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RABIES ANTISERUM

Equine Rabies Immunoglobulin

Category Passive immunizing agent.

          Rabies Antiserum is a sterile preparation containing the specific antirabies globulins from immunized horses or other mammals.

          The antiserum complies with the requirements stated under Antisera, with the following modifications.

Description Transparent or slightly opalescent faint brownish, yellowish or greenish liquid; odour, practically odourless or of the antimicrobial agent.

Warning See under Diphtheria Antitoxin, p. 227.

Expiration date The expiration date for Rabies Antiserum is not later than 3 years from the date of the last satisfactory test for potency.

Labelling Complies with the “General Information for Antisera”, p. 225. In addition the label on the container states (1) the recommended human dose; (2) the nature of the preparation, e.g. natural serum or purified immunoglobulin or treated immunoglobulin and, if purified or treated, the nature of the process.

Identification Specifically neutralizes the rabies virus rendering it harmless to susceptible animals. The potency test may serve as an identification test.

Assay The potency is determined by comparing the dose of Rabies Antiserum required to neutralize the infectivity of a rabies virus suspension with the dose of a reference preparation, calibrated in International Units, required to produce the same degree of neutralization.

          Carry out the determination as described in the “Immunochemical Method” (Appendix 14.5). The test is performed in sensitive cell cultures and the presence of unneutralized virus is revealed by immunofluorescence. Rapid Fluorescent-Focus Inhibition Test (RFFIT) is described as the reference immunofluorescent technique for rabies immunoglobulin. Carry out the test in suitable sensitive cells. It is usual to use the BHK 21 cell line, grown in the medium described below, between the 18th and 30th passage levels counted from the ATCC seed lot. Harvest the cells after 2 to 4 days of growth, treat with trypsin and prepare a suspension containing 500,000 cells per ml (cell suspension). Ten minutes before using the cell suspension, add 10 μg of diethylamino-ethyldextran per ml, if necessary, to increase the sensitivity of the cells.

          Use a fixed virus strain grown in sensitive cells, such as the CVS strain of rabies virus adapted to growth in the BHK 21 cell line (seed virus suspension). Estimate the titre of the seed virus suspension as follows.

          Prepare a series of dilutions of the viral suspension. In the chambers of cell-culture slides (8 chambers per slide), place 0.1 ml of each dilution and 0.1 ml of medium and add 0.2 ml of the cell suspension. Incubate in an atmosphere of carbon dioxide at 37º for 24 hours.

          Carry out fixation, immunofluorescence staining and evaluation as described below. Determine the endpoint titre of the seed virus suspension and prepare the working virus dilution corresponding to 100 CCID50 per 0.1 ml. For each assay, check the amount of virus used by performing a control titration: from the dilution corresponding to 100 CCID50 per 0.1 ml, make three tenfold dilutions. Add 0.1 ml of each dilution to four chambers containing 0.1 ml of medium and add 0.2 ml of the cell suspension. The test is not valid unless the titre lies between 30 CCID50 and 300 CCID50.

          Dilute the reference preparation to a concentration of 2 IU per ml using non-supplemented culture medium (stock reference dilution, stored below –80º). Prepare two suitable predilutions (1:8 and 1:10) of the stock reference dilution so that the dilution of the reference preparation that reduces the number of fluorescent fields by 50 per cent lies within the four dilutions of the cell-culture slide. Add 0.1 ml of the medium to each chamber, except the first in each of two rows, to which add respectively 0.2 ml of the two predilutions of the stock reference dilution transferring successively 0.1 ml to the other chambers.

          Dilute the preparation to be examined 1 in 100 using non-supplemented medium (stock immunoglobulin dilution) to reduce to a minimum errors due to viscosity of the undiluted preparation and make three suitable predilutions so that the dilution of the preparation to be examined that reduces the number of fluorescent fields by 50 per cent lies within the four dilutions of the cellculture slide. Add 0.1 ml of the medium to all the chambers except the first in each of three rows, to which add respectively 0.2 ml of the three predilutions of the stock immunoglobulin dilution. Prepare a series of two fold dilutions transferring successively 0.1 ml to the other chambers.

          To all the chambers containing the dilutions of the reference preparation and the dilutions of the preparation to be examined, add 0.1 ml of the virus suspension corresponding to 100 CCID50 per 0.1 ml (working virus dilution). Shake manually and allow to stand in an atmosphere of carbon dioxide at 37º for 90 minutes. Add 0.2 ml of the cell suspension, shake manually and allow to stand in an atmosphere of carbon dioxide at 37º for 24 hours.

          After 24 hours, discard the medium and remove the plastic walls. Wash the cell monolayer with phosphate buffered saline pH 7.4 and then with a mixture of 20 volumes of water and 80 volumes of acetone and fix in a mixture of 20 volumes of water and 80 volumes of acetone at –20º for 3 minutes. Spread on the slides fluorescein-conjugated rabies antiserum ready for use.

Allow to stand in an atmosphere with a high level of moisture at 37º for 30 minutes. Wash with phosphate buffered saline pH 7.4 and dry. Examine twenty fields in each chamber at a magnification of 250×, using a microscope equipped for fluorescence readings. Note the number of fields with at least one fluorescent cell. Check the test dose used in the virus titration slide and determine the dilution of the reference preparation and the dilution of the preparation to be examined that reduce the number of fluorescent fields by 50 per cent, calculating the two or three dilutions together using probit analysis. The test is not valid unless the statistical analysis shows a significant slope of the dose response curve and no evidence of deviation from linearity or parallelism.

          The stated potency is not less than 150 IU per ml. The estimated potency is not less than the stated potency and is not more than two times the stated potency. The confidence limits (P = 0.95) are not less than 80 per cent and not more than 125 per cent of the estimated potency.

CULTURE MEDIUM FOR GROWTH OF BHK 21 CELLS

          Commercially available media that have a slightly different composition from that shown below may also be used.

 

Sodium chloride 6.4 g
  Potassium chlorid 0.40 g
  Calcium chloride, anhydrous  0.20 g
  Magnesium sulfate  0.20 g
  Sodium dihydrogenphosphate, monohydrate  0.124 g
  Dextrose monohydrate  4.5 g
  Iron(III) nitrate nonahydrate 0.10 mg
  L-Arginine hydrochloride  42.0 mg
  L-Cystine  24.0 mg
  L-Histidine 16.0 mg
  L-Isoleucine 52.0 mg
  L-Leucine 52.0 mg
  L-Lysine hydrochloride 74.0 mg
  L-Phenylalanine 33.0 mg
  L-Threonine 48.0 mg
  L-Tryptophan 8.0 mg
  L-Tyrosine 36.0 mg
  L-Valine 47.0 mg
  L-Methionine 15.0 mg
  L-Glutamine 0.292 g
  i-Inositol 3.60 mg
  Choline chloride 2.0 mg
  Folic acid 2.0 mg
  Nicotinamide 2.0 mg
  Calcium pantothenate 2.0 mg
  Pyridoxine hydrochloride 2.0 mg
  Thiamine hydrochloride 2.0 mg
  Riboflavin 0.2 mg
  Phenol red 15.0 mg
  Sodium hydrogencarbonate 2.75 g
  Water to 1000 ml
 

The medium is supplemented with: Fetal calf serum

(heated at 56º for 30 minutes)

10 per cent
  Tryptose phosphate broth 10 per cent
  Benzylpenicillin sodium 60 mg/l
  Streptomycin 0.1 g/l

Usual dose Rabies Antiserum should be given as soon as possible after exposure, preferably within 24 hours. The dose of 40 IU of rabies antiserum per kg of body weight should be infiltrated around the wound(s) as much as possible, the remainder being injected intramuscularly. Active immunization should be started immediately after rabies antiserum has been given.

MONOGRAPHS • RABIES ANTISERUM
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หมายเหตุ / Note : TP II 2011 PAGE 230-232