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

Preparation and evaluation of an IgG-Lucifer Yellow carbohydrazide conjugate for the detection of measles antigen.

A conjugate, prepared by covalently coupling Lucifer Yellow carbohydrazide (LYCH) to monkey anti-measles IgG, was used to detect measles antigen in in...
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