AU2001100242B4 - Micro titre tray assembly and viral diagnostic kit including same - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR AN INNOVATION PATENT

(ORIGINAL)

Name of Applicant: Actual Inventor: Address for Service: Invention Title: The following statement is performing it known to me: Robert ALEXANDER Robert ALEXANDER DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

"MICRO TITRE TRAYASSEMBLYAND VIRAL DIA GNOSTIC KIT INCLUDING SAME" a full description of this invention, including the best method of Q:\OPER\Axd\2444926 215.doc 7/8/01 P:\OPER\AXD\ALEXANDER INNOVAPPLN.DOC 7/8/01 -2- MICRO TITRE TRAY ASSEMBLY AND VIRAL DIAGNOSTIC KIT INCLUDING SAME The present invention relates to a micro titre tray assembly and to a viral diagnostic kit comprising same. More particularly, the invention relates to a micro titre tray assembly which includes a plurality of individual sample wells or sample well units which are received by receptacles of a micro titre plate.

Conventional diagnostic procedures for identifying viruses include seeding containers with particular cell lines selected on their sensitivity to certain viruses and then inoculating the cell culture with a biological sample putatively containing a virus. Such biological samples include saliva, swabs and the like. The inoculated cell culture is then incubated and the cells examined for cytopathic effects induced by the virus. As certain viruses only grow on certain cells, the virus can be identified on the basis of the cell type in which it either induces a cytopathic effect or does not induce a cytopathic effect.

There are a number of alternative protocols to this procedure including removing cells which have been inoculated with a virus preparation and subjecting the cells to trypsonisation and detecting viruses by monoclonal antibodies specific for viral-derived polypeptides labelled with a reporter molecule such as an FITC molecule. A further alternative is to include a cover slip within a culture tube in order to enhance recovery of the cells.

The conventional (or traditional drum) method utilizes tubes which are seeded with appropriate cell lines. After reaching about 80% of cell confluency the tube is inoculated with appropriate specimen and monitored for CPE for up to three weeks. Daily monitoring of CPE is required for the 1 st week. Less frequent monitoring is necessary for the second and third week. Often, blind passage is required to enhance virus recovery.

One of the disadvantages of this method is that it is time and work consuming since daily monitoring is required. Generally, two people inspect the same tube for CPE by light microscope to avoid subjectivity. Furthermore, not all viruses cause CPE and those which do P:\OPER\AXD\ALEXANDER INNOVAPPLN.DOC 7/8/01 -3not are not detected by this method.

CPE formation monitored in this method depends on sensitivity of the cell lines and virus capability to produce visible CPE. Toxicity of the specimen may also disadvantageously produce changes similar to the viral CPE. Also, some viruses produce CPE only after a long period of time (Cytomagalovirus). As results are commonly based on CPE detection only and are not routinely confirmed by any other method, these factors can lead to inaccurate diagnosis.

Using their method it is also practically impossible to use more than 2 or 3 tubes per specimen due to the resulting accumulation of tubes (40 specimens per day creates 500 tubes in first week alone).

The shell vial method is currently the most advanced method for virus recovery. A 5ml plastic vial (16mm in diameter) with a translucent lid is used. After an appropriate treatment a round (13mm) coverslip is inserted into the vial. The vial is seeded with a sensitive cell line which grows a monolayer on the cover slip. When the monolayer reaches about 80-90% confluency, the medium is discarded and the vial is inoculated with the patient's specimen. Then, the incubated vial is monitored for CPE, followed by the removal of the cover slip. The slip can then be fixed to a microscope slide and stained by monoclonal antibodies.

This method has advantages in that virus recovery can be enhanced by centrifugation of vials after inoculation. Also, the time required to obtain results is reduced, since there is no need to wait for CPE. The cover slip can be removed on the second or third day and stained with appropriate monoclonal antibodies. Further, results (specific CPE) can be confirmed by using antibody antigen staining. Due to enhancement by centrifugation, the time needed to obtain viral results can advantageously be shortened to 10-14 days.

However, this method is time consuming as the cover slips required special treatment: multiple washings with detergent and acetone followed by washing in distilled water and sterilization.

The cover slips also have to be manually inserted into the vials.

PNOPERRAS\2456576 2nd p.dom.-11114/2006 -4- 0 o Another disadvantage of this method is that daily observation for CPE is necessary (the same Z procedure as seen in conventional method). Also, if immunofluorescent staining is necessary, the procedure required is complicated and time consuming. The medium from the shell vial has to be discarded and the cover slip manually (using specific forceps) removed, air dried and fixed to a microscope slide (using vacuum grease). The removal of cover slips is tedious, since the cover slips may be broken by rough manipulation or unintentionally turned and fixed to the microscope slide with the monolayer upside down. Another complication may arise due to the seeded cells growing also on the bottom of the cover slip thus fixing it to the vial. Removal of (i such cover slips is very labourious.

Practically, in this method it is impossible to use more than 2 or 3 tubes per specimen due to the accumulation of tubes (40 specimens per day creates 500 shell vials per week). Still further, a large amount ofmonoclonal antibodies is required for immunofluorescence staining in order to cover the round 13 mm cover slip.

The 96 well plate method is used only in limited cases for recovery of viruses growing on the same cell line (if the wells are seeded with LLC-MK2 cell line recovery of parainfluenza and also influenza viruses is possible). Monoclonal antibodies are used for diagnosis in conjunction with a 96 well plate.

This method has advantages in that the samples are relatively easy to manipulate when seeded with a cell line; large number of specimens can be inoculated onto the same plate; enhancement by centrifugation is possible; only a small amount of media is used (only 0.3ml instead of 1used in shell vials), antigen-antibody techniques may be used for confirmation of results and it enables easy to "read" monitoring of CPE.

However, in this method the whole plate must be used for antigen-antibody detection which is not generally practical. Also, the entire plate has to be used on the same day, even when the number of specimens is smaller than required for the whole plate. This means that for each day a new set of different plates must be used. Further, commonly only one or two different cell lines can be used per plate (the same type of specimen is inoculated onto the plate). As such, PAOPERRAS2456576 2nd I d- 1114/2006 o methods confirming the detection of viruses have to be done on the whole plate and at the same Z time. This disadvantageously results in a situation where, once the detection is completed, there are not remaining cells available for a repeat procedure in case of an error or after a prolonged incubation period.

The present invention aims to provide a rapid, efficient and inexpensive means for alleviating the disadvantages of the known micro titre tray assemblies, preferably which is easily modified Sdepending on the particular diagnosis which is to be conducted.

According to one aspect of the invention there is provided a micro titre tray assembly when used in a viral diagnostic assay comprising: a micro titre plate having a plurality of receptacles; and a plurality of sample wells which are complimentary with the receptacles, each of the wells being individually and separately retainable within and removable from a respective receptacle of the plurality of receptacles.

In a preferred embodiment, each of the wells is resiliently retainable in a respective receptacle.

For example, each of the wells may be resiliently retainable within a respective receptacle by virtue of a friction fit. That is, each well may be tapered so that the base of the well may be inserted into a respective receptacle, but due to increase in diameter of the well, the well is lodged into its respective receptacle. Other alternatives will be readily determined by a person skilled in the art. For example, each of the wells may include at least one ridge on an external surface thereof which engages an internal wall, or internal walls of the respective receptacle of the micro titre plate causing the friction fit.

The micro titre tray may be of any form and may include, for example, a conventional micro titre plate. Preferably, the micro titre plate is provided with identification means for identifying sample wells held in the receptacles. In particular, the identification means may include a reference grid wherein each row of the plurality of receptacles is provided with a corresponding letter code and each column of the plurality of receptacles is provided with a corresponding number code.

The micro titre plate is preferably provided with a complimentary cover, preferably adapted to P:IOPERLASU2456576 2rnd sp doc-1 1114/2006

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-6provide each of the sample wells with individual cover. In particular, the cover preferably Z includes a plurality of circular ridges each of which, when the cover is placed on the micro titre plate, encircle the opening of a respective sample well to substantially enclose a sample held in the well.

According to another aspect of the invention there is provided a micro titre tray assembly for use in a viral diagnostic assay, the assembly comprising: a micro titre plate having a plurality of receptacles; and a plurality of sample wells which are complimentary with the receptacles, each of the wells being individually and separately retainable within and removable from a respective receptacle of the plurality of receptacles, wherein each sample well is seeded with a cell line.

In an embodiment the sample wells are arranged in rows and columns, wherein at least two sample wells in the same row are seeded with the same cell line, and wherein the assembly comprises wells seeded with at least two different cell lines.

In another embodiment, the sample wells are not individually and separately retainable, but rather are retained as a small unit of sample wells. The units of sample wells are preferably only small, including up to four sample wells. Any more than this renders the units inappropriate in accordance with the invention insofar as the advantages of the invention may not be achieved, the above disadvantages relating to the 96 well plate method applying. That is, the necessity to test the whole unit at the same time.

According to yet another aspect of the invention, therefore, there is provided a micro titre tray assembly comprising: a micro titre plate having a plurality of receptacles; and a plurality of sample holding means, wherein each of the sample holding means includes up to four sample wells each of which is complimentary with a respective receptacle of the micro titre plate, and wherein each of the sample holding means is, as a unit, retainable within and removable from a number of receptacles of the plurality of receptacles corresponding to the number of sample wells of the sample holding means.

According to this embodiment, the sample holding means including up to four sample wells P.0PRRAS2456576 2nd gp x I 1114/2006 Q-7o may be configured depending on the particular diagnosis which is to be carried out. For Z example, it may be that it is desirable to present a sample in duplicate so that duplicate assays may be conducted. In a preferred embodiment, the micro titre plate is configured such that the number of receptacles in each row or in each column of the plate corresponds to the number of sample wells in each of the sample holding means.

The sample holding means may be manufactured as desired. In this regard, the sample wells of each of the sample holding means may be integrally formed, or may be detachable from each C other to form individual sample wells. In the latter of these two options, it should be understood that the connection of the individual sample wells may be achieved using any means available for detachably connecting, for example, plastic articles. Similarly, the micro titre plate itself may be attachable to other plates as desired, preferably by a "snap-fit" engagement. For example, the plates may be of a standard 8 x 2 arrangement which are attachable to each other to provide 8 x 4 or 8 x 12 arrangements. Variations of this will be readily understood by those in the art, and are considered to fall within the ambit of this invention.

According to yet another aspect of the invention, there is provided a viral diagnostic kit comprising a micro titre tray assembly as described above and forceps adapted to facilitate removal of the sample wells or sample holding means from the micro titre plate.

The forceps may take any suitable form provided that secure grasping of the individual sample wells or a sample holding means including up to four sample wells is facilitated. In a preferred embodiment the forceps are especially adapted for this purpose including a first portion to be inserted in the well and a second portion which cooperates with the first portion and grasps the outer surface of the well. The first portion is preferably circular in cross-section and of a size to provide only minimal clearance from the inner wall of the well when inserted in the well.

In another aspect of the invention there is provided the use of a micro titre tray assembly comprising: a micro titre plate having a plurality of receptacles; and a plurality of sample wells which are complimentary with the receptacles, each of the wells being individually and separately retainable within and removable from a respective receptacle of the plurality of P :PERRASU2456576 2nd Ip d-I I/141200D6

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-8o receptacles, in a diagnostic method for the detection of a plurality of viruses.

z The above described assemblies according to the invention provide specific advantages over the known methods and assemblies currently available. In particular, the assemblies of the invention facilitate increased sensitivity, using five or more highly specific cell lines for the same specimen. Also, enhancement by centrifugation is available. In this regard, centrifugal forces enhance the viral absorption and thus shorten the time for viral detection, in some cases by up to 10 times. Additionally, between 8-16 specimens can be inoculated on the one plate (i resulting in more specimens being handled by a single operator.

Increased objectivity may be provided by methods employing the assemblies of the invention using rapid detection technique by means of fluorescence or enzymatic labelling and producing confirmed results that are available (in 80% of common viruses) within 2-3 days. In this regard, the assemblies of the invention are versatile in that up to 12 different cell lines can be used allowing a large range of viruses to be detected using a combination of different monoclonal antibodies. CPE can also be monitored. Furthermore, since a specimen may be inoculated into 12 separate wells with 5-6 different cell lines, different monoclonals can be used on the different cell lines and within a different time frame. In case of an error separate wells with inoculated specimen are still available to enable additional testing.

Methods incorporating the use of the assemblies of the invention may also provide time saving results being available in 2-3 days. Such methods represent a large saving in hands on work.

The assemblies also enable cost effective testing, only a small amount ofmonoclonal antibodies being required for one test (20-30 tl against 60-80 il in shell vial method).

In use, the plate is seeded with a plurality of cell lines, each specimen being inoculated onto a different row of the plate. For example, if the plate is 8 x 12, eight different specimens may be inoculated onto each plate, each specimen being inoculated into twelve wells of any given row on up to six different cell lines. This advantageously facilitates the detection of at least twelve viruses. It will be recognised, however, that this 96 well plate configuration can be changed according to specific diagnostic requirements. This simply requires an appropriate modification P NOPERIRASUI45676 2nd p doc-i 11 4l7006 -9to the cell line selection to maintain specificity towards the viruses to be detected.

Reference will now be made to Figure 1 which illustrates a typical 96 well plate configuration including a listing of viruses to be detected, relevant cell lines and removal days for each line.

As will be seen from this Figure, the plate is seeded with different cell lines in the following order: Columns 1-3 Columns 4,5 Column 6 Column 7 Column 8 Columns 9-12 LLC-MK2

MDCK

Hep 2 A549 RK13 The removal of the cell lines, due to the individual nature of the wells, can be selective depending on the time schedule which is appropriate for the specific viral detection in question.

In particular, if the detection of PI 1-3 is desired, taking row A for example, wells A 1 to A3 are removed on day two. Similarly, if the detection of INF A, B is desired, wells A4 and A5 are removed on day two. However, if the detection of Entero is required, then well A 11 is removed on day 5. The specific nature of the individual wells facilitates this selective removal and viral detection which provides substantial advantages over the known methods of the prior art.

Reference will now be made to a particular procedure which may be followed using the assembly according to the invention, many steps of which may be optional and should not be considered to be limiting on the invention in any way.

Using vacuum and sterile glass pasteur pipettes, medium is aspirated from all wells to be inoculated. Disposal of pasteur pipettes in a large sharps container is advantageously facilitated.

Using a disposable pipette, an appropriate number of wells of the well plate are inoculated with approximately 150-200 jl specimen per well. The remaining specimen is stored at -70EC. The P:\OPER\RAS2456576 2d Ipl dn-l 1/14/2006 o lid is then replaced on the plate and the date written over the wells inoculated in the plate.

z The plate is then weighed on a digital balance and balanced with balance plates and cards until all plates are equivalent weights 0.5g) and can be balanced in a centrifuge. The centrifuge (-i is run at about 37EC and 3500 rpm for a period of 60 min. Using vacuum and sterile pasteur pipettes each specimen is then aspirated from each well, and using a fresh disposable pipette for each specimen, each well is filled according to cell type, i.e.

N MRC5 10% MEM (labelled A549 2% MEM (labelled 2%) MDCK and LLC-MK MEM with Trypsin (labelled Trypsin).

The specimens are then incubated in a humidified environment at 37EC in a CO 2 incubator by carefully placing the plates in the CO 2 incubator and incubating at 37EC for up to 7 days after inoculation of the last specimen.

Immunofluorescent staining is advantageously used for detection of specific viruses in single wells, using specific monoclonal antibodies. Generally, the following procedure is followed: Using vacuum suction, the medium is removed from the appropriate well(s) and the wells removed from the plate using special forceps and transferred into a different holder. These are then air dried for 10 minutes. 300 itl of cold acetone is then added to each well and allowed to fix for 15 minutes at -20EC. The fixative is then discarded and the sample again air dried for minutes. A specific monoclonal antibody (primary) is then added to each well and the cover plate put in place and the samples incubated for 30 minutes at 37EC. The samples are then removed from the incubator and each well filled with PBS. The PBS is then discarded. This process is repeated four more times. Again, the sample is air dried for 5 minutes, after which a secondary antibody is added to each well. Following this, incubation of the sample again takes place followed by repeated treatments with PBS as mentioned above. A small amount (1 drop) of a specially prepared mounting medium is then added and the results observed under fluorescent microscope.

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S-11- 0 Z EXAMPLES Clinical Specimens Collection and Processing Specimens included cerebrospinal fluids (CSF), urine, faeces, bronchoalveolar lavages (BAL); Snasopharyngeal aspirates (NPA) (all collected directly into sterile containers); whilst swabs from mouth, skin, genital, nasal and throat were collected into viral transport medium (VTM) SMinimum Essential Medium Eagle with Earle's Salt, L-Glutamine 200mM, 1 00x Non Essential Amino Acids New Born Calf serum Gentamicin 50ug/ml, Vancomycin Fungizone 2,5ug/ml, Apothecon, Bristol-Mayers Squibb Co, USA).

Upon arrival in the laboratory the specimens were prepared as follows: Respiratory fluids were divided into two, with one half transferred into a 5-ml vial containing VTM, and the second half was diluted in phosphate-buffered saline (PBS) and centrifuged. The pellet was resuspended in 2-3 drops of PBS and the cells were spotted onto a glass slide used for direct immunofluorescence (DIF) staining.

Swabs in VTM were vortexed, then the swabs removed.

Urine was used neat for inoculation.

CSF specimens were transferred into VTM at a dilution of 1:2 then used for inoculation.

Faeces approximately Ig of material was transferred into 4.5 ml of VTM, vortexed and filtered (0.22um) to remove bacteria.

All specimens were then sonicated for 30 sees at 80 kHz (FISHER dismembrator Model 300 DYNATECH Lab. Inc. USA) and were then centrifuged for 10 mins at 2,500g after which the supernatant was inoculated into tissue culture plates.

Preparation of Shell Vial Plate: A 48 well plate (COSTAR 3548 48 well cell culture cluster, Coming Inc. N.Y. USA) was used, with each well representing one shell vial. Each row contained a cell line i.e. Row A LLC- P \OPER\RAS\2456576 2nd doc-I 1/14/2006

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-12o MK 2 (original kidney, Rhesus monkey ATCC-CCL-7); Row B MDCK (Kidney, Canine Z ATCC-CCL-34); Row C HEp 2 (Epidermoid Carcinoma, Larynx, Human ATCC-CCL-23); Row D A 549 (Lung Carcinoma, Human ATCC-CCL-185); Row E and F MRC 5 (Lung, Human diploid ATCC-CCL-171). The prepared 48 well plate presented the possibility of S 5 inoculation of eight specimens on to five different cell lines. The plates were seeded with 0ml of cell suspension (concentration 300,000 cells/ml) per each well. When the monolayer reached 80 90% confluence, the growth medium MEM (Minimum Essential Medium Eagle O with Earle's Salt), L-Glutamine 2mM, NEAA 100x (Non Essential Amino Acids) 1%, C1 (Multicel, Trace Bioscience PTY, LTD, Australia). Foetal Bovine Serum 10%, (CSL Limited Australia), Gentamicin 50 ;g/ml (Gentamicin Inj. B.P. David Bull Laboratories, Australia), Vancomycin 40p.g/ml (Vancomycin Hydrochloride for i.v. infusion, David Bull Laboratories), was replaced by maintenance medium (the same as the Growth medium, but containing only 2% FCS). The plates were kept in a CO 2 incubator until inoculated. Each clinical sample was inoculated into six wells (the five cell lines as delineated above).

Preparation of 96 Well Plate Strips from the 96 well plate (COSTAR 9102 well cell culture cluster, Coming Inc., N.Y. USA) were removed and individual wells separated and then replaced into the plate holder under sterile conditions. The above mentioned cell lines were used for seeding the wells of the plate using a concentration of approximately 300,000 cells/ml. For each well 200 pl of cells diluted in growth medium was used. The layout of the 96 well plates was as follows: Row A-H Rows 1, 2, 3, 4 were seeded with LLC-MK2 cell line; Whilst: 5 and 6 were seeded with MDCK; 7 with HEp 2; 8 with A 549; and 9-12 with MRC-5 cell line.

P OPER\RAS\2456576 2rd isp doc.- 114/2006

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-13- O Prepared plates were placed in 5% CO 2 incubator at 37 0 C and left until the confluence of cells Z reached approximately 80-90% confluence. Then growth medium was replaced with maintenance medium and the plates were ready for inoculation.

5 Specimen Inoculation 0 O Maintenance medium was removed from wells by vacuum aspiration and 200 300 Pl of specimen supernatant was carefully deposited into each well. A separate plastic, sterile pipette was used for each specimen inoculation into a row of 12 wells of a 96 well plate and a row of six wells on a 48 well plate. Inoculated plates were placed into a sealed metal bucket and centrifuged (HETTICH ROTANTA 96 R HETTICH ZENTRIFUGEN, Germany) at 2,500g in 0 C for 50 60 mins. The supernatant was then removed by vacuum aspiration and replaced with maintenance medium containing MEM supplemented with L-glutamine (200 mM), nonessential amino acids antibiotics (Gentamicin 50 p.g/ml; Vancomycin 40 pg/ml), and 2% of foetal calf serum (FCS). The maintenance medium for LLC-MK2 and MDCK cells also contained Trypsin (2.5 pg/ml) whilst that for A549 and MRC-5 cells was supplemented by addition of 10- 5 M Dexamethazone. Plates were placed in 5% CO 2 incubator (US AUTOFLOW, NUAIRE, Model NU- 4500. USA).

Detection and IF Staining of 96 Well Plate On day two, the maintenance medium was removed by suction from appropriate wells of the 96 well plate for detection of respiratory viruses (respiratory syncitial virus, adenovirus, influenza virus A and B, parainfluenza viruses I, II, III), cytomegalovirus (CMV), HSV 1,2 plus any wells with CPE. Using specially designed forceps the required wells from the 96 well plate were removed and placed in a further 96 well plate holder where cells were air-dried. Wells were then filled with cold methanol-acetone mixture (1:2 ratio) and fixed for 15 mins at -20 0 C. The mixture was then removed, wells air-dried (15 mins) and specific monoclonal antibody (30-40kp 1) added to each well. For example, for detection of adenovirus the well containing A549 cell line had anti-adenovirus monoclonal antibody added and for CMV, wells containing cell line had anti-CMV monoclonal antibody added. Plates were incubated for 30 mins at 37 0

C,

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O -14- O monoclonal antibodies discarded and each well washed with PBS-Tween five times. After air- Z drying each well, 40-50ul of FITC-conjugated secondary antibody was added and plates Sincubated for a further 30 mins at 37 0 C, after which the antibody was discarded, wells washed four to five times with PBS-Tween mixture and once with distilled water. After air-drying, 1 2 5 drops of fluorescent mounting medium was added to each well and observed for 0immunofluorescence using an inverted fluorescence microscope (LEICA DM IRB, Leica, SGermany, and OLYMPUS IX 70, Olympus Optical Co. LTD, Japan).

(N Detection and IF Staining in 48 Well Plate Two days after inoculation the modified shell vial plates were observed for CPE. If none were evident, plates were read again at days five, seven and 10. If no CPE were observed, specimens were reported negative.

If CPE were observed, cells from appropriate wells were scraped, transferred into an Eppendorf tube filled with PBS and spun at 10,000 rpm for 30 seconds (Eppendorf centrifuge 5415C (Eppendorf GmbH, Germany). The supernatant was removed, cell pellet disrupted and an additional wash with PBS followed. After centrifugation, PBS was removed (leaving only 2 3 drops of suspension), the cell pellet disrupted and 10 15 tl of resuspended cells were spotted onto a 10 spot teflon coated slide. The cells were air dried for 10 15 mins then fixed for 10 mins using cold (-20 0 C) acetone. Following air drying, wells were stained using specific monoclonal antibodies as described for the 96 well plate method. The only difference was the washing step, in which the slides were washed three times for five minutes instead of the five instant washes employed for the 96 well plate method.

RESULTS

During July to December 1998 inclusive, 3,298 patient specimens were compared by RETCIF and modified shell vial method. The source of samples included BAL NPA (1,835), CSF Urine (130), faeces (139), mouth swabs (174), nose swabs (116), throat swabs (117), skin swabs (110), genital swabs (350), and tissue samples (156). Collectively, both or either method P.\OPER\RAS\2456576 2nd p. doc- 1114/2006 detected 779 viruses a positivity rate of 24% (Table Of these, 621 viruses were isolated by both RETCIF and shell vial methods. The RETCIF isolated an additional 100 viruses, thus isolating a total of 721 viruses of the total The modified shell vial method isolated an additional 58 viruses resulting in a positivity rate of 87%. The breakdown of viruses isolated by both/either method is: CMV 116 VZV 14 Adenovirus

RSV

Enterovirus HSV 1, 2 48 240 13 222 Influenza Virus A Parainfluenza Virus I Parainfluenza Virus II Parainfluenza Virus III The breakdown of viral types isolated by RETCIF or modified shell vial methods is depicted in Table A.

BLE A compared to the modified shell vial method. Detection of viral isolates by RETCIF as Modified Shell Modified Shell Total Vial Vial RETCIF-96 621 100 721 (92.5%) RETCIF 58 TOTAL: 679 100 779 (24%) PAOPER/RAWA256S76 2nd mp. don.I 1/112006 16- TABLE I Viruses isolated by RETCIF Modified shell vial method Figure I).

CMV 109/81 RSV 234/226 HSV1,2 196/219 Paral1 3/2 WzV 13/13 Para 2 7/1 Enterovirus 10/10 Para 3 22/12 Adenovirus 42/40 Inf A 85/75 TABLE II The number of viruses missed by RETCIF Mod shell vial method figure 11) CMV 7/35 RSV 6/14 HSVI1,2 26/3 Paral1 1/2 VZV I /I Para 2 0/6 Entero virus 3/3 Para 3 0/10 Adenovirus 618 Inf A 8/18 P OPER\RASl2456576 2nd sp. dxn. 111412006 17 TABLE II A comparison of detection of viruses on day 2 by RETCIF Mod. shell vial method Figure 111).

CMV 59/16 RSV 135/83 HSVI1,2 131/154 Para 1 1/1I VZV 0/0 Para 2 Entero virus 0/3 Para 3 Adenovirus 20/6 Inf A 441/19 TABLE IV The percentage of viruses isolated by RETCIF Mod shell vial method Figure IV).

CMV 94/70 RSV 97194 HSV 1,2 88/98 Paral1 75/50 VZV 93193 Para 2 100/86 Entero virus 77/77 Para 3 100/45 Adenovirus 88183 Inf A 91/81 P:\OPER\RASU456576 2nd pI dc- 1114/2006

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0 -18- O TABLE V Mean period in days for viral identification by RETCIF Modified shell vial Figure V).

CMV 3.2 /5.7 Adenovirus 3.4 5.2 HSV 1,2 2.6 2.4 RSV 2.8 VZV 5.2 6.2 Para 1-3 3.0/7.6 c Enterovirus 7.0 3.7 InfA 3.1/4.6

DISCUSSION

In a busy viral diagnostic laboratory processing approximately 10,000 clinical samples per annum and predominantly servicing a paediatric tertiary institution, but also a diverse adult population, the overall positivity rate over a 12 month period was 24%. Detection was of a wide range of viruses (CMV, HSV, VZV, enteroviruses, adenovirus and respiratory viruses) with a peak in respiratory viruses for the winter months (influenza, parainfluenza, RSV). We have developed a modified viral culture method in a microtitre plate system (RETCIF), which we have shown to be more sensitive and allows processing of a large number of samples, yet with a shorter turn around time to a result, as compared to a modified shell vial method.

Previously, the shell vial method has been the most rapid and accurate culture method for viral detection. Enhancement achieved by centrifugation of the specimen onto the cell monolayer allows faster viral detection. However, the disadvantage of this method is the tedious preparation of cover slips (including multiple washings and drying) and the individual insertion of cover slips into the shell vials. No less tedious is the process of acquiring the cover slips from shell vials for viral detection. Furthermore, the need for 5 shell vials with different cell lines for maximum viral recovery is labour intensive.

P: OPERIRAS\2456576 2nd p doc- 11/14/2006

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0 -19- The versatility and efficiency of the RETCIF method is met by the use of a large number of cell Z lines, all placed on one microtitre tray, centrifugation of the sample to the cell line, use of a number specific monoclonal antibodies for detection in an immuno-fluorescence system and less scientist "hands-on" time per clinical sample (unpublished data). This allowed for more rapid detection of most isolates (Figs IV, V, VI, VII) and as shown in Figs VI and VII, 0 particularly for CMV and influenza respectively. In addition, this method detected 5.5% (100 viruses) more isolates (Table I, Figure particularly evident for CMV and parainfluenza (Figs SII and III respectively).

Surprisingly, on analysis of results, we found the modified shell vial method superior to RETCIF in isolating HSV, a virus relatively easy to detect. To further investigate this disparity, we trialed 96 and 48 well plates in the same manner as for routine RETCIF but for specimens requiring HSV isolation only. Over a three month period 503 genital samples from a sexually transmitted diseases clinic were evaluated for HSV. Further evaluation of positive samples has shown no discrepancy (data not shown). At a positivity of 27%, no discrepancies were recorded. We concluded that human error in the data collection was responsible for the pattern of HSV isolation. After meticulous investigation we found out that erroneous data collection with consequent incorrect interpretation were responsible for faulty results.

The choice to examine plates (Modified shell vials) for VZV and enteroviruses at day five and day seven respectively was based on previous observations. Despite observation of CPE earlier than on day seven for enteroviruses, we have taken into consideration that 85% of enteroviruses are isolated on day seven. However, our own observation using RETCIF (enhancement by sonication, centrifugation and hormonal supplementation) will allow us to shorten the detection time to three to four days (unpublished data). In case of VZ virus isolation we suggest (unpublished data) that 72 hours will be adequate, especially when using additional confirmation on A549 cell line (Brumback et al., 1993).

It should be pointed out that the lower peaks on days three and four for RETCIF and days five and seven (Figs VI and VII) by shell plate, relate to the confounder of a limited weekend laboratory service, whereby only new specimens for inoculation are performed and therefore P.OPERA\S(2456576 2nd pa do.l (14/2006

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o specimens inoculated on the last three days of the week are stained on Monday i.e. on day Z three and four after inoculation respectively.

Overall, the preparation of 96 well plates is relatively simple and with good organisation specimens arriving into the laboratory can be immediately processed and inoculated the same day. Inoculation of 40-50 specimens takes approximately one hour (for a well-trained person).

Care must be adhered to with this step to ensure no cross-contamination occurs.

Thus prepared plates have all the advantages of the previous methods, resulting in: 1. increased sensitivity using 5 or more different highly specific cell lines for the same specimen 2. enhancement by centrifugation the centrifugal forces enhance the viral absorption and thus shorten the time for viral detection in some cases by up to ten times 3. additionally, between 8-16 or even more specimens can be inoculated on the same plate resulting in more specimens handled by one person 4. increased objectivity of the method is achieved by using rapid detection technique employing fluorescent or enzymatic labelling that produce confirmed results within 2-3 days (in 85% of common respiratory viruses) 5. versatility A up to 12 different cell lines can be used allowing a large range of viruses to be detected using a combination of different monoclonal antibodies. CPE can also be monitored 6. versatility B since the specimen is inoculated onto 12 separate wells with 5 -6 different cell lines, different monoclonals can be used on the different cell lines and within a different time frame. In case of an error separate wells with inoculated specimen are still available to enable additional testing.

7. time saving the results are available in 2 3 days, thus the method represents a large saving in hand on work 8. economically more viable requires less hands-on work and a smaller amount of monoclonal antibodies for one test (30 .1 against 6 0- 8 01 in shell vial method) P:FPERMRASQ436576 2nd p do- I 114/2006

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-21- Based on 6-day work week we recommend that the seeding of plates by cell lines be done once Z weekly on Friday and the plates then used on the following Monday (3 days old). In such a configuration the seeded plates used are never more than 8 days old. All the cells in use are from ATCC (American Tissue Culture Collection) and are controlled weekly for sensitivity to the above mentioned viruses. Every Monday, one of the plates is inoculated with the control virus stock. Positive results obtained on day 2 are assurance of cell sensitivity and monoclonal antibody specificity.

Overall, the RETCIF, in our hands is a rapid, sensitive and cost saving procedure with a higher isolation ratio than the shell vial method. The high isolation rate, along with early diagnosis provides invaluable information for diagnosis, initiation of treatment with antiviral agents and isolation of infective patients. The results achieved allow us to recommend the RETCIF method for use in any diagnostic virology laboratory.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or group of integers or steps.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Claims (3)

1. 3. A micro titre tray assembly according to claim 2, wherein the sample wells are arranged in rows and columns, wherein at least two sample wells in the same row are seeded with the same cell line, and wherein the assembly comprises wells seeded with at least two different cell lines.
2. 4. A viral diagnostic kit comprising a micro titre tray assembly according to any one of claims 1 to 3 and forceps adapted to facilitate removal of the sample wells from the micro titre plate.
3. 5. Use of a micro titre tray assembly comprising: a micro titre plate having a plurality of receptacles; and a plurality of sample wells which are complimentary with the receptacles, each of the wells being individually and separately retainable within and removable from a respective receptacle of the plurality of receptacles, in a diagnostic method for the detection of a plurality of viruses.