EP0438457A1

EP0438457A1 - A method for simultaneous detection of different types of antibodies and/or antigens produced by individual cells

Info

Publication number: EP0438457A1
Application number: EP89911411A
Authority: EP
Inventors: Anders Vahlne; Cecil Czerkinsky
Original assignee: Syntello AB
Current assignee: Syntello AB
Priority date: 1988-10-14
Filing date: 1989-10-13
Publication date: 1991-07-31
Also published as: KR900702371A; AU4346989A; WO1990004182A1

Abstract

Dans le procédé décrit, qui sert à la détection d'anticorps et/ou d'antigènes sécrétés et/ou libérés par des cellules individuelles, une suspension de cellules est mise en contact avec un support solide. Après ou pendant cette opération de mise en contact, on ajoute au moins deux types différents d'autres anticorps dirigés contre respectivement les anticorps ou les antigènes à détecter et ayant la capacité de se lier respectivement à ces anticorps ou à ces antigènes à détecter, ces autres anticorps étant en outre dotés d'enzymes différentes. On ajoute ensuite le nombre correspondant de substances indicatrices, lesquelles ont un effet colorant différent pour la détection simultanée des différents types d'anticorps et/ou d'antigènes par évaluation des tâches de couleurs distinctes restant sur le support solide. Selon le mode de réalisation décrit, on utilise deux supports solides, aux surfaces opposées desquels sont attachés des antigènes et/ou des anticorps du même type ou d'un type différent, la suspension de cellules étant appliquée entre ces surfaces.In the method described, which serves for the detection of antibodies and / or antigens secreted and / or released by individual cells, a suspension of cells is contacted with a solid support. After or during this contacting operation, at least two different types of other antibodies directed against the antibodies or the antigens to be detected and having the capacity to bind respectively to these antibodies or to these antigens to be detected, are added. other antibodies are also endowed with different enzymes. The corresponding number of indicator substances is then added, which have a different coloring effect for the simultaneous detection of the different types of antibodies and / or antigens by evaluation of the distinct color spots remaining on the solid support. According to the embodiment described, two solid supports are used, to the opposite surfaces of which antigens and / or antibodies of the same or of a different type are attached, the cell suspension being applied between these surfaces.

Description

A METHOD FOR SIMULTANEOUS DETECTION OF DIFFERENT TYPES

OF ANTIBODIES AND/OR ANTIGENES PRODUCED BY INDIVIDUAL CELLS.

Tne present invention refers to a method for detection o antibodies and/or antigens secreted and/or released by individua cells, at which a cell suspension is brought in contact with solid carrier, and after that or simultaneously therewith othe antibodies are added which are directed against and have a ability to bind to the antibodies or antigens that are to b detected, and which other antibodies are provided with an enzym reacting with an indicator substance for detecting the antibodie or antigens in question.

Background of the invention

Laboratory diagnosis of e.g. virus infections is mainly based o either detection of the infection-inducing virus itself or part thereof, so called antigens, in samples from patients, or whic is more common on indication of antibodies in the patient' blood directed against the virus. A coπuπon method for antibod detection is ELISA ("enzyme linked immunosorbent assays") Recently a variant of this technique has been developed, calle ELISPOT. According to the original ELISPOT-technique antibodie labelled with either alkaline phosphatase (AP) (J. Immunol Methods 1883, 57, 301; Sedgwich et al, "A solid phase immuno enzymatic technique for the enumeration of specific antibody secreting cells") or horseradish peroxidase (HRP) (J. Immunol Methods 1983, 65, 109; (Czerkinsky et al. "A solid phase enzyme linked immunospot (ELISPOT) assay for enumeration of specifi anti-body secreting cells") and corresponding chromogen subs trates are used for the detection of antibody-producing cells.

The object and most important features of the invention

The object of the present invention is to provide a method base on ELISPOT, by means of which it is possible to detect simul taneously at least two types of antibodies and/or antigens. Th;. has according to the invention been achieved by the fact that a least two types of said other antibodies are added directe against at least two different types of antibodies that are to b detected and which other antibodies are provided with differen enzymes, and that then the corresponding number of indicato substances are added having different staining effect, fo simultaneous detection of the different types of antibodie and/or antigens by evaluation of spots of distinct colour remaining on the solid carrier.

Description of the drawing

The invention will below be described more in detail wit reference to the accompanying drawing, which schematically show a developed plate having spots of different colours indicatin presence of two different types of antibodies and/or antigens said different colours being indicated as filled and unfille spots.

Description of the invention

According to the described example a combination of the two abov mentioned indicator systems in the form of AP- and HRP- labelle antibodies and corresponding chromogen substrate were used fo simultaneous detection of distinct types of antibody-producin cells. In the described example zones of IgG and IgA antibodie bound to a solid carrier and produced by distinct cells wer visualized in the form of blue and red spots resp. correspondin to either of these different types of antibodies.

As the test four adult human volunteers received an intramuscula injection of a trivalent influenza virus vaccine, types A and (Wyett Laboratories, PA). Peripheral blood mononuclear cell (PBMC) were collected from 5 to 12 days after immunization, tha is at a time when the frequency of antigenspecific spontaneou ASC in the circulation is known to be maximal. PBMC were isolate from heparinized venous blood by centrifugation on Ficoll-Hypaqu (BOyum, 1968). Interface PBMC were washed twice with isotoni phosphate-buffered saline (0.01 M phosphates, 0.15 M NaCl, pH 7.4) (PBS), and resuspended at the appropriate densities in assay culture medium. The latter consisted of RPMI 1640 (Gibco, Glasgow, Scotland) supplemented with 5% foetal bovine serum (FBS) (Irvine Scientific, Santa Ana, CA).

Reagents

AP- or HRP-cojugated affinity-purified goat anti-human IgA and goat anti-human IgG anti-bodies were purchased commersially. The AP chromogen substrate soultion, consisting of 5-bromo-4-chloro- 3-indolyl phosphate toluidine salt and p-nitroblue tetrazolium chloride (BCIP/NBT) was prepared according to the manufacturer's instructions (Bio-Rad Laboratories, Richmond, CA). Initially, 15 mg of BCIP reagent and 30 mg of NBT salt were separated dissolved with 1 ml of dimethylformamide (DMF) and then added to 100 ml of a solution of 0.1 M NaHC03, 1 mM MgCl2, pH 9.8. The HRP chromogen substrate solution was prepared by dissolving 25 mg of 3-amino-9- ethylcarbazole (AEC) (Sigma, -St. Louis, MO) in 2 ml of DMF, followed by addition of 95 ml of 0.05 M acetate buffer, pH 5.0

(± 0.2) and 4 μl of 30% H2O2. The above substrate solution were filtered (0.45 μm) to remove particulate matter (the AEC/H2O2 solution becomes colourless after filtration while the BCIP/NBT solution remains pale yellow). Both enzyme substrate solutions could be kept in the dark at 4^βC for up to 1 week.

Assay

The assay consists of five stages: 1) first, a solid phase immunσadsorbent is prepared;

2) incubation of the cell suspension;

3) addition of both AP- and HRP-conjugated antibodies; ) stepwise addition of AP and HRP chromogen substrates which will yield insoluble blue and red spots, respectively; 5) enumeration of spots.

The standard ELISPOT assay was modified by using nitrocellulose membranes as the solid support instead of polystyrene. Individual wells of nitrocellulose bottomed 96-well Millititer HA plates (Millipore, Bedford, MA) were filled with 0.075-0.1 ml of PBS containing 0.2 μg of influenza virus hemaggultinin ( yett Laboratories) . Unadsorbed proteins were removed by three successive manual washings with PBS and the plates were immersed in this buffer for 5 min. Wells were the emptied of wash buffer and the outer surface of the nitrocellulose membrane was carefully dried with absorbent paper towels. In order to saturate remaining protein binding sites on the solid support individual wells were filled with 0.2 ml of assay culture medium and the plates were incubated at 37^CC for at least 30 min in a humidified atmosphere with 7% CO2. Wells exposed to an irrelevant antigen, tetanus toxoid (TT) ( yett) (0.1 μg/well), were prepared in the same way for control purposes.

The content of the wells was replaced with 0.1 ml of cell suspensions containing various numbers of PBMC. Routinely, we used at least three sets of triplicate wells. Each set of wells recieved 2 x 10⁵, 10⁵ and 5 x 10⁴ PBMC/well. Plates were then incubated undisturbed for 3-4 h at 37°C in a CO2 incubator. In one experiment, PBMC were incubated for 5 h at 37^βC with various concentrations (5 x 10~⁴ M, 10"^ M, 2 x 10~3 M) ₀f cycloheximide (Sigma) in assay culture medium, washed, resupended and plated in cycloheximide containing medium.

At the completion of the cell incubation period, plates were rinsed three times manually with PBS and three times with PBS containing 0.05% Tween 20 (PBS-T) and were then immersed in PBS-T for 5 min. The dishes were emptied of wash buffer and the outer surface of the plate was blot-dired as described in the first stage. Next 0.1 ml of PBS-T containing 1% FBS and a mixture of goat anti-human IgG and goat anti-human IgA antibodies conjugate with AP and HRP, resp. or vice versa, was added to each well. Optimal concentrations of AP- and HRP-labelled antigloubulins were determined in preliminary experiments. Concentrations ranging from 0.5 μg/ml to 2.5 μg/ml were used for both types of enzyme conjugate. Plates were incubated for 3 h at room temper¬ ature or overnight at 4 ° C . Dishes were then rinsed four times with PBS and immersed in 0.05 M Tris buffer saline, pH 8.0, for 5 min prior to development.

Dishes were emptied for wash buffer and blot-dired as above. The wells were then exposed to 0.1 ml of BCIP/NBT substrate solution and examined for the appearance of blue spots. These reactions appeared usually within 5-10 min. Plates were allowed to develop for an additional period of 5-10 rain, after which they were rinsed with PBS and blot-dired. Next, 0.1 ml of AEC/H 0 substrate was added to each well, yielding red spots within 1-3 min. Plates were allowed to develop for a variable time (up to 10 min) and then throughly rinsed with tap water. As excessive background staining may result from over-long incubation times, it is important to monitor control wells (not exposed to cells) during the enzyme-substrate reaction.

Developed plates were dried and individuial wells were examined for the presence of blue and red spots (see drawings). These reactions were enumerated under low and magnification (x40 to x60). Under low magnification, positive reactions were defined as circular, well individualized, densely granulated foxi contiguous to the background. Their diameter ranged from 0.05 mm to 0.2 mm. Small dence particles could occasionally be observed particularl with over-exposure to either enzyme substrate. These non-granula dots generally appeared above the plane of the background and could be distinguished from true spots.

In table 1 below a comparison is made between one-colour and two- colour ELISPOT assays for enumerating antigen specific IgG an IgA-secreting cells.

Peripheral blood PBMC were obtained from one volunteer 7 day after immunization with influenza virus vaccine. Values ar expressed as spot-forming cells (SFC) numbers of quadroplicat assay wells. Table 1

SFC numbers/10⁶PBMC in wells developed with:

HRP anti IgG HRP anti IgA

5080(621)^a 3730 (470

AP anti-IgA 3920(420)^b

AP anti-IgG 3445(376) 4153(537) 4540(420

4860(515) 3636(354)

^a Upper values correspond to numbers of red spots developed wit HRP-conjugated anti-Ig antibodies and AEC/H2O2 chromoge substrate

^b Lower values indicated in bold type correspond to number o blue spots developed with AP-conjugated anti-Ig and BCIP/NB substrate.

This observations indicates that AP-BCIP/NBT reactions have n appreciable effects on subsequent development of HRP-AEC/H2O reactions. Further, the sensitivity of both enzyme-substrat systems appeared comparable since similar numbers of spot-formin cells (SFC) were detected when using the same anti-immunoglobi preparation labelled with either enzyme.

The quality of the enzyme-antibody conjugates employed in thi two-colour ELISPOT assay must be carefully considered as doubl (indigo) spots may appear with conjugates containing cross reactive antibodies. Thus, monochromatic (red or blue) spot provide an ideal internal control of the specificity of the assa for detecting antigenically distinct products secreted b different cells. Further, although two enzyme-antibody conjugate may be equally specific, they may exhibit different affinitie for binding to cognate immunoglobulins and/or variable enzyme activities, resulting in different staining intensity of the corresponding spots. In such situations, a correction factor (ratio of number of spots developed with a given antibody conjugated with HRP: number of spots developed with the same antibody preparation but labelled with AP) should be applied to adjust for possible differences in sensitivity.

Under optimal conditions PBMC-secreting IgG antibodies and PBMC- secreting IgA antibodies to influenza virus could be detected simultaneously in all four volunteers examined. Virus-specific

ASC were detected as early as 5 days after systemic immunization with influenza virus vaccine. Spot forming cell (SFC) numbers reached a maximum on day 7, by day 9-12 the frequency of virus- specific SFC markedly decreased (data not illustrated). Influenza virus specific IgG-SFC and IgA-SFC responses followed a similar kinetic pattern but differed in magnitude. Thus, in three individuals, IgG-SFC predominated (5520 ± 983 day 7 SFC/10⁶ PBM versus 140 ± 69 day 7 IgA SFC/10⁶; n= 3). In the fourth voluntee the magnitude of influenza virus-specific IgA response on day 7 was very high (3910/10⁶ PBMC ± 420 SFC) being almost comparabl to that of the IgG responses (5080 ± 621 SFC/10⁵ PBMC).

The specifity of the assay for simultaneous demonstration o influenza virus specific-IgA ASC and IgG ASC was documented b several observations. First, omission of cells, coating antigen, or labelled antibodies prevented subsequent development of spo formation. Second, plating PBMC in wells coated with an irrele vant antigen (tetanus toxoid) resulted in the absence o detectable spots (Table 2). Third incubation of influenza viru immune cells with graded amounts of influenza virus antige during cell plating inhibited, in a dose dependet manner spo formation (Table 2). It should be noted that such treatment no only resulted in s reduction of SFC numbers but also in decrease in the diameter of the remaining spots. In contrast, addition of tetanus toxoid had no effects. Finally, treatment o the cells with cycloheximide prior to and during the cel incubation period markedly inhibited influenza virus specifi IgG- and IgA-mediated spot formation (Table 2). That "double" (biochromatic) spots were never observed confirmed the high degree of specificity of the enzyme labelled antibody preparation employed as class specific reagents in this assay.

In table 2 below is shown the specificity of two-colour ELISPOT assay for simultaneous detection of influenza virus specific IgA- secreting PBMC and IgG-secreting PBMC. Peripheral blood PBMC were obtained from one donor on day 7 following immunization with influenza virus vaccine. PBMC were assayed by two-colour ELISPOT assay for numbers of virus-secreting ASC. IgG SFC and IgA SFC were developed with AP-conjugated anti-IgG and HRP-conjugated anti-IgA, respectively followed by BCIP/NBT (blue) and AEC/H₂02 (red) enzyme substrates. Values represent mean SFC numbers of quadroplicate assay wells/10⁵ PBMC. Data in parentheses indicate percentages of inhibition.

Table 2

Inhibitor added SFC numbers/10⁶ PBMC per assay well IgG (blue) (IgA) red

6560 220

Influenzavirus

15μg 120 (98%) 0 (100%)

3μg 1400 (79%) 45 (80%)

0.6μg 2720 (59%) 96 (57%)

0.12μg 3840 (42%) 240 (-11%)

Tetanustoxoid 6120 (6.7%) 197 (10.4%) lOOμg Cycloheximide

2 x 10- M 720 (89%) 20 (91%)

10 -3 M 2000 (69.5%) 70 (68.2%)

5 x 10 -4 M 2960 (54.8%) 100 (54.5%)

Treatment with cycloheximide did not affect cell viability (assessed by trypan blue dye exclusion) at all 3 consentrations of drug tested. In preliminary comparative experiments, the method according to the invention here appeared at least five times as sensitive as the original ELISPOT techniques performed on plastic surfaces and developed with either paraphenylene diamine/H2θ2 in agar (HRP system) or BCIP (AP system) amplified with NBT. The high binding and retention properties of nitrocellulose membranes in the ELISPOT assay, recently introduced (J. Immunol. Methods 1985, 79, 195; Mβller and Borrebaeck, "A filter immunoplaque assay for the detection of antibody-secreting cells in vitro") also minimize substantially the requirements of the original techniques for relatively large amounts of coating material. In addition, this modification appears simpler since it does not rely on the use of a gel overlay, as is the care with certain peroxidase chromogens on plastic surfaces.

The potential of the method according to the invention has been confirmed for detecting simultaneously cells secreting other Ig isotypes (IgM, IgG subclasses) in both human and murine systems. The method should also be applicable for detection of other types of antibody-producing cells, e.g. lymphokine-secreting cells, and for detection of antigens, i.e. the virus itself or parts thereof, and for simultaneous detection of antibodies and antigens. The antibodies or antigens to be detected can be of eucarytic, bacterial, viral or parasitic origin.

Further visualization systems, e.g. silver immunogold staining (J.Immunol. Methods 1987, 104, 281; Walker and Dave "The rapid and sensitive enumeration of antibody-secreting cells using immunogold/silver staining" ) can also be applicable for simul- taneous detection of multiple immunoreactive substances. It should by that be possible to investigate whether two or more antigenically distinct products originate from the same or different cells.

The receptors, e.g. antigens or antibodies (in case antigens are to be detected) bound to the carrier can be of one single type reacting with the different types of antibodies or antigens resp. to be detected. They can also be of different types reacting with a type each of antibodies and/or antigens to be detected. The solid carrier can also consist of two opposed plates, at which one type of antigens or antibodies is bound to one of the plates and another of antigens or antibodies is bound to the opposed plate, at which the cell suspension is applied between the plates. It is in this way possible to detect simultaneously several types of antigens and/or antibodies. For certain substances, e.g. hormones, the solid carrier does not have to have antigens or anitbodies bound thereto, since these substances can bind directly to the solid carrier, in case this is of a material having intrinsic bindning properties, e.g. nitro- cellulose, nylon or polyvinyl.

Claims

C L A I M S

1. A method for detecting antibodies and/or antigens secrete and/or released by individual cells, at which a cell suspensio is brought in contact with a solid carrier, and after that o simultaneously therewith other antibodies are added which ar directed against and have an ability to bind to the antibodie or antigens that are to be detected, and which other antibodie are provided with an enzyme reacting with an indicator substanc for detecting the antibodies or antigens in question, c h a r a c t e r i z e d i n, that at least two types of said other antibodies are adde directed against at least two different types of antibodies tha are to be detected and which other antibodies are provided wit different enzymes, and that then the corresponding number o indicator substances are added having different staining effect, for simultaneous detection of the different types of antibodie and/or antigens by evaluation of spots of distinct colour remaining on the solid carrier.

2. Method as claimed in claim 1, c h a r a c t e r i z e d i n, that the solid carrier has antigens and/or antibodies corres pondning to the antibodies and/or antigens to be detected boun thereto.

3. Method as claimed in claim 2, c h a r a c t e r i z e d i n, that the antigens and/or antibodies bound to the solid carrie is of one single type with ability to react with the differen antibodies and/or antigens to be detected.

4. Method as claimed in claim 2, c h a r a c t e r i z e d i n, that the antigens and/or antibodies bound to the solid carrie are of different type with ability to react with one type each o the antibodies and/or antigens to be detected.

5. Method as claimed in claim 2, c h a r a c t e r i z e d i n, that antigens and/or antibodies of the same or different type are bound to two opposite surfaces of two solid carriers, a which the cell suspension is applied between the two carriers.

6. Method as claimed in claim 1, c h a r a c t e r i z e d i n, that the solid carrier has intrinsic binding properties and e.g. is of a material selected from the groups nitrocellulose, nylo and polyvinyl.

7. Method as claimed in any of the preceding claims, c h a r a c t e r i z e d i n, that as indicator system is used antibodies labelled wit alkaline phosphatase and horseradish peroxidase resp. and th corresponding chromogen substrates.

8. Method as claimed in claim 7, c h a r a c t e r i z e d i n, that said chromogen substrates consists of 5-bromo-4-chloro-3 indolyl phosphate toluidine salt and p-nitroblue tetrazoliu chloride (BCIP/NBT) and 3-amino-9-ethyl-carbazole (AEC).

9. Method as claimed in any of the preceding claims, c h a r a c t e r i z e d i n, that the antibodies to be detected comprises different types o Ig-antibodies.

10. Method as claimed in any of the preceding claims, c h a r a c t e r i z e d i n, that the antibodies to be detected comprises products o eucaryotyc, bacterial, viral and parasitic origin.