WO1999040437A1 - Improved methods and apparatus for performing determinations of immune reactants in biological fluids - Google Patents

Abstract

A method and apparatus for performing determinations of allergen-specific IgE antibodies in biological fluids includes a kit with coated, color coded test units for insertion into respective reaction containers containing the test fluid. If there is an antibody specific to the allergen coated on any of the test unit tips, that antibody becomes bound to the associated allergen in the corresponding reaction container. The tips are rinsed and inserted into a second set of reaction containers with an enzyme labeled antibody conjugate. The test units are removed, rinsed and placed in a third set of reaction containers containing chromogenic substrate of hydrogen peroxide and TMB (tetra methyl benzidine). Upon positive reaction with a tip, the chromogenic substrate develops a specific color. In one embodiment, a sealed reagent kit is provided having predispensed, color coded, liquid reagents including an incubation medium of a buffered protein solution containing preservatives; a second liquid reagent is an antibody conjugate in a buffer with stabilizers and preservatives; a third liquid reagent is a chromogenic substrate, a stabilized and buffered solution of peroxide and TMB. The kit includes calibrator solutions and a plurality of tips.

Description

PATENT APPLICATION

For: Improved Methods and Apparatus for Performing Determinations of Immune Reactants in Biological Fluids

BACKGROUND OF THE INVENTION Field of the Invention:

The present invention relates to improved methods and apparatus for performing determination of immune reactants in biological fluids and, more particularly, for performing enzyme-immunoassay for both qualitative and

quantitative (i.e., instrument based) detection c ;ller-qen specific (e.g., IgE) antibodies in human biological fluids.

Discussion of the Prior Art:

It is known that, in humans, immediate type allergic reactions (e.g., hay fever,

extrinsic asthma, or atopic eczema) are mediated by reaginic antibodies belonging to the IgE class of immunoglobulins. Atopic individuals exposed to allergens such

as pollens, dust or animal danders produce specific IgE antibodies against these

allergens. The present invention is concerned with determining the presence of circulating allergen-specific IgE antibodies in the blood, plasma or serum of an

affected individual. One technique for accomplishing this result is known in the prior art as

described in U.S. Patent No. 3,720,760 (Bennich et al), the disclosure of which is

expressly incorporated herein, in its entirety, by reference. Bennich et al discloses an in-vitro method for analyzing a test sample (e.g., a body fluid such as a blood

serum or blood plasma) by contacting, in vitro, the test sample with a water insoluble polymer to which a test allergen has been bound. A reaction takes place between

the test allergen on the polymer and the reagin-lgE directed against the allergen so that the reagin-lgE is bound to the test allergen on the insoluble polymer. The

polymer, in sheet form, and the test allergen and the reagin-lgE attached thereto,

may be contacted with antibodies against reagin-lgE that have been labeled with a radiation emitting atom or group. The insoluble polymer sheet is separated from the fluid, whereupon the radiation emitted from the insoluble polymer with the

substances attached thereto, or the radiation er.drte- from the separated fluid, is

measured. If the reagin-lgE directed against the allergen is present in the sample,

labeled reagin-lgE is bound to the insoluble phase which then emits radiation. The latter increases with increasing concentration of the reagin-lgE in the test sample.

The radiation of the liquid phase decreases with increasing concentration of the reagin-lgE as more labeled reagin-lgE is bound to the insoluble phase. The

measured radiation values obtained in this procedure for the test sample can be

compared with values for control samples. If, instead of radio-immunoassay (RIA)

techniques, one were to use enzyme-immunoassay (EIA) techniques, color intensity, rather than radiation, becomes the measured parameter.

The use of polymer sheets as a vehicle to which the test allergen is bound, and to which the allergen-specific antibodies attach, becomes unwieldy in practice and so new methods for performing determinations in immune reactants and

biological fluids have been developed. In particular, U.S. Patent 5,494,830 to Hubscher, the inventor of the present invention, discloses such methods, the entire

disclosure of which is incorporated herein by reference. The method of the '830

patent includes multiple test units, each taking the form of an elongated rod having

an allergen-coated tip at its distal end. The proximal end of each rod is frictionally engaged in a respective aperture or through-hole in a support strip so that the test units are disposed in a position-identified linear array. Spacing between the test

units, as determined by the spacing between the through-holes and the support

strip, matches the spacing between reaction containers in an assembly of such containers. The test unit spacing is also selected to permit alternate test units to be removed from the support strip so that the remainino test units can be inserted into respective test tubes supported in a linear array in a test tube rack. Color coding, or

another visible indicator, is provided for the test unit so that the specific allergen

coating on the tip of each test unit can be readily identified. In the preferred embodiment of Hubscher's method, the color coding uses the combination of colors of the rod and tip, each combination being unique and identifying a particular

allergen as indicated in a chart supplied with the system. The tip of each test unit

may take the form of two frusto-conical sections joined by short cylindrical sections

disposed between their widest ends. The surfaces of the tip can be smooth or can

be multi-faceted. The coated and color-coded test units depending from the support

strip are inserted into respective reaction containers containing the biological sample to be analyzed (e.g., blood, serum, etc.) If there is an antibody specific to the allergen coated on any of the tips, that antibody becomes bound to the associated

allergen in the corresponding reaction container. The tips are then washed, dried

without rubbing, and inserted into a second set of reaction containers in which a suitable enzyme-labeled antibody conjugate has been poured. The test units are

removed and rinsed once again and then placed into a third set of reaction containers containing chromogenic substrate that, upon positive reaction, develops a specific color. After incubation, the test units are removed, thereby stopping the

enzymatic reaction and the remaining liquid in each reaction container is

quantitatively analyzed for color development and intensity by a suitable spectrophotometric analyzer (e.g., the LabDex ASR® brand spectrophotometric analyzer from Dexall Biomedical Labs, Inc. of Gaithersburg, Maryland).

A problem encountered in using the EIA method of the '830 patent is that a

significant amount of time is required for the incubation steps, thereby requiring excessive time to test for each individual allergen.

Using the method and materials of the '830 patent, a reagent kit is provided having a first color coded liquid reagent, an incubation medium in the form of a

buffered protein solution containing preservatives. A second liquid reagent, also color coded, is an antibody conjugate, preferably an affinity purified anti-human IgE

(goat) conjugated to horseradish peroxidase, in a buffer with stabilizers and

preservatives. A third liquid reagent, also color-coded, is a chromogenic substrate,

a stabilized and buffered solution of hydrogen peroxide and ABTS ® (a trademark of

Boehringer Mannheim, Inc.) brand 2.2-azino-di 3-ethyl-benzthiazoline sulfonate (6). When performing quantitative (i.e., instrument based) determinations, the reagent kit also includes a set of buffered and stabilized human serum solutions (calibrated in A.U./ml.), including a first blank (0 A.U./ml) solution and one or more calibrators

such as an S1 standard (2.5 A.U./ml) solution, an S2 (10 A.U./ml) solution and an S3 (20 A.U./ml) solution; (an A.U. is an Allerg-Ens® Unit, an arbitrarily defined unit of concentration). Preferably, plurality of tips are also provided for use with each kit.

Use of the old kits required three excessively long incubation periods; a first incubation period (for the tips in patient serum and incubation medium) of two hours

and thirty minutes, a second incubation period (for the tips in antibody conjugate) of one hour and a third incubation period (for the tips in chromogenic substrate) of thirty minutes. Thus, each test for an individual allergen requires in excess of four hours

to complete, since the three incubation periods account for four hours time.

OBJECTS AND SUMMARY OF THE INVENTION It is therefore an object of the present invention to perform visual, qualitative,

in vitro determinations of immune reactants, in biological fluids, in less time than

required using the methods and apparatus of the prior art.

It is also an object of the present invention to perform quantitative (i.e.,

instrument based), in vitro determinations of immune reactants, in biological fluids, in less time than required using the methods and apparatus of the prior art.

It is another object of the present invention to provide a novel immunoassay

kit with predispensed reagents permitting simultaneous multiple tests for respective

multiple allergen-specific antibodies in human serum or plasma. It is a more general object of the present invention to provide an improved technique for diagnosing specific allergens in humans in a manner more time

efficient than that described in U.S. Patent No. 5,494,830 (to Hubscher).

The aforesaid objects are achieved individually and in combination, and it is not intended that the present invention be construed as requiring two or more of the objects to be combined unless expressly required by the claims attached hereto. In accordance with one aspect of the present invention, coated and color

coded test units, supported to depend from a support strip, are inserted into

respective reaction containers containing predispensed reagents. Another aspect of the present invention is a new formulation of chromogenic substrate for use in the method of the present invention and having a stabilized and buffered solution of

hydrogen peroxide and TMB (tetra methyl benzidine). Upon positive reaction with a

tip the chromogenic substrate with TMB develops a sDecific color in less time than was required for the methods of the prior art.

In yet another aspect of the present invention, the liquid in the reaction

container can either be quantitatively analyzed for color development and intensity by the spectrophotometric analyzer (as discussed above), or, alternatively, the liquid

in the reaction containers may be visually, qualitatively analyzed by classifying and grading the color for each well.

Using the method and materials of the present invention, a reagent kit is

provided having a first color coded liquid reagent, an incubation medium in the form

of a buffered protein solution containing preservatives. A second liquid reagent, also

color coded, is an antibody conjugate, preferably an affinity purified anti-human IgE (goat) conjugated to horseradish peroxidase, in a buffer with stabilizers and preservatives. A third liquid reagent, also color coded (e.g., initially colorless and so

readily distinguished from the other initially colored liquid reagents), is a chromogenic substrate, a stabilized and buffered solution of hydrogen peroxide and TMB; this third reagent provides a significant improvement over the reagent kits of

the prior art, as discussed above. When used in qualitative determinations, the reagent kit of the present invention also includes a set of buffered and stabilized

human serum solutions (calibrated in A.U./ml.), including a first blank (0 A.U./ml)

solution and one or more of an S1 standard (2.5 A.U./ml) solution, an S2 (10 A.U./ml) solution and an S3 (20 A.U./ml) solution. Preferably, a plurality (e.g.,

twelve) test tips are also provided in each kit; however, the tips can be separately provided.

When used in qualitative determinations, the kit includes a color reference comparison chart including a registration template for assisting the user in

positioning the reaction containers adjacent a first region displaying a continuum of increasing color saturation. The continuum of color is white at one end,

corresponding to a "negative" result and increases to be saturated with the selected

color (e.g., blue) at the opposing end, corresponding to a "very high" determination.

The comparison chart also includes a plurality of (e.g., six) subregions positioned in

close proximity to the first region, each subregion including a single, distinct, level of color saturation and a written caption such as, for example "Negative" for a

subregion with no color saturation (e.g. , white), "low" for a slight level of color

saturation, "Moderate" for an increased level of color saturation, "High" for a further increased level of color saturation, and "Very High" for subregions having levels of color saturation increased yet further (e.g., darker blue), respectively.

Use of the new kit materials of the present invention requires three

substantially abbreviated incubation periods, as compared to the method and

materials of the prior art; using the quantitative method as an example, a first incubation period (for the tips in patient serum and incubation medium) of fifty minutes, a second incubation period (for the tips in antibody conjugate) of twenty

minutes, and a third incubation period (for the tips in chromogenic substrate with TMB) of fifteen minutes. Thus, each test for an individual allergen requires

significantly less than two hours to complete, where the three incubation periods account for eighty-five minutes time. Using the new qualitative method, the three incubation periods amount to only seventy-five minutes time.

The above and still further objects, features and advantages of the present invention will become apparent upon consideration of the following detailed

description of a specific embodiment thereof, particularly when taken in conjunction

with the accompanying drawings wherein like reference numerals in the various figures are utilized to designate like components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded view in perspective showing one embodiment of the apparatus of the present invention;

FIG. 2 is a top view in plan of a support strip utilized the apparatus of FIG. 1 ;

FIG. 3 is a front view in elevation of the support strip and test units employed in the apparatus of FIG. 1 ;

FIG. 4 is a bottom view in plan of one embodiment of a test unit tip

constructed in accordance with the present invention;

FIG. 5 is a side view in elevation of the test unit tip of FIG. 4; FIG. 6 is a bottom view in plan of an alternative embodiment of the test unit tip of the present invention;

FIG. 7 is a side view in elevation of the test unit tip of FIG. 6;

FIG. 8 is a sectional view of the tips illustrated in FIGS. 4 and 6 showing the

manner in which the tip is secured to its supporting rod; and

FIG. 9 is an exploded view in perspective showing how the test units and support strip of the apparatus of FIG. 1 may be employed in connection with test tubes rather than the reaction containers illustrated in FIG. 1.

FIGS. 10a-10i are schematic illustrations of a frame of containers having

predispensed reagents for use in the eight test method steps included in the qualitative or quantitative test methods of the present invention.

FIG. 11 illustrates a color reference comparison chart for use in qualitative

test method determinations.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring specially to Figs. 1-8 of the accompanying drawings, testing

apparatus constructed in accordance with the principles of the present invention includes an array 10 of multiple test units 11 through 22, inclusive. Although twelve

test units are provided in the preferred embodiment, it is to be understood that this

number is not an essential feature of the invention and that any array of three or more test units may be employed. Each test unit includes an elongated cylindrical rod 30 with a transversely expanded tip 31 secured to its distal end. Each rod 30 may be solid or hollow and has a section of reduced diameter at both its distal and

proximal ends. The distal end is received in a suitable hole or aperture at the top of

a respective tip 31 and is secured in place by adhesive, cement, or the like. The

proximal end of the rod of each test unit 11-22 is removably engaged by a friction fit in a respective circular aperture or through-hole 41-52, inclusive, defined in an elongated holder strip 32. The substantially identical apertures 41-52 are disposed

in a linear array along the length dimension of strip 32 with spaces of equal lengths

between successive apertures. The apertures are positionally identified, as by molding the strip with position-identifying numbers (e.g., M1-M12) adjacent each aperture. Alternatively, a printed gummed label may be attached to the strip to identify each aperture by position. The test units 11-22, therefore, with their rods 30

engaged in respective apertures 41-52, are supported in a linear array wherein the

test units are suspended in side-by-side relation with equal spacing between each

two adjacent test units. Holder strip 32 has handles/support brackets 33, 34 at its opposite ends to facilitate handling/support of the strip, and the supported test units, during test procedures.

Tip 31 is required to project or to extend to a transversely or radially larger

size than the diameter of the rod 30, in order to present a large surface area for

absorbing or otherwise bonding allergen material. As illustrated in Figs. 4 and 5, the

tip 31 may take the form of three solid integrally-formed sections 35, 36, and 37.

Section 35, the most proximal for the three sections, in frusto-conical with its smaller

10 end apertured to receive the distal end of rod 30. The larger end of frusto-conical

section 35 joins the axially shorter cylindrical section 36 which in turn joins the larger

end of second frusto-conical section 37. The two frusto-conical sections 35 and 37 are of substantially equal axial length and identical shape but are inversely oriented axially to provide substantial symmetry on opposite sides of section 36. The surfaces of section 35, 36 and 37 may be smooth, as illustrated in Figs. 4 and 5, or

multi-faceted, as illustrated in Figs. 6 and 7. In either case, the distal end of rod 30

is secured in the aperture defined in section 35 by means of suitable cement 38, adhesive, or the like, as illustrated in Fig. 8.

The test unit rod 30 and tip 31 are made of a suitable water-insoluble polymeric material that is both rigid and capable of having the described allergen

materials adsorbed on, or otherwise bonded to, the tip 31. The material should be a type which readily adsorbs the proteinaceous allergen materials. Examples of

suitable polymeric materials for use in manufacturing test units 11-22 are hydrocarbon polymers such as polystyrene, polyethylene, polypropylene, polybutylene, butyl rubber and other synthetic rubbers, as well as polyesters,

polyamides, vinyl and acrylic polymers such as polyvinyl chloride and polymethyl

methacrylate, cellulose and cellulose derivatives such as cellulose acetate. In

general, any organic polymeric material which adsorbs protein in relatively large

amounts will be acceptable. The preferred substrate material for the test unit is

impact grade polystyrene. The coating on tips 31 is achieved by dipping the tips into

the antigen/allergen solution desired and thereafter following known procedures for suitable adsorption and/or chemical bonding.

11 As illustrated in Fig. 1 , the suspended array 10 of test units 11-22 is used in conjunction with a linear array or strip 60 of reaction containers or wells 61-72, respectively, formed as an integrally-molded, one-piece, clear plastic unit. Containers 61-72 are preferably positioned in abutting side-by-side relation with their

centers spaced by the same spacing provided between adjacent through-holes 41-

52, respectively, in strip 32. Consequently, test units 11-22 may be simultaneously

inserted into, or withdrawn from, respective reaction containers 61-72 by hand or automated process. To this end, the open tops of the containers are very much wider than the transverse dimension of tips 31 , thereby providing adequate

clearance during insertion and withdrawal. Plural arrays 60 may be supported in

adjacent rows in a frame 75 so that each of the supported arrays may receive its own array of test units 11-22 simultaneously. Alternatively, different arrays 60 may be used successively in connection with the same array of test units during performance of the test procedure described below. As a further alternative, the

reaction containers may be formed as a one-piece unit integrally molded with tray

75.

Each of the test units 11-22 in the array 10 are color coded to be visibly

identifiable and distinguishable from other test units in the same array. The coding permits a technician to correlate the visible identifier for each test unit with the

particular allergen adsorbed on the tip 31 of that test unit. This is accomplished by

means of a chart or table correlating each visible identifier with a corresponding allergen. The visible identifier may be the color of the tip 31 , the color of rod 30, the

combination of colors used for the rod and tip, a number of dots along the rod

12 length, a number of colored circumferential stripes on the rod, etc. In the preferred

embodiment described herein, the coded visible identifier is the color combination of

the rod and tip. Typical color combinations employed for the test units are represented in Table 1 which is a typical example of a color-coded allergen correlation chart supplied with the apparatus of the present invention. The chart includes a first column listing test unit positions 11-22 (as referenced in Figs. 1 and

3), a second column listing rod and tip colors for each test unit, and a third column listing the respective allergens adsorbed on each test unit tip.

TABLE 1

Test Unit (Figs. 1 , 3) Rod/Tip Color Allergen

11 Red/Red Blank calibrator

12 White/White Reference calibrator

13 Blue/Purple Oak tree

14 Green/Orange Bermuda grass

15 Green/Blue Kentucky blue grass

16 Yellow/Purple Ragweed, short

17 Purple/Red Cat epithelium

18 Purple/Green Dog dander

19 Black/Orange Altemaria mold

13 20 Black/Blue Cladosporium mold

21 Purple/Orange House dust

22 Purple/Blue Dust mite (D. Farinae)

The general principles of the test procedures in which the above-described apparatus is employed may be understood from the following brief description, a

specific allergen, coated to a solid phase support on each tip 31 , reacts with allergen-specific IgE antibodies in the patient's serum during a first incubation

period. After washing away nonspecific reactants, enzyme-labeled (peroxidase) anti- human IgE (i.e., the antibody conjugate reagent) reacts with the allergen-bound human IgE during a second incubation period. After further washing, the bound

complex "allergen" - "IgE" - "peroxidase anti-human IgE" is caused to react with a chromogenic substrate specific for the peroxidase enzyme for a third incubation period. This results in the development of a blue color. The intensity or saturation of

the developed color is proportional to the amount of circulating allergen-specific IgE

antibodies. Quantation of these antibodies is achieved spectrophotometrically, and

results are expressed in arbitrarily defined Allerg-Ens® Units (A.U.), as discussed

above.

A typical quantitative test procedure using a method and apparatus in

accordance with the present invention (but not in pre-dispensed kit form) is set forth

in the following steps:

1. For each patient serum to be tested, one array 60 of reaction containers is

14 set in place. Each reaction container is identified as to its position (i.e., positions one through twelve).

2. One hundred micro liters of a first calibrator standard solution (blank, 0

A.U./ml) is placed in first container 61 of array A by means of a pipette (this and other dispensing steps may be skipped when using a kit with pre-dispensed reagents). The first calibrator standard may be buffered and stabilized human serum solution containing a known concentration (i.e., 0 A.U./ml) of antibodies.

3. One hundred micro liters of a second calibrator standard solution (10

A.U./ml) is placed in container 62 of array 60 by means of a pipette. The second calibrator standard may be buffered and stabilized human serum solution containing a known concentration (i.e., 10 A.U./ml) of antibodies.

4. One hundred micro liters of test serum is then placed in all of the other

reaction containers 63-72 of array A and all containers 61-72 of the other arrays B-

G. It is remembered that each array 60 (i.e., A, B, C, D, E, F, and G) is dedicated to

a particular patient and that only the test serum for that patient is employed in a particular array (e.g., array B).

5. Fifty micro liters of incubation medium is then placed in all reaction

containers 61-72 of each array A-G. A typical incubation medium would be a buffered protein solution containing preservatives.

6. The arrays 10 of test units, supported by respective holder strips 32, are

then removed from the plastic bags in which they are supplied as part of the kit.

Care is taken not to touch the tips 31 which are coated with specific allergen

material. It is also important that the test units not be removed from the holder strip

15 32 unit the end of the procedure.

7. Test units 11-22 are then inserted simultaneously into reaction containers 61-72, respectively. The test units may be moved up and down slightly to ensure proper mixing.

8. The inserted test units are permitted to remain in place to achieve incubation for approximately fifty minutes at room temperature.

9. The test units, still supported by the holder strip 32, are then transferred

into a rinsing dish where the test units are rinsed for at least one minute by filling the

dish with washing solution (e.g., buffered salts and detergent and/or distilled water ). Three rinsing cycles should be performed, with all of the water drained between

cycles; (when using a kit with pre-dispensed reagents, containers for rinsing are included).

10. The test units are then placed on an adsorbent paper towel with care being taken not to rub the tips 31.

11. The previously used arrays 60 are discarded and a new set of arrays 60

(one for each array 10) are set in place.

12. One hundred twenty five micro liters of antibody conjugate is placed, by a pipette into each reaction container 61-72 of each array. The antibody conjugate

may be an affinity purified anti-human IgE (goat) conjugated to horseradish

peroxidase in a buffer with stabilizers and preservatives.

13. Drained test units 11-22 are then inserted simultaneously into reaction

containers 61-72, respectively. The test units may be moved up and down slightly to

ensure proper mixing.

16 14. The inserted test units are permitted to remain in place to achieve a second incubation for approximately twenty minutes at room temperature.

15. The test units are removed from the reaction containers and washed and

dried by repeating steps 9 and 10, described above. 16. The just-used arrays 60 are then discarded and a new set of arrays 60

are set in place.

17. Each reaction container 61-72 receives two hundred micro liters of

chromogenic substrate, with tetra methyl benzidine (TMB), by means of a pipette.

18. The test units 11-22 are gently tapped on the paper towels and placed

into their appropriate reaction containers 61-72, respectively, containing the chromogenic substrate.

19. A third incubation is permitted to occur for fifteen minutes at room

temperature.

20. The test units are gently removed from the reaction containers with care

being taken not to spill the contents of those containers.

21. The reaction containers of array 60 are removed from the frame 75 and placed in a spectrophotometric analyzer such as the LabDex ASR® brand spectrophotometric analyzer sold by Dexall Biomedical Labs, Inc., of Gaithersburg,

Md. The quantitative results displayed by such a reader are direct concentrations of

allergen-specific antibodies in the patient sera expressed in allergen units per milliliter (A.U./ml). Readings can be taken immediately or within eighteen hours, in

which case the arrays of containers 61-72 should be sealed and kept refrigerated.

The test units may be employed with larger containers, such as test tubes, in

17 the manner illustrated in Fig. 9. Specifically, a rack 80 for supporting test tubes 81 , 83, 85, 87, 89 and 91 in a linear array has suitable supporting apertures having their centers spaced at twice the spacing between apertures defined in strip 32. In order

to utilize the array of test units with the supported test tubes, alternate test units are removed from the strip 32. Thus, as illustrated in Fig. 9, the remaining test units 11 , 13, 15, 17, 19 and 21 are positioned to permit their simultaneous insertion into test

tubes 81 , 83, 85, 87, 89 and 91. The openings of the test tubes are sufficiently wide to permit the tips of the test units to be inserted and removed without scraping.

In a typical embodiment, the components of the invention described above have the following dimensions. Holder strip 32 is 4.712 inches long, 0.250 inches

wide and 0.100 inches thick. The twelve apertures 41-52 have diameters of 0.062 inches and are spaced, on center, by 0.355 inches. Rods 30 are 3.375 inches long

with an outside diameter at its widest point of 0.0925 inches. The proximal end of rod 30 has a reduced diameter of 0.062 inches. Tip 31 has an axial length of 0.2187 inches. Cylindrical section 36 has an axial length of 0.032 inches and an outside

diameter of 0.220 inches. The frusto-conical sections 35 and 37 taper at a 45° angle

and are of substantially equal configuration with the exception of the rod-receiving aperture defined in section 35. That aperture extends to a depth of approximately

0.109 inches and tapers slightly at an angle of one degree in an inward direction

from a diameter of 0.062 inches. The individual containers in array 60 are cylindrical

and typically have an inside diameter opening of approximately 0.279 inches.

It is to be understood that the dimensions set forth above are by way of example and not limiting on the scope of the present invention.

18 Turning now to the sequence of steps shown schematically in Figs. 10a through 10i, another variation of the quantitative method of the present invention is illustrated. Prefabricated, on-piece, molded, light transmissive plastic contiguous

strips 100 of twelve containers 102 each are arrayed in a frame or tray 104 and

include predispensed reagents. The light transmissive plastic containers 102 are sealed within the frame 104 by a two-part, fluid-tight, resilient, adhesively applied,

flexible sealing film membrane 106. A typical test procedure using the apparatus of Figs. 10a-10i is set forth in the following steps:

1. The first and larger film membrane segment 106a is rolled back from a

first frame edge 108 and removed by use of a key 110, as shown in Fig. 10a, thereby uncovering strips or rows A-G. The containers 102 of strip or row A contain fifty micro liters of predispensed incubation media. The containers of row D contain one hundred twenty five micro liters of predispensed antibody conjugate. The

containers of rows B, C, E, F and G contain two hundred micro liters of

predispensed washing buffer solution. The containers 102 of row H remain covered

under the second smaller film segment 106b and contain two hundred micro liters of predispensed chromogenic substrate with TMB.

2. One hundred micro liters of patient test serum is then placed in all of

the other reaction containers or wells 102 of row A using a pipette 112.

3. An array of test units or tips 31, supported by a holder strip 32, is then

removed from the plastic bag in which it is supplied as part of the kit. Care is taken

not to touch the tips 31 coated with specific allergen material.

4. The test units 31 are then inserted simultaneously into reaction

19 containers 102 of row A. The test units 31 may be moved up and down slightly to ensure proper mixing.

5. The inserted test units are permitted to remain in place to achieve incubation for approximately fifty minutes at room temperature. 6. The test units are then removed from the containers of row A and placed on an absorbent paper towel with care being taken not to rub the tips.

7. The test units, still supported by the holder strip 32, are then transferred sequentially into a rinsing containers of rows B and C, the test units are rinsed for at least one minute in each row, such that two rinsing cycles are performed.

8. The test units are then removed from the containers of row C and placed on an absorbent paper towel with care being taken not to rub the tips.

9. The drained test units are then inserted simultaneously into the row D reaction containers of antibody conjugate. The test units may be moved up and down slightly to ensure proper mixing.

10. The inserted test units are permitted to remain in place to achieve a second incubation for approximately twenty minutes at room temperature.

11. The test units are removed from the reaction containers and blotted (not

rubbed) on an absorbent paper.

12. The test units are then sequentially dipped, rinsed and transferred in the containers of rows E, F and G, agitating and standing for one minute in the

containers of each row.

13. The test units are gently tapped on the paper towels.

14. The key is used to remove the second segment 106b of the adhesive

20 film covering row H.

15. The test units are placed into the reaction containers of row H, containing the chromogenic substrate.

19. A third incubation is permitted to occur for fifteen minutes at room

temperature.

20. The test units are gently removed from the reaction containers of row H

with care being taken not to spill the contents of those containers.

21. The reaction containers 102 of row H are removed from the frame as

shown in Fig. 10h and placed in a spectrophotometric analyzer 120 (e.g., the

LabDex ASR® brand spectrophotometric analyzer from Dexall Biomedical Labs, Inc. of Gaithersburg, Maryland), as shown in Fig. 10i. The quantitative results displayed by spectrophotometric analyzer 120 are direct concentrations of allergen-specific antibodies in the patient sera expressed in A.U. per milliliter (A.U./ml). Readings can be taken immediately or within eighteen hours, in which case the arrays of

containers should be sealed and kept refrigerated.

In yet another, qualitative, variation of the method of the present invention,

steps 1-4, 6-9, 11-20 are performed, as above; incubation steps 5 and 10 differ slightly in length, with the incubation period of steps 5 and 10 being thirty minutes

duration. After step 20, the containers 102 of row H are visually, qualitatively

evaluated, whereupon the remaining fluid in each container 102 is evaluated and

classified for the darkness or grade of the resulting color, as compared to the color

of fluid in the negative control well or container, H1. In the example of Fig. 10h, fluid in container H2 is a darker (more saturated) color than the color of the fluid in

21 negative control well, H1 ; whereas fluid in container H3 is as light a color as the color of the fluid in negative control well or container, H1

FIG. 11 illustrates a color reference comparison chart 120 for use in

qualitative test method determinations. Preferably, a kit includes color reference comparison chart 120 which has printed indicia including a registration template 122 for assisting the user in positioning the reaction containers (e.g., H1-H3) adjacent a

first region 124 displaying a continuum of increasing color saturation. The

continuum of color is white at one end 126, corresponding to a "negative" result and increases to be saturated with the selected color (e.g., blue) at the opposing end 128, corresponding to a "very high" determination. The comparison chart also

includes a plurality of (e.g., six) subregions 130, 132, 134, 136, 138, 140 positioned

in close proximity to first region 124, each subregion including a single, distinct, level of color saturation and a written caption such as, for example "Negative" for

subregion 130 with no color saturation (e.g. , white), "low" for a slight level of color saturation (e.g., 132), "Moderate" for an increased level of color saturation (e.g.,

134), "High" for a further increased level of color saturation (e.g., 136), and "Very High" for subregions having levels of color saturation increased yet further (e.g.,

darker blue, 138, 140), respectively. Comparison chart 120 preferably also

includes a space for marking a date and patient name.

The invention, as described in terms of the preferred embodiment, utilizes an

antibody labeled with peroxidase enzyme and a chromogenic substrate with TMB

specific thereto. It is to be understood, however, that this is by way of example only

and not a limitation on the scope of the invention. Another example would be

22 alkaline-phosphatase enzyme with its corresponding paranitrophenyl substrate (although providing a slower reaction).

The invention as thus far described is specific to testing for allergen-specific antibodies in human bodily fluids. It is to be understood that the apparatus described herein, and its method of use, apply to testing other antigens, in animal and human fluids, such as bacterial, viral, or auto-antigens (i.e., lupus, DNA, etc.).

From the foregoing description it will be appreciated that the invention makes available a novel apparatus for rapidly performing determinations of allergen-specific antibodies in human serum as well as a time efficient step-by-step procedure for making such determinations.

Having described preferred embodiments of a new and improved method and apparatus for performing determinations of allergen-specific IgE antibodies in human serum in accordance with the present invention, it is believed that other

modifications, variations and changes will be suggested to those skilled in the art in view of the teachings set forth herein. In as much as the present invention is subject

to various modifications and changes in detail, the above description of a preferred

embodiment is intended to be exemplary only and not limiting. It is believed that other modifications, variations and changes will be suggested to those skilled in the

art in view of the teachings set forth herein. It is therefore to be understood that all

such variations, modifications and changes are believed to fall within the scope of the present invention as defined by the appended claims.

23

Claims

WHAT IS CLAIMED IS:

1. A method for performing determinations of allergen-specific IgE antibodies in human serum, including the steps:

a) providing a first plurality of at least two reaction containers; b) providing a first calibrator standard human serum solution containing a known concentration of antibodies in a first of the reaction containers;

c) providing a human test serum in all of the other reaction containers; d) providing an incubation medium in all of the first plurality of reaction

containers; e) providing a plurality of at least two test units having tips coated with

specific allergen material;

f) inserting the test unit tips into the first plurality of reaction containers; g) incubating the test units for a first incubation period; h) rinsing the test units in wash solution;

I) providing a second plurality of at least two reaction containers containing antibody conjugate;

j) inserting the test units into the second plurality of reaction containers; k) incubating the test units for a second incubation period; I) rinsing the test units in wash solution;

m) providing a third plurality of at least two reaction containers containing

chromogenic substrate fluid, with tetra methyl benzidine (TMB);

n) inserting the test units into the third plurality of reaction containers

24 containing the chromogenic substrate fluid; o) incubating the test units for a third incubation period; and

p) evaluating concentrations of allergen-specific antibodies in the patient sera as a function of color development of the fluid in the third plurality of reaction containers.

2. The method of claim 1 , wherein said first incubation period of step f) is

not more than fifty minutes.

3. The method of claim 1 , wherein said second incubation period of step k) is not more than thirty minutes.

4. The method of claim 1 , wherein said third incubation period of step o) is not more than fifteen minutes.

5. The method of claim 1 , wherein step p), evaluating concentrations of allergen-specific antibodies in the patient sera as a function of color development of

the fluid in the third plurality of reaction containers, comprises visually, qualitatively,

grading the color of the fluid in each reaction container.

6. The method of claim 1 , wherein step p), evaluating concentrations of

allergen-specific antibodies in the patient sera as a function of color development of

the fluid in the third plurality of reaction containers, comprises spectrophotometrically

25 measuring the color of the fluid in each reaction container, and providing a quantitative analysis therefore.

7. A kit for performing determinations of allergen-specific IgE antibodies in human serum, including:

a quantity of a calibrator standard human serum solution containing a known concentration of antibodies; a fluid incubation medium;

a plurality of at least two test units having tips coated with specific al'ergen material; a quantity of antibody conjugate solution; and a quantity of chromogenic substrate fluid, with tetra methyl benzidine (TMB).

8. The kit of claim 7, further including:

a frame having an upper surface;

a plurality of reaction containers and a sealing membrane; wherein said

calibrator standard human serum solution, said fluid incubation medium, said antibody conjugate solution, and said chromogenic substrate fluid with TMB are predispensed in said reaction containers;

said reaction containers being supported within said frame; and

said reaction containers and said frame being in sealed relation with said

sealing membrane.

26

9. The kit of claim 7, further including a color reference comparison chart including printed indicia with areas of color calibrated for use in qualitative test method determinations.

10. A method for performing determinations of allergen-specific IgE antibodies in human serum, including the steps:

a) removing a seal from a first plurality of at least two reaction containers; including a predispensed first calibrator standard human serum solution containing a known concentration of antibodies in a first of the reaction containers;

b) providing a plurality at least two test units having tips coated with

specific allergen material;

c) inserting the test unit tips into the first plurality of reaction containers; d) incubating the test units for a first incubation period; e) rinsing the test units in wash solution; f) providing a second plurality of at least two reaction containers

including predispensed antibody conjugate;

g) inserting the test units into the second plurality of reaction containers; h) incubating the test units for a second incubation period; I) rinsing the test units in wash solution;

j) providing a third plurality of at least two reaction containers including

predispensed chromogenic substrate fluid, with tetra methyl benzidine (TMB);

k) inserting the test units into the third plurality of reaction containers containing the chromogenic substrate fluid;

27 I) incubating the test units for a third incubation period; and m) evaluating concentrations of allergen-specific antibodies in the patient sera as a function of color development of the fluid in the third plurality of reaction

containers.

11. The method of claim 10, wherein step m), evaluating concentrations of allergen-specific antibodies in the patient sera as a function of color development of

the fluid in the third plurality of reaction containers, comprises visually, qualitatively, grading the color of the fluid in each reaction container.

12. The method of claim 11 , wherein said step of visually, qualitatively, grading the color of the fluid in each reaction container includes comparing said fluid to a color comparison chart indicia.

13. The method of claim 10, wherein step m), evaluating concentrations of

allergen-specific antibodies in the patient sera as a function of color development of the fluid in the third plurality of reaction containers, comprises spectrophotometrically measuring the color of the fluid in each reaction container, and providing a

quantitative analysis therefore.

28