Pathology (1979), 11, pp. 393-9


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S. M. GARLAND, S. A. LOCARNINI AND I. D. GUST Virus Laboratory, Fair$eld Hospital for Communicable Diseases, Melbourne

Su rnmary An enzyme-linked immunosorbent assay was established for detection of antibodies to rubella virus. In this system commercially available rubella antigen was attached to the wells of polystyrene microtitre plates after which sera were added and incubated to allow the formation of antigen-antibody complexes. The presence of bound antibody was detected by adding anti-human globulin coupled to horseradish peroxidase and visually observing the colour change produced after addition of an appropriate substrate. The test was reproducible and simple to perform and had a similar sensitivity to the widely used haemagglutination inhibition system.

INTRODUCTION Whilst in utero rubella infection remains an important cause of congenital defects, the clinical diagnosis of the disease is notoriously difficult (Avery et al., 1965) and decisions on whether to continue with or terminate the pregnancy are usually made on the basis of specific laboratory tests. Whilst serological diagnosis of rubella infection is performed by most laboratories, each of the tests in use suffers from some limitations in terms of speed, specificity or simplicity. For example, the widely used haemagglutination inhibition test (HAI) requires an adsorption procedure to rid the test sera of non-specific inhibitors of haemagglutination and non-specific agglutinins. Likewise the virus neutralization test, whilst extremely specific, is tedious and can only be performed in laboratories equipped for tissue culture and then takes up to two weeks to produce a result. The introduction of enzyme-linked immunosorbent assay (ELISA) by Engvall & Perlmann (1972) offered an attractive new approach to the problem. Because of their sensitivity and simplicity these assays have been applied to a number of systems (Wisdom, 1976) including the serodiagnosis of viral infections (Voller et al., 1976; Wisdom, 1976; Yolken et al., 1977). The aims of the current study were twofold: firstly, to establish a micro ELISA test for rubella antibody along the lines suggested by Voller & Bidwell (1975, 1976) and determine the optimal conditions for test performance; secondly, to compare the sensitivity and specificity of micro ELISA with the HA1 test.



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Pathology (1979), 11, July


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Patients und specimens Test samples were selected from sera which had been referred to the Virus Laboratory at Fairfield Hospital for Communicable Diseases over a 2 yr period. All sera had been stored at 4'C prior to testing. These specimens included 80 sera from healthy adult females which had been forwarded by local doctors for assessment of the patients' immune status, eight sera from patients with suspected rubella which had been forwarded for detection of rubella specific immunoglobulin M (IgM) and 18 pairs of acute and convalescent sera obtained from patients with serologically confirmed (by HAI) rubella. Six of these paired sera had been obtained from patients involved in a recent outbreak, and the remaining twelve pairs of sera had been stored for 18 mth. Huemugglutination inhibition test ( H A I ) HA1 tests for the detection of rubella virus antibody were performed in duplicate using both the kaolin and the manganous chloride-heparin methods of removing non-specific inhibitors (Gust. 1973). Enzyme-linked inimunosorbenr ussuy ( ELISA )

Rubella antigen was coupled to a solid phase support and test sera thought to contain antibody incubated in this sensitized carrier. After washing away unbound serum components, an enzyme-labelled antiglobulin was added and incubated to allow time for the formation of an antigen-antibody-conjugate sandwich. After further washing the amount of bound conjugate was assessed by observing the degree of degradation of the substrate added. Solid phase

Microtitre plates were chosen for use because they are relatively cheap, utilize small quantities of reagent and are suitable for large-scale use. The following commercially available plates were tested: Polystyrene MTP-I (Disposable Products, South Australia); Linbro Polystyrene IS-FB096 (Ramsay Laboratories, Victoria); Cooke Polyvinyl 22C-24, Cooke Polystyrene M220-24A. Cooke Polystyrene M220-29ART, and Cooke Polystyrene M29AR plates (Dynatech Laboratories, Alexandria. Va.). Coating with antigen A variety of commercially available antigens were tested. The correct amount of coating was determined by checkerboard titrations with known positive and negative sera. The following antigens were evaluated: haemagglutination inhibition antigens, Gibco Diagnostics (Madison, Wisconsin, U.S.A.); Behringwerke (Marburg, Germany); Commonwealth Serum Laboratories (CSL, Melbourne), Flow Laboratories (Rockville, U.S.A.) Wellcome Research Laboratories (Beckenham, England); and rubella complement fixation antigen (Flow Laboratories, Rockville. U.S.A.).

Test parameters A variety of different washing fluids and periods of incubation were evaluated. Sheep anti-human IgG (Silenus Laboratories, Melbourne), gamma chain specific. was rendered monospecific by immunoadsorption. The immunoadsorbent was prepared by cross-linking purified human IgG (CSL, Melbourne) with glutaraldehyde (Taab, Reading. England) using the method of Avrameas & Ternyck (1969). The antiserum was incubated with adsorbent, eluted with 0.1 M glycine-HCI, pH 2.8, and then concentrated by negative-pressure dialysis (Sartorius Membrane Filter. Gottingen, Germany). Horse-radish peroxidase (RZ approximately 3.0, Type VI, Sigma Chemical Co., St Louis, Mo.. U.S.A.) was coupled to the IgG by periodate oxidation according to the method of Nakane & Kawaoi (1974). Aliquots of each conjugate were stored in the presence of 10 mg bovine serum albumin (CSL. Melbourne) at -20°C. and diluted with PBS-Tween (PBS, pH 7.ltO.05"~ v,'v Tween 2 0 Fisher ScientificCo., Fairlawn, N.J.) to working strength, prior to use. The optimal working strengths of the conjugate and antigen for the test procedure were determined by checkerboard titration test using known antibody positive and negative sera (Yolken et al., 1977). Suhstrute Two substrates were evaluated: (1) 5-amino-salicylic acid. This substrate was prepared by dissolving 80 mg of 5amino-salicylic acid (Merck Chemicals, Munchen, Germany) in 100 ml of hot (80'C) distilled water. Immediately prior to use, the pH was adjusted to 6.0 with IN NaOH, and to 9 parts of acid was added 1 part of 0.05;; (vjv) H,O, (British Drug Houses, England). (2) 0-phenylene diamine. 100 mg of 0-phenylene diamine (OPD) (British Drug



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Houses, England) was dissolved in 10 ml of methanol (Fisher Scientific Co., Fairlawn, N.J., U.S.A.). Immediately before use, 2 ml of OPD solution was added to 0.2 ml of 3% (v/v) H,O, and 98 ml of distilled water. ELISA test procedure The ELISA method used was a modification of the method of Voller & Bidwell (1975). The wells of each microtitre plate were filled with 100 pl of rubella antigen diluted in 0.05 M carbonate buffer, pH 9.6, and allowed to stand overnight at 4°C. The plates were then washed 3 times with PBS-Tween, allowing a contact time of 3 min per wash. Serial doubling dilutions (1:8 to 1:4096) of each serum under test were made in PBS-BSA (PBS, pH 7.1,O.1% (w/v) bovine serum albumin, Cohn fraction V, CSL, Melbourne) and 50 pl aliquots were added to the wells. Controls containing buffer only or antigen only were included in each test in addition to known HA1 positive and negative sera. Each test was performed in duplicate. The plates were incubated for 1 h at 37"C, then washed 3 times with PBS-Tween and shaken dry. Fifty pl of enzyme-labelled conjugate was added to each well and plates were incubated for a further 1 h at 37 "C. After washing and drying as above, 50 p1 of substrate was added to each well and the plates were incubated at room temperature in the dark for 1 h. The results of the enzyme-substrate reaction were then read visually after the reaction was stopped by adding 0.5% (w/v) sodium azide (British Drug Houses, England) and scored on a 0-3 + scale. The ratings were 0 = no colour, 1 + = a light brown-purple colour, 2 + = medium colour, and 3 + = a strong colour reaction. All readings were performed under code and read by two observers. Statistics

Statistical analysis using correlation coefficient (r) and significant values (P) was performed as described by Armitage (1971).

RESULTS Solidphase Of the 6 types of microtitre plates tested, only the Cooke polystyrene plates (M220-29ART and M29AR) and the Linbro polystyrene plate (IS-FB096) bound sufficient antigen to function satisfactorily. Because of their greater reliability, Cooke M29AR plates were used in all subsequent tests. Coating with antigen While each of the 6 commercially available rubella antigens adsorbed satisfactorily to the microtitre plates, the Wellcome HA1 antigen was chosen for use in this study because it consistently demonstrated a higher optimal dilution on checkerboard titration. Plates sensitized with this antigen were found to retain their activity after storage at 4°C for at least one week. Washing procedures Different washing procedures were investigated. Water or saline was compared to PBS-Tween, both with and without overnight incubation of BSA after antigen adsorption. The polysorbate Tween 20 was found to be an essential component in the washing fluid to reduce non-specific binding of serum proteins to the wells. Tween 20 was especially important if an overnight incubation of BSA was not included following antigen adsorption. Incubation time Incubation times of 0.5, 1.0 and 2.0 h were assessed for each serum, conjugate and substrate step. It was found that a 1 h incubation time for each step provided the optimum level of sensitivity and specificity. Conjugate Optimal conditions for storage were found to be at - 20 "C. It was necessary to store the conjugate in small aliquots as repeated freezing and thawing led to a reduction in working titre. Once thawed, each aliquot was stable at 4°C for at least one week. Substrate 5-amino-salicylic acid was preferred to 0-phenylene diamine because the purplebrown colour produced after reaction with horseradish peroxidase was easier to read and score than the yellow colour produced by 0-phenylene diamine. Because colour continued to




et al.

Pathology (1979), 11, July

Comparison of titres of rubella antibody by HA1 and ELISA



ELISA titre*



No. of sera

HA1 titre*





2 4 3



512-1024 20484096 ~~

26 15 10

13 6



5 9 4 1

128-256 5 12-1024 20484096


3 4 8 2

2 3 2



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* Titres shown as reciprocal of serum dilution

develop in weakly positive reactions after the addition of 1N NaOH, it was found preferable to terminate the reaction with 0.5% NaN,. Reproducibility of results Each serum was tested in duplicate and each test was read independently under code by two observers. On no occasion was a greater than 1 difference observed between duplicate tests on the same serum and the results obtained by the two observers were remarkably consistent. In addition, twelve sera were tested by ELISA on 2 occasions 8 days apart. No significant differences were noted in the titres obtained on each occasion.


Comparison with HAI

The titres obtained on 80 sera tested by both HA1 and ELISA are shown in Table 1. In general, sera which produced low HA1 titres had low ELISA titres and sera with high HA1 titres also had high titres by ELISA. The correlation coefficient (r) between the two tests was


FIG. 1 Correlation of titres as determined by HA1 and ELISA values shown as log, of reciprocal titre



HAI titre*

Interval between sera (days) 1 Recent outbreak: 10 10 10 10 10 13 Pathology Downloaded from by RMIT University on 03/14/15 For personal use only.


Comparison of HA1 and ELISA titres obtained on paired sera from 18 patients with proven rubella


A solid-phase enzyme-linked immunosorbent assay (ELISA) for detection of antibody to rubella virus.

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