406
Replacement for Radioimmunoassay?
ELISA:
a
RADIOIMMUNOASSAY is a versatile and excellent laboratory tool, but three factors put its future in doubt. The first two are the expense of reagents and equipment, and the potential health risks from isotopes. The third is the advent of ELISA (enzyme-linked immunosorbent assay), the brainchild of the Swedish researchers ENGVALL and
Fig. 2-Double-antibody-sandwich ELISA
only minute amounts of serum or plasma, usually highly diluted for the procedure. Even samples from finger-pricks are usable-a property which makes ELISA particularly suitable for epidemiological purposes.
these
Fig. 1-Indirect ELISA This and fig. 2 are redrawn from the the authors and publishers.
originals9
17
PERL MANN. 12 In this system enzymes
by permission
of
linked to the isotopes
are
antibodies or antigens, thus replacing used in R.I.A. The enzyme/antibody conjugates have both immunological and enzymic activity, and they can be quantified by their ability to degrade a suitable substrate. The substrate is chosen so that the final result is a colour change which can be assessed visually or objectively in a simple spectrophotometer. For measurement of antibody, an indirect ELISA system (fig. 1) can be used in which the enzyme is linked to an antiglobulin ; antigen can be detected by the doubleantibody-sandwich ELISA (fig. 2) in which the enzyme is linked to a specific antibody. Most of 1. 2.
Engvall, E., Perlmann, P. Immunochemistry, 1971, 8, Engvall, E., Perlmann, P. J. Immun. 1972, 109, 129.
871.
ELISA
tests
take
The test can be done with sensitised carrier surfaces in the form of tubes, beads, or plates. Disposable polystyrene or polyvinyl microhxmagglutination plates are particularly convenient for mass processing since they enable hundreds of samples to be dealt with simultaneously. Many different enzymes could be used for indicator conjugates, the
only requirements being stability, high reactivity, ready availability, cheapness, and safety. Horseradish peroxidase has been advocated, but its substrates may present a carcinogenic hazard. A good alternative is alkaline phosphatase which is slightly more expensive but has an apparently safe substrate. Early experience suggests that ELISA has the same range of sensitivity as R.I.A. and results which are as reproducible. The reagents seem to be stable for much longer than most of those used in R.I.A. ELISA is expected to develop along two main paths. The first should lead to replacement of radioimmunoassay in centralised facilities such as area laboratories and blood-transfusion centres, In this context high-precision ELISA systems will be 3.
Voller, A., Bidwell, D. E., Huldt, G., Engvall, E. Bull. 51, 209.
Wld Hlth Urg 1974
407
needed, possibly incorporating automation. The ,
, ’
obvious applications are hormonal assays,4 assays for hæmatological factors,S and HBsAg and cx-fetoprotein screening.6 In Nice, ELISA is already routinely used for a-F.P. measurement, with results as precise and reproducible as those of R.I.A. The second line of development may be towards cheaper, less precise ELISA assays with results read visually or with simple equipment. This type of assay could be provided in kit form, possibly with as the ultimate simplification. of the routine Many antibody assays for viral and bacterial disease could be based on ELISA; the existing wide variety of techniques is uneconomical in staff and equipment. To date, antibodies to rubella,’ have been measured in similar microplate ELISA systems, so one can envisage ELISA screening for the more important infectious diseases of pregnancy. Tests for syphilis,10 brucellosis," salmonellosis,12 and cholera13 have shown promise, and ELISA may have a special part to play in tropical diseases where low-cost serological tests are needed for mass use. Preliminary observations 14 on malaria, schistosomiasis, Chagas’ disease, and sleeping sickness are encouraging. Veterinaryls and agricultural diagnostic problems may also yield to the method, and the perspective is still broadening. Enzyme immunoassays are expected to overtake radioimmunoassays within a few years.16
dipstick system
a
LACTATION, FERTILITY, AND CONTRACEPTION
j ’
WOMEN who are breast-feeding their infants tend to have a later return of menstruation and to be less fertile than mothers who are not lactating. The reasons are complex and the statistical evidence is inconsistent and difficult to interpret. Thomson et al. 18 in a review for the World Health Organisation, point out that behavioural as well as physiological factors must be taken into account. In some societies there may be taboos prohibit-
4 Van-Weemen, B. K. PH.D. thesis, Groningen University, 1974. 5. Bartlett, A., Dormandy, K., Hawkey, C., Stableforth, P., Voller, A. Br. med.
J. 1976, i, 994. 6 Masseyeff, P., Maiolini,
R. in
Immunoenzymatic Techniques (edited by
G.
Feldman, P. Druet, J. Bignon, and S. Avrameus); p. 199. Amsterdam, 1976.
7 Voller, A., Bidwell, D. E., Br. J. exp. Path. 1975, 56, 338. 8 Voller, A., Bidwell, D. E. ibid. 1976, 57, 243. 9 Voller, A., Bidwell, D. E., Bartlett, A., Fleck, D. G., Perkins, M., Oladehin,
B. J. clin. Path. 1976, 29, 150. 10
Veldkamp, J., Visser, A. M. Br. J. ven. Dis. 1975, 51, 227. 11 Engvall, E., Carlsson, H. E. in Immunoenzymatic Techniques (edited by G. Feldman, P. Druet, J. Bignon, and S. Avrameus); p. 135. Amsterdam, 1976.
12. Carlsson, H. E., Lindberg,
A.
A., Hammarstrom,
S.
Infect.
Immun.
1972,
6, 703.
13 Holmgren, J., Svennerholm, A. M. ibid. 1973, 7, 759. 14Voller, A.,
Bartlett, A., Bidwell, D. E.
Trans. R. Soc. trop. Med.
Hyg. 1976,
70, 98, 15. Ruttenberg, E. J., matic
16
Steerenberg, P. A., Borsi, B. J. M., Buys, J. ImmunoenzyTechniques (edited by G. Feldman, P. Druet, J. Bignon and S.
Avrameus); p. 149. Amsterdam, 1976. Broadhurst, I. Proc. I Symp. non-isotop. Immunoass.,
paper 6.
Brussels,
1976.
17 Voller, A., Bidwell,
D.
E., Bartlett,
A. Bull. Wld Hlth Org. (in the press). Wld Hlth Org. 1975, 52,
18 Thomson, A. M., Hytten, F. E., Black, A. E. Bull. 337
intercourse for a given time after parturition; and, even in the absence of formal constraints, women who lactate may have a lessened sexual drive. Thus care must be taken in the interpretation of data from different cultural groups. The physiological changes involved in the return of menstruation and fertility have not been fully definedlargely because of the difficulty in determining the exact moment at which a woman regains her fertility. The only absolute evidence would be a further pregnancy. Usually the return of menstruation is accepted as indicating renewed fertility, but this definition may have pitfalls because of possible confusion with bleeding caused by retained products of conception or infection, or associated with an intrauterine device. However, even if it were possible to ensure that the bleeding was menstrual (in the sense that it arose from endometrial shedding), fertility could not be assumed. In the early weeks of lactation, women are usually amenorrhoeic, but menstruation and even conception are compatible with prolonged breast-feeding. A host of hormonal relationships is therefore possible during this time, and the report by Delvoye and colleagues’9 is a first step towards clarifying them. Mothers in Central Africa who were breast-feeding their infants up to 15 months post-partum had raised plasma-prolactin levels whether or not menstruation had returned. The prolactin concentrations were higher in the amenorrhoeic than in menstruating mothers, but both groups had very much higher levels than the men used as controls. The plasma F.S.H. and L.H. concentrations were not significantly different between the two groups of women, suggesting (at least for these African women) that the early menstrual cycles were anovulatory. But many questions remain to be answered. Do all breast-feeding mothers, amenorrhoeic or menstruating, have high prolactin levels as long as lactation is established? Does prolactin in such mothers inhibit F.s.H. and L.H. production or inhibit the action of these hormones at ovarian level? What percentage of women who are successful breast-feeders will ovulate early, and what will the post-partum time interval be? Are there ethnic and cultural differences? Lactation is probably associated with reduced fertility in the population sense, but is not a reliable form of contraception for the individual. In those situations where efficient and reliable contraception is required while breast-feeding is maintained, we need to know the effects of any contraceptive measure upon lactation. Hormonal agents are the main cause for concern, but it is very hard to organise controlled trials to assess their effects. A double-blind trial, in which patients were randomly allocated to groups which took either a contraceptive agent or a placebo, would be unethical; contraceptive agents can not be forced on women who do not want them, nor can placebos be given to women who think they are using a reliable contraceptive agent. Trials have been undertaken during the first days or weeks post-partum, when the risk of pregnancy is reduced and in societies where no subsidiary problems are likely to arise, but they are really needed in developing countries, with mothers in fully established lactation. Such a trial needs to show clearly whether the agent under study affects establishment or maintenance of lactation. Unin-
ing
19.
Delvoye, P., Delogne-Desnoeck, J., Robyn, C. Lancet, 1976, ii, 288.
Replacement for Radioimmunoassay?
ELISA:
a
RADIOIMMUNOASSAY is a versatile and excellent laboratory tool, but three factors put its future in doubt. The first two are the expense of reagents and equipment, and the potential health risks from isotopes. The third is the advent of ELISA (enzyme-linked immunosorbent assay), the brainchild of the Swedish researchers ENGVALL and
Fig. 2-Double-antibody-sandwich ELISA
only minute amounts of serum or plasma, usually highly diluted for the procedure. Even samples from finger-pricks are usable-a property which makes ELISA particularly suitable for epidemiological purposes.
these
Fig. 1-Indirect ELISA This and fig. 2 are redrawn from the the authors and publishers.
originals9
17
PERL MANN. 12 In this system enzymes
by permission
of
linked to the isotopes
are
antibodies or antigens, thus replacing used in R.I.A. The enzyme/antibody conjugates have both immunological and enzymic activity, and they can be quantified by their ability to degrade a suitable substrate. The substrate is chosen so that the final result is a colour change which can be assessed visually or objectively in a simple spectrophotometer. For measurement of antibody, an indirect ELISA system (fig. 1) can be used in which the enzyme is linked to an antiglobulin ; antigen can be detected by the doubleantibody-sandwich ELISA (fig. 2) in which the enzyme is linked to a specific antibody. Most of 1. 2.
Engvall, E., Perlmann, P. Immunochemistry, 1971, 8, Engvall, E., Perlmann, P. J. Immun. 1972, 109, 129.
871.
ELISA
tests
take
The test can be done with sensitised carrier surfaces in the form of tubes, beads, or plates. Disposable polystyrene or polyvinyl microhxmagglutination plates are particularly convenient for mass processing since they enable hundreds of samples to be dealt with simultaneously. Many different enzymes could be used for indicator conjugates, the
only requirements being stability, high reactivity, ready availability, cheapness, and safety. Horseradish peroxidase has been advocated, but its substrates may present a carcinogenic hazard. A good alternative is alkaline phosphatase which is slightly more expensive but has an apparently safe substrate. Early experience suggests that ELISA has the same range of sensitivity as R.I.A. and results which are as reproducible. The reagents seem to be stable for much longer than most of those used in R.I.A. ELISA is expected to develop along two main paths. The first should lead to replacement of radioimmunoassay in centralised facilities such as area laboratories and blood-transfusion centres, In this context high-precision ELISA systems will be 3.
Voller, A., Bidwell, D. E., Huldt, G., Engvall, E. Bull. 51, 209.
Wld Hlth Urg 1974
407
needed, possibly incorporating automation. The ,
, ’
obvious applications are hormonal assays,4 assays for hæmatological factors,S and HBsAg and cx-fetoprotein screening.6 In Nice, ELISA is already routinely used for a-F.P. measurement, with results as precise and reproducible as those of R.I.A. The second line of development may be towards cheaper, less precise ELISA assays with results read visually or with simple equipment. This type of assay could be provided in kit form, possibly with as the ultimate simplification. of the routine Many antibody assays for viral and bacterial disease could be based on ELISA; the existing wide variety of techniques is uneconomical in staff and equipment. To date, antibodies to rubella,’ have been measured in similar microplate ELISA systems, so one can envisage ELISA screening for the more important infectious diseases of pregnancy. Tests for syphilis,10 brucellosis," salmonellosis,12 and cholera13 have shown promise, and ELISA may have a special part to play in tropical diseases where low-cost serological tests are needed for mass use. Preliminary observations 14 on malaria, schistosomiasis, Chagas’ disease, and sleeping sickness are encouraging. Veterinaryls and agricultural diagnostic problems may also yield to the method, and the perspective is still broadening. Enzyme immunoassays are expected to overtake radioimmunoassays within a few years.16
dipstick system
a
LACTATION, FERTILITY, AND CONTRACEPTION
j ’
WOMEN who are breast-feeding their infants tend to have a later return of menstruation and to be less fertile than mothers who are not lactating. The reasons are complex and the statistical evidence is inconsistent and difficult to interpret. Thomson et al. 18 in a review for the World Health Organisation, point out that behavioural as well as physiological factors must be taken into account. In some societies there may be taboos prohibit-
4 Van-Weemen, B. K. PH.D. thesis, Groningen University, 1974. 5. Bartlett, A., Dormandy, K., Hawkey, C., Stableforth, P., Voller, A. Br. med.
J. 1976, i, 994. 6 Masseyeff, P., Maiolini,
R. in
Immunoenzymatic Techniques (edited by
G.
Feldman, P. Druet, J. Bignon, and S. Avrameus); p. 199. Amsterdam, 1976.
7 Voller, A., Bidwell, D. E., Br. J. exp. Path. 1975, 56, 338. 8 Voller, A., Bidwell, D. E. ibid. 1976, 57, 243. 9 Voller, A., Bidwell, D. E., Bartlett, A., Fleck, D. G., Perkins, M., Oladehin,
B. J. clin. Path. 1976, 29, 150. 10
Veldkamp, J., Visser, A. M. Br. J. ven. Dis. 1975, 51, 227. 11 Engvall, E., Carlsson, H. E. in Immunoenzymatic Techniques (edited by G. Feldman, P. Druet, J. Bignon, and S. Avrameus); p. 135. Amsterdam, 1976.
12. Carlsson, H. E., Lindberg,
A.
A., Hammarstrom,
S.
Infect.
Immun.
1972,
6, 703.
13 Holmgren, J., Svennerholm, A. M. ibid. 1973, 7, 759. 14Voller, A.,
Bartlett, A., Bidwell, D. E.
Trans. R. Soc. trop. Med.
Hyg. 1976,
70, 98, 15. Ruttenberg, E. J., matic
16
Steerenberg, P. A., Borsi, B. J. M., Buys, J. ImmunoenzyTechniques (edited by G. Feldman, P. Druet, J. Bignon and S.
Avrameus); p. 149. Amsterdam, 1976. Broadhurst, I. Proc. I Symp. non-isotop. Immunoass.,
paper 6.
Brussels,
1976.
17 Voller, A., Bidwell,
D.
E., Bartlett,
A. Bull. Wld Hlth Org. (in the press). Wld Hlth Org. 1975, 52,
18 Thomson, A. M., Hytten, F. E., Black, A. E. Bull. 337
intercourse for a given time after parturition; and, even in the absence of formal constraints, women who lactate may have a lessened sexual drive. Thus care must be taken in the interpretation of data from different cultural groups. The physiological changes involved in the return of menstruation and fertility have not been fully definedlargely because of the difficulty in determining the exact moment at which a woman regains her fertility. The only absolute evidence would be a further pregnancy. Usually the return of menstruation is accepted as indicating renewed fertility, but this definition may have pitfalls because of possible confusion with bleeding caused by retained products of conception or infection, or associated with an intrauterine device. However, even if it were possible to ensure that the bleeding was menstrual (in the sense that it arose from endometrial shedding), fertility could not be assumed. In the early weeks of lactation, women are usually amenorrhoeic, but menstruation and even conception are compatible with prolonged breast-feeding. A host of hormonal relationships is therefore possible during this time, and the report by Delvoye and colleagues’9 is a first step towards clarifying them. Mothers in Central Africa who were breast-feeding their infants up to 15 months post-partum had raised plasma-prolactin levels whether or not menstruation had returned. The prolactin concentrations were higher in the amenorrhoeic than in menstruating mothers, but both groups had very much higher levels than the men used as controls. The plasma F.S.H. and L.H. concentrations were not significantly different between the two groups of women, suggesting (at least for these African women) that the early menstrual cycles were anovulatory. But many questions remain to be answered. Do all breast-feeding mothers, amenorrhoeic or menstruating, have high prolactin levels as long as lactation is established? Does prolactin in such mothers inhibit F.s.H. and L.H. production or inhibit the action of these hormones at ovarian level? What percentage of women who are successful breast-feeders will ovulate early, and what will the post-partum time interval be? Are there ethnic and cultural differences? Lactation is probably associated with reduced fertility in the population sense, but is not a reliable form of contraception for the individual. In those situations where efficient and reliable contraception is required while breast-feeding is maintained, we need to know the effects of any contraceptive measure upon lactation. Hormonal agents are the main cause for concern, but it is very hard to organise controlled trials to assess their effects. A double-blind trial, in which patients were randomly allocated to groups which took either a contraceptive agent or a placebo, would be unethical; contraceptive agents can not be forced on women who do not want them, nor can placebos be given to women who think they are using a reliable contraceptive agent. Trials have been undertaken during the first days or weeks post-partum, when the risk of pregnancy is reduced and in societies where no subsidiary problems are likely to arise, but they are really needed in developing countries, with mothers in fully established lactation. Such a trial needs to show clearly whether the agent under study affects establishment or maintenance of lactation. Unin-
ing
19.
Delvoye, P., Delogne-Desnoeck, J., Robyn, C. Lancet, 1976, ii, 288.
