Biologicals
(1990) 18, 45-48
The Development of a Latex Agglutination Inhibition Assay for the Determination of Foetal Calf Serum in Viral Vaccines S. Sladoljev,*
Andja TreSCec, Maja CvetkoviC,
hsrirure of Immunology,
M. Beck and Mirjam
JuzbaSiC
4 100 Zagreb, Yugoslavia
Abstract.
A simple but specific, sensitive and reproducible latex agglutination inhibition assay for the determination of foetal calf sera in viral vaccines has been developed and standardized. The detection limit was at nanogram level. The assay procedure requires two pipetting steps, a short centrifugation stage and the use of a spectrophotometer
Introduction
Latex
The production of viral vaccines involves the use of sera of bovine origin in the culture medium, foetal calf serum (FCS) being commonly preferred. The presence of such animal proteins in vaccines for human use can lead to sensitization1,2 and, therefore, manufacturers are required to reduced this impurity in their products. The currently used assays for FCS are immunodiffusion,2 counter current electrophoresis3 and, most recently, a double antibody sandwich ELISA.4 The aim of this study was to establish a simple and sensitive assay for the determination of FCS both during vaccine production and in final vaccines. The method chosen was a widely used latex agglutination inhibition assay (LAIT).5 The LAIT was standardized and evaluated with our reagents.
Uniform latex particles, ULP, 150 and 180 nm in diameter (lot 97-l and lot 5412-88, Institute of Immunology, IZ) were used. Buffer and vaccine medium
Phosphate buffered saline without Ca or Mg (PBS) was used as the washing and dilution buffer. Eagle’s Minimum Essential Medium with Hank’s Salts (MEM Hank’s Gibco, U.K.) containing gelatine and sorbitol was the vaccine medium. Antiserum
Rabbit anti-FCS (Institute of Immunology, IZ) was used. Rabbits were bled after three booster doses when the serum reached a sufficient titre (1:1024000) as demonstrated by passive hemagglutination of sheep red cells coated with FCS. Laboratory glassware
Materials
and methods
Antigen
FCS mixture was obtained from three different suppliers: Microbiological Associates, U.S.A., Batch No. 97310; Gibco, U.K., Batch No. 20 Q 4240A: Seromed, W. Germany, Batch No. 3 Q 05. Control antigens
Horse serum, rabbit serum and human serum (Institute of Immunology, IZ, Zagreb, Yugoslavia) were used. * To whom correspondence should be addressed. 1045-1056/90/010045+04
$03.00!0
All glassware was treated with chromium sulphuric acid and extensively washed prior to use. LAIT procedure
The assays were done in 2-ml round-bottomed testtubes as shown in Fig. 1. A standard curve was constructed from the means optical density values (OD& of titrations in triplicate of the standard solution using a log-log transformation (Fig. 2). Coating of latex particles 0.4 ml FCS was added to each ml of a 05% latex suspension diluted in PBS and incubated overnight at 37°C. For the stabilization of the antigen-coated latex @ 1990 The International
Association of Biological Standardization
I.
46
The freeze-dried vaccine wos dissolved in re-distilled water (usually 0.5 ml per dose).
u l
S. Sladoljev
.
25~1 of the optimol dilution of FCS ontiserum wos odded ond the mi,.ture wos held for 3 h at room temperature.
et a/.
particles 20% glycine in PBS (w/v) was added to the suspension in the proportions 1:lO (v/v>, and the mixture incubated for 2 h at room temperature. The coated ULPs were washed five times in PBS containing 0.5% Tween 80 (30 min, 20000 x g, 4”C, Beckman L2 ultracentrifuge) and then resuspended in PBS containing 0.02% (w/v> sodium aside and 0.001% (w/v> merthiolate. This preparation was used for at least 6 months when stored at 4°C. Negative control
Vaccine control.
w@ I
50~1 of the optimal dilution of cooted latex suspension wos added ond the mixture wos incubated overnight at room temperature.
4.
After incubation centrifugoted 2000-2500 of the supernote
the mixture wos for IO min at x g ond the optical read 380 nm.
medium
without
FCS was used as the
Reading and evaluation
density
The clearing of latex particles from the reaction mixture was expressed as the difference between the ODsso of supernates of the negative control and that of the analyte. These values were measured manually at 380 nm (0.5 cm cuvette) in a PerkinElmer Lambda 3 spectrophotometer. During the assay procedure care was taken to avoid ‘drifts’ in absorbance caused mainly by agitation of test-tubes and consequent disintegration of the pellet. Results
5. FCS content colculoted stondord curve.
l
=FCS
--
(1990) 18, 45-48
The Development of a Latex Agglutination Inhibition Assay for the Determination of Foetal Calf Serum in Viral Vaccines S. Sladoljev,*
Andja TreSCec, Maja CvetkoviC,
hsrirure of Immunology,
M. Beck and Mirjam
JuzbaSiC
4 100 Zagreb, Yugoslavia
Abstract.
A simple but specific, sensitive and reproducible latex agglutination inhibition assay for the determination of foetal calf sera in viral vaccines has been developed and standardized. The detection limit was at nanogram level. The assay procedure requires two pipetting steps, a short centrifugation stage and the use of a spectrophotometer
Introduction
Latex
The production of viral vaccines involves the use of sera of bovine origin in the culture medium, foetal calf serum (FCS) being commonly preferred. The presence of such animal proteins in vaccines for human use can lead to sensitization1,2 and, therefore, manufacturers are required to reduced this impurity in their products. The currently used assays for FCS are immunodiffusion,2 counter current electrophoresis3 and, most recently, a double antibody sandwich ELISA.4 The aim of this study was to establish a simple and sensitive assay for the determination of FCS both during vaccine production and in final vaccines. The method chosen was a widely used latex agglutination inhibition assay (LAIT).5 The LAIT was standardized and evaluated with our reagents.
Uniform latex particles, ULP, 150 and 180 nm in diameter (lot 97-l and lot 5412-88, Institute of Immunology, IZ) were used. Buffer and vaccine medium
Phosphate buffered saline without Ca or Mg (PBS) was used as the washing and dilution buffer. Eagle’s Minimum Essential Medium with Hank’s Salts (MEM Hank’s Gibco, U.K.) containing gelatine and sorbitol was the vaccine medium. Antiserum
Rabbit anti-FCS (Institute of Immunology, IZ) was used. Rabbits were bled after three booster doses when the serum reached a sufficient titre (1:1024000) as demonstrated by passive hemagglutination of sheep red cells coated with FCS. Laboratory glassware
Materials
and methods
Antigen
FCS mixture was obtained from three different suppliers: Microbiological Associates, U.S.A., Batch No. 97310; Gibco, U.K., Batch No. 20 Q 4240A: Seromed, W. Germany, Batch No. 3 Q 05. Control antigens
Horse serum, rabbit serum and human serum (Institute of Immunology, IZ, Zagreb, Yugoslavia) were used. * To whom correspondence should be addressed. 1045-1056/90/010045+04
$03.00!0
All glassware was treated with chromium sulphuric acid and extensively washed prior to use. LAIT procedure
The assays were done in 2-ml round-bottomed testtubes as shown in Fig. 1. A standard curve was constructed from the means optical density values (OD& of titrations in triplicate of the standard solution using a log-log transformation (Fig. 2). Coating of latex particles 0.4 ml FCS was added to each ml of a 05% latex suspension diluted in PBS and incubated overnight at 37°C. For the stabilization of the antigen-coated latex @ 1990 The International
Association of Biological Standardization
I.
46
The freeze-dried vaccine wos dissolved in re-distilled water (usually 0.5 ml per dose).
u l
S. Sladoljev
.
25~1 of the optimol dilution of FCS ontiserum wos odded ond the mi,.ture wos held for 3 h at room temperature.
et a/.
particles 20% glycine in PBS (w/v) was added to the suspension in the proportions 1:lO (v/v>, and the mixture incubated for 2 h at room temperature. The coated ULPs were washed five times in PBS containing 0.5% Tween 80 (30 min, 20000 x g, 4”C, Beckman L2 ultracentrifuge) and then resuspended in PBS containing 0.02% (w/v> sodium aside and 0.001% (w/v> merthiolate. This preparation was used for at least 6 months when stored at 4°C. Negative control
Vaccine control.
w@ I
50~1 of the optimal dilution of cooted latex suspension wos added ond the mixture wos incubated overnight at room temperature.
4.
After incubation centrifugoted 2000-2500 of the supernote
the mixture wos for IO min at x g ond the optical read 380 nm.
medium
without
FCS was used as the
Reading and evaluation
density
The clearing of latex particles from the reaction mixture was expressed as the difference between the ODsso of supernates of the negative control and that of the analyte. These values were measured manually at 380 nm (0.5 cm cuvette) in a PerkinElmer Lambda 3 spectrophotometer. During the assay procedure care was taken to avoid ‘drifts’ in absorbance caused mainly by agitation of test-tubes and consequent disintegration of the pellet. Results
5. FCS content colculoted stondord curve.
l
=FCS
--
