WO1989000290A1

WO1989000290A1 - Capillary device for immunoassay of multiple analytes

Info

Publication number: WO1989000290A1
Application number: PCT/US1988/002132
Authority: WO
Inventors: Kenneth A. Kelly
Original assignee: In Vitro Technologies, Inc.
Priority date: 1987-07-02
Filing date: 1988-06-23
Publication date: 1989-01-12
Also published as: AU2087388A; EP0367794A4; EP0367794A1; ES2009312A6; IN171202B; JPH02504188A

Abstract

A device is described for simultaneously testing in vitro a biological fluid for the presence of antibodies and/or antigenic substances to a plurality of antigenic substances and/or antibodies. The fluid sample is made to react with immunological reagents attached to water-insoluble segments (4, 5, 6, 7, 8 and 9) encased within a capillary tubing (3). Analytes from the sample insolubilized to reactive segments (4, 5, 6, 7, 8 and 9) through immune complexing are detected by subsequent reaction with labeled second antibodies specific to the analytes.

Description

CAPILLARY DEVICE FOR IMMUNOASSAY OF MULTIPLE ANALYTES

Background of the Invention The present invention relates to a device and method for the in vitro screening of human subjects for allergic sensitivity. By this invention serum, plasma or other bo fluid from the individual is tested in a solid phase im u assay, simultaneously in a single test apparatus, for the presence of antibodies to a variety of allergens as well for the level of Total IgE immunoglobulin in the fluid. This invention also relates to a method of performing mul pie tests for antibodies and/or antigens simultaneously i single device and kits therefor, which employ conventiona immunoassay techniques using radioactive-, enzyme- or fluorescent-labeled reagents.

Antibodies of the immunoglobulin classes, IgE and Ig are known to be responsible for the allergic hypersensi- tivity in humans and animals. Individuals predisposed to allergies, either genetically or through exposure to en¬ vironmental allergens, by virtue of an imbalance in their immune system usually have levels of Total IgE elevated f the norm. Proper diagnosis and treatment of allergic dis orders, therefore, require not only a measure of the Tota IgE level but more so a measure of the levels of antibodi directed against specific allergens (Dockhorn, R.J., Ann. Allergy, 49, 1-7, 1982). Methods for the detection of allergen-specific anti¬ bodies involve both iri vivo and ;Ln vitro techniques. In vitro techniques consist of challenging the patient eithe directly into the skin or by inhalation of allergen aeros with aqueous extracts of the allergens (Nelson, H.S., Ann Allergy, 51, 411-417, 1983; Pelikan, Z., ibid., 51, 395-4 1983) . These iri vivo techniques are usually discomfortin to the patient and are not always applicable as in childr the elderly and patients with skin disorders. Bennich, K et al., U.S. Patent 3,720,760, disclose a radioimmunoassa for the in_ vitro detection of antibodies to specific al¬ lergens. The method comprises reacting serum from a pati with single allergens attached to a water-insoluble soli phase. The antibodies present in the patient's serum bi to the solid phase-allergen and are detected by subseque reaction with a radioactively-labeled second antibody re active against the patient's antibody. This general in vitro technique is termed "RAST" for Radio-Allergo-Sorbe Test. Modifications of the test include coupling indivi allergens to paper discs and other cellulosic materials as Sepharose or cellulose, and polymeric materials such polyvinyl or polystyrene microtiter wells. By use of an enzyme-labeled second antibody to human IgE, the test ha been converted to a nonisotopic assay. Numerous referen describe the correlation among these ill vitro enzyme-and radioimmunoassays and in vivo methods for diagnosing all gies (Ali, M-. and Ramanarayanan , M.P., Ann. Allergy, 53,

143-150, 1984; Seltzer, J.M., Malpern, G.M. and Tsay, Y. ibid, 54, 25-30, 1985) .

For the diagnosis of allergies and their treatment, new patient or one with a continuing allergic disorder i subjected to either ill vivo or in vitro tests against a panel of 50 or more different allergens by a specialist. The testing is very expensive and time-consuming to both patient and the physician. Merrett, T.G. et al. (Allerg 35, 491-501, 1980) have proposed that patients be screen for allergies to select only those likely to benefit fro detailed investigation. An efficient and convenient scr would reduce the unnecessary expenditure of time and mon at the allergy clinic for most mild sufferers. Merrett al. have demonstrated that by screening a patient for To IgE and IgE antibodies to three of the most common aller such as grass, house dust mite and cat hair, as high as (261 out of 275) of RAST-positive patients, out of a tot of 425 tested, with allergies could be predicted. The concept and advantages of an allergy screen have been su ported further by Atkinson, P., Henderson, G. . and Kirw P. (The Lancet, March 26, 1983) who advocate enlarging t screening panel to ten or more allergens to minimize fal negatives.

Commercial screening tests for allergies are now av able which utilize (1) single or mixed allergens coupled paper discs for reaction with sample and subsequently wi radioactive- or enzyme-labeled second antibody to human (Kallestad Laboratories, Inc.). To test a single sample against more than one disc requires accurate dispensatio reagents into separate reaction chambers for each disc a expensive instrumentation for measurement. Other tests utilize (2) single or mixed allergens insolubilized on o plastic wells for reaction with sample and enzyme-labele second antibody to human IgE (Ventrex Laboratories, Inc.; Immunomedics, Inc.). This technology requires dispensin reagents into separate test chambers, careful handling o the test device to avoid spilling, and a moist chamber t reduce evaporation during the long incubations necessary.

Efforts have been made to enable multiple simultane testing for allergen induced antibodies. U.S. Patent 3,941,876 to Marinkovich discloses a method for determini the allergic hypersensitivity to a large number of allerg which are coated to cellulose threads fixed within a sing test chamber. The chamber is filled with serum sample an incubated for 16-24 hours, then washed 3 times and filled with radioactively labeled antilgE antibody reagent. Aft another 16-24 hours incubation, the chamber is washed aga and bound radioactivity to specific allergen bands measur by exposure onto photographic film. A further developmen of this technique utilizes a che ilumine-scent-labeled an IgE antibody reagent (Brown, C.R. et al., Clin. Che . , 31 1500-1505, 1985). This technique is more a quantitative assay, too elaborate and costly to be used as an allergy screen. Both modifications, utilizing either radioactive or luminescent-labeled antibody, require instrumentation measurement. Both modifications require 24 to 48 hours t complete the test. Chandler, H.M. and Hurrell, J.G.R. (Clin. Chim. Acta 121: 225-230, 1982) describe a device for use in detecti or determining the presence of antigenic or haptenic sub¬ stances or antibodies in a sample comprising a plurality tubular or capillary elements each having antibodies, ant genic or haptenic substances attached to the internal sur face thereof, and means for causing fluids to pass simul¬ taneously or sequentially through the plurality of capill elements. A method and test kit for detecting and deter- mining the presence of antigenic or haptenic substances, e.g., snake venoms, in a sample by an enzyme-linked immun sorbent assay technique is described as using urease as t enzyme in an antibody-enzyme or antigen-enzyme conjugate, with urea being used as the enzyme substrate wherein the presence of ammonia is detected or determined using di- bromo-O-cresolsulfophthalein indicator. Similar capillar immunoassays have been described by Friedel in Acta Endo- crinol♦ Suppl. 202:33-35 (1976) "Methodological principle of the capillary radioimmunoassay" and Heinrich in German Offenlegungschrift 1,598,224. The use of capillary tubin offers the advantages over the device described in U.S. Patent 3,941,876 of requiring much smaller volumes of re¬ agents and, by virtue of the much larger surface area to volume ratio, much faster reaction kinetics. This capill device described by Chandler and Hurrell (1982) utilizes glass capillary tubes which break easily in shipment and handling. In addition, assembling such a device of multi capillaries into a single tubular chain is difficult and time-consuming with the connections likely to leak reagen fluid during the test.

Summary of the Invention The present invention describes a device and its app cation for testing a fluid sample simultaneously and rapi for the presence of antibodies to a plurality of antigens for the presence of antigens to a plurality of antibodies combinations thereof. The device consists of a capillar tubing encasing a series of water-insoluble segments, ea having different antigens and/or antibodies attached to surface, and a means of filling and emptying the device reagents. The analytes in the sample, whether antibody antigen, are immobilized through immunocomplexing with specific reagents on the segment surface and are measure subsequent reaction with labeled immunochemical reagents. Measurement of bound analyte is possible by monitoring radiation emitted through the wall of the tubing about specific segments.

Description of the Drawings Figure 1 is a sectional view through the device (sc of 1) depicted as a particular embodiment for use in scr ing for allergies. A capillary tubing, 3, contains with it six reactive water-insoluble segments 4, 5, 6, 7, 8 a separated by non-reactive water-insoluble segments 10. syringe, 1, is connected to the capillary tubing through means of attachment, 2. At the other end of the capillar tubing, a tubing, 11, of narrower bore is attached. Fig 2 is an enlarged view (scale of 4) of a portion of the device. Each of the six reactive segments has a differen immunochemical reagent attached to its surface as follows Segment 4 - A 1st mixture of allergens e.g. grasses Segment 5 - A 2nd mixture of allergens e.g. mite/mou Segment 6 - A 3rd mixture of allergens e.g. weeds Segment 7 - A 4th mixture of allergens e.g. trees Segment 8 - A 5th mixture of allergens e.g. cat and allergens Segment 9 - Anti- IgX antibodies e.g. anti-human IgE antibodies

Detailed Description The test of the present invention is particularly suited for analyzing a single biological fluid for the presence of antibodies to a panel of antigens and/or for presence of antigens to a panel or antibodies. Reactions are carried out within a capillary tubing chamber which, because of a high surface area to volume ratio, requires small reagent volumes and much shorter reaction times tha conventional immunoassays. In a specific embodiment of t invention, the device is used to detect IgE antibodies to defined allergens as well as Total IgE levels in serum or plasma. In a nonisotopic enzyme i munoassay, reaction wi substrate produces color changes that can be recorded vis ually through the clear encasing capillary tubing. The t is ideally suited for use in a physician's office since i requires little technical skill to perform, is completed less than three hours and requires no instrumentation for testing or measurement.

,In the particular application as a test for allergie it is advantageous to detect the presence of antibodies t variety of allergens as well as to obtain an estimate of level of Total IgE in the sample. Based on the findings Merrett et al., clinically accurate diagnosis of severe allergies can be obtained by testing against a few of the major allergens such as a major grass, weed, tree, animal mould allergen. Reducing the number of allergens and hen segments in the device to a small number (less than 10) compared to 30-70 allergens in the RAST device (Marinkovi et al.) reduces the cost of the test to the physician and patient, while satisfying the purpose for which the test designed, identification of severe allergies. Since a patient may on occasion have restricted allergies to one more allergens, the probability of false negatives is red by utilizing a mixture of two-to-four of the major allerg in a grass mix, or weed, tree, animal or mite/mould mixes immobilized on the solid surface.

Since floral allergens are more or less indigenous t restricted geographical regions and flourish at different seasons, the three floral allergen mix segments in the te device, i.e., grass, weed, and tree mixes, in specialize regional allergy screens will contain the major floral allergens specific for a season and geographical region. The remaining cat, dog, mite and mould allergens would b common to regional screens across the U.S.A. In general the major allergens in any restricted region throughout world should be used in the test device for clinically diagnosing IgE antibodies and hence allergies in patient within that region. More specialized screens can be ass bled wherein the allergens in a single device are restri to grasses only, or weeds, trees, moulds, foods, insect stings, only. Single allergens can be coupled to each s segment as well as mixtures of allergens.

Preparation of the device of this invention involve coupling allergens, antigens or antibodies to the surfac a water-insoluble solid phase. Exemplary materials of t solid phase include glass, hydrocarbon polymers such as polystyrene, polyethylene, polyvinyl chloride, polypro¬ pylene, nylon, etc. or copolymers. The surfaces may be treated in such a way to form reactive functional groups such as H₂, COOH, isocyanate, to facilitate the conjuga of reagent to the solid surface. The antigens or antibo may be attached to the solid phase surface by known tech niques such as passive adsorption and covalent bonding. exact procedure will depend on the composition and conce tration of the material to be coupled and its affinity o bonding to the water-insoluble surface. Thus, some aller gens of high potency or concentration can be coupled rea to plastic surfaces by passive adsorption whilst others o lower potency or coatability may require covalent couplin through some chemical coupling agent. Exemplary procedur of covalent coupling involve treating the polymer surface with glutaraldehyde prior to reaction with the allergen extract solution in a 2-steρ procedure, or reacting a mix ture of allergen and glutaraldehyde with the polymer in a step procedure. Since allergen extracts can differ in potency from lot to lot and from one allergen to another, each extract solution should be coupled at different con¬ centrations to the solid phase and tested with sera con¬ taining antibody to the allergen to define conditions pro ducing maximum reactive capacity. When a mixture of alle gens is to be coated to a single solid surface, concentra tions of individual allergenic components in the mixture should be similarly optimized. By limiting the number of components in a mixture to two-to-four major allergens, steric interference of binding of antibody to any one com ponent can be minimized. It is preferred, though not essential, after coupling of the antigen or antibody to t solid surface, to treat the surface with an inert materia such as gelatin or albumin, to saturate any residual acti sites on the surface. This treatment can be utilized to stabilize the coated surface and to reduce nonspecific adsorption to the surface during the test.

In a particular embodiment of the device, solid poly vinyl chloride (PVC) rod-like segments, 1 cm long by 0.18 diameter are activated with a 1% (v/v) aqueous glutaralde hyde solution, washed, then reacted with allergen or anti body solution for 16-24 hours, washed, then postcoated to stabilize the immobilized allergen or antibody. Segments coated with different allergens and anti-IgE^" antibody (he inafter referred to as "reactive segments") are inserted into a clear PVC capillary tubing of 0.23 cm inner diamet with inert segments, 10, acting as spacers separating the reactive segments. The inert segments may be cut from th same PVC material as the reactive segments but used un- treated as spacers. To one end of the capillary tubing i attached a narrower capillary tubing, 11, which serves to retain the segments and through which reagents are drawn into the apparatus. The distal end of the capillary tubi is connected to a syringe which is used to fill and empty the device of reagents. The cross-sectional dimension of the segments should be large enough to prevent one segmen overlapping another within the capillary tubing. The le of the capillary tubing should be sufficient to contain segments arranged end-to-end. A small inner diameter of capillary tubing, 3, is preferred for rapid kinetics of reaction and to minimize the volume of reagents required the test. The capillary tubing of optically clear mater is preferred to facilitate the detection of positive, spe fic changes in color or fluorescence about reactive segme within the device. In an enzyme immunoassay, wherein a change in color or fluorescence occurs in a liquid substr reagent within the capillary tubing, inert spacers sepa¬ rating reactive segments are preferred and should be of sufficient length to restrict diffusion of colored produc from one reactive segment to another, leading to false positives within the desired time of measurement. As a specific example, 1.5 cm spacers have been used to allow measurement of specific positive signals up to at least o hour after filling the tubing with substrate. In contras the lengths of reactive segments are not critical and may vary between 0.5 and 2 cm. In a quantitative radioimmuno assay wherein the labeled reagent bound specifically to t surface of reactive segments is measured, the reactive segments should be of equal length and inert spacers may omitted. in a typical embodiment of the apparatus depicted in Figure 1 as used for allergy screening in an enzyme immun assay, the capillary tubing, 3, is 18.5 cm long and 0.23 inner diameter, contains within six 1 x 0.18 cm reactive segments, 4-9, separated by seven 1.5 x 0.18 cm inert spacers, 10. The outlet tubing, 11, is 6 cm long, 0.23 c outer diameter and 0.15 cm inner diameter, and is attache by gluing to the capillary tubing, 3, to prevent leakage reagents. The syringe connector, 2, may also be glued in the capillary tubing to prevent leakage. One embodiment of the invention involves drawing the sample fluid into the capillary tubing which contains six reactive segments coated with a grass allergen mix, a weed allergen mix, a tree allergen mix, a mixture of cat and do hair allergens, a mixture of house dust mite and mould allergens and anti-Ige antibody as illustrated in Figure 1 The apparatus stands at ambient temperature for a predeter mined time to allow IgE antibodies to react with the coate segments, forming insolubilized immune complexes on their surfaces. The reaction time and temperature may vary wide from 5 minutes to 24 hours and 0°C to 50°C, respectively. Most preferably, because of the rapid kinetics of reaction within capillaries (Friedel, R., Acta Endocrinol., Suppl. 202, 33-35, 1976), the reaction is carried out for 5 to 60 minutes at 18° to 30°C. By means of the syringe the sampl fluid is then expelled and the apparatus rinsed internally with water, or buffer of neutral pH (pH 5 - 9) or detergen in water or buffer, to remove protein not specifically adsorbed to the surface of reactive segments within the device. Labeled second antibody, specific to IgE from the sample which is complexed to the reactive surface, is next drawn into the apparatus to fill the capillary tubing.

Reaction is allowed to proceed at ambient temperature for predetermined time, dictated principally by the affinity a concentration of the second antibody. High affinity anti¬ bodies require short reaction times of 5-30 minutes, while low affinity antibodies require longer times.

Where the labeled antibody is a monoclonal antibody, is possible to premix this reagent with the sample prior t drawing the mixture into the apparatus. This modified procedure eliminates the need for a second reaction period with the labeled antibody.

The apparatus is rinsed again to remove excess labele antibody in solution or nonspecifically adsorbed to sur¬ faces.

If the label used is a radioactive isotope such as

125 3 14 I, H, C, etc., the reactive segments are then remove and specifically bound radioactivity measured in an appro- -U

priate gamma or scintillation counter.

If the label used is an enzyme conjugated to the se antibody, after incubating with labeled second antibody washing, substrate reagent is drawn into the apparatus t fill the tubing. Enzyme-antibody complexed with IgE on reactive surface catalyses the conversion of substrate t product which in the presence of chromogen leads to a vi ble color change around reactive segments to which IgE antibody from the sample has complexed. Several enzyme- substrate systems can be utilized in the test. Typical examples are an antibody-horseradish peroxidase conjugat which with hydrogen peroxide (substrate) and tetramethyl benzidine (chromogen) produces a colorless to blue color change, and an antibody-urease conjugate which with urea (substrate) and bromocresol purple (chromogen) produces yellow to purple color change.

In a qualitative test for allergies, for example, a filling the tubing with substrate reagent, the apparatus observed for several minutes for color development while undisturbed to prevent mechanical mixing of contained fl reagents. The intensity of color developed is directly proportional to the amount of enzyme-second antibody com plexed to the surface, which in turn is proportional to amount of analyte, in this case IgE, complexed to reagen (allergens) coupled to the surface of the reactive segme Strong positive reactions are easily visible as rapid, intense color development within the capillary tubing ar a specific reactive segment. Weak positive reactions are indicated by slower color development. Negative reactio around reactive segments and inert spacers show no color change within the prescribed period of observation.

In addition to i munoassay systems which produce vis ble color changes, other immunoassay systems may be emplo that yield detectable fluorescent, luminescent or colored reaction products around or upon the surface of reactive segments. Qualitative applications of the test device ca be realized where the positive signal can be recorded by visual examination, such as in the case of fluorescent or colored products. Quantitative applications of the cest device are possible with suitable instrumentation for mea- suring light energy emitted through the wall of the capil¬ lary tubing.

In general, the device of this invention can be appli to a variety of immunoassay procedures, of which the fol¬ lowing are some examples: 1. Antibody detection:

(a) Antigens or low mol. wt. haptens coupled to the segments react with first antibodies in the sample which in sequence are made to react with labeled second antibody specific to the first antibodies in the sample.

(b) Sandwich method, differing from 1(a) in tha after reacting with insolubilized antigen o hapten, the antibody from the test sample i made to react with labeled antigen or hapte In a multiple screen, the labeled reagent i a mixture of the different antigens or hap¬ tens used in the test that have been labele 2. Antigen or hapten detection:

(a) Sandwich method. Antigens in the sample ar made to react with specific antibodies in¬ solubilized onto the solid surface, and are measured by reaction with labeled antigen- specific antibodies. Again, in a multiple screen the labeled reagent is a mixture of antibodies, each with specificity to one of the antigens to be detected.

(b) Competitive inhibition method. Antigens or haptens in the sample compete with labeled antigens or haptens for binding to insolu- bilized antigen- or hapten-specific antibod and are measured bv the extent of inhibitio of binding of labeled antigen or hapten t its homologous antibody on a particular segment. Thus, the device and method of this invention can b utilized both as a qualitative and quantitative test, no only for antibodies mediating allergies, but for other multiple analytes, such as a panel of cancer antigens o viral antigens, e.g.. Hepatitis A, B, HTLV-III, or bacte antigens, or small molecules or haptens such as drugs of abuse, as well as for the presence of antibodies to these antigens or haptens.

The invention also contemplates a kit for testing a sample for Total IgE and IgE antibodies to allergens. Preferably, the kit will contain the assembled capillary tubing device and syringe, labeled second antibody reagen (and substrate reagent if an EIA) , wash reagent and instr tions for use.

The present invention is further illustrated by the following examples:

EXAMPLE I

This example demonstrates the preparation of the cap lary tubing device.

(a) Preparation of Reactive Segments: Aqueous alle gen extracts in 50% glycerol (Hollister-Stier, Spokane, W are dialyzed in membranes of MW CO 3,500 against 0.01 M phosphate buffer, pH 7, to remove glycerol and low molecu material. Solid, rod-like polyvinylchloride segments, 1 long by 0.18 cm diameter, are washed in 0.01 M pH 7 phos¬ phate buffer, then reacted with 1% (v/v) glutaraldehyde i the same pH 7 buffer for 60 + 10 minutes at ambient tempe ature (18-30°C) with gentle rotation. The segments are washed rapidly in pH 7 buffer to remove excess glutaralde hyde, then reacted with the allergen or a mixture of alle gens at an appropriate dilution in 0.01 M pH 7 buffer for + 4 hours at ambient temperature with gentle rotation. Th segments are washed in pH 7 buffer, then reacted with 0.1% (w/v) human serum albumin (HSA) s_^/ 4 hours at ambient temperature to block residual glutaraldehyde-active sites their surface. Finally, the segments are treated with a 2.5% (w/v) Sucrose solution in physiological saline /~\_^* 4 hours at ambient temperature. The Sucrose solution is removed and the segments allowed to dry at ambient temper¬ ature. Similarly, 1 cm segments are coated with monospecific anti-(human IgE) IgG antibody by activation with 1% glu¬ taraldehyde, reaction with the IgG antibody at an appro¬ priate dilution, followed by treatment with HSA and Sucros solutions, then dried. In this manner, separate lots of segments are coated with (1) a mixture of Bermuda, Kentucky Blue, Timothy and Perennial Rye grass allergens, (2) a mixture.of Short Rag¬ weed, English Plantain and Lamb's Quarters weed allergens, (3) a mixture of Cottonwood, Elm, Mesquite and Mountain Cedar tree allergens, (4) a mixture of D. farinae mite an Alternaria tenuis mould allergens, (5) a mixture of cat a dog allergens, and (6) with anti-IgE antibody.

To optimize the analytical performance of reactive segments, the allergen or antibody solution is coupled to the segments at increasing concentrations, then tested wi highly allergic human sera of appropriate specificity or Total IgE level and labeled ( 125I- or urease enzyme-) mono specific anti-IgE antibody to determine the optimum coati concentration of the allergen or antibody solution. (b) Assembly of the Device:

Optically clear, semirigid PVC capillary tubing, 18.5 cm long by 0.23 cm inner diameter, is first fitted wi a syringe connector at one end. The tubing is then loade with one each of the dried reactive segments coated with grass mix, mite/mould mix, weed mix, tree mix, cat/dog mi and anti-IgE antibody. A narrower tubing, 6 cm long, is attached to the other end of the capillary tubing for in sertion into reagents. Finally, a syringe (1 ml capacit is connected to the capillary tubing device. The assemb apparatus is illustrated in Figure 1. If the device is to be used in an enzyme immun assay, inert untreated PVC segments, 1.5 cm long and cut from the same or similar material as the reactive segmen are inserted alternately with the reactive segments to a as spacers between the segments. If the device is to be used in a radioimmunoassay, the inert spacers may be omi ted.

EXAMPLE 2

This example demonstrates the application of the de as a quantitative screen for allergies in a radioimmuno- assay.

(a) Materials: PVC segments are coated with mixes allergens and anti-IgE antibody, then assembled as the screening device, as described in Example 1. The allerge mixes are also coupled to cyanogen bromide-activated pape discs, 5 mm diameter, cut from Whatman No. 541 filter pap as described by Ceska and Lundkvist (Immunochem. 9:1021-3 1972) .

Affinity purified goat anti-human IgE antibody (Atla t c Antibodies Scarborough, ME) is labeled with 125I usin the chloramme-T method. The 125I-antibody is isolated b

Sephadex G25 gel filtration and rendered monospecific for human IgE by absorption with a heat-inactivated immunosor bent of normal human serum coupled to CNBr-activated seph rose (Pharmacia, Ltd) . Human serum samples are selected from nonatopic indi viduals and individuals with mild, moderate and severe allergies, characterized by quantitative assay in the Rad AllergoSorbent Test or RAST. (b) Procedure: (i) Quantitative paper disc RAST. 100 jul volu of each serum sample is reacted with the allergen-discs f 3 hours at ambient temperature, washed 3 times with 1 ml

0.2% (v/v) Triton X-100, 0.05 M NaCl in water, then react with 100 ul of 125I-anti-human IgE reagent for 16-24 hour at ambient temperature. The discs are again washed 3 tim and the radioactivity bound to each disc is measured.

(ii) Quantitative device RAST. With the aid of the attached syringe, the sample is drawn into the device to the syringe connector. The apparatus is allowed to si at ambient temperature for 2 hours. The sample is dispel from the tubing and the syringe plunger removed completel Using a squirt bottle 1 ml of the Triton X, NaCl wash sol tion is poured into the syringe barrel and allowed to dra through the device washing the segments.

The plunger is replaced in the syringe and the capillary tubing filled with 125I-anti-human IgE. Th apparatus is allowed to sit at ambient temperature for 16 hours. The tracer is dispelled, the syringe plunger remo and the device washed three times with 1 ml wash solution

The segments are carefully removed from th capillary tubing in order, placed into test tubes and the bound radioactivity measured.

(c) Results: The bound radioactivity (counts per minute, CPM) measured on the paper discs and the reactive segments of the device are listed in Table 1(a) for three serum samples. The CPM bound in the conventional paper d RAST for the individual allergen components in the mixtur as well as for the mixtures for samples 1 and 3 demonstra that by using a mixture of allergens on a single solid phase, one can obtain a representative value for the leve of IgE antibodies to the allergens examined. Thus, the results of sample 1 indicate high allergies to grasses, moderate to low allergies to weeds and trees, respectivel and no allergies to cat, dog, mite and Alternaria. Pro¬ portional signals are also obtained in the device RAST fo these 5 allergen mixes.

The CPM bound on the reactive segments are much lowe because (1) there is a higher density of the allergens on the discs compared to the plastic segments, and (2) the volumes of reagents (sample and tracer) in the paper disc RAST are more than 6 times those involved in the reaction with each segment in the device. If the CPM bound speci¬ fically is expressed as a percentage of the Total CPM of

125 I-antibody involved, then the close correlation become evident (Table lb) . Thus, using 125I-labeled antibody, the device of the invention can be used to quantitatively assay IgE antibod to a variety of allergens in a single test procedure, wit good correlation to the conventional RAST with respect to intensity and specificity of signals but with much less hands-on time and technical skill required by the procedu

TABLE 1 (a) Comparison of signals (CPM bound) between the Paper RAST and the Device RAST

Sample 1 Sample 2 Sample 3 Allergen Paper Device Paper Device Paper Devi

RAST RAST RAST RAST RAST RAS

31584 23973 31554 31263

8826 860 179 43696 85

Short Ragweed 13950 8930

English Plantain 6877 7514

Lamb's Quarters 1931 3170 Weed Mix 13362 1323 750 197 12748 13

3197 6696 5715 5722 688 656 145 9987 10

5202 13929 266 571 306 11198 14

33006 1493

395 675 146 28824 61 (b) Comparison of signals (% Total Counts bound) betwee the Paper RAST and the Device RAST

This example demonstrates the application of the cap lary device as a visual allergy screen in an 'enzyme immun assay (EIA) . (a) Preparation of the Enzyme-Antibody conjugate:

Monospecific anti- (human IgE) goat IgG isolated from serum by (NH. SO. precipitation and molecular sievin through an AcA34 column is coupled to Type VII Urease enzy using N-Succinimidyl 3- (2-pyridyldithio) propionate (SPDP) The active conjugate is purified by filtration of the re¬ action product on an AcA22 (1.5 x 120 cm) column. The conjugate is diluted in physiological saline containing protein stabilizers, detergent and EDTA to the optimum concentration required in the test. The substrate/indicator solution is prepared by dissolving 40 mg Bromocresol Purple indicator in 7.4 ml 0. M NaOH and diluting to 450 ml with deionized water. 500 m Urea substrate and 37 mg disodium EDTA are then added to t BCP solution and the volume adjusted to 500 ml with water. The pH of the substrate/indicator solution is adjusted to 4.8, if necessary.

(b) Serum Samples: Serum samples tested in the devi in an EIA have been characterized for the level of IgE antibodies to the various allergens by the quantitative paper disc RAST and for levels of Total IgE by a commer¬ cially available kit, the Kallestad QuantiCLONE RIA for Total IgE.

(c) Test Procedure:

1. By means of the attached syringe, fill the capillary tubing up to the syringe connecto with sample.

2. Allow the device to react for 60 minutes at ambient temperature (18° - 30°C) .

3. Dispel the sample, remove the syringe plung • and wash the device by dispensing 1 ml dete gent-saline wash solution into the syringe barrel. Replace the syringe plunger.

4. Fill the capillary tubing with the enzyme- antibody conjugate and allow to react for 6 minutes at ambient temperature.

5. Dispel the conjugate and wash the device as described in Step 3 hereinabove four times with 1 ml wash solution. Replace the syrin plunger. 6. Fill the capillary tubing with the substrat indicator solution, lay the device down on the bench and observe for the occurrence of specific yellow to purple color changes in the solution about each reactive segments within the next hour.

(d) Results :

To relate to the data listed in Table 2, the signals (CPM bound) obtained in the RAST have been rated a follows: CPM Bound Score

In this way, the quantitative results obtained in the RAST can be more easily compared with the subjective, semi quantitative results, scored visually on a scale of 0 to 4 in the Allergy Screen EIA. The results of the RAST are compared with those of the EIA scored after 30 minutes incubation with the substrate/indicator.

The date in Table 2 demonstrate the good correlation between the conventional quantitative RAST which is an overnight procedure and the more rapid, semiquantitative E performed in the device of the invention in its applicatio as an allergy screen. Elevated levels of IgE antibody in sample produce an intense yellow to purple coloration abou a specific reactive segment almost immediately on filling the capillary tubing with substrate/indicator. Moderate t low levels of IgE antibody cause a specific color change which takes progressively longer. In this particular appl cation of the device, the system has been optimized to yie relative levels of IgE antibody from low to high after 30 minutes with substrate/indicator. The Total IgE segment h been optimized so that normal levels of IgE, 50-150 I.U./m would yield a score of 3 after 30 minutes. The user of th test can then estimate samples to have low, normal or ele¬ vated levels of Total IgE. TABLE 2 Correlation between the RAST(R) and Allergy Screen EIA(E

Obviously, if left sufficiently long, continued catal ysis of substrate by enzyme and diffusion will cause the fluid throughout the device to turn purple, hence the need to define the lengths of the inert spacers within the devi and the period of observation for measurement after fillin with substrate/indicator. It should also be obvious to those experienced in the immunochemistry of immunological reactions that optimum incubation times for each reaction step will depend on the affinity of the antibodies involved and the concentration the reagents in the test. Measurement of analytes at high concentrations can be accomplished within very short incu¬ bation times, 5-10 minutes, whereas measurement of analyte at extremely low concentrations such as IgE antibody in test, will require hours of incubation.

Claims

What is claimed is:

1. A device for simultaneously detecting or quanti- tating a plurality of analytes in a biological fluid sampl which device comprises: (a) a capillary tubing,

(b) a plurality of water-insoluble reactive segments located in said capillary tubing, each segment having different immunochemica reagents attached to its surface, (c) nonreactive water-insoluble segments locate in said capillary tubing for separating sai reactive segments from one another, with sa reactive and nonreactive segments alternati serially in said capillary tubing, (d) ^• means of attachment at both ends of the capillary tubing which serve to retain said segments within said capillary tubing, eac of said means of attachment having a bore i fluid connection with said capillary tubing and

(e) means for causing fluids to pass into said capillary tubing.

2. The device of claim 1 wherein a syringe is con¬ nected to said capillary tubing via one of said means of attachment at one end thereof and serves as said means for causing fluids to pass into said capillary tubing.

3. The device of claim 1 wherein said means of at¬ tachment at the other end of said capillary tubing compris an end tubing.

4. The device of claim 1 wherein said water-insolub segments are of glass or polymeric plastic materials or derivatives thereof.

5. The device of claim 1 wherein the dimensions of said segments are of large enough in cross-section to pre¬ vent overlapping of segments within said capillary tubing.

6. The device of claim 1 wherein said immunochemica reagents may be antibodies, antigens or low molecular hap tens which can be attached to the surface of said water- insoluble segments by passive or covalent bonding.

7. A method for simultaneously detecting or determ a plurality of analytes in a biological fluid sample with the use of an apparatus comprising a syringe connected to capillary tubing with a plurality of water-insoluble re- active segments located in said capillary tubing, each segment having attached to its surface a different immuno chemical reagent that binds specifically with said sample analytes, with nonreactive segments alternating serially with said reactive segments in said capillary tubing, the method comprising the steps of:

(a) filling said capillary tubing with the flu sample,

(b) allowing the analytes in said fluid sample react with said specific immunochemical reagents on said reactive segments,

(c) removing said fluid sample from said capil lary tubing,

(d) washing said capillary tubing internally t remove sample components not specifically bound to reactive segments,

(e) filling said capillary tubing with a label second immunochemical reagent capable of reacting with one of the components in ste (b) on the surface of each reactive segmen and allowing the reaction to take place,

(f) removing said labeled second immunochemical reagent from said capillary tubing,

(g) washing said capillary tubing internally to remove said labeled second immunochemical reagent not specifically bound to reactive segments, (h) measuring the amount of said labeled second immunochemical reagent specifically bound t reactive segments.

8. The method of claim 7 wherein said second immuno chemical reagent is labeled with a radioactive isotope, fluorescent molecule, luminescent molecule, or enzyme.

9. The method of claim 7 wherein said immunochemica reagents attached to reactive segments are antigens or low molecular haptens for the detection of a plurality of spec fic antibodies.

10. The method of claim 7 wherein said immunochemica reagents attached to reactive segments are antibodies for the detection of a plurality of specific antigens or hap¬ tens.

11. The method of claim 7 wherein said immunochemica reagents attached to reactive segments are antigens, hap¬ tens, and/ or. antibodies, though on separate segments in said capillary tubing, for the simultaneous determination a plurality of analytes in a single sample, which analytes comprise antibodies to said attached antigens or haptens, and antigens or haptens to said attached antibodies.

12. The method of claim 7 wherein said plurality of analytes are immunoglobulins of the class IgX, where X is selected from the group A, D, E, G, and M, and said labele second immunochemical reagent is labeled second antibody specifically reactive with said IgX immunoglobulin.

13. A method for the simultaneous detection or dete mination of both IgE antibody to various allergens and To IgE immunoglobulin in a biological fluid sample with the of an apparatus comprising a syringe connected to a capil- lary tubing with a plurality of water-insoluble reactive segments located in said capillary tubing, one of said reactive segments having attached thereto anti-IgE anti¬ bodies for the determination of Total IgE and the remainde of said reactive segments having attached thereto differe allergens for the determination of IgE antibodies, with nonreactive segments alternating serially with said react segments in said capillary tubing, the method comprising steps of:

(a) filling said capillary tubing with said flui sample,

(b) allowing IgE in said fluid sample to bind specifically with said reactive segments,

(c) removing said fluid sample from said capil¬ lary tubing, (d) washing said capillary tubing internally to remove sample components not specifically bound to said reactive segments,

(e) filling said capillary tubing with enzyme- labeled anti-IgE antibody and allowing it to react with analyte bound specifically to sai reactive segments,

(f) removing said enzyme-labeled antibody reagen from said capillary tubing,

(g) washing said capillary tubing internally to remove enzyme-labeled antibody not bound specifically to reactive segments, (h) filling said capillary tubing with substrate for the determination of enzyme activity bound specifically to reactive segments.