SOLID PHASE IMMUNOASSAY

Abstract: In a solid phase immunoassay, e.g. RAST or PRIST, the sensitivity can be substantially increased by reducing the volume of liquid in which the indicator (e.g. <125> I anti-IgE) is contained, preferably to the saturation volume of the paper carrier. If the volume of sample is also similarly reduced, the incubation time can be reduced to as little as 5 minutes without loss of sensitivity in comparison with conventional tests. A washing device for the paper carrier is also disclosed, together with immunoassay kits for use in the method of the invention.

Description: - 1 - SOLID PHASE IMMUNOASSAY This invention relates in one aspect to a method of solid phase immunoassay, comprising contacting a sample liquid containing an anaiyte with an absorbent carrier having a binding material immobilised therein or thereon, whereby anaiyte in the liquid becomes bound to the binding material, allowing a labelled indicator to contact the bound anaiyte, whereby the indicator becomes bound to the bound anaiyte, removing unbound indicator from the carrier, and detecting the remaining bound indicator. Another aspect of the invention relates to a method of solid phase competitive immunoassay, wherein a sample liquid containing an anaiyte and a labelled indicator are contacted with an absorbent carrier having a binding material immobilised therein or thereon, the anaiyte and the indicator competing to bind with the binding material, and the quantity of indicator bound is detected. The invention relates in yet another aspect to a washing device for use in solid phase immunoassay, comprising a washing liquid supply, a washing liquid receiving chamber, and a holding plate therebetween, the holding plate comprising a plurality of wells in the upper side thereof, and each well having an outlet communicating with the underside of the holding plate. Several different types of solid phase immunoassay are currently used. Examples are RAST (radio allergosorbent test) and ELISA (enzyme-linked immunosorbent assay). Typically, in RAST, cellulose paper discs are used as the carriers, to which an allergen is bound. A sample of the test liquid containing antibodies to the allergen is added to the disc in a microtitre plate and the plate is incubated, for example for eighteen, hours. For a disc having a liquid capacity of 3 μl, for example, the sample volume is typically 50 μl. After washing and removal of surplus liquid, 50 μl of a liquid containing a radio labelled antibody specific to the antibody to be assayed is added to the disc in the plate, and a further incubation is carried out for eighteen to twenty four hours. After washing the disc, its residual radioactivity is counted to give a measure of the quantity of the antibody bound to the disc, and thus the quantity in the original sample. RAST is a sensitive test, particularly useful for allergen-specific antibodies, and is easily adapted to a wide range of allergens. A disadvantage, however, is the relatively long time required to conduct the test. Other solid phase immunoassay, such as PRIST (paper radio immunosorbent test, in which an antibody to IgE is bound to the paper disc), ELISA, class-capture and two-site assays, all require significant incubation times to achieve satisfactory sensitivity. It has now been found that by reducing the volume of the liquid in which the indicator is contained, relative to the liquid capacity of the disc, a significant increase in sensitivity can be achieved, permitting positive detection of a lower concentration of anaiyte in a sample than with conventional tests, and the incubation time with the indicator liquid can also be reduced significantly. It has also been found that if the volume of sample liquid is also reduced, relative to the liquid capacity of the disc, the incubation time can be reduced to as little as five minutes while achieving a sensitivity comparable with that of conventional tests. In GB-A-1420916, there is disclosed a radio- immunoassay test conducted in glass fibre filter material using 0.1 ml of liquid, which is approximately equal to the filter material capacity. However, the test is of the competitive type r using a significantly larger volume of liquid than the tests of the type with which the present invention is concerned. The first aspect of the present invention is characterised in that the labelled indicator contained in a volume of liquid less than ten times the saturation volume of the carrier. The volume of liquid is preferably less than five times the saturation volume of the carrier, more preferably less than twice the saturation volume. Most preferably, the volume of liquid is substantially equal to the saturation volume. Where the concentration of the anaiyte is very low, but quantities are not restricted, for example in tissue culture supernatants, umbilical cord blood samples, and body fluids such as saliva and nasal washings, the volume of sample liquid added and the incubation time are suitably chosen to be comparable with those in conventional tests. Where a higher concentration of anaiyte is present in the' sample liquid, for example antibodies in serum, a volume of sample liquid less than ten times the saturation volume of the carrier is used. Preferably less than five times the saturation volume is used, more preferably less than twice the saturation volume. Most preferably, a volume substantially equal to the saturation volume is used. With reduced volumes of the sample liquid, substantially smaller incubation times are required, and the assay may therefore be completed in a substantially shorter time than is required for conventional tests, for example as little as 65 minutes for IgE antibodies. The method of solid phase competitive immunoassay of the invention is characterised in that a carrier is used which has a liquid saturation volume of less than 0.1 ml, preferably 10 μl or less, more preferably 5 μl or less, and the total quantity of liquid contacted therewith is less than ten times the saturation volume of the carrier. Preferably the quantity of liquid is less than five times, more preferably less than twice, and most preferably substantially equal to, the saturation volume of the carrier, which is suitably a cellulose paper carrier. The use of small sample volumes in accordance with the invention permits . economical use of reference reagents. This makes it possible to hold supplies of a wide range of reference reagents, in particular serum samples, which is impractical for conventional RAST assay methods using large volumes of sample. Accordingly, the invention also provides an immunoassay kit for use in the immunoassay method of the invention, comprising a plurality of absorbent carrier members having a binding material immobilised therein or thereon, a supply of a solution containing a labelled indicator, and a plurality of different reference samples containing known concentrations of known analytes. A further aspect of the invention provides an immunoassay kit for use in the immunoassay method of the invention, comprising a plurality of absorbent carrier members having a binding material immobilised therein or thereon, and a supply of a solution containing a labelled indicator, the volume of the solution being approximately equal to the aggregate liquid capacity of the carrier members. In non-competitive tests such as RAST it is advantageous or essential to wash the carrier discs after each incubation period, and a known washing device for this purpose has a plate which can be mounted between a washing liquid supply and a vacuum chamber, the plate having a plurality of wells, each of which receives a filter disc, the device permitting a plurality of discs to be washed simultaneously. The discs remain in the wells during each stage of the processing, but are removed for the radioactive testing step. Typically, 96 wells are provided in each plate, permitting 96 discs to be processed simultaneously in each batch. While the conventional washing device permits rapid processing of the batches, the testing step requires the careful removal of each disc from its well, and this can slow the operation. Accordingly, the washing device of the invention is characterised in that the holding plate is removably mountable between the supply and the receiving chamber. In one embodiment of the invention, a holder is removably located in each well, the holder having a drain hole therein corresponding with the outlet, and the washing liquid supply is arranged to supply liquid to the holders. Preferably, the drain hole in each holder is sized such that liquid in the holder is not lost therethrough under gravity. The holders may be in the form of cups or tubes with a drain hole in the base thereof. The cups or tubes are suitably formed of plastics material or glass and are dimensioned and arranged to be a close, but readily removable, fit within the wells. The outlets from the wells are suitably capillaries of the same size as the drain holes in the holders. The holding plates are more readily handled than the individual filter discs they contain, and thus the processing of batches of filters is facilitated. In some circumstances the use of holders in the wells is preferred. Each holder may, if desired, be suitably labelled to identify an individual disc. Reference is made to the drawings, in which: Figure 1 is a series of graphs illustrating the effect of reducing sample volumes (Figure 1A) indicator volume (Figure IB) and sample and indicator volumes (Figure 1C), and increasing indicator concentration with reduced volumes of indicator and sample (Figure ID); Figure 2 is a graph showing the correlation between the results obtainable by the method of the invention and those obtainable by conventional RAST; Figure 3 is a pair of graphs showing rate of binding of anaiyte (IgE antibody) (Figure 3A) and a rate of binding of indicator ( 125I anti-IgE) (Figure 3B) for conventional RAST; Figure 4 is a pair of graphs showing the corresponding rates for the assay of the invention; Figure 5 is a graph illustrating the effect of sample volume on the sensitivity of an assay in accordance with the invention; Figure 6 is a pair of graphs illustrating the effect of reduced indicator volume on sensitivity at three sample volumes (Figure 6A), compared with conventional RAST procedure (Figure 6B) . Figure 7 is a series of graphs showing the rate of binding of IgG antibody with various dilutions of high titre (Figure 7A) and low titre (Figure 7B) IgG antibody samples, and the rate of binding "" of the indicator ( I anti IgG) for the same dilutions of high and low titre samples (Figure 7C and 7D); Figure 8 is a pair of graphs showing the effect of sample volume (Figure 8A) and 125I anti IgG volume (Figure 8B) in assay of IgG antibody to bee venom according to the invention; Figure 9 is a graph showing the effect of reducing 125 I, anti-IgE volume In PRIST; Figure 10 is a graph which shows the effect on sensitivity of IgE ELISA using Fab' anti-IgE as indicator, and with enzyme amplification; Figure 11 shows a washing device according to the other aspect of the invention, with the holding plate removed therefrom; and Figures 12 and 13 are respectively side elevation and top plan views of the holding plate for the device shown in Figure 11. For the " following examples, serum samples were collected from patients allergic to bee venom, grass pollen and castor bean, which had previously be shown to contain IgE antibodies. For most experiments a pool of positive sera was used. Preparation of 125I anti-IgE Rabbit anti-IgE was prepared by immunisation of New Zealand white rabbits with 0.25 mg IgE PS (from myeloma patient PS). In complete Freund's adjuvant intra-muscularly followed by three two-weekly boosts with 0.25 mg IgE PS in incomplete Freund's adjuvant. The serum was separated and absorbed extensively with normal human serum (20 ml serum/10 ml CNBr-Sepharose 4B and human gamma globulin (100 mg/10 ml sepharose 4B (agdrose). The anti IgE was then affinity purified over a column containing IgE WT (from myeloma patient WT). Agarose (10 mg/lOml CNBr-sepharose 4B). the purified antibody was labelled with 125 Nal, supplied by Amersham International P C, UK. COMPARATIVE EXAMPLE 1 RAST In a conventional RAST method, allergen coated discs having a liquid capacity of 3 μl where washed with phosphate buffered saline (PBS) 0.05 M, pH 7.4 + 0.05% Tween 20 in a known washing device as hereinbefore described, which removes all surplus liquid from the washed discs. The test sample (50 μl), diluted, if necessary, in horse serum, was incubated with an allergen coated disc in a flat bottomed microtitre plate, (Linbro, Flow Labs Limited, UK) for eighteen hours. The discs were washed as before and 10 ng 125I anti-IgE (50 μl), prepared as hereinbefore described, were added. The following day the discs were again washed and the residual radio acitivity counted for one minute in a multi channel gamma counter (LKB-Sweden) . Intra-assay variation at 1/5 dilution of serum pool equals 5.7%, n=14. The test was repeated using 25 μl, 10 μl and 5 μl of the test sample, with all the other conditions remaining the same. Further tests were then conducted using test samples diluted to a tenth, a hundredth and a thousandth of the original strength. The results are plotted in Figure 1A. It will be seen that the reduction in sample volume produces a reduction in the number of counts measured for each dilution, ie. sensitivity of the test is reduced when sample volume is reduced. Example 1 The # tests conducted in the Comparative Example were repeated, keeping the sample volume constant at 50 μl and varying the indicator volume from 50 μl down to 5 μl in the same steps for varying dilutions of sample. The concentration of 125 I anti-IgE was increased ten fold so that 10 ng was added in " 5 μl. It may be seen from Figure IB that reducing the I anti-IgE volumes increased the sensitivity of the test, ie. increased the number of counts per minute measured. Example 2 Using the same first and second incubation volumes (5 + 5, 10 + 10, 25 + 25 and 50 + 50) it was possible to achieve comparable results with as little as 5 μl of sample (Figure 1C). The use of small volumes of 125I anti-IgE at the same concentration as it is normally used in RAST (0.4 μg/ml) resulted in low radio active counts bound, which necessitated prelonged counting. Example 3 The tests conducted in Example 2 were repeated, raising the concentration of the 125 I anti-IgE to 4 μg/ml. Similar results were obtained, and the 5 μl assay compares very favourably with the conventional assay procedure using 50 μl of sample and of indicator (Figure ID) . COMPARATIVE EXAMPLE 2 Forty eight different samples of sera taken from patients allergic to castor bean were tested by the conventional RAST. Example 4 The tests conducted in Comparative Example 2 were repeated using the method of the present invention, as described in Example 3, and for each sample, the counts per minute measured in the conventional test were plotted against the counts per minute measured in the test according to the invention (Figure 2) . It will be seen from Figure 2 that a close correlation is obtained between the two methods (r = 0.96). COMPARATIVE EXAMPLE 3 Tests were conducted using conventional RAST to determine the increase -in rate of binding of IgE antibody with time at different dilutions of the sample. The results are plotted in Figure 3A. It can be seen that in the first incubation, the time taken for IgE antibody to bind is greater at high than at low dilution, but that even at low dilution it may take four to five hours for equilbrium to be reached. COMPARATIVE EXAMPLE 4 Tests were also conducted in accordance with conventional RAST, measuring the rate of binding of 125 I anti-IgE at varying dilutions of the indicator, the results being plotted as Figure 3B. The rate at which the 125 I anti-IgE binds is normally very slow and a plateau is not reached even after twenty-four hours. Example 5 The tests of Comparative Example 3 were repeated using the method of the present invention, varying the dilution of the IgE antibody serum sample. The results are plotted a"s Figure 4A, and it may be seen that equilbrium is reached very quickly at each dilution. In the method of the present invention, the distances which the antibodies have to diffuse before coming into contact with the antigen are much shorter, and this is reflected in the increased rate at which the IgE antibody binds, as compared with the conventional assay (Figure 3A). Equilibrium is reached within ten minutes, and subsequent experiments have shown that the plateau is reached in about five minutes. With a smaller sample, say 3 μl, it is expected that equilibrium would be reached in less than five minutes. Example 6 The test conducted in Comparative Example 4 was repeated using the method of the present invention. The rate of binding of the 125 I anti-IgE is much more rapid, as may be seen from Figure 4B, than in the conventional test (Figure 3B) both with 0.4 and 4 μg/ml of 125 I anti-IgE. Example 7 To determine the absolute lower limit to the sample size in the method of the present invention, a wider range of volumes from 50μi down to 1 μl were used with a fixed volume of 125 I anti-IgE (5 μl) . The results are plotted in Figure 5. For practical purposes, the lower limit can be seen to be about 3 μl. At greater than 5 μl, sensitivity was increased significantly, especially at high sample dilution. Example 8 Tests using reduced 125 I anti IgE indicator volume (5 μl) in accordance with the invention and using different serum sample volumes at different dilutions were conducted. The results were plotted as Figure 6A, and it can be seen that, using 300 μl of neat sample, up to 80% of added radioactive indicator could be bound. COMPARATIVE EXAMPLE 5 The tests of Example 8 were repeated using 50 μl of the indicator with the same quantity of antibody and therefore the same number of radioactive counts as in the 5 μl quantity of indicator used in Example 8. It can be seen from Figure 6B that the sensitivity is substantially reduced, with 300 μl of sample giving only slightly higher number of counts than that for 5 μl in the test according to the invention (Figure 6A). The increased sensitivity that results from bringing 125 I anti-IgE into closer contact with the IgE antibody bound to the disc, and the greater ef iciency with which IgE antibody can be bound, makes it possible to use very small amounts of serum (as little 3 μl) with equal sensitivity to that obtained in conventional tests. The method of the invention has been used to assay total IgE and IgE antibodies in blood samples taken from babies by heel prick. The same economy of serum is also useful when testing rats, where serum is also in short supply. Example 9 A series of measurements of IgG antibodies to bee venom in serum samples of high and low titre was. carried out using cellulose paper discs of about 3 μl liquid capacity coated with bee venom. The discs were incubated with samples at dilutions from 1/100 to 1/10000 for the high titre samples and of 1/100 and 1/300 for the low titre samples. * A range of incubation times was used, and at the end of the incubation each sample was washed and then incubated with 125I anti-IgG (Pharmacia (GB) pic), followed by further washing. The residual radioactivity was counted in a gamma counter, the results being plotted as Figure 7A (high titre) and Figure 7B (low titre). It can be seen that for high dilutions, a plateau is reached very quickly, within about one minute, while at lower dilutions for the high titre sample the time taken is about five minutes. Example 10 A further series of measurements of IgG antibodies to bee venom in serum samples of high and low titre was carried out as in Example 9, but with a constant incubation time, sufficient to achieve equilibrium. The 125 I anti-IgG indicator was used as in Example 9 and the counts measured for various different incubation times. The results are plotted in Figure 7C (high titre) and Figure 7D (low titre). It will again be seen that equilibrium is reached rapidly, especially for higher dilutions of sample, being within about ten minutes. COMPARATIVE EXAMPLE 6 A procedure similar to that of Example 9, but using different quantities of serum, each of several different dilutions and a constant incubation time sufficient to ensure equilibrium, was used to produce a series of graphs for high (solid lines) and low (broken lines) titres of serum. The graphs are shown as Figure 8A, and it can be seem that, with lower quantities, sensitivity is reduced. Example 11 By contrast, a series of tests similar to those of Comparative Example 6 were carried out, but using a constant quantity of serum, in varying dilutions, and constant incubation times, while using different volumes of 125I anti-IgG. It can be seen from Figure 8B that, where the 125 I anti-IgG volume is reduced to 3 μl, there is a further increase in sensitivity so that up to 83% of added counts bind. Example 12 A PRIST assay was carried out in accordance with the invention. PRIST is a 2-site assay in which a cellulose paper disc is coated in anti-IgE which binds all the IgE in the sample. Specific bound IgE is then detected using a radiolabelled anti-IgE indicator. (An enzyme-labelled anti-IgE can be used as an alternative). Sample and indicator volumes of 5 μl, 10 μl and 25 μl were used with a disc of 3 μl capacity. For comparison, a test with 50 μl was also conducted. A range of different IgE concentrations in the sample was tested. The results are shown in Figure 9. It will be seen that, in terms of signal-to-noise, the results obtained using 5 μl are as good as those obtained with 50 μl, but with a much higher signal level in the^'case of the 5 μl sample. Example 13 The effect of Fab' anti-IgE conjugated to alkaline phosphatase on a total IgE ELISA was tested. The assay procedure has been described previously using whole anti-IgE (Kemeny D M and Richard R, "ELISA for the detection of total serum IgE: speed and sensitivity." in "Immunological Techniques in Microbiology", Blackwells, Oxford, 1987). Briefly, plastics microtitre plates (Nunc Imrrtuno 1) were coated with more monoclonal anti-IgE (kind gift from Professor A. Saxon, UCLA, USA) which had been isolated by 45% saturated ammonium sulphate precipitation and gel filtration through a column containing AcA34 (LKB Ltd). The monoclonal antibody was used at 1 μg /ml. Sample was added and incubated for three hours, washed and 2-5 μg/ml alkaline phosphatase (AP)labelled anti-IgE added and incubated for three hours followed by substrate, p-nitrophenyl phosphatase and optical density read at 405 nm. Alternatively, with Fab' anti-IgE AP at 0.1 - 0.3 ml was used with the amplified substrate (AMPAK Novo Bio Ltd) and optical density read at 492 nm. It may be seen from Figure 10 that using the Fab' portion of the rabbit anti-IgE conjugated to alkaline phosphatase (AP) followed by enzyme amplification (AMPAK, trade mark of Novo Bio Ltd, UK) results in a ten-fold increase in sensitivity. The reason for this is that inherently lower non specific binding of the detector (Fab' anti IgE-AP) to the solid phase permits greater amplification. It is to be expected that the advantages would also be obtained in the method of the present invention. Referring now to the washing device illustrated by Figures 11 to 13, and in particular first to Figure 11, the washing liquid supply chamber 1 is in the form of a rectangular perspex tank 3 having a sealed lid 4 through which there is an opening 5 for introduing the washing liquid. The chamber 1 has a thickened base plate 6 through which passageways 7 extend to provide washing liquid outlets. The passageways 7 are arranged in the base plate in an evenly spaced 8 by 12 array, similar to the arrangement of wells in a conventional microtitre plate. The internal opening of the passageways 7 are formed by conical recesses 8 machined into the upper surface of the base plate 6, and the external ends of the passageways 7 communicate with shallow cylindrical recesses 9 in a lower surfaces of the base plate 6. The shallow cylindrical recesses 9 provide seatings for synthetic rubber o-rings 11 which act as seals. The end overlaps of the base plate 6 have bore holes 12 extending through them, two at one end and one at the other, to receive locating pegs, while the edge overlaps have bore holes 13 to receive clamping bolts to enable the supply chamber to be clamped to the receiving chamber 2.1 The receiving chamber 2 is of similar construction to the supply chamber 1, essentially consisting of a closed rectangular perspex washing liquid receiving tank 15 having a thickened top plate 16 with an 8 by 12 array of drain passages 26 corresponding to the passageways 7. The upper end of each drain passage 26 opens into a recessed 25 in which a rubber o-ring 27 is seated to act as a seal. The end overlaps of the top plate 16 are provided with up-standing pegs 17, two at one end and one at the other, which locate in the bore holes 12 when the chambers 1 and 2 are clamped together with the holding plate therebetween. The edges of the overlaps of the top plate 16 have bore holes 18 extending through them, their lower ends being fitted with bushes 19 to receive the clamping bolts. Referring to Figures 12 and 13, the holding plate 30 comprises a rectangular perspex block having an evenly spaced 8 by 12 array of wells corresponding in position to the passageways 7 in the base plate 6 of the supply chamber 1. Each well 20 comprises a cylindrical bore, extending through a major part of the thickness of the,plate 21, and a capillary 22 extending through the remainder of the thickness of the plate. In each well 20 a cup 23, having a capillary hole 24 in the base thereof, may be removably seated. The holding plate 30 has bore holes corresponding with those of the base plate 6 to ensure accurate location of the plate between the chambers 1 and 2. The interior of the washing liquid receiving chamber 2 is provided with an outlet 28 towards it base, constituting an outlet for spent washing liquid and for applying suction to the chamber. The outlet 28 is conveniently attached direct to a vacuum line connected to vacuum source, preferably by way of a trap to receive spent washing liquid. When the supply chamber and the receiving chamber are clamped together with the holding plate therebetween, the o-rings 11 are brought to bear on the upper face of the plate 30 or on the rims of the cups 23, if used, thereby sealing the outlets 7 to the chamber 1, while the o-rings 27 in the recesses 25 ensure that the capillaries 22 are in sealed communication with the receiving chamber 2 via the drain passages 26. Washing liquid is introduced into the supply chamber 1 through the opening 5, the outlet 28 of o the receiving chamber 2 is attached to the vacuum source, and the vacuum is applied. Suction due to the vacuum causes washing liquid to be drawn through the outlet 7 into the cups 23, if used, or into the wells 20, passing through the filter paper discs therein, and thence through the outlet holes 24, capillaries 22 and a drain passage 26, and into receiving chamber 2. The filter discs can be partly dried using the device, for example by isolating the device from the vacuum source after emptying the supply chamber 1 of washing liquid, to allow the vacuum to build up and then reconnecting the device to the vacuum source so that the rush of air through the filter paper discs causes them to be partly dried. The discs are then removed from the holding plate by removing the cups 23, and radioactive counting can be conducted on the discs while they are still in the cups 23. In an alternative technique, instead of radio active labelling of the antigen, enzyme labelling is used. The resulting bound enzymes are then used to catalyse a reaction producing a colour change which can then be detected. For such a test, the cups are conveniently placed into receiving members (eg. wells in a conventional titre plate) .arranged to seal the holes 24 so that the liquid poured into the cups does not escape. The washing device of this aspect of the invention is conveniently utilized in the immunoassay methods of the invention.

Claims: CLAIMS 1. A method of solid phase immunoassay, comprising contacting a sample liquid containing an anaiyte with an absorbent carrier having a binding material immobilised therein or thereon, whereby anaiyte in the liquid becomes bound to the binding material, allowing a labelled indicator to contact the bound anaiyte, whereby indicator becomes bound to the bound anaiyte, removing unbound indicator from the carrier, and detecting the remaining bound indicator, characterised in that the labelled indicator is contained in a volume of liquid less than ten times the saturation volume of the carrier. 2. A method according to claim 1, characterised in that the indicator is contained in a volume of liquid less than five times the saturation volume of the carrier. 3. A method according to claim 2, characterised in that the indicator is contained in a volume of liquid less than twice the saturation volume of the carrier. 4. A method according to claim 3, characterised in that the indicator is contained in a volume of liquid substantially equal to the saturation volume of the carrier. 5. A method according to any preceding claim, characterised in that the volume of the sample liquid contacted with the absorbent carrier is less than ten times the saturation volume of the carrier. 6. A method according to claim 5, characterised in that the volume of the sample liquid is less than five times the saturation volume of the carrier. 7. A method according to claim 6, characterised in that the volume of the sample liquid ■ is less than twice the saturation volume of the carrier. 8. A method according to claim 7, characterised in that the volume of the sample liquid is substantially equal to the saturation volume of the carrier. 9. A method according to any preceding claim, characterised in that the saturation volume of the carrier is less than 0.1 ml. 10. A method according to claim 9, characterised in that the saturation volume of the carrier is 10 ul or less. 11. A method according to claim 10, characterised in that the saturation volume of the carrier is 5 ul or less. 12. A method according to any preceding claim, characterised in that the anaiyte is an antibody and the binding material is an antigen specific to the antibody. 13. A method according to claim 12, characterised in that the antibody is IgE. 14. A method according to claim 12, characterised in that the antibody IgG» 15. A method according to any preceding claim, characterised in that the indicator is a radioactive labelled indicator, and the remaining bound indicator is detected by deleting the radioactivity thereof. 16. A method according to claim 15, characterised in that the radio-labelled indicator has a specific activity of less than 5 μCi/μg. 17. A method according to claim 16, characterised in that the radio-labelled indicator has sufficient radioactivity to permit counting of the radioactivity to detect remaining bound indicator in 1 to 2 minutes. 18. A method according to any of claims to 1 to 14, characterised in that the • indicator is an enzyme-labelled indicator, and the remaining bound indicator is detected by detecting a change in the optical properties of a solution of a substrate resulting from action of the enzyme thereupon. 19. A method according to any preceding claim, characterised by the steps of: applying the sample liquid to the carrier and incubating together; washing the sample liquid from the carrier and substantially drying the carrier; applying to the carrier a liquid containing the indicator and incubating together; washing the sample liquid from the carrier; and then detecting the bound indicator. 20. A method according to any preceding claim. characterised in that the absorbent carrier is a paper element. 21. A method according to any preceding claim, characterised by washing and substantially drying the carrier before bringing the carrier and the sample liquid into contact with each other. 22. A method of solid phase competitive immunoassay, wherein a sample liquid containing an anaiyte and a labelled indicator are contacted with an absorbent carrier having a binding material immobilised therein or thereon, the anaiyte and the indicator competing to bind with the binding material, and the quantity of indicator bound is detected, characterised in that the carrier has a liquid saturation volume of less that 0.1 ml and the total quantity or liquid contacted therewith is less than ten times the saturation volume. 23. A method according to claim 22, characterised in that the saturation volume of the carrier is 5 μl or less. 24. A washing device for use in solid phase immunoassay, comprising a washing liquid supply, a washing liquid receiving chamber, and a holding plate, therebetween, the holding plate comprising a plurality of wells in the upper side thereof, and each well having an outlet communicating with the underside of the holding plate, characterised in that the holding plate is removably mountable between the supply and the receiving chamber. 25. A device according to claim 24, characterised in that a holder is removably located in each well, the holder having a drain hole therein corresponding with the outlet, and in that washing liquid supply is arranged to supply liquid to the holders. 26. A washing device according to claim 24 or 25, characterised in that the drain hole in each holder is sized such that liquid in the holder is not lost therethrough under gravity. 27. An immunoassay kit for use in a method according to claim 1 or 22, comprising a plurality of absorbent carrier members having a binding material immobilised therein or thereon, a supply of a solution containing a labelled indicator, and a plurality of different reference samples containing known concentrations of known analytes. 28. An immunoassay kit for use in a method according to claim 1, comprising a plurality of absorbent carrier members having a binding material immobilised therein or thereon, and a supply of a solution containing a labelled indicator, the volume of the solution being approximately equal to the aggregate liquid capacity of the carrier members.

Inventors: KEMENY DAVID MICHAEL, KEMENY, DAVID, MICHAEL
Applicants: KEMENY DAVID MICHAEL, KEMENY, DAVID, MICHAEL
Application year: 1987
Main classification: G01N33
IPC codes: G01N33