GB2329018A - Immobilisation of monolayers in boric acid containing buffer - Google Patents

Abstract

There is described a method of immobilising cells, preferably erythrocytes, on solid supports in which the cells are formed in the presence of a boric acid containing buffer and then subsequently fixed, preferably with glutaraldehyde. Examples involving competitive ELISA's and a radioimmunoassay using anti-D antibodies are shown.

Description

Improvements in or Relating to Xmmobilisation of Cells Field of the Invention This invention relates to a method of immobilising cells upon a solid support, and to a method of performing an assay -using cells immobilised upon a solid support, and to a solid support having cells immobilised thereon.

Background of the Invention Many routine tests and assays require the immobilisation of cells upon a solid support, such as a microtitre plate. The cells are generally eukaryotic cells and frequently of mammalian origin. It is often desirable for cells to be immobilised in what is known as a monolayer: a layer which is substantially the thickness of a single cell, upon a solid support. The monolayer is desirably substantially confluent (i.e. the monolayer covers most of the surface of the solid support, or that portion thereof to which cells are applied).

It is know to form a monolayer of red blood cells (RBC) or erythrocytes in the wells of a microtitre plate, by activating the inert plastics surface of the wells with coupling agents such as poly-L-lysine, concanavalin A, or anti-RBC antibody (Plapp et al, 1984 Am. J.

Clin. Pathol. 82, 719-721). A simpler method of forming an immobilised RBC monolayer has also been described (Llopis et al, 1996 Vox Sanguinis 70, 152-156), in which there is no requirement for the use of coupling agents; the method involves suspending RBC in a boric acid-containing cell fixation buffer (CFB) and allowing the cells to settle (preferably assisted by centrifugation) onto the surface of the microtitre wells. The authors report that the method allows the formation of stable RBC monolayers, which can be stored at 4"C for up to 1 month without deterioration of RBC antigens.

RBC monolayers can be used in many assays including, for example, the detection of anti RBC antibodies. The presence of such antibodies in patients' serum is clinically significant in ensuring compatibility of blood transfusions.

Suininary of the Invention The present inventors have surprisingly found that, contrary to the teaching of the prior art, monolayers prepared by the boric acid CFB method of Llopis et al are not particularly stable: washing of the monolayers with typical washing buffers (such as phosphatebuffered saline, or PBS) causes sections of the monolayer to become detached from the solid support. The inventors appear to be the first to have appreciated this problem and have provided a solution thereto.

Thus, in a first aspect, the invention provides a method of forming a stable cell monolayer immobilised upon a solid support, the method comprising the steps of: forming a monolayer of cells upon the solid support in the presence of a boric-acid containing buffer and fixing the monolayer so formed by adding a suitable amount of fixing reagent.

The boric acid-containing buffer preferably comprises at least 0.01M boric acid, preferably at least 0.02M boric acid, although the actual concentration used may vary considerably within sensible limits.

In one particular embodiment, the boric acid-containing buffer has the following formulation: 0.1M glucose; 0.07M NaCl; 0.02M boric acid; 0.035M trisodium citrate; 0.002M tetrasodium EDTA with water up to 1L; at pH 7.2-7.3. The precise concentration of the ingredients is not critical and can vary within about 2056 without significantly decreasing the performance of the buffer.

The fixing reagent is preferably glutaraldehyde, which fixes the monolayer effectively without substantially altering its antigenic properties, which is important if the monolayer is to be used in immunological assays. A "suitable amount" of fixing agent will be apparent to those skilled in the art by routine trial and error - there must be sufficient fixing reagent effectively to fix the monolayer but not so much as to alter significantly the antigenic properties of the monolayer. A suitable amount of glutaraldehyde is in the range 0.05 - 1.0%, conveniently in the range 0.0625 - 0.25%. Other fixing agents known to those skilled in the art, and which may be used at similar concentrations, include formaldehyde and paraformaldehyde, but glutaraldehyde is preferred.

The cells are desirably RBC: they may be human RBC (which are preferred), or from some other mammalian species, such as a sheep, pig, cow, horse, rat or mouse. The solid support typically comprises an inert synthetic plastics material and conveniently takes the form of a microtitre plate formed with a plurality of wells (e.g. 96).

The cells forming the monolayer are generally RBC obtained from blood samples.

Conveniently the cells are washed several times (e.g. 36 times) in a standard physiological solution (such as saline, or phosphate-buffered saline), and (optionally) washed again (usually just a single time) in the boric acid-containing buffer. The RBC may also have been previously treated with enzymes e.g. papain to enhance binding of antibodies. The packed RBC are then suspended in the boric acid-containing buffer: a cell concentration in the range 0.05-5.0% (v/v) is found suitable, with a preferred cell concentration in the range 0.1 1-1.0 0.1-1.0% (v/v).

The suspended cells may then be placed upon the surface of the solid support upon which they are to be immobilised. For example, if the solid support is a microtitre plate, the cell suspension may be placed in aliquots in the wells of the plate. The cells may then be left to settle on the surface of the support under the influence of gravity, or more preferably, subjected to centrifugation (e.g. about 3 ruins at 350g) to facilitate monolayer formation.

Once the monolayer has been formed, the fixing reagent is added. This may either be added in the presence of the boric acid-containing buffer, or else the buffer may be removed prior to addition of the fixing reagent. In one particular embodiment, a microtitre plate (the wells of which are coated with an RBC monolayer), is immersed in cold (4"C) PBS comprising 0.25% - 1% (v/v) of a 25% aqueous solution of glutaraldehyde for 10 minutes, in order to fix the monolayers.

Other suitable fixation protocols are taught by Heusser et al, (1985 Methods in Enzymol.

73, 406919).

The inventors have found that monolayers of RBCs prepared by the method of the present invention are far more stable than those prepared by the prior art method of Llopis et al.

Whilst Llopis et al describe the monolayers produced by their method as stable, they are used only in a haemagglutination-type assay, which does not include any step where the monolayer is washed. The inventors have found that washing steps will disrupt monolayers prepared by the method of Llopis et al, but not those monolayers prepared by the method of the present invention. In addition, monolayers in accordance with the invention substantially retain their native antigenic properties, even after storage at 4"C, which characteristic is extremely useful if the monolayers are to be used in irnmunologically-based assays.

In a second aspect therefore, the invention provides a method of performing an assay to determine the presence of a substance of interest in a sample, the method comprising forming a cell monolayer immobilised upon a solid support according to the method of the first aspect of the invention defined above; contacting the sample with the monolayer so as to generate a detectable signal; and detecting the signal. The method may be qualitative or, more preferably, quantitative. Typically the method comprises the inclusion of one or more washing steps, which will generally be performed after contacting the sample with the monolayer but before detection of the signal.

The invention also provides in a third aspect a method of performing an assay to determine the presence of a substance of interest in a sample, the method comprising contacting the sample with a stable RBC monolayer immobilised upon a solid support, performing one or more washing steps, and detecting a signal generated as a result of an interaction between the sample and the immobilised monolayer. Typically the monolayer is formed by the method of the first aspect of the invention.

Generally the substance of interest in the sample will bind specifically to an antigen present on the monolayer. Frequently, the substance of interest will be an antibody, typically with binding specificity for a human blood group antigen. The washing step(s) may be employed to remove excess unbound material present in the sample or to reduce the amount of weakly (non-specifically) bound material adhering to the monolayer.

Assay methods which may be employed in performing the second or third aspects of the invention include radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) or other techniques which require one or more washing steps to obtain accurate and/or reproducible results.

The assays may, if desired, be competitive assays, in which the substance of interest in the sample competes with a labelled reagent for binding to an antigen present on the monolayer. The reagent may be radio-labelled or enzyme-labelled, for example. Binding of the substance of interest to the monolayer, in preference to the labelled reagent, will reduce the amount of label associated with the monolayer, which feature can be used to detect binding of the substance of interest.

Alternatively, the assay may be non-competitive. For example, a sandwich ELISA-type assay can be envisaged in which the substance of interest is captured on the monolayer (e.g. by specific binding to an antigen by an antibody of interest), and a second, labelled antibody is added (following a washing step) which binds specifically to the substance of interest.

It will be apparent to those skilled in the art that the nature of the signal detection step will depend on the type of assay being performed. Typically binding of the substance of interest to the monolayer will be detected indirectly by immunological means (e.g. capture of a labelled antibody) or by capture of a labelled ligand.

In a fourth aspect the invention provides a solid support having immobilised thereon an RBC monolayer which is resistant to disruption by washing but which does not require the use of coupling agents. Such a monolayer in accordance with this aspect of the invention remains substantially intact (over 95% intact) following a first or second washing step (i.e.

contact with excess liquid), and significantly intact (over 90% intact) following a third or fourth washing step. Washing, in the context of the present invention, means immersion in excess quantities of substantially static washing liquid (e.g. water, or aqueous buffer solutions), although gentle agitation may optionally be included as part of the washing process.

The solid support and/or the methods of the invention can be used in direct and indirect solid-phase binding assays for the qualitative or quantitative detection of RBC surface antigens (such as human or mammalian animal blood group antigens) using antigenspecific reagents, or for the detection of antibodies reactive with RBC antigens (especially blood group antigens).

The invention will now be further described by way of illustrative examples, and with reference to the accompanying drawings, in which: Figure 1 is a graph (cpmxl03 bound against reciprocal of dilution) showing the effect of different cell suspension media on the immunoreactivity of stored RBC monolayers; Figure 2 shows dose/response results from a competitive ELISA plotted on a graph of absorbance (nm) against Logs0 Dose; Figure 3 is a graph of anti-D potency (IU/ml) as measured by a competitive ELISA in accordance with the invention, against anti-D potency (manufacturers' results obtained using prior art assay techniques), showing agreement between the different assay formats.

Examples Example 1 - Formation of an immobilised RBC monolayer on a microtitre plate.

A monolayer was first formed substantially in accordance with the method described by Llopis et al, (1996, cited above). Briefly, the appropriate red cells were washed six times in normal saline (0.9% Nail) or buffered saline followed by a final wash in cell fixation buffer (CFB).

The CFB was made up as follows: glucose 0.1M, NaCI 0.07M, boric acid 0.02M, trisodium citrate 0.035M, tetrasodium EDTA 0.002M with water up to 1 litre; pH 7.27.3.

A 0.2% (vol/vol) suspension of red cells was made in CFB and 50y1 of this dispensed into wells of a microtitre assay plate; The plate was then centrifuged at 350g for 3 min.

Subsequent fixation of the monolayer was carried out by immersion for 10 min at 4"C in cold PBS containing 0.25% gluataraldehyde, according to the method of Heusser et al, (1985 cited above).

Wells were blocked using CFB containing 3% protein blocking reagent (e.g. Marvel > , albumin or casein).

Plates can be stored like this at 4"C for several weeks or washed and used immediately.

Example 2 - Anti-D Quantitation using RBC immobilised on Microtitre Plates.

Anti-D quantitation is necessary to ensure the potency of anti-D immunoglobulin preparations used for the prevention of haemolytic disease of the newborn. Although automated haemagglutination using autoanalyzers is the reference method described in the European Pharmacopoeia, this technique is unsuitable for intermittent use in national control and manufacturers' laboratories.

The inventors have previously developed a competitive radioimmunoassay using monoclonal anti-D antibodies for anti-D quantitation (Thorpe & Heath 1995 Trans. Med.

5, 97-103) in which 'ZSI-labelled monoclonal anti-D and unlabelled anti-D samples compete for erythrocyte binding. This assay is routinely used to determine anti-D potency of clinical grade immunoglobulin preparations at the National Institute for Biological Standards and Control (NIBSC). The inventors have now adapted the original tube procedure (using erythrocytes in suspension) to devise a competitive ELISA for use with erythrocyte-coated microtitre plates.

MATERIALS AND METHODS Anti-D Preparations The monoclonal anti-D used as calibrand was Brad-5 (kindly provided by the Bio Products Laboratory, Elstree, Herts). Purified Brad-5 was iodinated using Chloramine T, according to the method of Greenwood et al, (1963 Biochemical Journal 89, 114-123) and was used to assess the retention of immunoreactivity of the immobilised RBC monolayers (see Figure 1). Clinical grade anti-D immunoglobulin preparations were obtained from seven manufacturers. They were assayed against the International Reference Preparation (IRP) for anti-D immunogloblin (68/419; from NIBSC).

Formation of Ervthrocvte Monolavers in Wells of Microtitrate Plates Two erythrocyte suspension media, alone or in combination, were tested for their effect on: a) bringing about initial erythrocyte monolayer adhesion; b) the subsequent stability of the monolayer during washing procedures and storage at 4"C; and c) retaining anti-D immunoreactivity.

Monolayer Formation RBC monolayers were formed as described in example 1: R2R2 red blood cells were washed 6 times in normal saline. For immobilisation in CFB, the cells were then washed once in CFB and diluted to 0.2% (vol/vol) in CFB. For immobilisation using glutaraldehyde, saline-washed cells were diluted to 0.2% (vol/vol) in 0.25% glutaraldehyde.

50 l of the cell suspensions were dispensed into wells of U-bottomed flexible microtitre plates (Falcon 38911 microtest m plate, Becton Dickinson Labware). The plates were then centrifuged at 350 g for 3 min.

The effect of subsequent glutaraldehyde fixation on monolayers prepared using CFB was also investigated. Fixation was carried out by immersing the erythrocyte-coated plates in PBS containing 0.25% glutaraldehyde at 4"C for 10 min. Monolayers were tested for their ability to withstand numerous washing procedures by visual examination.

Assessment of Anti-D Binding Erythrocyte-coated wells were blocked to prevent non-specific adsorption of reagents by incubation with PBS containing 3 % (w/v) Marvel for 30 min at room temperature (RT).

The plates were then washed 3 x with PBS, then incubated with serial 1 in 2 dilutions of 25I-labelled Brad-5 in PBS for 1 hour at RT.

Competitive ELISA for Anti-D Ouantitation using RBClmmobilised on Microtitre Plates PRINCIPLE: Varying amounts of unlabelled anti-D immunoglobulin and a constant amount of biotinylated monoclonal anti-D compete for red cell binding. The lower the unlabelled anti-D content, the more biotinylated monoclonal anti-D is bound. Binding of biotinylated monoclonal anti-D is then detected by incubation with a constant amount of alkaline phosphatase labelled Extravidin and carrying out the enzyme reaction. The intensity of colour is related to the amount of biotinylated monoclonal anti-D bound and hence to the unlabelled anti-D immunoglobulin content. Dose-response curves are constructed from the absorbance versus unlabelled anti-D dose (i.e. dilution) and potencies of samples calculated relative to the International Reference Preparation (IRP) for anti-D immunoglobulin by standard parallel line analysis methodology.

Competitive ELISA for Anti-D Ouantitation Procedure A. Formation of Immobilised Red Cell Monolayers 1. A 0.1 % (v/v) suspension of papain-treated R2R2 (other D-positive phenotypes may be acceptable) cells was prepared in cold Cell Fixation Buffer (CFB). 50y1 of the suspension was dispersed into each well of a flexible U-bottomed 96-well microtitre plate (Falcon 38911 microtest m plate, Becton Dickinson Labware).

2. The plates were spun in a centrifuge at 1400 rpm for 3 min (Sorvall 6000B rotor or equivalent), at 4"C (equivalent to approximately 350 g for 3 min).

3. Without removing supernatant, plates were immersed in cold PBS containing 0.259E glutaraldehyde (from a 2596 aqueous solution giving a final glutaraldehyde concentration of approx. 0.063%) for 10 min (using a pipette to displace any air bubbles from wells and tilting the plates once or twice to aid dispersion of CFB from the wellsj.

4. The plates were then washed 3x with PBS, 2-3 minutes each wash, and stored at 4"C in CFB until used to carry out the competitive ELISA as described below.

B. Competitive ELISA Procedure 5. CFB was removed from the plates and the wells blocked with PBS containing 2% (w/v) BSA for 20-30 min at RT.

6. Separately, serial 1 in 2 dilutions of anti-D immunoglobulin standard and samples were prepared in PBS. A replicate series of dilutions (eg 75iLl of each dilution) was prepared.

7. An aliquot of each of the above dilutions (eg 60y1) was mixed with an equal volume of biotinylated monoclonal anti-D (Brad-5, Bio Products Laboratory, Elstree, Herts) at 0.25 yg/ml.

8. Plates were drained and duplicate 50y1 aliquots of each of the above mixtures were dispersed into the erythrocyte-coated wells and the plates incubated for 1 hr at RT.

9. Wells were then washed 3x with Tris-buffered saline (TBS) (2-3 min each wash), and then; 10. Incubated with 50Z1 of alkaline phosphatase labelled Extravidin (Sigma, E-2636) diluted 1/2000 in TBS for 30 min.

11. The plates were again washed 3x with TBS and incubated with 10owl of solutions prepared (according to the manufacturer's instructions) from Sigma Fast pNPP tablets (N1891) for 10 min.

12. 25y1 of 3M NaOH was added to the wells to stop the reaction and 105y1 transferred out of each well into a flat-bottomed ELISA plate for reading at 405 nm.

Blank mixtures were prepared by substitution of PBS/BSA for the anti-D mixture (step 8), and positive controls by substitution of PBS/BSA for unlabelled anti-D immunoglobulin (step 6).

The "blank" readings were subtracted from sample readings and dose-response curves constructed from absorbance (nun) versus dose (i.e. dilution). The potency of samples was calculated by standard parallel line analysis (Armitage 1971 "Statistical Methods in Medical Research", Blackwell Scientific Publications, Oxford; Finney 1978 "Statistical Methods in Biological Assay" 3rd edition Griffin, London; Williams et al 1978 Scientific Foundations of Clinical Biochemistry vol 1 William Heinemann Medical Books Ltd, London).

RESULTS Monolaver Formation and Adhesion Cell monolayers were formed by dispensing the erythrocyte suspension in CFB and centrifuging the plate. However, monolayers formed in this way did not remain intact after repeated washing necessary for the RIA. The effect of the fixatives formaldehyde, paraformaldehyde and glutaraldehyde on monolayer adhesion were therefore investigated, and glutaraldehyde was found to be the most effective in promoting adhesion.

Glutaraldehyde fixation did not result in loss of immunoreactivity as judged by the ability of monolayers to bind 1251-labelled Brad-5.

Storage and Immunoreactivi of Monola ers An advantage of the erythrocyte monolayer-coated microtitre plates is that they may be made up in batches and stored at 4"C for use at a later date. Monolayer-coated plates were stored at 4"C in 3 different ways in order to ascertain which were the best conditions for storage for use in the competitive RIA: a) Monolayers were prepared in CFB and subsequently fixed in PBS containing 0.25% glutaraldehyde; b) Monolayers were prepared in PBS containing 0.25% glutaraldehyde; or c) Monolayers were prepared in CFB containing 0.25% glutaraldehyde.

In each case, prepared plates were washed with saline and stored in CFB/Marvelm' for 3 days.

The effect of these conditions on anti-D binding is shown in Figure 1. In Figure 1, preparation and storage as method (a) above gave the results indicated by lozenges; method (b) above gave the results indicated by squares; and method (c) gave the results indicated by triangles. The combination of monolayer formation in CFB followed by glutaraldehyde fixation gave the best results overall (Table 1).

TABLE 1 Effect of Different Cell Sus 'ision Media on~E hroc e Monola ers

Cell Suspension Monolayer Immunoreactivity Immunoreactivity medium stability immediately following following storage at 40C monolayer forms 0.25% +++ ++ + glutaraldehyde in PBS CFB only + + + NT CFB+ +++ ++ ++ Glutaraldehyde (o.zs a > CFB followed by +++ +++ +++ Glutaraldehyde fixation * difficult to assess due to loss of cells.

Competitive ELISA for Anti-D Ouantitation using RBC Immobilised on Microtitre Plates Results Dose-response curves for the International Reference Preparation for anti-D immunoglobulin and the anti-D immunoglobulin samples were parallel, as shown in Figure 2.

Figure 2 shows the dose-response plots for the International Reference Preparation (square symbols) and two commercially-available anti-D immunoglobulin preparations (triangle and lozenge symbols).

The competitive ELISA was performed twice on each of 13 samples of clinical grade anti D immunoglobulin product. The results are shown in the Table below. Also shown are the manufacturers' potency estimations (using other methodology). Figure 3 shows the comparison graphically.

Comparison of potencies obtained using competitive ELISA with manufacturers' potencies of clinical grade anti-D immunoelobulin DIeparations MANUFACTURERS' POTENCY COMPETITIVE ELISA POTENCY (IU/ML) INDIVIDUAL ASSAYS AVERAGE 276 277 299 288 283 297 283 290 287 301 294 298 150 147 147 147 164 145 147 146 269 267 284 276 252 275 271 273 513 658 655 657 577 581 605 593 557 576 602 589 805 901 864 883 805 779 767 773 530 550 550 550 CONCLUSIONS 1. The use of CFB followed by gluraraldehyde fixation allows the formation of erythrocyte monolayers that withstand numerous washing procedures and storage at 4"C without loss of anti-D immunoreactivity.

2. Anti-D potencies obtained by competitive ELISA using immobilised erythrocyte monolayers were similar to those obtained by the manufacturers of anti-D immunoglobulin products using other anti-D potency tests.

3. The microtitre plate ELISA was quick and easy to perform, and allowed analysis of several samples in the same assay nin.

4. Erythrocyte monolayer-coated plates have potential for widespread use for anti-D quantitation and other direct and indirect immunoassays.

Claims (18)

Claims

1. A method of forming a stable cell monolayer immobilised upon a solid support, the method comprising the steps of: forming a monolayer of cells upon the solid support in the presence of a boric-acid containing buffer and fixing the monolayer so formed by adding a suitable amount of fixing reagent.

2. A method according to claim 1, wherein the monolayer comprises red blood cells (RBC).

3. A method according to claim 1 or 2, wherein the boric acid containing buffer comprises boric acid at a concentration of 0.01M or greater.

4. A method according to any one of claims 1, 2 or 3, wherein the boric acid containing buffer comprises: 0.1M glucose; 0.07M NaCI; 0.02M boric acid; 0.035M trisodium citrate; and 0.002M tetrasodium EDTA, all in aqueous solution at pH 7.2-7.3.

5. A method according to any one of the preceding claims, wherein the fluxing reagent comprises glutaraldehyde.

6. A method according to any one of the preceding claims, wherein the fixing reagent comprises 0.05 - 1.0% glutaraldehyde.

7. A method of performing an assay to determine the presence of a substance of interest in a sample, the method comprising the steps of: forming a cell monolayer immobilised upon a solid support according to the method of any one of the preceding claims; contacting the sample with the monolayer so as to generate a detectable signal; and detecting the signal.

8. A method according to claim 7, wherein the signal is generated directly or indirectly by an interaction between the sample and the irrimobilised monolayer.

9. A method according to claim 7 or 8, wherein the method comprises one or more washing steps prior to detection of the signal.

10. A method according to any one of claims 7, 8 or 9, wherein the substance of interest binds specifically to an antigen present on the monolayer.

11. A method according to any one of claims 7-10, wherein the substance of interest is an antibody.

12. A method according to any one of claims 7 - 11, wherein the substance of interest is an anti-D antibody.

13. A method according to any one of claims 7-12, wherein the immobilised monolayer comprises RBC.

14. A solid support having immobilised thereon an RBC monolayer which does not require the use of coupling agents for its formation and is resistant to disruption by washing.

15. A solid support according to claim 14, wherein the RBC monolayer substantially retains native antigenic properties.

16. A method of forming a stable monolayer substantially as hereinbefore described.

17. A method of performing an assay substantially as hereinbefore described.

18. A solid support having imnobilised thereon an RBC monolayer substantially as hereinbefore described.