IE41633B1 - Viral anti-body detection - Google Patents

Abstract

The essential component of the diagnostic composition for detecting antibodies against a specific virus in body fluids is a gel which contains intact erythrocytes with the corresponding virus on their surface. It is moreover possible for the virus particles either to adhere directly to the surface of the erythrocytes or else to be connected to the erythrocytes by means of a coupling member. The gel expediently contains a complement in an amount sufficient to ensure lysis of the erythrocytes. In addition, the gel can also contain bactericidal and/or fungicidal agents as well as agents which prolong the life of the erythrocytes. The diagnostic composition can be used in particular for the diagnosis of influenza, parainfluenza, mumps, measles and rubella and of a number of other viral diseases.

Description

This invention relates to a system for diagnosing viral diseases in humans, animals and plants, and in particular for measuring the amount of antibodies produced as a result thereof.

The most common method used for the diagnosis of certain types of viral disease is the haemigglutination-inhibition (H.I.) te..t. This test has the disadvantage that it depends on the fact certain viruses will only agglutinate certai types of red blood cells, e.g. influenza, mumps and parainfluenza viruses agglutinate chicxer human and guinea-pig erythrocytes, adenovir es rat and monkey erythrocytes, and reo·.ir„s.-s -.r-c many enteroviruses only human erythrocytes, lhe test is performed by mixing virus with ene a .□ropriate erythrocytes in the presence of patient's serum, i.e. the fluid which is expressed when blood coagulates to form a clot. If antibodies to the virus are present in the serum, tne virus will be unable to agglutinate the cells.

The haemagglutination inhibition test has the further disadvantage that it is tedious and not accurate as doubling dilution series need to be used with the consequent loss of accuracy, which makes it impossible to detect small differences in antibody titre. Furthermore the sera m>be pretreated to remove non-specific inhibitors .hut -241633 prevent the virus binding to the erythrocytes and therefore result in a false positive reading of the test.

Recently a new test for detecting influenza 5 virus antibodies, called the single radialdiffusion test has been proposed by Schild et al. (Symp. Series Immunobiol. Standard, Vol.20, pp.39-46) in which the appropriate influenza virus (67 to 100 ^.g purified virus parti cles/ml gel) is incorporated into agarose gel. When the gel has set circular wells are cut out and the patient's or animal's serum to be tested is introduced into the wells.

The presence in the test serum of viral antibodies is detected by the appearance of zones of opalesc15 ence surrounding the wells. The opalescence is caused by the diffraction of light owing to a halo of antibody molecules surrounding each virus particle.

The single radial diffusion test has better resol2o ution than the H.I. method and is quicker to perform.

However, it does have the disadvantage that the influenza virus preparation used for the purpose has to be purified and highly concentrated in the gel, which makes the text expensive. Additionally large amounts of virus are required for the test. -341633 It has now been found that by incorporating into a gel erythrocytes, which have virus adhering to their surfaces, allowing a sample of body fluid, which may contain specific anti5 bodies to the specific virus to diffuse into the gel, introducing complement into the -ys am, which is fixed, i.e. comhines, where specif.>_ antibodies have interacted with the virus, visible circular zone of lysis will result, the io size of which is dependent on the amount of antibody present. Lysis is the rupture of erythrocyte with the resultant liberation of haemoglobin. 2u According to the present invention in one aspect therefore there is provided a diagnostic system for detecting the presence of antibodies to a specific virus in body fluids such as blood, serumplasma, nasal washings, cerebrospinal fluid, saliva or milk, which comprises a thin layer of gel containing intact erythrocytes which have the specific virus adhering to the surface thereof. Optionally, the system as hereinbefore defined also comprises complement.

The gel that is used is preferably made from O.s to 2.0% agarose, but agar, gelatin or any suitable matrix which allows diffusion and does not have anti-complement activity can alternatively be used. -441633 The gel concentration should preferably be strong enough so that it is easy to handle, but not so concentrated that diffusion cannot take place.

The erythrocytes may be obtained by venipuncture 5 of a vertebrate animal and collected in a medium that prevents clotting. The concentration of erythrocytes having virus adhering to their surfaces may be from 0.5 to 5.0%. Several types of erythrocytes may suitably be used for the purposes io of the present invention, for instance bovine erythrocytes can be used with certain viruses. Sheep erythrocytes, are usually used, however they have the disadvantage of having the Forssman erythrocyte antigen on their surface, and therefore the fluid to be tested may be treated prior to testing with sheep erythrocytes at 37°C for 30 minutes in order to absorb antibodies to the Forssman antigen.

Other types may be preferred in association with particular viruses, for example when measles virus 20 is being used Patas monkey erythrocytes are usually very convenient and preferred although certain erythrocytes can be too fragile or larger than necessary causing less defined zones of lysis.

On storage erythrocytes tend to become more fragile -5 with age and more susceptible to spontaneous lysis, therefore in order to prolong the life of the -541633 erythrocytes chemical substances may be incorporated into the gel in order to stabilise the cells Alternatively, the erythrocytes may be pretreated with a chemical substance such as glutaraldenyde, in order to stabilise them.

Another method of prolonging the life of the erythrocytes is to incorporate dimethylsulphoxide (DMSO) into the gel at the pouring stage and then freeze the gel in the vapour above liquid nitroge lo The concentration of DMSO in the gel may be from 0.5 to 20% preferably 10% at which concentration the integrity of the gel is maintained.

The virus to be used in the system may be prepared by conventional techniques from animals, 15 egg or tissue culture, see for example, Textbook of Virology by A.J. Rhodes and C.E. Van Rooyan, 5th Edition published by Williams and Wilkins, Baltimore, U.S.A. 1968.

With certain types of viruses the erythrocytes 2o require pretreatment with a chemical substance that promotes the binding of the virus particles to the erythrocyte surface, for example, influenza virus will agglutinate erythrocytes but this is followed by elution at 37°C, i.e. the virus particles will soon detach themselves from the errthrocytes spontaneously, owing to the action -541633 of the viral enzyme neuraminidase on the erythrocytes receptors. In order to ensure that the influenza virus remains permanently attached to the erythrocytes, they may be pretreated with potassium periodate solution, the concentration of which will be dependent on the species of erythrocytes used and will vary from 1.25 to 5.5xlO^M. This results in the formation of aldehyde or similarly modified groups on the erythrocytes surface and prevents the 10 action of neuraminidase. With other types of virus, e.g. rubella, pre-treatment of the erythrocytes with periodate, or any other chemical agent such as chromic chloride or carbodiimide may not be required.

Certain micro-organisms will not adhere directly to the erythrocytes and in such cases it may be possible to couple the organisms to the erythrocytes indirectly, using compounds having an affinity for erythrocytes, such as lectins or lipo20 polysaccarides.

Complement, which in one particular type of the system is already incorporated into the gel, comprises a complex group of serum proteins, most of which have the characteristic of interacting with antibody molecules once these have combined with -741633 an antigen, the effect of such combination being to bring about lysis where the antigen concerned is a cell such as an erythrocyte. The complement required for the purpose of the present invention is conveniently vertebrate complement, which may preferably be obtained commercially from Wellcome Reagents Ltd. or prepared by allowing blood to clot, removing the serum and storing it at 4°C or below, preferably at -20°C. It is incorporated 10 into the system in an amount sufficient to ensure lysis.

Alternatively the complement may be provided for incorporation in the system, for instance in a sealed glass or plastics vial, together with 15 appropriate instructions for use with the gel.

In addition it is convenient to provide two further sealed and sterilized vials containing reference sera one of which contains antibodies to the microbe which has been incorporated into the system, and 2o the other being free from such antibodies. All these essential and optional components of the system for testing body fluids for specific microbial antibodies may be presented and packaged in a box or container, together with instructions for use. It can therefore be emphasised that such a presentation of the system, comprising the -341633 gel composition, the complement and instructions as well as other optional components and aids, is considered to fall within the scope of the present invention and accordingly provide a testing kit.

The system can be adapted to detect a class of antibody, for example IgQ, by using a gel containing in addition to the specific virus an anti-IgG serum. The zone produced by IgG can thereby be prevented by incorporating the anti-immunoglobulin serum into the system.

Alternatively a well is cut in the centre of the gel, into which the test fluid is placed and at a distance from it another well is cut into which anti-IgG serum is deposited. The gel is incubated to allow a zone of lysis to develop and where a zone of no haemolysis occurs, at the edge of the haemolysis zone near the adjacent well, this shows that IgG antibodies were present in the test fluid and have therefore been neutralized. 2o In a further particular aspect therefore the system may comprise an effective amount of an anti-immunoglobulin serum.

Such anti-immunoglobulin sera, may be obtained, -941633 commercially from Wellcome Reagents Ltd., or by purifying sera from myeloma patients, for instance on DEAE cellulose in order to remove unwanted immunoglobulins, adding adjuvant and injecting into animals. The animals are later bled and the sera are removed and can be used for incorporation into the gel.

The system may also be adapted to detect particular haemagglutinin and neuraminidase antigens on influ10 enza virus particles, by using various recombinart strains of influenza virus.

The vessel into which the gel is poured conveniently comprises a plastics or glass plate which has a plain rectangular recess suitable for containing the gel layer, and a flat, transparent rectangular glass or plastics plate which can be placed over the plate with the recess, in order to keep the gel moist, sterile and prevent evaporation. Alternatively a Petri dish or any suitable con20 tainer can be used as long as the thickness of the gel layer is not less than about 0.1 mm and not greater than 5 mm. In addition the gel must have an even thickness.

The vessels used are normally packed under sterile conditions and therefore require no further ster-1041633 iTizing treatment. In order to prevent the unwanted growth of bacteria or fungi on the finished gel a bactericidal and fungicidal agent such as sodium azide or merthiolate is incorpor5 ated into the gel. Therefore the sodium azide has the property of prolonging the storage life of the complement if it is incorporated into the gel.

The sample of body fluid for testing with the system as hereinbefore defined, may be obtained from the patient or animal by several different methods depending on the particular fluid being tested. For instance blood can be obtained by venipuncture. If serum is to be used for the test, it is preferably heated to 56°C and held at that temperature for 30 minutes in order to remove any complement present. The samples can be applied to the gel either by deposition in a well cut out of the gel, or adsorption onto a circle of filter paper having a diameter of 4-5 mm, which in turn is placed on the surface of the gel.

Where specific microbial antibodies are present in the sample of fluid, a circular and visible zone of lysis is produced, whenever the complement is already present in the gel or introduced subsequent to the fluid, the size of which is positively -1141633 related to the logarithm of the antibody titre.

In another aspect of the invention there is provided a method of detecting the presence of antibocJ in body fluids to a specific virus wnich comoi ts applying a sample of body fluid, such as clooa, serum, plasma, nasal washings, cerebrospinal flud, saliva or milk from a patient or animal, t. a diagnostic system, as hereinbefore defined, allowing the fluid to diffuse into the gel and interact with the components of the system which also contain complement, to provide a visible zone of erythrocyte lysis corresponding to the amount of micrcoial antibody in the body fluid. Alternatively the complement is not incorporated into the system but may be added after the fluid has been allowed to diffuse into the gel.

The method of detecting microbial antibodies described may be used for diagnosing many diseases, for example viral diseases such as influenza, botn A & B, rubella, measles, parainfluenza, cytomegalovirus and louping ill. The test is especially useful for detecting those people or animals who have no immunity or low levels of immunity to a particular disease and therefore are at risk and requ re vaccination. The test can also be used for -1241633 diagnosing virus diseases in plants. A preparation of diseased plant tissue is injected into an animal and after a period of time serum, which contains antibodies to the plant virus, is removed from the ‘j animal and used in the test. The test can also be used for detecting antibodies to psittacosis, mycoplasmas as well as bacteria.

The advantages of this method in the viral field over previously used methods are that it is quick, simple and easy to carry out, and it is very specific since it can distinguish between antisera to closely related strains of a virus, particularly strains of influenza virus, and may even differentiate between primary and secondary infection. The advantage of the system pro15 vided by the invention is that, compared with the Schild technique, no purification or concentration of the virus may be needed, indeed with influenza virus crude allantoic fluid may be used. In addition the visible area of the lysis provides antibody levels within an accuracy of 5 to 202.

A Hyland plate suitable for containing the gel according to the invention will now be described with reference to the accompanying drawing. The Hyland plate 2 comprises a plain rectangular base 4 and a lid 6, both made from a transparent plastics material.

The base 4 has a rectangular recess 8 into which a gel 10 is poured. Wells 12 are cut out of the gel 10 -1341633 in order to receive the body fluid samples. The lid 6 fits tightly over the base 4 in order to protect the gel from drying and physical damage. The lid 6 is surrounded by a rim 14 which engages a lip which sur5 rounds the base 4.

The invention will now be illustrated in the following examples: Example 1 Equal volumes of washed packed sheep erythrocytes and J-θ a 5.5 x 10~Sl solution of potassium periodate in .aline were mixed and allowed to stand at room temperature for 10 minutes. Allantoic fluid, from chicken eggs, containing influenza virus strain A2 (haemagglutination titre approximately 4 x 10 /ml) was added, the volume of 15 the fluid being ten times that of the packed erythrocytes.

The mixture was left at room temperature for 10 minutes, the coated erythrocytes were then washed three times in barbitone buffered saline i.e. saline containing barbitone and buffered at pH 7.2, to remove free virus not attachca to the erythrocytes, and finally made up to a 50% v/v suspension in buffered saline. 1% Agarose was made up in barbitone buffered saline, and an aliquot (1.5 ml) of this was poured into a Hyland plate to form a base layer (1 mm thick). A sample (100 μΐ) of -1441633 the 50% erythrocytes suspension was mixed with agarose (1.5 ml) at 45°C and the resultant suspension was poured on top of the base layer and allowed to solidify to form a test system having an even thickness (2 mm).

Example 2 The 50% v/v virus coated erythrocyte suspension was prepared as in Example 1. A portion (100 ρΛ) of this was added to agarose (3 ml) and to this was added guinea pig complement solution (100 pi of 1:160 haemolytic titre) Ιθ obtainable from Wellcome Reagents Ltd. The resultant suspension was poured into a Hyland plate and allowed to solidify to give an even layer (2 mm thick).

Example 3 A 1% solution of Agarose was made up in barbitone buffered saline and to this was added sufficient sodium azide to give a 0.1% solution. A suspension containing virus coated erythrocytes and guinea pig complement was then prepared as in Example 2, using the agarose solution and aliquot (3 ml) was then poured into a Hyland plate.

Example 4 A virus coated erythrocyte suspension was prepared as in Example 1. After leaving at room temperature for 10 minutes the cells were washed once, then 1 ml of the -1541633 packed cells were treateG with 0.5 ml of 1% glutaraldehyde solution for 7| minutes. The cells v. then washed four times to remove excess glutaraldehyde and a gel plate was then prepared as described in Example 1.

Example 5 A gel was prepared as in Example 1, except that the virus used was rubella, 8HK grown Thomas strain (haemagglutination titre approximately 1024/ml). The lo erythrocytes and rubella virus were allowed to s arfor 1 hour at 4°C to allow for adsorption. In aGdit'ur· 0.1% sodium azide was incorporated into the agarose gel.

A sample of patients blood was placed on an area of filter paper (Whatman, Trade Mark) in an amount sufficient that discs (4 mm diameter) cut out of it each contained 2.3 x 10 ml of blood adsorbed thereon. The blood soaked discs were then placed on top of the gel left at 4°C overnight and then incubated at 37°C for 3 hours, at the end of which time any zone 2o of lysis under each filter paper disc indicated that the patient has antibodies in their blood to rubella.

Example 6 A gel was prepared as in Example I except that tne virus used was louping ill (haemagglutination titre 1:256) -1641633 The erythrocytes were treated with an equal volume _3 of 10 M potassium periodate before being left to stand with the virus for 45 minutes at 4°C to allow for adsorption. The standard procedure for preparation of the plate, as described in example 1, was then carried out.

Example 7 A gel was prepared as in Example 1 except that the erythrocytes used were Patas Monkey cells and the virus used was measles virus (haemagglutination titre 1:1024). The Patas Monkey erythrocytes are treated with an equal _3 volume of 10 M potassium periodate before being left to stand with the virus for 15 minutes at 37°C. The standard procedure for preparation of the plate, as described in Example 1, was then carried out.

Example 8 A gel was prepared as in Example 1 except that the micro-organism used was Psittacosis (complement fixation titre 1:64). Erythrocytes and Psittacosis organisms were allowed to stand for 1 hour to allow for adsorption The standard procedure for preparation of the plate, as described in example 1, was then carried out.

Example 9 A gel was prepared as in Example 1 except that the micro-organism used was Myeopla.-.ma gallisepliaum (haemagglutination titre 1:64). The periodate treated -1741633 erythrocytes were mixed with 2.0 ml of a sonicated 1 in 5 dilution of the Mycoplasma preparation and left to stand for 20 minutes. The standard procedure for preparation of the plate, as described in Example 1, was then carried out.

Example 10 A gel was prepared as in Example 1 except that the micro-organism used was Bordetella pertussis. The erythrocytes and Bordetella organisms were allowed 10 to stand for an hour at 4°C, with intermittent snaking, to allow for adsorption. In addition 0.1% sodium azide was incorporated into the agarose gel. The standard procedure for preparation of the plate, as described in Example 1, was then carried out.

Example 11 A gel was prepared as in Example 10 except that the micro-organism used was Vibrio. choleras Inaba.

Example 12 A patient suspected of suffering from influenza strain A2 provided samples of blood.

A test system was prepared as in Examples 1, 2 and 3 and wells (3 mm diameter) were punched out. To each well was added an aliquot (10 Jt,l) of the test body -1841633 fluid e.g. serum, which had previously been heated to 56°C and held at that temperature for 30 minutes in order to inactivate it. The system was kept overnight in a moist box at a reduced temperature (4°C) 'j in order to allow the completion of diffusion. After diffusion the system was covered with a solution of guinea pig complement (1 ml of a freeze dried preparation obtainable from Wellcome Reagents Ltd., and reconstituted with distilled water according to the lo instructions on the container) and then incubated (37°C) until zones of lysis developed (2-3 hours) which indicated that the patient has at some time 2 suffered from type A influenza. The system was then washed to remove complement and released haemoglobin and it was finally fixed in Formal Saline (10%) for storage and reference purposes. The diameter of the zones of lysis produced were then measured, and compared with calibrated reference zones.

Example 13 20 A gel was prepared as in Example .2, except that the virus used was Rubella, and a well (3mm diameter) was cut out of the centre. Two further wells were cut out at equal distances from the central well. Into the centre well an aliquot (10 μ,Ι) of the test fluid zs was put, and into the other wells a volume (10 μΐ) of anti-IgG serum was deposited.

The gel was incubated until zones of lysis developed. -1941633 The zones of lysis were examined and in the part of the zone nearest the IgG well an half moon portion pointing towards the test fluid well of no haemol'sis was visible, which showed that IgG immunoglobulin molecules were present in the test fluid and had been neutralized.

Example I4 A kit for use in diagnosing influenza A2 or for detecting the presence of influenza A2 antibodies is 10 composed of two vials each containing a freeze-driec preparation of reference serum, one serum containing specific influenza A2 antibodies and the other not. Also contained in the kit are Hyland plates containing the system as prepared in either Example 1 or 4 together with instructions for use as set out in Example 5.

Example 15 A kit for use in diagnosing influenza A2 strain or for detecting the presence of influenza A2 antibodies is composed of two vials each contains a freeze-dried preparation of reference serum, one containing specific influenza A2 antibodies and the other not, together with Hyland plates containing the system as prepared in any of Example 2, 3 or 4, together with 5 instructions for use.

Claims (11)

1. WHAT WE CLAIM IS:1. A diagnostic system for detecting the presence of antibodies to a specific virus in body fluids comprising a thin layer of gel containing intact erythrog cytes which have the specific virus adhering to the surface thereof.

2. A diagnostic system as claimed in claim 1 comprising a gel containing intact erythrocytes having the specific virus adhering directly to the surface thereof.

3. A diagnostic system as claimed in claim 1 comprising a gel containing intact erythrocytes having the specific virus adhering indirectly to the surface thereof. 15

4. A diagnostic system as claimed in claim 3 wherein the viral particles are coupled by means of a lectin or 1 ipopol.ysaccaride to the erythrocytes.

5. A diagnostic system as claimed in any one of the preceding claims wherein the gel also contains comple20 ment.

6. A diagnostic system as claimed in claim 5 wherein the complement is that of guinea piq.

7. A diagnostic system as claimed in either claim 5 -2141633 or claim 6 wherein the complement is present in the qel in an amount sufficient to ensure l.ysis of all the erythrocytes.

8. A diaqnostic system as claimed in any precedinq r claim wherein the qel comprises from 0.5 to 2% aqarose. □

9. A diaqnostic system as claimed in any one of the precedinq claims wherein the concentration of erythrocytes having virus adhering to their surfaces, present in the gel is from 0.5 to 5,0%. io 10. A diagnostic system as claimed in any one of the preceding claims wherein the erythrocytes are bovine erythrocytes. 11. A diagnostic system as claimed in any one of claims 1 to 9 wherein the erythrocytes are those of 15 sheep. 12. A diagnostic system as claimed in any one of claims 1 to 9 wherein the erythrocytes are those of Patas Monkey. 13. A diagnostic system as claimed in any one of the 0 preceding claims wherein the gel also contains a preservative for the erythrocytes. 14. A diagnostic system as claimed in claim 13 wherein the preservative is dimethylsulphoxide. -2241633 15. A diagnostic system as claimed in claim 14 wherein the concentration of the dimethysulphoxide in the gel is 0.5 to 20%. 16. A diagnostic system as claimed in any one of the 5 preceding claims wherein the gel also contains a bactericidal and/or fungicidal agent. 17. A diagnostic system as claimed in claim 16 wherein the bactericidal and/or fungicidal agent is sodium azide or merthiolate. lo 18. A diagnostic system as claimed in any one of the preceding claims wherein the virus is measles virus. 19. A diagnostic system as claimed in any one of claims 1 to 17 wherein the virus is influenza virus. 20. A diagnostic system as claimed in any one of claims 15 1 to 17 wherein the virus is rubella- virus. 21. A diagnostic system as claimed in any one of claims 1 to 17 wherein the virus is cytomegalovirus. 22. A diagnostic system as claimed in claim 1 wherein the gel contains sheep erythrocytes having influenza 2o virus adhering to the surface thereof. ίο 23. A diagnostic system as claimed in claim 1 wherein the gel contains sheep erythrocytes having rubella virus adhering to the surface thereof. 24. A diagnostic system as claimed in claim 1 wr.ere the gel contains Patas Monkey erythrocytes na. ng ea. virus adhering to the surface thereof. 25. A diagnostic system as claimed in any one of the preceding claims wherein the gel also contains an eui immunoglobulin serum. 26. A diagnostic system as claimed in claim 25 wherein the anti-immunoglobulin serum is anti-IgG. 27. A diagnostic system as claimed in one of the preceding claims wherein the gel is in the form of a thin layer which is contained within an enclosed plate. 28. A diagnostic system for detecting the presence of antibodies to a specific virus in body fluids substantially as hereinbefore described, with p.;- J cular reference to any one of Examples 1 o 11. 29. A method of preparing a diagnostic system fo. detecting the presence of antibodies in body fluids to a specific virus comprising preparing a suspenslc intact erythrocytes which have the specific virus aane to the surface thereof and incorporating the -2441633 suspension into a gel. 30. A method as claimed in claim 29, wherein the erythrocytes are pret’reated with a coupling agent. 31. A method as claimed in claim 30 wherein the 5 couplinq agent is a lectin or a lipo-polysaccharide. 32. A method as claimed in claim 29 wherein the erythrocytes are pretreated with a substance that promotes adherence of the virus to the erythrocytes. 33. A method as claimed in claim 32 wherein the sub 10 stance is potassium periodate, chromic chloride or carbodiimide. 34. A method as claimed in any one of claims 29 to 33 which comprises the additional step of mixing complement into the gel. 35. A method as claimed in claim 34 wherein the com15 plement is that of guinea pig. 36. A method as claimed in claim 34 or claim 35 wherein the complement is incorporated into the gel in an amount sufficient to ensure lysis of all the erythrocytes. 20 37. A method as claimed in any one of claims 29 to 36 wherein the gel comprises from 0.5 to 2% agarose. -2541633 38. A method as claimed in any one of claims 29 to 37 wherein the concentration of erythrocytes, having virus adhering to their surfaces, incorporated into the gel is from 0.5 to 5.0%. 5 39. A method as claimed in any of claims 29 to 38 wherein the erythrocytes are bovine erythrocytes. 40. A method as claimed in any one of claims 29 to 38 wherein the erythrocytes are those of sheep. 41. A method as claimed in any one of claims 29 to 3 io wherein the erythrocytes are those of Patas Monkey. 42. A method as claimed in any one of claims 29 to 41 which comprises the additional step of incorporating into the gel a preservative for the erythrocytes. 15 43. A method as claimed in claim 42 wherein the preservative is dimethysulphoxide. 44. A method as claimed in claim 43 wherein the dimethyl sulphoxide is incorporated into the gel in a concentration of 0.5 to 20%. 20 45, A method as claimed in any of claims 29 to 44 which comprises the additional step of incorporating into the gel a bactericidal and/or fungicidal agent. -2641633 46. A method as claimed in claim 45 wherein the bactericidal and/or fungcidal agent is sodium azide or merthiolate. 47. A method as claimed in any one of claims 29 to 5 46 wherein the virus is measles virus. 48. A method as claimed in any one of claims 29 to 46 wherein the virus is influenza virus. 49. A method as claimed in any one of claims 29 to 46 wherein the virus is rubella virus. io 50. A method as claimed in any one of claims 29 to 46 wherein the virus is cytomegalovirus. 51. A method as claimed in claim 29 wherein the sheep erythrocytes are mixed with influenza virus. 52. A method as claimed in claim 29 wherein sheep 15 erythrocytes are mixed with rubella virus. 53. A method as claimed in claim 29 wherein Patas Monkey erythrocytes are mixed with measles virus. 54. A method as claimed in any one of claims 29 to 53 which comprises the additional step of mixing anti20 immunoglobulin serum into the gel. -2741633 55. A method as claimed in claim 54 wherein the anti-immunoglobulin serum is anti-IgG. 56. A method of preparing a diagnostic system for detecting the presence of antibodies in body iluius 5 to a specific virus substantially as hereinbefore described with particular reference to any one of Examples 1 to 11. 57. A test kit, for use in the detection of the presenc of antibodies in body fluids to a specific virus comio prising a flat container enclosing a thin layer of gel containing intact erythrocytes which have the specific virus adhering directly or indirectly to the surface thereof, and also containing complement, together with instructions for use. 15 58. A test kit, for use in the detection of the presence of antibodies in body fluids to a specific virus comprising a kit of two containers, the first container enclosing a thin layer of gel containing intact erythrocytes which have the specific virus 20 adhering directly or indirectly to the surface thereof, and a second container with complement therein, together with instructions for use. 59. A test kit as claimed in either claim 57 or 58 which comprises two additional vessels containing -2841633 reference sera, one of the vessels containing reference serum containing antibodies to the virus which has been incorporated into the gel, and the second vessel containing reference serum free from such antibodies. 5 60. A method of detecting the presence of antibodies in body fluids to a specific virus, comprising applying a sample of body fluid from a patient or animal to a thin layer of a diagnostic system as claimed in any one of claims 5 to 28 and allowing the fluid to diffuse into the

10. Gel and interact with the diagnostic system to provide a visible zone of erythrocyte lysis. 61. A method of detecting the presence of antibodies in body fluids to a specific virus comprising applying a sample of body fluid from a patient or animal to a thin

11. 15 layer of a diagnostic system as claimed in any one of claims 1 to 4 and 8 to 28, allowing the fluid to diffuse into the gel and interact with the diagnostic system and introducing complement onto the gel, to provide a visible zone of erythrocyte lysis.