DE2639569A1 - PROCEDURES FOR VIRUS DETERMINATION - Google Patents

Description

SEPTEMBER 2, 1378 15 778

MERCK & CO., INC.
Rahway, New Jersey 07065, V.St.A.

Procedure for virus "determination

The invention relates to a harrow-automated method for Determination of virus infectivity and the level of neutralizing antibodies in the serum.

To carry out the virus determination, dilutions of a virus preparation, which are preferably produced electro-mechanically with a suitable tissue culture cell suspension, which is preferably automatically transferred to the wells of a microtiter plate have been pipetted mixed. The cells are then incubated on the plate under suitable conditions. The liquid is then withdrawn from the plate. After staining the cells with a protein stain, this follows Reading without optical magnification.

In the neutralization test to determine the antibody content in the serum, dilutions of a serum sample to be determined are preferred electro-mechanically mixed with a pathogen virus. A suitable tissue culture cell suspension, preferably automatically pipetted into the wells of a microtiter plate

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has been mixed with the preincubated serum / virus mixture. After the 'incubation of the cells on the microtiter plate below under suitable conditions, the liquid is withdrawn from the plate. The cells are stained with a protein stain and the reading is taken without optical magnification.

The Yirus determination according to the invention can be applied to any Viruses, for example viruses that infect humans or animals, use. Examples of such viruses are rubella, measles, mumps, herpes, polio, varicella and Marek viruses.

I. Viral mood A. Preparation of the identification plates

Sterile tissue culture plates, transfer plates and lids are unwrapped. The tissue culture and transfer plates are then made assembled in the intended manner and both covered with a lid. On the lids and the tissue culture plates the cells used, the day on which the test was started and a test number are noted in each case. For every sample a test sheet is created.

B. Preparation of samples

Frozen samples and aliquots of standard comparative samples are quickly thawed and placed in a bath partially filled with cold tap water. The samples are immediately before Thawed before use and left to stand in the cold water bath until removed for determination.

Lyophilized samples are reconstituted by adding the required The amount of diluent is added and the sample is also placed in a cold water bath until the start of the determination lets stand.

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C. Prepare the pipetting device

A single-use plastic container is placed at the bottom of the automatic pipetting device. Of the The pipetting head is brought into a preliminary position, after which its tip is provided with a rubber vacuum diaphragm. Then the pipetting head covered with the diaphragm is brought into its final position and locked. Afterward add about 70 ml of diluent to the container, depending on the is refilled according to requirements, pipetted using a sterile 100 ml volumetric pipette.

D. Prepare the dilution device

A single-use plastic container is filled with sterile, distilled water to a height of 9 _? 5 is now filled, which is sufficient for complete immersion of the tips of the micro-dilution device.

The micro-diluent kit is removed from the dilution device, briefly dipped in the water and placed on a sheet of paper. Then the individual microdilution devices Flamed with a bunsen burner. The heated micro-diluents are then put back into the water immersed, placed on top of the flow sheet, and returned to its position in the thinner.

After each plate, the microfluidic diluent kit is removed Removal of residual virus suspension placed on the flow sheet, then immersed in water for rinsing and finally extinguished again. The immersion and extinguishing cycle is repeated at least twice. After every third record remove the micro-diluent set, place it on the blotting paper, immerse it in water, erase it on the above described manner subsided, immersed again and extinguished. The same procedure is followed at the end of each determination day carried out.

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E. Titration of the samples

The transfer plate is removed from the unit consisting of the tissue culture plate, the transfer plate and the cover and placed in the transfer plate holder. The lid is placed back on the tissue culture plate. The transfer plate holder is located in the automatic pipetting device. In the individual wells (there are preferably 96 wells) 0.075 ml of diluent (3 drops with 0.025 tablespoon each) are added. The transfer plate holder is then removed from the pipetting device. 1 drop (0.025 ml) of the sample to be tested is carefully added to each of the 12 wells in row A of the transfer plate using a sterile pipette. A fresh pipette is used for each sample. The transfer plate holder is then placed in the automatic dilution device, which is then operated according to the manufacturer's instructions. Thereby resulting seven 1: 4 Eeihenverdünnungen (0.6 log ^ iQ P ^ro dilution means). The transfer plate is then removed from the transfer plate holder, returned to the tissue culture plate and covered with the lid. Venn is assumed that the sample has a titer of more than 4.2 log ^ _Q, it should be immediately before the determination with the Verdü nn ungsmittel be diluted at least 10-fold by this concentration.

Once all samples and comparative samples have been diluted in the manner described above, the cell suspension becomes as follows admitted. The transfer plate is removed, placed in the transfer plate holder and covered with the lid. The tissue culture plate is placed in the automatic pipetting device. (Immediately before that, it becomes the thinner containing containers intended for single use by another intended for single use Replaces the container that holds the cell suspension in a Concentration of about 160,000 to 260,000 cells per ml, preferably about 200,000 cells per ml. The pipetting

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device is filled several times for rinsing and emptied into the container). In the wells of the tissue culture plate 0.075 ml each of Zeil suspension (3 drops with 0.025 ml each) given.

The tissue culture plate is then removed from the pipetting device removed. The transfer plate is placed in the tissue culture plate and then lifted out, thereby transferring the Virus dilution, en. in the transfer plate on the

Cell suspension in the tissue culture plate is made possible. the The transfer plate is then set aside (for autoclaving). The lid is carefully placed on the tissue culture plate watch out. The other plates are treated in a similar manner and then stacked on top of one another and placed in the incubator Put where an incubation at the required temperature, generally about 30 to about 39 ° C, for that for the virus optimal duration is made.

After the incubation time optimal for the individual virus, the plates are removed from the incubator. The content will emptied into a large container with a jerky movement of the wrist, keeping the hollows down. The plate is returned to the automatic pipetting device, the container of which has been filled with a protein stain, e.g. Carbol fuchsin, is filled.

The carbol fuchsin stain can be used in a concentrated form be washed out after dyeing. The dye can also be used in diluted form, no washing is required after dyeing. Each well is mixed with 0.075 ml of dye (3 drops with each 0.025 ml). After 1/2 minute or later, the stain is removed from the plate (as described above). The plate is then used to rinse off the excess dye immersed in a deep container of tap water. The liquid is removed as described above.

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(If a more dilute dye is used, the Rinsing process). The plate is then covered with a paper towel dried and covered with their lid.

F. Reading

The plates are macroscopically read under exposure to a light source without optical magnification. Appropriately xirden the plates to a fluorescent light source placed. The wells that have a cytopathic see effect (CPE), can be easily recognized by the fact that they are not colored. Uninfected hollows show a uniform red matrix on. On the test sheet, the infected wells are shown as positive (+) and the uninfected cells as negative (-) noted.

The infected and total uninfected wells in each line of the determination plate using a calculation sheet. As indicated on the sheet, the titer is determined on the basis of a calculation according to Reed-Muench or Karber. In short, the minus signs from above and the plus signs from below are added together. The percentage of plus signs of the individual dilution levels is as follows shown, calculated.

II. Neutralization test of the antibodies in the serum A. Preparation of the determination plates Sterile tissue culture plates, transfer plates and lids are unwrapped. Sodar m are put together, the tissue culture plates and the transfer plates in the intended Veise and both covered with a lid. The cells used, the day on which the test was started and the test number are noted on the lids and tissue culture plates. A test sheet is created for each sample.

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B. Preparation of samples

The sera to be determined are inactivated at 56 ° C. for 30 minutes and cooled to room temperature before use.

C. Prepare the pipetting device

A single-use plastic container is placed at the bottom of the automatic pipetting device. The pipetting head is brought into a preliminary position, after which the tip of the pipetting head with a rubber vacuum diaphragm is provided. Then the pipetting head covered with the diaphragm is in its final position brought and locked. Then about 70 parts of the dilution medium are poured into the container again, depending on the requirements is filled, pipetted using a sterile 100 ml volumetric pipette.

D. Prepare the dilution device

A single use plastic container is now filled with sterile distilled water up to a height of 9.5. filled, which is sufficient for complete immersion of the tips of the microdilution device.

The micro-diluent kit is removed from the dilution device, briefly dipped in the water and placed on a sheet of paper. Then the individual micr ο dilution devices are flamed with a Bunsen burner. The heated ones Micro-diluents are then re-immersed in the water, placed on top of the flow sheet, and back into theirs Positioned in the dilution device.

After each plate, the micro-diluent set is removed to remove any remaining virus suspension on the I-sheet put on, then immersed in water for rinsing and finally extinguished again. The immersion and extinguishing cycle is repeated at least 2 times. After every third record

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if the micro-thinner set is removed, placed on the blotting paper , immersed in water, extinguished, flamed in the manner described above, re-immersed and extinguished. The same procedure is carried out at the end of each determination day.

E. Titration of the samples

The transfer plate is removed from the unit consisting of the tissue culture plate, the transfer plate and the lid and placed in the transfer plate holder. The lid is placed back on the tissue culture plate. The transfer plate holder is located in the automatic pipetting device. In each 0.025 ml (1 drop) of diluent is added to the 96 wells. The transfer plate holder is then used by the automatic Pipetting device removed. 1 drop (0.025 ml) of the sample to be examined (serum). will. using a sterile Pipette carefully placed in 2 adjacent wells in row A of the transfer plate. Accordingly, with Proceed with 5 further sera to be examined. Then the Transfer plate holder placed in the automatic thinner, which is then operated according to the manufacturer's instructions. This results in seven Ί: 2 serial dilutions. The transfer plate holder is then removed from the automatic thinner and placed back in an automatic pipetting device, whose container is filled with a suspension of the pathogen virus. 1 drop (0.025 ml) of the suspension of the pathogen virus is then placed in each well of the transfer plate. The one containing the mixtures of diluted serum samples and virus The transfer plate is then removed from the holder, placed on the tissue culture plate, covered with the lid and kept for 1 hour 36 + 1 ° C, 5 percent GOp and 95 percent relative humidity. At the end of the incubation period, the Transfer plate placed on the transfer plate holder and with the Cover covered. The tissue culture plate is then placed in the automatic pipetting device, its container with a suitable tissue culture suspension (preferably 160,000

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Vero cells per ml) is filled. In each of the hollows in the Tissue culture plate are each 0.075 ml (3 drops) of the ZeilSuspension given. Then the tissue culture plate away from the pipetting device. The transfer plate is placed in the tissue culture plate and then lifted out, causing a transfer of the virus dilutions in the transfer plate on the cell suspension in the tissue culture plate will. The transfer plate is then set aside (to "piano" the car). The lid is carefully placed on the Tissue culture plate watch out. The other panels are treated in a similar manner, then stacked on top of each other and in Put the incubator where incubation at the required temperature, generally at about 30 to about 39 ° C for the optimal duration is made for the virus.

J According to the optimal incubation time for the individual virus the plates are removed from the incubator. The contents are enlarged by a jerky movement with the wrist Vessel emptied with the hollows held down. The plate is returned to the automatic pipetting device brought whose container is filled with a protein stain such as carbol fuchsin.

The carbol fuchsin stain can be in concentrated form can be used, washing off after dyeing. The dye can also be used in diluted form, no washing is required after dyeing. Each well is then mixed with 0.075 ml of dye (3 drops with 0.025 ml each). After 1/2 minute or more it will Remove stain from plate (as described above). The plate is then used to rinse off the excess dye placed in a deep container of tap water and the liquid removed in the manner described above. (If a diluted dye is used, the rinsing process is not necessary). The plate is then covered with a paper towel dried and covered with their lid.

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G. Serum Control ·. .

In order to determine whether the examined sera are toxic for the cells used for the titration, another plate is produced, where a diluent is used instead of the pathogen virus, but whose conditions otherwise exactly match the conditions correspond to the test plates.

H. Virus titration

The pathogen virus used in this method is simultaneously determined according to the virus determination method described above. ! For the dilution used in the serum neutralization test, the virus contains 20 to 50 TCIDn _n P ^ o 0.025 ml.

The examples illustrate the invention. All temperature data relate to ⁰ C.

example 1 A. Tissue culture

Cell suspensions of a continuous cell line / rabbit kidneys are made on the day of use. The concentration is 200,000 cells per ml of EMEM (Eagle essential minimal medium) + 10 percent (v / v) fetal calf serum (not inactivated by heat) + 1 percent (v / v) of a 200 millimolar solution of L-glutamine + 0.05 percent of a 100 mg / ml keomycin solution (the L-glutamine is added just before use). About 8 ml of cell suspension are per sample necessary. The cells are stirred until used.

B. Growing Medium

Dilution medium: EMEM + 2 percent (v / v) fetal calf serum (not inactivated by heat) + 0.05 percent (Vol./VaI.) of a 100 mg / ml neomycin solution + 1 percent (v / v) one 200 millimolar solution of L-glutamine (the L-glutamine is added immediately before use).

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C. Concentrated carbol fuchsin staining solution

10 ml fuchsine stock solution (10 g basic fuchsine is stirred for minutes in 95 ^ l hot, 95 percent ethanol). 70 ml of 95 percent ethanol and
320 ml of phenol with a water content of 5 percent.

D. Virus samples

1. Frozen samples are stored until analysis at -70 ⁰ C.

2. Lyophilized samples are stored ^{at 2 to 5 ° C. until the examination.}

3. Frozen aliquots of a standard virus suspension for comparison are stored until examination at -70 ^0C. An aliquot is used for each determination.

The determination plates, the pipetting device and the dilution device are prepared as explained above in the general description. Samples of red virus (frozen or lyophilized) are prepared according to Section B of the general description. The sample is then in given the wells of row A of the transfer plate. The sample is titrated, incubated and stained according to Section E of the general description. Incubation is at 32 + 1 ° C, 5 percent CO2 and 95 percent relative humidity 10 days carried out. The staining is done with concentrated carbol fuchsin staining solution performed.

The plates are then placed on a fluorescent light source placed and read. The infected wells are recorded as positive (+) and the uninfected as negative (-). The test sheet looks like this:

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sample

1 2 3 + + 6th 7th 8th 9 10 11 12 A + _{+ +} + + _{+ + +} + + + + B + _{+ +} + + _{+ + +} + + ■ + + C + _{+ + + + + +} + + + + D + _{+ + + + +} + + '+ + E - _{+ +} • a » + + F - _ _m + + _{+ mm} _ ._ " G - ^ _{- 1 M} , ^ _{- B} ". ^ H - - mm - «· _M ^. _

The titer of the sample is determined by the method according to Reed-Muench or Karber carried out according to the following calculation sheet:

of the diluted sample Ρ / Ν Ή P% P

A. 0.6 12/0 0 20th 100 B. 1.2 12/0 8th 8th 50 C. 1.8 12/0 17th 4th 19th D. 2.4 12/0 28 1 3 E. 3.0 4/8 40 0 0 F. 3.6 3/9 G 4.2 1/11 H 4.8 0/12

The titer of this sample is 3 * 0 logy] Q per 0.025

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Example 2 Neutralization test for mumps antibodies in the serum A. Tissue culture

Vero cell suspensions (a cercopitheque continuous monkey kidney cell line) is produced on the day of the experiment. The concentration 160,000 cells per ml of medium is 199 + 10 percent (v / v) fetal calf serum (not inactivated) + 0.05 percent (V / v) of a 100 mg / ml neomycin solution. About 8 ml Cell suspensions are required per plate. The cells are stirred until used.

B. Diluent

Medium 199 + 20 ml agamma calf serum (inactivated) + 85 ml pro Liter of 2.8 percent NaHCO ^ solution + 0.05 percent (vol./vol.) a 100 mg / ml neomycin solution.

C. Diluted carbol fuchsin dye solution 10 ml fuchsin stock solution,

175 i & l 95 percent ethanol and

800 ml of phenol with a water content of 5 percent.

D. Serum samples

All samples are archived prior to use for 30 minutes at 56 ⁰ C inactivated.

E. pathogen virus

MSD Mumps Standard No. 3 is diluted 1:20 with dilution medium before use diluted (other corresponding mumps virus preparations can also be used).

G. procedure

(1) The determination plates, the pipetting device and the dilution s device are according to the general description prepared. A transfer plate held in the transfer plate holder is placed in the pipetting device. In each of the 96 wells add 1 drop (0.025 ml) of diluent to each

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given. 1 drop (0.025 ml) of the serum to be examined is placed in 2 adjacent wells in row A of the transfer plate. The same procedure is used for 5 other sera to be examined. The transfer plate is then placed in the automatic dilution device, where seven 1: 2 serial dilutions of the sera to be examined are made. The transfer plate in the holder is then returned to the pipetting device (immediately before this, the container intended for single use is filled with the suspension of the pathogen virus). 1 drop of the pathogen virus is placed in each well. The transfer plate is then placed in the tissue culture plate, covered with the cover and kept for 1 hour in an incubator at 36 + 1 ⁰ C, 5 percent CO2 and 95 percent relative humidity. The transfer plate is then removed from the unit, placed in the transfer plate holder and covered with the lid. The tissue culture plate of the unit is then placed in the automatic pipetting device (immediately before the single-use container is filled with the cell suspension). 0.075 ml cell suspension is added to each of the 96 wells of the tissue culture plate. The tissue culture plate is then removed from the pipetting device. The transfer plate is placed in the tissue culture plate and then lifted out, allowing the mixtures of serum dilutions and viruses in the transfer plate to be transferred to the cell suspensions in the tissue culture plate. The transfer plate is then set aside. The lid is fitted onto the tissue culture plate. The remaining panels are treated in a similar way. All plates are left to stand for 7 days at 36 + 1 ° C. and 5 percent COp. The plates are stained using the dilute carbol fuchsin staining solution, de-fluidized but not washed and read (as in Example 1).

(2) Serum Control

To determine whether the sera used for the titration

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Cells are toxic, another plate is made, called hot which is used instead of the pathogen virus dilution medium, while the conditions are the same.

(3) virus titration

The pathogen virus is determined according to Example 1 at the same time. For the dilution used for the serum neutralization test, the virus solution contains 20 to 50 TCID ₅₀ ^per °> ⁰² 5 ^m1

Q) Test sheet

Serum 1 Serum 2 Serum 3 Serum 4- Serum 5 Serum 6

1 2 3 M- 5 6 7 8 9 10 11 12 A + + + + B + + - - + + - - + + C + + - - + + - - + + D + ₊ _ - + + - - + + + + E + + + ■: + + + - - + + + + F + + + + + + - - + + + + G + _{+ +} + + + + + + + + + H + + + + + + + + + + + +

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15778 A. 1: 2 Results Serum 2 Serum 3 Serum 4 2639569 Serum 6 Calculation of B. 1: 4 Serum 1 0/2 0/2 0/2 0/2 dilution C. 1: 8 2/0 0/2 2/0 0/2 Serum 5 0/2 D. 1:16 2/0 0/2 2/0 0/2 2/0 0/2 E. 1:32 2/0 0/2 2/0 0/2 2/0 2/0 J7 1:64 2/0 2/0 2/0 0/2 2/0 2/0 G 1: 128 2/0 2/0 2/0 0/2 2/0 2/0 H 1: 256 2/0 2/0 2/0 2/0 2/0 2/0 Titer 2/0 2/0 2/0 2/0 2/0 2/0 Example 3 2/0 1:16 1: 2 1:64 2/0 1: 8 C1: 2 2/0 -1: 2

A herpes virus neutralization test is carried out on 192 sera Example 1 carried out. One worker is therefore 2 days employed. There are 48 Auto-CPE plates and about 500 ml of reagents required. To carry out the same determination according to a conventional plaque determination ■ method (PEU) would be 20 working days, 2000 CPE plates and about 25OO ml of reagents required.

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Claims (9)

PATENT CLAIMS 1. Method for determining the cytopathic effect with a Tissue culture plate, characterized in that a tissue culture plate which is infected with a virus and Contains cell culture stained with a protein stain, exposed to a light source and see the cytop athi effect determined without optical magnification. 2. The method according to claim 1, characterized in that one used as protein stain carbol fuchsin. 3. The method according to claim 1, characterized in that one the cell culture is stained by bringing it into contact with a protein stain for a sufficient length of time and then the excess stain removed. 4. Virus determination method, characterized by a) an automatic dilution of the virus samples, b) an automatic supply of the cell suspension, c) a simultaneous infection and formation of the cell layer during the incubation of the cell culture and d) a staining and determination of the cytopathic effect without optical magnification. 5. The method according to claim 1, characterized in that one used the rubella virus. 6. Procedure according to. Claim 1, characterized in that one used the mumps virus. 7. The method according to claim 1, characterized in that the measles virus is used. 8. The method according to claim 1, characterized in that the herpes virus is used. - 17- . 709815/1139 ORIGINAL INSPECTED 9. A method for determining the antibody content of serum, characterized by a) an automatic dilution of the serum samples, b) an automatic addition of the pathogen virus, c) a pre-incubation of the mixtures of serum and virus, d) an automatic supply of the cell suspension, e) a simultaneous infection and formation of the cell layer during the incubation of the tissue culture and f) a staining and determination of the cytopathic effect without optical magnification. 709815/1 139