World
Journal
of Microbiology
& Biotechnology
12,295-296
Short Communication: Preparation and evaluation of an IgG-Lucifer carbohydrazide conjugate for the detection of measles antigen
Yellow
M. Njayou* and C. Mouliom A conjugate, prepared by covalently coupling Lucifer Yellow carbohydrazide (LYCH) to monkey anti-measles IgG, was used to detect measles antigen in infected cell monolayers and in nasopharyngeal swabs from children. Compared with fluorescein isothiocyanate, LYCH is not only easier to couple to antibodies but also produces better fluorescence. Key words:
Antigen,
fluoroscein isothiocyanate,
IgG, immunofluorescence,
Measles is a widespread and highly infectious disease, occurring most frequently among children (Harry 1981). Although measles in developed countries is mostly a benign disease of older children, the disease tends to occur in younger children and have more serious consequences in poorer, developing countries (Guillozet 1979). In some African countries, for example, the disease is responsible for about 25% of infant mortality (Ofusu-Amaah 1983). lmmunofluorescence is a common technique used to detect various antigens (Fulton & Middleton 1975). Although fluorescein isothiocyanate is often used as the fluorescent marker, Lucifer Yellow carbohydrazide (LYCH; Figure I), which is similar in terms of its ease of use and fluorescence intensity, emits at a longer, redder, wavelength (540 nm compared with 515) which contrasts more strongly with tissue autofluorescence (Stewart 1981). The absorption maximum for LYCH is at 430 nm and the compound labels proteins rapidly and covalently under mild conditions. In the present study, in an attempt to develop a more reliable, direct, immunofluorescent test for measles diagnosis, antimeasles IgG was conjugated to LYCH.
Materials Cell &/lure
Vero (Cercopithecoid monkey kidney) cells were seeded at 1 X lo6 to 2 X IO6 cekd25 cm’ and maintained in MEM medium containing 5% inactivated calf serum and IO5 U penicillin and The
strain
of measles
virus
IS(YIS), was isolated (Njayou 1987) during the in Cameroon in December 1993.
used, Yaounde measles epidemic
The authors are with Laboratoire de Biotechnologie Microbienne, Facult& des Seances, Nkolbisson, YaoundB, Cameroon; fax: 237 214310. M. Njayou is also with the Laboratoire de Biochimie Microbienne at the same address. ‘Corresponding author.
@ 1996 Rapid
Science
II
NH -c-NH-NH, I N
0
0
66
Li+O,S
/
\
\
/\
SO-,Li +
I
NH,
Figure
1. The structure
Production
of Lucifer
Yellow
carbohydrazide
of Conjugate
Monkey antiserum to the measles virus was prepared as described by Vaitukaitis (1981). Anti-measles IgG was precipitated from the serum Quash
Facultk gentle
and Viral Strain
measles.
0
with (NH,),SO, and oxidized with NaIO, (Njayou & 1991). The oxidized IgG, 1 nmol in 0.1 M phosphate buffer
(pH 6) was then incubated with Professor G. Quash (Laboratoire
and Methods
40 mg gentamycin/l.
Lucifer Yellow carbohydrazide,
de MCdecine,
Lyon-Sud,
nmol LYCH, a gift from d’Immunochemie des Virus,
10
France),
for
2 h at 4”C,
with
stirring. Any free LYCH was then removed by overnight dialysis at 4OC, against 50 mM phosphate buffer, pH 6.
Evaluation of the Conjugate The conjugate was used in direct immunofluorescence (DI) tests (Njayou et al. 1991) in an attempt to detect measles virus in cells recovered from swabs from 50 sick children in a measles epidemic and 50 healthy controls. Sterile swabs, soaked in phosphate-buffered saline (PBS) were used to collect nasopharyngeal specimens. Each swab was then cut up and vortexed with I ml PBS for 30 min. The resultant suspension was decanted off and any cells in it recovered and washed twice, using 0.2 ml PBS and centri-
fugation at
1000
x g for 5 min each time.
Publishers World Journal of Mmbiology
6 Biotechnology.
Vol 12. 1996
295
M.
Njayou
and C. Mouliom
Results
Figure an
swab, specific
Figure focus
2. A cell
uninfected
cell
infected (left
under fluorescent conjugate.
3. Vero of intensely
cells
with arrow)
measles virus from a positive
illumination
stained fluorescing,
after
(right arrow) nasopharyngeal
staining
with
with the IgG conjugate, virus-infected cells.
Half (25) of the swabs from the six children but none of those from the controls were found positive for measles virus by the DI test. An example result for a positive nasopharyngeal swab is shown in Figure 2. Similarly, all 50 slides of infected monkey cells (Figure 3) but none of the control monolayers were positive by Dl. The DI test appears more sensitive and specific than indirect tests and is simple, fast and cheap. The present conjugate was developed to reduce the costs of the test and make it easily available in rural areas. A similar conjugate, based on mouse, not monkey, anti-measles IgG can also be employed (unpublished work).
and
the virus-
showing
References
a
The conjugate was also used in DI tests to detect virus in infected Vero-cell monolayers grown on 50 glass slides and fixed in acetone for 1 h at 37°C in a humidified atmosphere. The same number of monolayers of healthy, uninfected cells was used as a control.
296
World~ourmd
of Mmbiology
& B&chnology,
Vol 12, 1996
and Discussion
Fulton, R.E. & Middleton, PJ. 1975 Immunofluorescence in diagnosis of measles infections in children. ]oumal of Pediatrics 86, 17-22. Guillozet, N. 1979 Measles in Africa: a deadly disease. Some personal comments. ClinicuI Pediatrics 18, 95-100. Harry, T.O. 1981 Antimeasles IgG in healthy adult Nigerians. ]ownal of Tropical Medicine and Hygiene 84, 171-173. Njayou, M. 1987 Doctorat d’Etat es-sciences, Universite de Lyon 1, France. Njayou, M., Balla, A. & Kapo, E. 1991 Comparison of four techniques of measles diagnosis: virus isolation, immunofluorescence, immunoperoxidase and ELISA. Indian ]ournal of Medical Research 93, 34&344. Njayou, M. & Quash, G. 1991 Purification of measles virus by affinity chromatography and by ultracentrifugation. A comparative study. ]ournal of Virological Methods 32, 67-77. Ofosu-Amaah, S. 1983 The control of measles in tropical Africa: a review of past and present efforts. Reviews of Infectious Diseases 5,546-553. Stewart, W.W. 1981 Lucifer dyes - highly fluorescent dyes for biological tracing. Nuttrre 292, 17-21. Vaitukaitis, J.L. 1981 Production of antisera with small doses of immunogen: multiple intradermal injections. Methods in Enzymology 121, 717-725. (Received
in
December
1995)
revised
form
14
December
19%;
accepted
20