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A simple immunoelectrophoretic method for detecting specific antigens in mixtures of antigens
Journal of Immunological Methods 3(1973) 105-106 © North Holland Publishing Company
Short c o m m u n i c a t i o n A SIMPLE IMMUNOELECTROPHORETIC FOR DETECTING
METHOD
S P E C I F I C A N T I G E N S IN M I X T U R E S O F A N T I G E N S F. de St. J. VAN DER RIET and Mike VILJOEN 3/£R.C. and U.C. T Virus Research Unit, Medical School, Cape Town, South AJ?ica
Received 24 March 1973
Accepted 30 March 1973
A fetal-specific antigen was detected in newborn serum by a modification of Mead's procedure of preparative immunoelectrophoresis.
Mead (1958) described a procedure which he called preparative immunoelectrophoresis for isolating specific antigens from biological extracts consisting of a mixture of antigens. Tile antigens in the extract were allowed to migrate electrophoretically through agar containing antibodies to all the constituents except to the required antigen. The unwanted antigens were each precipitated with their homologous antibodies in the appropriate zones of equivalence but the remaining antigen migrated free. We have modified Mead's method to detect a fetal-specific antigen I in the serum of newborn mice. Rabbit antiserum to adult mouse serum (RAAMS), either undiluted or diluted to the desired concentration in veronal buffer (0.05 M, pH 8.5) was mixed at about 45°C with an equal volume of 2% agarose in the same buffer. This was poured onto one third of a microscope slide (2.5 × 7.6 cm) to form a 2 mm thick layer (fig. I, section A). The remainder of the slide was covered to the same thickness with 1% agarose in buffer only (fig. 1, section B). Newborn mouse serum was deposited in a well made in section A and a voltage gradient of about 0.6 V/cm at 2 mA applied. Bromophenol blue was used as tracer dye. After the dye had migrated to the anode end of the slide, electrophoresis was stopped and two narrow longitudinal troughs were cut as shown in fig. 1. One was filled with rabbit antiserum to newborn mouse serum (RANMS) and the other with RAAMS. hnmunodiffusion and precipitation were then allowed to proceed at room temperature overnight in a humid atmosphere. 1 By fetal-specific antigens is meant those antigens which occur mainly in the fetus, frequently in young (immature) animals and may be detected in trace quantities in normal adult animals, e.g., a-fetoprotein.
106
I". de St.J. van der RIET and M. VILJOEN
--
A
.
,
B
,
i
Fig. 1. Id entificat ion of a fetal-specific antigen in newborn mouse serum. The agarose of section A of the upper slide contained rabbit antiserum to adult mouse serum (RAAMS) which was not previously diluted and of the lower slide the same antiserum previously diluted 1 : 10 in buffer. In both slides the well contained newborn mouse serum, the upper trough, rabbit antiserum to n ewb orn mouse serum, and the lower trough RAAMS.
Tire upper slide in fig. 1 shows the result of an immunoelectrophoretic run in which section A contained a 1 : 2 dilution of antiserum. Immunoprecipitation of all the antigens common to both the neonatal and the adult serum (non-fetal-specific antigens) occurred in this region. A fetal-specific antigen (probably a-fetoprotein) migrated into section B (fig. 1) where it formed a precipitin arc with its homologous antibody which diffused from the upper trough containing RANMS. The lower trough contained RAAMS. Tile lower slide in fig. 1 shows the outcome of a similar experiment to that described above using a 1 : 20 dilution of antiserum in section A. In this instance, the precipitation of non-fetal-specific antigens in section A was incomplete and, therefore, some of these constituents migrated into section B where they formed precipitin arcs with corresponding antibodies which diffused from both of the troughs. These arcs served as reference locations for the fetal-specific antigen arc. The technique described here should be useful in demonstrating the presence of specific antigens in other antigen mixtures, e.g., in the detection of hitherto unrecognized fetal proteins and demonstrating hepatitis antigens in serum.
REFERENCE Mead, T.H., 1958, Biochem. J. 69, 48P.
Short c o m m u n i c a t i o n A SIMPLE IMMUNOELECTROPHORETIC FOR DETECTING
METHOD
S P E C I F I C A N T I G E N S IN M I X T U R E S O F A N T I G E N S F. de St. J. VAN DER RIET and Mike VILJOEN 3/£R.C. and U.C. T Virus Research Unit, Medical School, Cape Town, South AJ?ica
Received 24 March 1973
Accepted 30 March 1973
A fetal-specific antigen was detected in newborn serum by a modification of Mead's procedure of preparative immunoelectrophoresis.
Mead (1958) described a procedure which he called preparative immunoelectrophoresis for isolating specific antigens from biological extracts consisting of a mixture of antigens. Tile antigens in the extract were allowed to migrate electrophoretically through agar containing antibodies to all the constituents except to the required antigen. The unwanted antigens were each precipitated with their homologous antibodies in the appropriate zones of equivalence but the remaining antigen migrated free. We have modified Mead's method to detect a fetal-specific antigen I in the serum of newborn mice. Rabbit antiserum to adult mouse serum (RAAMS), either undiluted or diluted to the desired concentration in veronal buffer (0.05 M, pH 8.5) was mixed at about 45°C with an equal volume of 2% agarose in the same buffer. This was poured onto one third of a microscope slide (2.5 × 7.6 cm) to form a 2 mm thick layer (fig. I, section A). The remainder of the slide was covered to the same thickness with 1% agarose in buffer only (fig. 1, section B). Newborn mouse serum was deposited in a well made in section A and a voltage gradient of about 0.6 V/cm at 2 mA applied. Bromophenol blue was used as tracer dye. After the dye had migrated to the anode end of the slide, electrophoresis was stopped and two narrow longitudinal troughs were cut as shown in fig. 1. One was filled with rabbit antiserum to newborn mouse serum (RANMS) and the other with RAAMS. hnmunodiffusion and precipitation were then allowed to proceed at room temperature overnight in a humid atmosphere. 1 By fetal-specific antigens is meant those antigens which occur mainly in the fetus, frequently in young (immature) animals and may be detected in trace quantities in normal adult animals, e.g., a-fetoprotein.
106
I". de St.J. van der RIET and M. VILJOEN
--
A
.
,
B
,
i
Fig. 1. Id entificat ion of a fetal-specific antigen in newborn mouse serum. The agarose of section A of the upper slide contained rabbit antiserum to adult mouse serum (RAAMS) which was not previously diluted and of the lower slide the same antiserum previously diluted 1 : 10 in buffer. In both slides the well contained newborn mouse serum, the upper trough, rabbit antiserum to n ewb orn mouse serum, and the lower trough RAAMS.
Tire upper slide in fig. 1 shows the result of an immunoelectrophoretic run in which section A contained a 1 : 2 dilution of antiserum. Immunoprecipitation of all the antigens common to both the neonatal and the adult serum (non-fetal-specific antigens) occurred in this region. A fetal-specific antigen (probably a-fetoprotein) migrated into section B (fig. 1) where it formed a precipitin arc with its homologous antibody which diffused from the upper trough containing RANMS. The lower trough contained RAAMS. Tile lower slide in fig. 1 shows the outcome of a similar experiment to that described above using a 1 : 20 dilution of antiserum in section A. In this instance, the precipitation of non-fetal-specific antigens in section A was incomplete and, therefore, some of these constituents migrated into section B where they formed precipitin arcs with corresponding antibodies which diffused from both of the troughs. These arcs served as reference locations for the fetal-specific antigen arc. The technique described here should be useful in demonstrating the presence of specific antigens in other antigen mixtures, e.g., in the detection of hitherto unrecognized fetal proteins and demonstrating hepatitis antigens in serum.
REFERENCE Mead, T.H., 1958, Biochem. J. 69, 48P.