INFECTION

AND

Vol. 20, No. 3

IMMUNITY, June 1978, p. 660-664

0019-9567/78/0020-tE60$02.00/0 Copyright © 1978 American Society for Microbiology

Printed in U.S.A.

Enzyme-Linked Immunosorbent Assay for Measurement of Serological Response to Respiratory Syncytial Virus Infection LINDA S.

RICHARDSON,`*

ROBERT H. YOLKEN,' ROBERT B. BELSHE,' ENA CAMARGO,' HYUN W. KIM,2 AND ROBERT M. CHANOCK'

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20014,'and Children's Hospital National Medical Center, Washington, D.C. 200102 Received for publication 31 October 1977

An enzyme-linked immunosorbent assay was applied to the detection of serum antibodies against respiratory syncytial virus. The end points of the various sera tested in the assay were approximately 100 times higher than in the complementfixation test and 2 to 4 times higher than in the plaque reduction test. In addition, the immunosorbent assay appeared to be more efficient than the plaque reduction and complement-fixation techniques for detecting a serological response in young infants (1 to 6 months old) with serious respiratory syncytial virus lower respiratory disease. The simplicity, sensitivity, and rapidity of the enzyme-linked immunosorbent assay make it a useful tool for immunological studies with respiratory syncytial virus. The immunological response to respiratory syncytial (RS) virus infection has been measured in the past by the detection of a rise in complement-fixation (CF) antibodies in the convalescent serum of the infected individual (1). Failure to detect a CF antibody rise in young infants during convalescence from severe respiratory disease caused by RS virus has variously been ascribed to immunological immaturity of the infant or to the presence of preexisting maternal antibodies which mask an immune response or impair the ability of the infant to mount an immunological response (5-7). More recently, the plaque reduction test has been used for assaying RS virus neutralizing antibodies in human sera. This technique is more sensitive than the CF test; however, the difficulty of performing the plaque assay, as well as the longer time necessary to carry out the test, has limited its general use (4). We report here a sensitive and rapid assay for serum antibodies to RS virus, utilizing the enzyme-linked immunosorbent assay (ELISA) technique, that should have wide applicability in diagnostic and seroepidemiological investigations.

and amphotericin (10 ug/ml). When cytopathic effects of the virus were maximal (3 to 4 days), the cells and fluid were harvested together by quick freezing and stored at -70'C. Virus suspensions made in the above manner contained 105 to 106 plaqueforming units of RS virus per ml. A dilution of this virus preparation was used without further manipulation in the ELISA test to provide both internal and surface viral antigens for interaction with antibodies. Enzyme conjugate. Goat anti-human immunoglobulin G, y-chain-specific serum was obtained from Antibodies Inc., Davis, Calif. The globulin fraction was prepared by sodium sulfate precipitation and coupled to alkaline phosphatase (Sigma type VII) by the method of Engvall and Perlman (3) using 5 mg of enzyme to 2 mg of immunoglobulin. The activity and specificity of the enzyme-linked antiglobulin was tested as previously described (11). ELISA procedure. The method used was modified from Voller et al. (8). The optimal dilutions of reagents were determined by checkerboard titration. The antigen (RS A2 virus grown in HEp-2 or GMK cells) was diluted 1:10 in carbonate buffer (pH 9.8), and 75,ul was added to the wells of round-bottom polyvinyl microtiter plates (Cooke Laboratory Products, Alexandria, Va.). Uninfected cells treated in the same manner as virus-infected cells served as a control. Plates coated with antigen were then stored for at least 14 h at 40C in a moist chamber. Additional storage at 40C for at least 3 months did not result in measurable loss of activity. At the time of testing, the plates were washed three times in a solution of phosphate-buffered saline containing polysorbate (Tween 20) at a concentration of 0.5 ml/liter (PBS-Tween). Fourfold dilutions of serum were made in the antigen-coated plates using PBSTween supplemented with 1% fetal calf serum and 10% uninfected cell suspension. The calf serum and control cell suspension were added to the diluent to

tug/ml),

MATERIALS AND METHODS CF and plaque reduction assays. Procedures have previously been described for the CF and plaque reduction assays (2, 4). Antigen for ELISA. The A2 strain of RS virus (4) was grown in HEp-2 or primary African green monkey kidney (GMK) cells (Flow Laboratories, Rockville, Md.) maintained in Eagle minimal essential medium supplemented with 2% agamma calf serum, 0.002 M glutamine, penicillin (200 U/ml), streptomycin (200 660

VOL. 20, 1978

ELISA FOR RS VIRUS SERUM ANTIBODIES

reduce the nonspecific binding of the test serum (9). The final volume of diluted serum in each well was 75 pl. After an overnight incubation at 40C, the plates were washed three times with PBS-Tween, and a 75of a 1:400 dilution of the enzyme-linked pl samplefraction of goat anti-human immunoglobulin globulin G serum (made in the same diluent as used for the dilution of patients' sera) was added. This was allowed to react for 2 h at 370C. The plates were again washed three times with PBS-Tween, and 75 ul of p-nitrophenyl phosphate substrate (Sigma 104), diluted to contain 1 mg in 1 ml of diethanolamine buffer (pH 9.8), was added (8). After 15 min of incubation at 370C, the amount of yellow color produced by the action of the enzyme (bound to the solid phase) on the substrate was measured in a colorimeter, which determined the absorbance at 400 nm through the bottom of the microtiter plate (10). The ELISA titer of each serum was determined by comparing the absorbance value of each serum dilution with the value of a known positive human serum standard diluted 1:1,600. In each case the absorbance value of the serum in the control well was subtracted from the value obtained in the RS antigen-coated wells. The 1:1,600 dilution of the standard serum was considered the end point in the ELISA test since it had an absorbance value of approximately 0.35, while the absorbance value of the serum at a 1:6,400 dilution was only 0.05. Therefore values greater than 0.35 were considered positive. The standard serum at 1:1,600 dilution was run in each plate to eliminate plate-to-plate variation. Serum specimens. Acute and convalescent sera were obtained from patients admitted to the Children's Hospital National Medical Center of the District of Columbia with severe RS virus respiratory disease. The diagnosis of RS virus disease was established by isolation of RS virus from the oropharynx and/or a fourfold rise in RS virus serum CF antibodies during convalescence. Each of the 1- to 3-month-old infants whose sera were used in the study shed RS virus.

661

Thus, the titer of this serum was

Enzyme-linked immunosorbent assay for measurement of serological response to respiratory syncytial virus infection.

INFECTION AND Vol. 20, No. 3 IMMUNITY, June 1978, p. 660-664 0019-9567/78/0020-tE60$02.00/0 Copyright © 1978 American Society for Microbiology Pr...
695KB Sizes 0 Downloads 0 Views