood, MA) was used to calibrate either "direct sandwich" or "indirect tertiary complex" Troponin I immunoassays. Ten-fold concentrated Sample Treatment Buffer (STB for) "wet" assays is comprised of

0.5 M EPPS buffer (pH 8.0) supplemented with 6.25 % (v/v) of Tween-20 (cat. P 1379; Sigma Chemical Company), 2 % (w/v) of BSA and 0.1 % (w/v) OfNaN₃.

For "wet" assays for Troponin I, a 14 X 100 mm strip of the capture zone membrane is affixed centrally on an adhesive opaque strip. The opaque backing is a 23 X 350 mm strip of 5 mil. white mylar laminated with 3M 9502 transfer adhesive. The absorbent-which is a 10 X I 00 mm rectangle of Whatman 31ET cellulose paper (Whatman, Inc., Fairfield, NJ)~is affixed distal to the capture zone pad with 0.5 mm overlap. The sample zone pad composed of 7 X 100 mm cellulose acetate (Part # 12301R101X50M; Sartorius Corporation, Santa Clara, CA) is then placed next to the capture zone membrane with 0.5 mm overlap. In "wet" assays for Troponin I in a "direct sandwich" format, 9 μl of a respective Opus^®

TnI calibrator is mixed sequentially in a test tube with lμl of 10-fold concentrated stock of SXB and 1 μl of labeling MPs prepared at 0.54 % (w/v) solids. Subsequently, the "wet" assay strip assembled as just described is placed into the tube, allowed to develop for 7 min, then removed and the intensity of the band is measured with a Gretag reflectance densitometer (Model #D19C; Gretag Color Control Systems, Regensdorf, Switzerland). Increasing values from the Gretag instrument indicate increasing color intensity, which corresponds to increasing analyte concentration.

In "wet" assays for Troponin I in an "indirect tertiary complex" format, a 9 μl of specimen sample is mixed sequentially in a test tube with lμl of 10-fold concentrated stock of STB, 1 μtl of fragment of mouse monoclonal anti-Tnl antibody and 1 μl of labeling MPs prepared at 0.54 %

(w/v) solids. Subsequently, the "wet" assay strip assembled as just described is placed into Hie tube, allowed to develop for 7 min, then removed and the intensity of the band is measured with a Gretag reflectance densitometer. Similarly as described above, increasing values from the Gretag instrument indicate increasing color intensity, which corresponds to increasing analyte concentration. Fig. 3 shows a comparison of dose response curves obtained in "direct sandwich" or "indirect tertiary complex" format for the respective immunoassays for Troponin I. The results indicate that an "indirect tertiary complex" format is superior to a "direct sandwich" format for Troponin I immunoassay, in terms of sensitivity.

Example 2

To verify effectiveness of an "indirect Streptavidin/biotinylated binding member antibody tertiary complex" format for the clinically relevant immunoassays for low levels of Troponin I in serum specimens obtained from human subjects suspected of myocardial infarction, the following studies were undertaken. To accommodate a Troponin I-specific analysis of human serum specimens in a one-step lateral flow immunoassay the composition of the immunoassay was modified, in accordance with the findings described in the co-pending US Patent Application # filed on May ., 1998. To this end, first, a chemically modified BSA reagent (namely per-acetylated BSA thereinafter described) was prepared to assist in blocking of unspecific interactions associated with an introduction of human serum components into a one-step lateral flow immunoassay, along with its stabilizing and dispersing properties. Secondly, a modified sample treatment buffer (STB) composition was introduced, in accordance with an experimental evidence presented in the just mentioned US Patent application. To prepare Acetylated BSA, Acetic anhydride (AA) is a reagent of choice for acetylating protein amino groups. At neutral or mildly alkaline pH (7-9.5), AA reacts with unprotonated α- and ε-NH₂ groups rendering them electrically neutral. Thus, 2.1g of BSA was dissolved at RT in 70 ml of 0.2 M carbonate buffer (pH 8.7) and 825.7 μl of acetic anhydride (cat. # A-6404; Sigma Chemical Company) was added with stirring. After 2 hours, 45.3 μl of ethanolamine was added and allowed to react at RT for 30 min. The resultant reaction mixture was buffer exchanged into

25 mM Tris buffer (pH 8.0) containing 0.1 % (w/v) NaN₃ using Sephadex G-25 Fine resin to a final concentration of 10 mg/ml.

To modified Sample Treatment Buffer (STB) for Troponin I "wet" assays for human serum specimens, ten-fold concentrated STB for Troponin I "wet" assays that is suitable for analysis of human serum specimens comprised of 0.5 M Tris buffer (pH 8.0) supplemented with 10 mg/ml of acetylated BSA, 0.3-0.6 mg/ml of heterophilic IgG block (Heteroblock; P/N 70506; Omega Biologicals, Inc., Bozeman, MT), 5% (v/v) of bovine serum (Cat. # B 8655; Sigma Chemical Company), 4 M urea (Cat. # U 5128, Sigma Chemical Company) and 0.1 % (w/v) OfNaN₃ was used .

To activate the labeling beads, one ml of 0.403 um Dark Blue latex particles at 10% (w/v) solids are combined with 1 ml of 0.5 M MES buffer (pH 6.0), 5.5 ml of deionized H₂0, 2.3 ml of 50mg of NHS per ml deionized H₂O and 0.2 ml of 5 mg of EDC in deionized H₂O. The resultant mixture is sonicated on ice for 40 sec and then allowed to react on a shaker at RT for 30 min. The activated MPs are then centrifuged at 10⁰C at 10,000xg and washed three times with cold 50 mM MES buffer (pH 6.0) by resuspension and centrifugation cycles. In a typical procedure, the final pellet of MPs is suspended in 3.67 ml of 50 mM MES buffer (pH 6.0), a 5 ml mixture of 0.6-1.2 mg antigen-specific antibody such as e.g. goat anti-Troponin I peptide 3 antibody (Product code # G-129-C; BiosPacific/Fortron BioScience Inc.) and 2.5 mg of mouse IgG in the same buffer is added with mixing, followed by an addition of 5 ml of 0.1 M borate buffer (pH 8.5). The mixture is allowed to incubate at RT for 2 hrs and then centrifuged as described above. Subsequently, 10 ml of 50 mM borate buffer (pH 8.5) containing 5 mM ethanolamine is added to the pellet, MPs are suspended, incubated at RT for 30 min, and the suspension is centrifuged as described above. The remaining hydrophobic sites on MPs are then blocked with acetylated BSA solutions (10 mg/ml) described above, and the pellet is resuspended to a final particle concentration of 0.5 % (w/v) solids.

To prepare the capture zone membranes, nitrocellulose having a pore size of >5 um is affixed to an XY-plotter table. A Streptavidin-BSA capture band is dispensed in a 2.0 mm zone at the distal end of the nitrocellulose membrane using Streptavidin-BSA conjugate at 2.57 mg/ml. The solution is dispensed with an IVEK Digispense dispensing system. After air drying at 45⁰C, the membrane is placed into a tray containing the membrane blocking solution comprised of acetylated BSA solution at 10 mg/ml for 20 minutes at RT. The membranes are then removed and blotted for 5 minutes. The membranes are air dried at 45°C for 5 minutes, and then placed at less than 5.0 % RH overnight. Processed capture membranes remain at less than 5.0 % RH until assembly. For "wet" assays for Troponin I, a 14 X 100 mm strip of the capture zone membrane is affixed centrally on an adhesive opaque strip. The opaque backing is a 23 X 350 mm strip of 5 mil. white mylar laminated with 3M 9502^'transfer adhesive. The absorbent-which is a 105C 100 mm rectangle of Whatman 3 IET cellulose paper (Whatman, Inc., Fairfield, NJ)~is affixed distal to the capture zone pad with 0.5 mm overlap. The sample zone pad composed of 7 X 100 mm cellulose acetate (Part # 12301R101X50M; Sartorius Corporation, Santa Clara, CA) is thea placed next to the capture zone membrane with 0.5 mm overlap.

Serum samples were collected at a hospital in California from patients suspected of • myocardial infarction, and the samples were analyzed at the site for Total-CK, CK-MB and. Myoglobin levels. Upon receiving, the specimens were analyzed at Metrika, Inc. for Troponin I levels by Behring Opus^® Plus Troponin I Reference Quantitative Assay, and the respective values were assigned. Table 1 summarizes the just mentioned data.

In "wet" assays, 9-10 μl of specimen sample is mixed sequentially in a test tube with lμl o> f 10- fold concentrated stock of modified STB, 1 μl of biotinylated F(ab')₂ fragment of goat anti-Tnl peptide 69-80 antibody (Code # 9099A, HTI Bio-Products, Inc., Ramona, CA) and 1 μl of labeling MPs prepared at 0.5 % (w/v) solids. Subsequently, the "wet" assay strip assembled as just described is placed into the tube, allowed to develop for 7 min, then removed and the intensity of the band is measured with a Gretag reflectance densitometer. Increasing values from the Gretag instrument indicate increasing color intensity, which corresponds to increasing analyte concentration.

Figure 4 shows a typical Metrika TnI calibration curve generated with Opus^® TnI calibrators and the modified STB just described.

Table 1 shows the results of 33 patient samples tested with the Metrika TnI assay and the Behring TnI assay. The Metrika patient samples were tested using the modified STB formulation.The raw reflectance density for each sample test strip is recorded as Metrika GDU. This density is converted to clinical TnI ng/mL using the calibration curve in Figure 3, and this clinical value is recorded in the Metrika TnI (ng/mL) column. The Behring Opus TnI assay was used to determine reference values. Figure 5 shows the Metrika and Behring TnI patient sample correlation for the samples in Table 1. Correlation to the Behring TnI assay is 0.928 with a slope of θ.95. Table 1. Troponin I Patient Sample Results

Patient Metrika Metrika Behring

Sample # GDU TnI (ng/mL) TnI (ng/mL)

46 0.49 23.8 36.6

47 0.22 ^' 0.0 0.0

48 0.40 11.5 11.5

49 0.28 2.4 9.8

50 0.47 21.2 31.3

51 0.23 0.0 0.0

52 0.24 0.9 0.0

53 0.57 42.2 37.5

54 . 0.20 0.0 0.0

55 0.21 0.0 0.0

56 0.60 50.5 41.2

57 0.23 0.0 0.0

59 0.23 0.0 0.8

60 0.22 0.0 0.0

61 0.22 0.0 0.0

62 0.22 0.0 2.0

63 0.24 1.0 0.0

64 0.64 61.2 76.6

65 0.23 0.8 0.0

66 0.23 0.0 0.0

67 0.23 0.8 0.5

68 0.66 68.0 54.6

69 0.22 0.0 ^■ 0.5

70 0.21 0.0 0.0

71 0.24 0.9 0.9

72 0.22 0.0 0.0

73 0.24 0.9 0.0 74 0.23 0.0 O.O

75 0.21 0.0 O.O

76 0.26 1.5 3.0

77 0.25 1.3 1.9

78 0.22 0.0 0.0

79 0.22 0.0 0.0

Example 3

It became apparent that in addition to an appropriate selection of a composition suitable for the most efficient "indirect Streptavidin/biotinylated antibody tertiary complex" format, a process of creating a streptavidin capture test zone is quite important. To this en.<l, alternate methods for preparation of the streptavidin capture zone for Troponin I assays were tested, utilizing application of biotinylated BSA to nitrocellulose followed by strepta-vidin solution. To prepare the capture zone, Biotinylated BSA is prepared as follows. BSA is dissolved at 6 mg/ml in 1 ml of 0.1 M sodium phosphate buffer (pH 7.45) and 59 μl of sulfo-NHS-LC-biotin dissolved at 35 mg/ml in anhydrous DMF is added with stirring at RT. After 1 hr, the reaction mixture is buffer exchanged into 50 mM Tris/HCl buffer (pH 8,0) containing 0.1 % (w/v) NaN₃ using PD-10 columns. In the first process (referred to as Process A in Fig. 6 below), nitrocellulose having a pore size of >5 um is affixed to an XY table. A biotinylated BSA capture band is dispensed in a 2.0 mm zone at the distal end of the nitrocellulose membrane using biotinylated BSA at 2.0 mg/ml. The solution is dispensed with an IVEK Digispense dispensing system. Tlie membrane is air dried at 45°C for 15 min and placed into a tray containing the membrane blocking solution comprised of acetylated BSA solution at 10 mg/ml for 20 minutes at RT. The membranes are then removed and blotted for 5 minutes. The membranes are air dried at 45°C for 15 minutes and streptavidin dissolved at 2 mg/ml in 50 mM Tris/HCl buffer (pH 8.0) containing O.I % (w/v) NaN₃ is dispensed with an IVEK Digispense dispensing system. The membranes are then air dried at 45°C for 15 minutes, washed for 5 min with 50 mM Tris/HCl buffer (pH 8.0) containing 0.1 % (w/v) NaN₃. Finally, the membranes are placed again into a tray containing the membrane blocking solution comprised of acetylated BSA solution at 10 mg/ml for 20 minutes at RTT. The membranes are then removed and blotted for 5 minutes. The membranes are air dried at 45°C for 15 minutes and then placed at less than 5.0 % RH overnight. Processed capture membranes remain at less than 5.0 % RH until assembly. In the second process (referred to as Process B in Fig. 6 below), nitrocellulose having a pore size of >5 um is again affixed to an XY table. A biotinylated BSA capture band is dispensed in a 2.0 mm zone at the distal end of the nitrocellulose membrane using biotinylated BSA at 2.0 irαg/ml. The solution is dispensed with an IVEK Digispense dispensing system. The membrane is air dried at 45°C for 15 min and streptavidin dissolved at 2 mg/ml in 50 mM Tris/HCl buffer (pH 8.0) containing 0.1 % (w/v) NaN₃ is dispensed with an IVEK Digispense dispensing system. The membranes are then air dried at 45⁰C for 15 minutes, washed for 5 min with 50 mM Tris/KCl buffer (pH 8.0) containing 0.1 % (w/v) NaN₃ and placed into a tray containing the membrane blocking solution comprised of acetylated BSA solution at 10 mg/ml for 20 minutes at RX. The membranes are then removed and blotted for 5 minutes. The membranes are air dried at 45⁰C for 15 minutes and then placed at less than 5.0 % RH overnight. Processed capture membranes remain at less than 5.0 % RH until assembly.

To activate the labeling beads, one ml of 0.403 um Dark Blue latex particles at 10% (w/v) solids are combined with 1 ml of 0.5 M MES buffer (pH 6.0), 5.5 ml of deionized H₂O, 2.3 ml of 50mg of NHS per ml deionized H₂O and 0.2 ml of 5 mg of EDC in deionized H₂O. The resultant mixture is sonicated on ice for 40 sec and then allowed to react on a shaker at RT for 30 rnin. The activated MPs are then centrifuged at 10⁰C at 10,000xg and washed three times with cold 50 mM MES buffer (pH 6.0) by resuspension and centrifugation cycles. In a typical procedure, trie final pellet of MPs is suspended in 3.67 ml of 50 mM MES buffer (pH 6.0), a 5 ml mixture of O.6-1.2 mg antigen-specific antibody such as e.g. goat anti-Troponin I peptide 3 antibody (Product code # G-129-C; BiosPacific/Fortron Bio Science Inc.) and 2.5 mg of mouse IgG in the same buffer is added with mixing, followed by an addition of 5 ml of 0.1 M borate buffer (pH 8.5). The mixture is allowed to incubate at RT for 2 hr and then centrifuged as described above. Subsequently, 10 ml of 50 mM borate buffer (pH 8.5) containing 5 mM ethanolamine is added to the pellet, MPs are suspended, incubated at RT for 30 min, and the suspension is centrifuged as described, above. The remaining hydrophobic sites on MPs are then blocked with acetylated BSA solutions (10 mg/ml) described above; the pellet is resuspended to a final particle concentration of 0.5 % (w/v) solids.

For "wet" assays for Troponin I, a 14 X 100 mm strip of the capture zone membrane is affixed centrally on an adhesive opaque strip. The opaque backing is a 23 X 350 mm strip of 5 mil. white mylar laminated with 3M 9502 transfer adhesive. The absorbent-which is a 10 X 100 mm rectangle of Whatman 31ET cellulose paper (Whatman, Inc., Fairfield, NJ)--is affixed distal to the capture zone pad with 0.5 mm overlap. The sample zone pad composed of 7 X 100 mm cellulose acetate (Part # 12301R101X50M; Sartorius Corporation, Santa Clara, CA) is then placed next to the capture zone membrane with 0.5 mm overlap. Serum samples were drawn at Metrika, Inc. from apparently healthy asymptomatic volunteers. The specimens were then analyzed by Behring Opus^® Plus Troponin I Reference Quantitative Assay and/or by Dade Stratus ^® Cardiac Troponin-I Fluorometric Enzyme Assay, and they were found to be negative for Troponin I. Subsequently, a Troponin I+C complex (cat. # T5124; Scripps Laboratories) was used as an exogenous source of Troponin I which was added to the serum specimens.

For Sample Treatment Buffer (STB) for "wet assays, ten-fold concentrated STB for "wet" assays comprised of 0.5 M Tris buffer (pH 8.0) supplemented with 10 mg/ml of acetylated BSA, 0.3-0.6 mg/ml of heterophilic IgG block, 0.1 % (w/v) OfNaN₃, 0.5 M Urea, and 5 % (v/v) of bovine serum was used. In "wet" assays, 10 μl of specimen sample is mixed sequentially in a test tube with 3.4 μl of lϋ-fold concentrated stock of STB, 1 μl of biotinylated F(ab')₂ fragment of goat anti-Tnl peptide 69-80 antibody (Code # 9099A, HTI Bio-Products, Inc.) and 1 μl of labeling MPs prepared at 0.5 % (w/v) solids. Subsequently, the "wet" assay strip assembled as just described is placed into the tube, allowed to develop for 10 min, then removed and the intensity of the band is measured with a Gretag reflectance densitometer. Increasing values from the Gretag instrument indicate increasing color intensity, which corresponds to increasing analyte concentration. The results depicted in Figure 6 shows that Process A is superior to Process B. Example 4:

In order to substantiate further an advantage of using a general concept of tag/anti-tag or ligand/receptor "indirect tertiary complex" over a "direct sandwich" format in one- step lateral flow immunoassays, the following example is provided. Here, a "direct sandwich" format for Troponin I is compared to an "indirect anti-fluorescein/fluorescein labeled antibody tertiary complex" approach in a one-step lateral flow immunoassay. Figs. 7 and 8 depict an overview of the two formats.

Preparation of the device for immunoassays:

Preparation of covalentlv coated labeling beads:

To activate the labeling beads, one ml of 0.403 um Dark Blue latex particles at 10% (w/v) solids are combined with 1 ml of 0.5 M MES buffer (pH 6.0), 5.5 ml of deionized H₂O, 2.3 ml of 50mg of NHS per ml deionized H₂O and 0.2 ml of 5 mg of EDC in deionized H₂O. The resultant mixture is sonicated on ice for 40 sec and then allowed to react on a shaker at RT for 30 min. The activated MPs are then centrifuged at 10⁰C at 10,000xg and washed three times with cold 50 mM MES buffer (pH 6.0) by resuspension and centrifugation cycles. In a typical procedure, the final pellet of MPs is suspended in 3.67 ml of 50 mM MES buffer (pH 6.0), a 5 ml mixture of 0.6-1.2 mg antigen-specific antibody such as e.g. goat anti-Troponin I peptide 3 antibody (Product code # G-129-C; BiosPacific/Fortron Bio Science Inc.) and 2.5 mg of mouse IgG in the same buffer is added with mixing, followed by an addition of 5 ml of 0.1 M borate buffer (pH 8.5). The mixture is allowed to incubate at RT for 2 hrs and then centrifuged as described above. Subsequently, 10 ml of 50 mM borate buffer (pH 8.5) containing 5 mM ethanolamine is added to the pellet, MPs are suspended, incubated at RT for 30 min, and the suspension is centrifuged as described above. The remaining hydrophobic sites on MPs are then blocked with acetylated BSA solutions (10 mg/ml) described above; the pellet is resuspended to a final particle concentration of 0.5 % (w/v) solids.

Preparation of the Capture Zone: /. Preparation of "direct sandwich" immunoassay:

To prepare the capture zone membrane, nitrocellulose having a pore size of >5 urn is affixed to an XY-plotter table. A goat anti-Tnl peptide 69-80 antibody (Code # 9099A, HTI Bio-Products, Inc., Ramona, CA) prepared at 1.0 mg/ml in PBS is dispensed in a 2.0 mm zone at the distal end of the nitrocellulose membrane using an IVEK Digispense dispensing system. The memt>ranes are then air dried at 45⁰C for 15 minutes, washed for 5 min with 50 mM Tris/HCl buffer (pH 8.0) containing 0.1 % (w/v) NaN₃ and placed into a tray containing the membrane blocking solution comprised of acetylated BSA solution at 10 mg/ml for 20 minutes at RT. The membranes are then removed and blotted for 5 minutes. The membranes are air dried at 45°C for 15 minxites and then placed at less than 5.0 % RH overnight. Processed capture membranes remain at less than 5.0 % RH until assembly.

2. Preparation of "indirect anti-fluorescein/fluorescein labeled antibody tertiary complejc" test zonefor immunoassay:

To prepare the capture zone membrane, nitrocellulose having a pore size of >5 um is affixed to an XY-plotter table. A conjugate of fluorescein-labeled BSA (prepared as described beloΛv) is dispensed in a 2.0 mm zone at the distal end of the nitrocellulose membrane using an IVEK

Digispense dispensing system. The membranes are then air dried at 45°C for 15 minutes, washed for 5 min with 50 mM Tris/HCl buffer (pH 8.0) containing 0.1 % (w/v) NaN₃ and placed into a tray containing the membrane blocking solution comprised of acetylated BSA solution at 10 mg/ml for 20 minutes at RT. The membranes are then removed and blotted for 5 minutes . The membranes are air dried at 45°C for 15 minutes and then placed at less than 5.0 % RH overnight. Processed capture membranes remain at less than 5.0 % RH until assembly.

A conjugate of fluorescein-labeled BSA is prepared as follows: One ml of 6.6 mg of BSA prepared in 0.1 M NaP buffer (pH 7.4) is allowed to react at RT for 45 min with 50 μl of 15.83 mg of sulfo-NHS-LC-SPDP dissolved in ml ofDMSO. Subsequently, fifty μl of 47.34 rng of 5- (and-6) carboxylfluorescein succinimidyl ester (5(6)-FAM, SE: Product # C-1311; Molecular Probes, Inc., Eugene, OR) dissolved per ml of anhydrous DMF is added with stirring at RT and allowed to react for the additional 45 min. Subsequently, the mixture is buffer exchanged into 0.1 M sodium acetate buffer (pH 4.5) using PD 10 column.

Preparation of anti-fluorescein antibody/anti-Troponin I antibody conjugate for an "indirect fluorescein/anti-fluorescein tertiary complex" immunoassay:

A conjugate of anti-fluorescein antibody and anti-Troponin I antibody is prepared as follows: One ml of an affinity purified goat anti-fluorescein antibody (Product code # G-148-C; BiosPacific) prepared in PBS is allowed to react at RT for 1 hr with 50 μl of 4.2 mg of sulfo- NHS-LC-SPDP dissolved in ml of DMSO. Subsequently, the mixture is buffer exchanged into 0.1 M sodium acetate buffer (pH 4.5) using PD 10 column. The SH-groups are released from the conjugate by adding IM DTT to a final concentration of 50 mM at RT for 30 min, following by a buffer exchange into 0.1 M NaP buffer (pH 7.4) using PD 10 columns. Simultaneously, one ml of affinity purified goat anti-Tnl peptide 69-80 antibody (Code # 9099A, HTI Bio-Products) prepared at 2.0 mg/ml in 0.1M NaP buffer (pH 7.4) is allowed to react at RT for 45 min with 50 μl of 3.2 mg of SMCC dissolved in 1 ml of anhydrous DMF, and the mixture is buffer exchanged into 0.1 M NaP buffer (pH 7.4) using PD column. The resultant products from the two reactions are combined at the (w/w) ratio of 1 : 1 with stirring at RT and allowed to react for 1 hr at RT. Finally, the conjugate reaction mixture is buffer exchanged into 50 mM Tris/HCl buffer (pH 8.0) containing 0.1 % (w/v) NaN₃ _ using Sephadex G-25F resin, yielding the final anti-fhiorescein- antibody/anti-Troponin I antibody conjugate product.

Carrying out immunoassays in "direct sandwich" or "indirect fluorescera/anti-fluorescem tertiary complex" format:

Analyte sources for Troponin I calibration curves for either format for Troponin I immunoassays:

A plasma pool obtained from apparently healthy asymptomatic volunteers was spiked with Troponin I+C complex (cat. #T5124; Scripps Laboratories). The specimens were then analyzed by Behring Opus^® Plus Troponin I Reference Quantitative Assay and/or by Dade Stratus^® Cardiac Troponin-I Fluorometric Enzyme Assay.

Sample Treatment Buffer (STB^{^}) for "wet" assays:

Ten-fold concentrated STB for "wet" assays comprised of 0.5 M Tris buffer (pH 8.0) supplemented with 10 mg/ml of acetylated BSA, 0.3-0.6 mg/ml of heterophilic IgGblock, 0.1 % (w/v) OfNaN₃, 5.0 M Urea, and 5 % (v/v) of bovine serum.

Assembly of the device for "wet" assays in either format:

For "wet" assays for Troponin I, a 14 X 100 mm strip of the capture zone membrane is affixed centrally on an adhesive opaque strip. The opaque backing is a 23 X 350 mm strip of 5 mil. white mylar laminated with 3M 9502 transfer adhesive The absorbent-which is a 10 X 1OO mm rectangle of Whatman 3 IET cellulose paper (Whatman, Inc., Fairfield, NJ)-is affixed distal to the capture zone pad with 0.5 mm overlap. The sample zone pad composed of 7 X 100 mm cellulose acetate (Part # 12301R101X50M; Sartorius Corporation, Santa Clara, CA) is then placed next to the capture zone membrane with 0.5 mm overlap.

"Wet" immunoassays for Troponin I in a "direct sandwich" format:

In "wet" assays for Troponin I in a "direct sandwich" format, 9 μl of a respective plasma TnI calibrator is mixed sequentially in a test tube with 3.2μl of 10-fold concentrated stock: of STB and 1 μl of labeling MPs prepared at 0.54 % (w/v) solids. Subsequently, the "wet" assay strip assembled as just described is placed into the tube, allowed to develop for 5 min, then removed and the intensity of the band is measured with a Gretag reflectance densitometer. Increasing values from the Gretag instrument indicate increasing color intensity, which corresponds to increasing analyte concentration.

"Wet" immunoassays for Troponin I in an "indirect fluorescein/anti-fluorescein tertiary complex" format: In "wet" assays for Troponin I in an "indirect tertiary complex" format, a 9 μl of specimen sample is mixed sequentially in a test tube with 1 μl of 10-fold concentrated stock of STB, 1 μl of fragment of mouse monoclonal anti-Tnl antibody and 1 μl of labeling MPs prepared at 0.54 % (w/v) solids. Subsequently, the "wet" assay strip assembled as just described is placed into the tube, allowed to develop for 7 min, then removed and the intensity of the band is measured with a Gretag reflectance densitometer. Similarly as described above, increasing values from the Gretag instrument indicate increasing color intensity, which corresponds to increasing analyte concentration.

Results:

Fig. 9 shows a comparison of dose response curves obtained in "direct sandwich" or "indirect fluorescein/anti -fluorescein tertiary complex" format for the respective immunoassays for Troponin I. Similarly as it has been shown above for streptavidin/biotinylated antibody format, the results indicate that an "indirect tertiary complex" format is superior to a "direct sandwich" format for Troponin I immunoassay, in terms of assay sensitivity.

Example 6:

Relative effectiveness and selectivity (i.e. relative clinical sensitivity and specificity) of a "direct sandwich" vs. "indirect streptavidin/biotinylated antibody tertiary complex" approach for one- step lateral flow immunoassays were compared in a high-sensitivity immunoassay for steroid hormones (namely hCG) using human specimens derived from whole blood. Fig. 10 and 11 depict an overview of the two formats.

To prepare "reductively aminated" (methylated) BSA for low molecular weight product: 990 mg of BSA was dissolved at RT in 30 ml of 0.2 M carbonate buffer (pH 8.7), 242 μl of formaldehyde (37.3 % solution; cat. G-5250; Sigma Chemical Company) was added with stirring, followed by an addition of 3.33 ml of IM sodium cyanoborohydride (cat. # 15-615-9; Aldrich Chemical Company) dissolved in the same buffer. After 2 hours, the resultant reaction mixture was buffer exchanged into 25 mM Tris buffer (pH 8.0) containing 0.1 % (w/v) NaN₃ using Sephadex G-25 Fine resin to a final concentration of 10 mg/ml and for high molecular weight product: 57.6 g of BSA was dissolved at RT in 960 ml of 0.2 M carbonate buffer (pH 8.7), and 3.27 ml of glutaraldehyde (Grade I; 25 % aqueous solution; cat. # G-5882; Sigma Chemical Company) was added with stirring, followed by an addition of 96 ml of IM sodium cyanoborohydride dissolved in the same buffer. After 4 hours, 104 ml of formaldehyde was added with stirring and allowed to react overnight at RT. The reaction was terminated by adding 106 ml of ethanolamine and allowing to react at RT for 30 min. The resultant mixture was exchanged into 50 mM Tris buffer (pH 8.0) containing 0.1 % (w/v) NaN₃ using Sephadex G-25 F resin to a final concentration of 10 mg/ml.

The sample receiving zone is prepared from Ahlstrom 1281 or 8975 (Ahlstrom Filtration Inc., Mt. Holly Springs, PA) materials. The selected material is saturated with a blood separating solution at 45 ul/cm² containing 2.5 mg/ml rabbit anti-human red blood cells (Code 209-4139; Rockland Immunochemicals, Gilbertsville, PA) antibody diluted in acetylated bovine serum albumin (AcBSA) or methylated BSA. (mBS A) prepared at 10 mg/ml. The membrane is frozen at -70° C for at least one hour and then lyophilized in a Virtis Genesis (Virtis, Gardiner, NY) overnight. The treated sample receiving zone is cut into 7.0 X 7.0 mm squares and stored at less than 5.0 % RH until assembly. The sample treatment zone for "direct sandwich" hCG immunoassay is prepared from Ahlstrom 1281 material. The material is treated with a sample treatment buffer (STB) at 45 ul/cm². STB is composed of 50 mM Tris buffer containing 2.0 mg/ml non-specific mouse IgG (P/N 9902; Intergen Company, Milford, MA), and 1.67 mg/ml heterophilic IgG block. Optionally, the STB is supplemented with 0.5 M sodium perchlorate. The pad of Ahlstrom material is frozen at -70° C for at least one hour and then lyophilized in the Virtis Genesis overnight. The sample treatment zone is then cut into 3.5 X 3.0 mm rectangles and stored at less than 5.0 % RH until assembly. The sample treatment zone for "indirect streptavidin/biotinylated antibody tertiary complex" hCG immunoassay is prepared similarly as just described above except for an addition of biotinylated mouse anti-hCG antibody prepared in 50 mM Tris buffer (pH 8.0) containing 0.1 % (w/v) NaN₃ and added to a final concentration of 0.02 mg of antibody per ml. Biotinylated anti-hCG antibody is prepared as follows: Antibody (Clone # MC079; Scripps

Laboratories, Inc.) solution is buffer exchanged into 0.1 M sodium borate buffer (pH 8.5) using PD-IO columns and then diluted to a concentration of 1 mg/ml. Subsequently, thirteen μl of 3.5 mg of sulfo-NHS-LC-biotin dissolved per ml of anhydrous DMF is added with stirring at RT per each ml of the antibody solution. After 1 hr, the reaction is stopped by an addition of 30 mM glycine solution at a ratio of 19 μl per each ml of the reaction mixture. The final solution is buffer exchanged into 50 mM Tris/HCl buffer (pH 8.0) containing 0.1 % (w/v) NaN₃ using Sephadex G-25F resin columns.

To prepare the labeling beads, 0.50 ml of 0.36 μm blue latex particles (P/N LC9786; Emerald Diagnostics Inc., Eugene, OR) at 2.5 % solids are combined with 0.50 ml of mouse monoclonal anti-hCG (Clone #5008; Oy Medix Biochiemica Ab, Kauniainen, Finland) antibody prepared in 25 mM Tris buffer at 1.0 mg/ml. The solution is allowed to react passively on an orbital rotator at RT overnight. After centrifugation at 10₅OOO rpm for 5 minutes, the supernatant is aspirated. The pellet is resuspended in highly polymerized bovine serum albumin (polyBSA) (P/N 99-012- 5; Bayer Corporation) solution at 10 mg/rnl. The particles are allowed to block for one hour at RT on an orbital rotator. After centrifugation at 10,000 rpm for 5 minutes, the supernatant is aspirated. The pellet is resuspended in polyBSA solution (10 mg/ml). The particles are allowed to block for one hour at RT on an orbital rotator. After centrifugation at 10,000 rpm for 5 minutes, the supernatant is aspirated. The pellet is resuspended in acetylated or methylated BSA solution (10 mg/ml) to a final particle concentration of 1.0 % solids. To prepare the labeling zone solution, the labeling beads are diluted to a concentration of 0.1 % solids in 10 mg/ml AcBSA or mBSA, prepared in 50 mM Tris buffer, pH 8.0, with 0.1% (w/v) NaN₃. Sucrose in 50 mM Tris buffer is added to a final concentration of 2.0 %. The resultant mixture is stirred and dispensed onto Whatman F075-14 (Whatman, Inc., Fairfield, NJ) material at 60 ul/cm². The material is frozen at -70° C for at least one hour and then lyophilized in Virtis Genesis overnight. The label-containing pads are cut into 3.5 X 3.0 mm rectangles and stored at less than 5.0 % RH until assembly.

To prepare the "direct sandwich" capture zone membrane, nitrocellulose having a pore size of 8- 12 um (Schleicher and Schuell) is affixed to an XY-plotter table. An hCG capture band is dispensed in a 2.0 mm zone at the distal end of the nitrocellulose membrane using mouse monoclonal anti-hCG antibody (Clone MC097; Scripps Laboratories) at 1.0 mg/ml. The solution is dispensed with an IVEK Digispense dispensing system. After air drying at 45⁰C, the membrane is placed into a tray containing blocking solution (10 mg/ml AcBSA or mBSA) for 20 minutes at RT. The membrane is removed and blotted for 5 minutes. The membrane is air dried at 45°C for 5 minutes and then placed at less than 5.0% RH overnight. Processed capture membranes remain at less than 5.0% RH until assembly.

To prepare the "indirect sandwich" capture zone membrane, nitrocellulose is affixed again to an XY-plotter table. A streptavidin capture band is dispensed in a 2.0 mm zone at the distal end of the nitrocellulose membrane using Streptavidin-BSA conjugate (prepared in accordance -with the Example 1) at 2.0 mg/ml. Similarly as just described, the solution is dispensed with an IVEX Digispense dispensing system. After air drying at 45⁰C, the membrane is placed into a tray containing blocking solution (10 mg/ml AcBSA or mBSA) for 20 minutes at RT. The membrane is removed and blotted for 5 minutes. The membrane is air dried at 45°C for 5 minutes and then placed at less than 5.0% RH overnight. Processed capture membranes remain at less than 5.0% RH until assembly.

To assemble the device, a 3.0 X 7.0 mm strip of the capture zone membrane is affixed centrally on an adhesive opaque strip. The opaque backing is a 350 X 23 mm strip of ARCare mylar made adhesive with 3M 9502. The pad containing visible label is affixed next to the capture zone pad with 0.5 mm overlap. The sample treatment zone pad is then placed next to the label containing pad with 0.5 mm overlap. The device is provided with an absorbent, which is a 3.5 X 3.0 mm rectangle of Whatman 31ET (Whatman, Inc.) membrane. It is placed distal to the capture membrane with 0.5 mm o^~verlap. The resultant test strip on the opaque backing is then placed membrane side down in the MPl unit such that the sample treatment pad is overlapped by the sample receiving pad by 1 .0 mm. The strip is aligned such that the label capturing zones oix the capture membrane are visible through the optical aperture of the device. Finally, the top co^ver is placed together with the bottom casing such that the sample well is aligned over the sample receiving pad. Biological samples including whole blood, plasma or serum were collected from apparently healthy, asymptomatic donors. The specimens were analyzed on Dade Stratus® to determine endogenous hCG levels. Analyte was spiked into the specimens at varying concentrations and levels were confirmed on the Reference Quantitative Assay. Quantitative hCG values were assigned and the specimens were assayed as follows: the device is placed flat on the bench top and 75 ul of sample is applied to the sample receiving zone. The liquid is allowed to flow through the four zones of the assay strip and collect in the absorbent pad. If hCG is present in the sample at least 25 mIU/mL, a blue band in the capture region will appear. The intensity of the band is measured with a Gretag reflectance densitometer. Increasing values from the Gretag indicate increasing color intensity.

The performance of mBSA compared to AcBSA as a blocking reagent in the "clirect sandwich" hCG assay system using hCG plasma calibrators is shown in Figure 12. The mBSA block gives much better curve separation than the AcB SA block.

Table 2 shows the assay results of 19 different male (m) and female (f) donors "that are negative for hCG. None of the samples gave a false positive.

Table 2. Screening of hCG Negative Serum Samples

Donor # Sex GDU FP?

180 f 0.20 No

181 f 0.20 No

184 m 0.20 No

185 m 0.20 No

186 f 0.25 No

187 m 0.20 No

188 m 0.28 No

189 f 0.21 No

190 m 0.20 No

179 m 0.21 No

183 f 0.22 No

191 m 0.22 No

192 f 0.22 No

193 f 0.21 No

194 m 0.19 No

195 f 0.20 No

196 m 0.20 No

197 m 0.21 No

198 m 0.20 No

Table 3 shows the results of the screening of 14 different female donors that tested positive for hCG by the Dade assay. All 14 samples also test positive by the Metrika assay.

Table 3. Screening of hCG Positive Serum Samples

Metrika Dade Donor # mIU/mL Positive? mlU/mL Positive?

2 4867 Yes 2965 Yes

5 5911 Yes 3011 Yes

6 1280 Yes 1699 Yes

9 6387 Yes 4905 Yes

52 7009 Yes 6113 Yes

64 7500 Yes 6646 Yes

96 346 Yes 689 Yes

82 4333 Yes 4051 Yes

87 4867 Yes 2740 Yes

83 382 Yes 220 Yes

19 127 Yes 70 Yes

97 155 Yes 86 Yes

91 461 Yes 196 Yes

24 491 Yes 274 Yes

Fig. 13 shows a comparison of dose response curves obtained in "direct sandwich" vs. "indirect tertiary complex" format for the respective immunoassays for h.CG using human plasma calibrators. The results indicate that an "indirect tertiary complex" format is superior—in terms of analytical sensitivity~to a "direct sandwich" format for hCG immunoassay.

Example 7

Relative effectiveness and selectivity (i.e. relative clinical sensitivity and specificity) of a "direct sandwich" vs. "indirect streptavidin/biotinylated antibody tertiary complex" approach for one- step lateral flow immunoassays were compared in a high-sensitivity immunoassay with a high clinical utility potential, such as prostate specific antigen (PSA). An early identification of a presence of this marker is indicative of a specific clinical stage of benign or invasive prostate tumor, depending on the levels in the specimens derived from whole blood samples obtained from the asymptomatic or cancer-suspect subjects. Figs. 14 and 15 depict an overview of two formats.

The sample receiving zone is prepared from AMstrom 1281 or 8975 (Ahlstrom Filtration) materials. The selected material is saturated with a blood separating solution at 45 ul/crn² containing 2.5 mg/ml rabbit anti-human red blood cells antibody diluted in irxBSA prepared at 10 mg/ml. The membrane is frozen at -70° C for at least one hour and then lyopiiilized in a Virtis Genesis lyophilizer overnight. The treated sample receiving zone is cut into 7.0 X 7.0 mm squares and stored at less than 5.0 % RH until assembly.

The sample treatment zone for "direct sandwich" free PSA immunoassay is prepared from Ahlstrom 1281 material. The material is treated with a sample treatment buffer (STB) at 45 ul/cm². STB is composed of 50 mM Tris buffer containing 0.1 % (w/v) NaN_{3 #} 10 mg mBS A/ml, 2.0 mg/ml non-specific mouse IgG, and 1.67 mg/ml heterophilic IgG block. The pad of Ahlstrom material is frozen at -70° C for at least one hour and then lyophilized in the Virtis Genesis lyophilizer overnight. The sample treatment zone is then cut into 3.5 X 3.0 mm rectangles and stored at less than 5.0 % RH until assembly. The sample treatment zone for "indirect streptavidin/biotinylated antibody tertiary complex" free PSA immunoassay is prepared similarly as just described above except for an addition of biotinylated mouse anti-free PSA antibody prepared in 50 mM Tris buffer (pH 8.0) containing 0.1 % (w/v) NaN₃ and added to a final concentration of 0.02 mg of antibody per ml. Biotinylated anti-free PSA antibody is prepared as follows: Mouse monoclonal anti-free PSA antibody (Cat # MP077; Clone # BP0045 ; Scripps Laboratories) solution is buffer exchanged into 0.1 M sodium borate buffer (pH 8.5) using PD-10 columns and then diluted to a concentration of 1 mg/ml. Subsequently, thirteen μl of 3.5 mg of sulfo-NHS-LC-biotin dissolved per ml of anhydrous DMF is added with stirring at RT per each ml of the antibody solution. After 1 hr, the reaction is stopped by an addition of 30 mM glycine solution at a ratio of 19 μl per each ml of the reaction mixture. The final solution is buffer exchanged into 50 mIVl Tris/HCl buffer (pH 8.0) containing 0.1 % (w/v) NaN₃ using Sephadex G-25F resin columns.

To prepare the labeling beads, 0.50 ml of 0.36 μm blue latex particles (P/N LC9786; Emerald Diagnostics) at 2.5 % solids are combined with 0.50 ml of mouse monoclonal anti-PSA antibody (Clone # BPOOl; Cat. # MP077; Scripps Laboratories) prepared in 25 mM Tris buffer at 1.0 mg/ml. The solution is allowed to react passively on an orbital rotator at IRT overnight. After centrifugation at 10,000 rpm for 5 minutes, the supernatant is aspirated. The pellet is resuspended in mBSA solution prepared in 50 mM Tris buffer (pH 8.0) with 0.1% (w/v) NaN₃ at 10 mg/ml. The particles are allowed to block for one hour at RT on an orbital rotator. After centrifugation at 10,000 rpm for 5 minutes, the supernatant is aspirated. After centrifugation at 10,000 rpm for 5 minutes, the supernatant is aspirated. The pellet is resuspended in mBSA solution (10 mg/ml) to a final particle concentration of 1.0 % solids.

To prepare the labeling zone solution, the labeling beads are diluted to a concentration of 0.05- 0.1 % solids in 10 mg/ml of mBSA, prepared in 50 mM Tris buffer, pH 8.0, with 0.1% (w/v) NaN₃. Sucrose in 50 mM Tris buffer is added to a final concentration of 2.0 %. The resultant mixture is stirred and dispensed onto Whatman F075-14 (Whatman, Inc., Fairfield, NJ) material at 60 ul/cm². The material is frozen at -70° C for at least one hour and then lyophilized in Virtis Genesis lyophilizer overnight. The label-containing pads are cut into 3.5 X 3.0 mm rectangles and stored at less than 5.0 % RH until assembly.

To prepare the "direct sandwich" capture zone membrane, nitrocellulose having a pore size of^" 8- 12 um (Schleicher and Schuell) is affixed to an XY-plotter table. An anti-free PSA capture ba/nd is dispensed in a 2.0 mm zone at the distal end of the nitrocellulose membrane using mouse monoclonal anti-free PSA antibody (Clone # BP0045; Scripps Laboratories) at 1.0 mg/ml. The solution is dispensed with an IVEK Digispense dispensing system. After air drying at 45°C, ttie membrane is placed into a tray containing blocking solution (10 mg/ml of mBSA) for 20 minutes at RT. The membrane is removed and blotted for 5 minutes. The membrane is air dried at 45⁰C for 5 minutes and then placed at less than 5.0% RH overnight. Processed capture membranes remain at less than 5.0% RH until assembly.

To prepare the "indirect sandwich" capture zone membrane, nitrocellulose is affixed again to an XY-plotter table. A streptavidin capture band is dispensed in a 2.0 mm zone at the distal end of the nitrocellulose membrane using Streptavidin-BSA conjugate (prepared in accordance with "the Example 1) at 2.0 mg/ml. Similarly as just described, the solution is dispensed with an IVEK Digispense dispensing system. After air drying at 45°C, the membrane is placed into a tray containing blocking solution (10 mg/ml of mBSA) for 20 minutes at RT. The membrane is removed and blotted for 5 minutes. The membrane is air dried at 45⁰C for 5 minutes and then placed at less than 5.0% RH overnight. Processed capture membranes remain at less than 5.0Vo RH until assembly. To assemble the device, a 3.0 X 7.0 mm strip of the capture zone membrane is affixed centrally on an adhesive opaque strip. The opaque backing is a 350 X 23 mm strip of ARCare mylar made adhesive with 3M 9502. The pad containing visible label is affixed next to the capture zone pad with 0.5 mm overlap. The sample treatment zone pad is ttαen placed next to the label containing pad with 0.5 mm overlap. The device is provided with an absorbent, which is a 3.5 X 3.0 mm rectangle of Whatman 31ET (Whatman, Inc.) membrane. It is placed distal to the capture membrane with 0.5 mm overlap. The resultant test strip on the opaque backing is then placed membrane side down in the MPl unit such that the sample treatment pad is overlapped by the sample receiving pad by 1.0 mm. The strip is aligned sucti that the label capturing zones on the capture membrane are visible through the optical aperture of the device. Finally, the top cover is placed together with the bottom casing such that the sample well is aligned over the sample receiving pad.

The calibrators used for free PSA immunoassays are Behring Opus^® PSA Calibrators (Cat. # 502-006; Behring Diagnostics), and the values for free PSA content are assigned using CanAg Free PSA EIA assay.

The device is placed flat on the bench top and 75 μl of sample is applied to the sample receiving zone. The liquid is allowed to flow through the assay strip and collect in the absorbent pad. If free PSA is present in the sample at the level of at least .0. 1 ng/ml, a blue band in the capture region will appear. The intensity of the band is measured "with a Gretag reflectance densitometer. Increasing values from the Gretag indicate increasing color intensity.

Fig. 16 shows a comparison of dose response curves obtained in "direct sandwich" vs. "indirect tertiary complex" format for the respective immunoassays for free PSA using Behring Opus^® PSA Calibrators (Behring Diagnostics). The results indicate that an "indirect tertiary complex" format is superior—in terms of analytical sensirWity--to a "direct sandwich" format for free PSA immunoassay.

Example 8

A "direct sandwich" format for CK-MB is compared to an. "indirect Streptavidin/biotinylated antibody tertiary complex" approach in a one-step lateral (i.e. immunochromatographic) immunoassay. Figs. 17 and 18 depict an overview of a "direct sandwich" or an "indirect tertiary complex" immunoassay formats, respectively, for CK-MB, an important marker for myocardial infarction.

The sample receiving zone of the self-contained device for CK-MB immunoassays is prepared from Ahlstrom 1281 or 8975 (Ahlstrom Filtration Inc., Mt. Holly Springs, PA) materials. The selected material is saturated with a blood separating solution at 45 ul/om² containing 2.5 mg/ml rabbit anti-human red blood cells (Code 209-4139; Rockland Immunochemicals, Gilbertsville, PA) antibody diluted in acetylated bovine serum albumin (AcBSA) or methylated BSA (mBSA) prepared at 10 mg/ml. The membrane is frozen at -70° C for at least one hour and then lyophilized in a Virtis Genesis (Virtis, Gardiner, NY) overnight. The treated sample receiving zone is cut into 7.0 X 7.0 mm squares and stored at less than 5.0 % RHL until assembly.

The sample treatment zone for "direct sandwich" CK-MB immunoassay is prepared from ^• Ahlstrom 1281 material. The material is treated with a sample treatment buffer (STB) at 45 ul/cm². STB is composed of 50 mM Tris buffer containing 2.0 mg/ml non-specific mouse IgG (P/N 9902; Intergen Company, Milford, MA), and 1.67 mg/ml heteropliilic IgG block. Optionally, the STB is supplemented with 0.5 M sodium perchlorate. The pad of Ahlstrom material is frozen at -70° C for at least one hour and then lyophilized in the Virtis Genesis overnight. The sample treatment zone is then cut into 3.5 X 3.0 mm rectangles and stored at less than 5.0 % RTf until assembly. The sample treatment zone for "indirect streptavidin/biotinylated antibody tertiary complex" CK- MB immunoassay is prepared similarly as just described above except for an addition of biotinylated mouse anti-CK-MB antibody (Clone # MC079; Scripps Laboratories, Inc.) prepared in 50 mM Tris buffer (pH 8.0) containing 0.1 % (w/v) NaN₃ and added, to a final concentration of 0.02 mg of antibody per ml. Biotinylated anti-CK-MB antibody (Clone # MC079) is prepared as follows: Antibody solution is buffer exchanged into 0.1 M sodium borate buffer (pH 8.5) using PD-10 columns and then diluted to a concentration of 1 mg/ml. Subsequently, thirteen μl of 3.5 xng of sulfo-NHS-LC- biotin dissolved per ml of anhydrous DMF is added with stirring at RT^* per each ml of the antibody solution. After 1 hr, the reaction is stopped by an addition of 30 mM glycine solution at a ratio of 19 μl per each ml of the reaction mixture. The final solution is buffer exchanged into 50 mM Tris/HCl buffer (pH 8.0) containing 0.1 % (w/v) NaN₃ using Sephadex G-25F resin columns. To activate the labeling beads, one ml of 0.403 urn Dark Blue latex particles at 10% (w/v) solids are combined with 1 ml of 0.5 M MES buffer (pH 6.0), 5.5 ml of deionized H₂O, 2.3 ml of 50mg of NHS per ml deionized H₂O and 0.2 ml of 5 mg of EDC in deionized H₂O. The resultant mixture is sonicated on ice for 40 sec and then allowed to react on a shaker at RT for 30 min. The activated MPs are then centrifuged at 10⁰C at 10,000xg and washed three times with cold 50 irM MES buffer (pH 6.0) by resuspension and centrifugation cycles. In a typical procedure, the final pellet ofMPs is suspended in 3.67 ml of 50 mM MES buffer (pH 6.0), a 5 ml mixture of 0.6-1.2 mg antigen-specific antibody such as e.g. goat anti-CK-MB peptide 3 antibody (Product code # G-129-C; BiosPacific/Fortron BioScience Inc.) and 2.5 mg of mouse IgG in the same buffer is added with mixing, followed by an addition of 5 ml of 0.1 M borate buffer (pH 8.5). The mixture is allowed to incubate at RT for 2 hr and then centrifuged as described above. Subsequently, 10 ml of 50 mM borate buffer (pH 8.5) containing 5 mM ethanolamine is added to the pellet, MPs are suspended, incubated at RT for 30 min, and the suspension is centrifuged as described above. The remaining hydrophobic sites on MPs are then blocked with acetylated BSA solutions (10 mg/ml) described above, and the pellet is resuspended to a final particle concentration of 0.5 % (w/v) solids.

To prepare the labeling zone solution, the labeling beads are diluted to a concentration of 0.1 % solids in 10 mg/ml AcBSA or mBSA, prepared in 50 mM Tris buffer, pH 8.0, with 0.1% (w/v) NaN₃. Sucrose in 50 mM Tris buffer is added to a final concentration of 2.0 %. The resultant mixture is stirred and dispensed onto Whatman F075-14 (Whatman, Inc., Fairfield, NJ) material at 60 ul/cm². The material is frozen at -70° C for at least one hour and then lyophilized in Virtis Genesis overnight. The label-containing pads are cut into 3.5 X 3.0 mm rectangles and stored at less than 5.0 % RH until assembly. To prepare the "direct sandwich" capture zone membrane for CK-MB immunoassay, nitrocellulose having a pore size of 8-12 um (Schleicher and Schuell) is affixed to an XY-plotter table. A CK-MB capture band is dispensed in a 2.0 mm zone at the distal end of the nitrocellulose membrane using mouse monoclonal anti-CK-MB antibody (Clone MC097; Scripps Laboratories) at 1.0 mg/ml. The solution is dispensed with an IVEK Digispense dispensing system. After air drying at 45⁰C, the membrane is placed into a tray containing blocking solution (10 mg/ml AcBSA or mBSA) for 20 minutes at RT. The membrane is removed and blotted for 5 minutes. The membrane is air dried at 45°C for 5 minutes and then placed at less than 5.0% RH overnight. Processed capture membranes remain at less than 5.0% RH until assembly.

To prepare the "indirect sandwich" capture zone membrane for CK-MB immunoassay, nitrocellulose is affixed again to an XY-plotter table. A streptavidin capture band is dispensed in a 2.0 mm zone at the distal end of the nitrocellulose membrane using Streptavidin-BSA conjugate (prepared in accordance with the Example 1) at 2.0 mg/ml. Similarly as just described, the solution is dispensed with an IVEK Digispense dispensing system. After air drying at 45°C, the membrane is placed into a tray containing blocking solution (10 mg/ml AcBSA or mBSA) for 20 minutes at RT. The membrane is removed and blotted for 5 minutes. The membrane is air dried at 45°C for 5 minutes and then placed at less than 5.0% RH overnight. Processed capture membranes remain at less than 5.0% RH until assembly.

To assemble the device, a 3.0 X 7.0 mm strip of the capture zone membrane is affixed centrally on an adhesive opaque strip. The opaque backing is a 350 X 23 mm strip of ARCare mylar made adhesive with 3M 9502. The pad containing visible label is affixed next to the capture zone pad with 0.5 mm overlap. The sample treatment zone pad is then placed next to the label containing pad with 0.5 mm overlap. The device is provided with an absorbent, which is a 3.5 X 3.0 mm rectangle of Whatman 31ET (Whatman, Inc.) membrane. It is placed distal to the capture membrane with 0.5 mm overlap. The resultant test strip on the opaque backing is then placed membrane side down in the MPl unit such that the sample treatment pad is overlapped by the sample receiving pad by 1.0 mm. The strip is aligned such that the label capturing zones on the capture membrane are visible through the optical aperture of the device. Finally, the top cover is placed together with the bottom casing such that the sample well is aligned over the sample receiving pad. Biological samples including whole blood, plasma or serum were collected from apparently healthy, asymptomatic donors. The specimens -were analyzed on Dade Stratus® to determine endogenous CK-MB levels. Analyte was spiked into the specimens at varying concentrations and levels were confirmed on the Reference Quantitative Assay. Quantitative CK-MB values were assigned and the specimens were assayed as follows: the device is placed flat on the bench top and 75 μl of sample is applied to the sample receiving zone. The liquid is allowed to flow through the four zones of the assay strip and collect in the absorbent pad. If CK-MB is present in the sample at least 25 mlU/mL, a blue band in the capture region will appear. The intensity of the band is measured with a Gretag reflectance densitometer. Increasing values from the Gretag indicate increasing color intensity.

Fig. 19 shows a comparison of dose response curves obtained in "direct sandwich" vs. "indirect tertiary complex" format for the respective immunoassays for CK-MB using human plasma calibrators. The results indicate that an "indirect tertiary complex" format is superior— in terms of analytical sensitivity--to a "direct sandwich" format for CK-MB immunoassay.

Claims

What is claimed is:

1. A diagnostic immunoassay format for assessing ischemic heart events at early onset of patient chest pain, comprising: a one step lateral immunochromatographic immunoassay configured to compare at least one of Troponin, mitochondrial enzymes or low molecular weight polypeptide ischemic markers to an indirect Streptavidin/biotinylated antibody tertiary complex.