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Studies of ferritin conjugates used in immune electron microscopy
587
BIOCHIMICA ET BIOPI-IYSICA ACTA
Short Communications Studies of ferritin conjugates used in immune electron microscopy The introduction of immune electron microscopy 1 has presented a method for the detection of antigen-antibody reactions at the subcellular level. The specificity of the conjugates used can be ascertained b y immunofluorescent techniques ~, but there are no simplified methods for the determination of the conjugation of the ferritin label to the immune sera. Further, it would be desirable to add additional ferritin molecules to the antibody so that the immune reaction could be more easily observed. This investigation reports our results with paper electrophoresis to determine the degree of conjugation with ferritin in varying amounts. Horse-spleen ferritin, prepared according to GRANICK 3, w a s conjugated to a whole immune serum of Staphylococcus aureus (conj. I) according to SMITH1. A duplicate aliquot was then conjugated with an amount of ferritin calculated to provide for the attachment of twice the ferritin molecules to the antibody protein as follows: I.o g of horse-spleen ferritin (IO ml) was diluted to 30 ml with 0.5 M carbonatebicarbonate buffer (pH 9.o) at 4 °. 1.2 ml m-xylylene di-isocyanate was added dropwise with slow stirring, to prevent frothing. This mixture was stirred for 45 min at the same temperature. The supernatant (Felso) was removed by centrifugation, then retained at 4 ° for I h. An equal volume of the Felso was then added to the diluted immune sera for conjugation under the same conditions as conj. I (conj. II). Paper-electrophoretic studies were carried out in veronal buffer (pH 8.6,10.075) for 16 h at 2. 5 mA in a Durrum-type cell (Spinco Model R) using a o.oI6-ml sample. Quantitation was then made using a Spinco RB analytrol with a B- 5 cam. Ultracentrifugation studies were made on the Spinco Model E, using the analytical Rotor A with a I 2 - m m cell. U.V. and visible spectra were determined on a Beckman recording spectrophotometer. As a control, a mixture of ferritin, immune serum, phosphate buffer and saline was prepared in the same proportions as for conjugation. AIb.
Fern
_
Glob.
Fig. I. Normal r a b b i t serum.
Fig. 2. F e r r i t i n - i m m u n e sera mixture. Alb. + Fern
Alb. + Fern
Fig. 3. I m m u n e c o n j u g a t e d sera (conj. I).
Fig. 4. I m m u n e c o n j u g a t e d sera (conj. II).
Biochim. Biophys. Acta, 47 (1961) 587-588
588
SHORT COMMUNICATIONS
Spectrophotometry of the conjugates and ferritin-serum mixtures failed to reveal a difference in the absorption patterns in either the u.v. or visible range. When the electrophoretic pattern of a normal rabbit serum (Fig. I) was compared with the ferritin-immune serum mixture (Fig. 2), a ferritin-apoferritin peak in the e2-globulin position is evident. A comparison of the normal rabbit serum with the conj. I (Fig. 3) shows an increase in the albumin and globulin peaks, without a distinguishable ferritin peak. When the conj. I I (Fig. 4) is then compared with conj. I, a quantitative increase can be seen in the protein fractions. Ultracentrifugation studies of the ferritin solution revealed a broad colored band which separated rapidly from a slower moving boundary of apoferritin which had a sedimentation coefficient Szo,w = 17 as found b y ROTHEN4. The sedimentation coefficient of ferritin in the ferritin-serum mixture was S2o,w-- 66 and in conj. I it w a s s20,w ~
60.
Although u.v. and visible spectrophotometry could not differentiate free and conjugated ferritin, paper eleetrophoresis presented a method in which differentiation could be obtained. ROTHEN4 states that ferritin consists of a homogeneous protein portion, apoferritin, with the associated micelles of iron complex but he found that both have the same electrophoretic mobility. This is confirmed by the distinct peak found by paper electrophoresis. Conj. I has a pattern in which unbound ferritin is negligible as indicated by the absence of a ferritin peak. The pattern of conj. I I indicates that the area of the peak is directly proportional to the amount of ferritin molecules attached. The specificity of both conjugates have not been altered as determined by immune electron microscopy and immunofluoreseenee. Ultracentrifugation indicates the absence of free ferritin in conj. I. The decrease in the sedimentation coefficient is due, most probably, to an increase in resistance of the ferritin-protein complex. These observations indicate that ferritin is conjugated to the immune serum and that detectable amounts of free ferritin are not present. Paper electrophoresis presents a reliable and reproducible method to confirm the labelling of an immune serum with ferritin. Unattached ferritin presents a specific peak, while a comparison of an unconjugated with a conjugated serum will demonstrate the attachment of ferritin by a linear increase in the albumin and y-globulin peaks. Antibody can be labelled with a variable amount of ferritin as indicated by a quantitative relationship in the protein peaks. This will alter the number of ferritin molecules discernible in an immune reaction by electron microscopy.
Bacteriology, Immunology and Infectious Disease Branch, Armed Forces Institute of Pathology, Washington 25, D.C. (U.S.A.)
(~,HAUNCEY W . SMITH .JOSEPH 1:i'. METZGER
1 C. W. SMITH, J. F. I~IETZGER, S. I. ZACKS AND A. KASE, Pro& Soc, Exptl. Biol. Med., Io 4 (196o) 336. a j . D. MARSHALL, W. C. EVELAND ANn C. W'. SMITH, ProP. Soc. Exptl. Biol. 2lied., io2 (1958) 898. 3 S. GRANICK AND g. MICHAELIS, J . Biol. Chem., 147 (1943) 91. 4 A. ROTHEN, J. Biol. Chem., 152 0944) 679.
Received October I7th, 196o Biochim. Biophys. Acla, 47 (1961) 58"7 588
BIOCHIMICA ET BIOPI-IYSICA ACTA
Short Communications Studies of ferritin conjugates used in immune electron microscopy The introduction of immune electron microscopy 1 has presented a method for the detection of antigen-antibody reactions at the subcellular level. The specificity of the conjugates used can be ascertained b y immunofluorescent techniques ~, but there are no simplified methods for the determination of the conjugation of the ferritin label to the immune sera. Further, it would be desirable to add additional ferritin molecules to the antibody so that the immune reaction could be more easily observed. This investigation reports our results with paper electrophoresis to determine the degree of conjugation with ferritin in varying amounts. Horse-spleen ferritin, prepared according to GRANICK 3, w a s conjugated to a whole immune serum of Staphylococcus aureus (conj. I) according to SMITH1. A duplicate aliquot was then conjugated with an amount of ferritin calculated to provide for the attachment of twice the ferritin molecules to the antibody protein as follows: I.o g of horse-spleen ferritin (IO ml) was diluted to 30 ml with 0.5 M carbonatebicarbonate buffer (pH 9.o) at 4 °. 1.2 ml m-xylylene di-isocyanate was added dropwise with slow stirring, to prevent frothing. This mixture was stirred for 45 min at the same temperature. The supernatant (Felso) was removed by centrifugation, then retained at 4 ° for I h. An equal volume of the Felso was then added to the diluted immune sera for conjugation under the same conditions as conj. I (conj. II). Paper-electrophoretic studies were carried out in veronal buffer (pH 8.6,10.075) for 16 h at 2. 5 mA in a Durrum-type cell (Spinco Model R) using a o.oI6-ml sample. Quantitation was then made using a Spinco RB analytrol with a B- 5 cam. Ultracentrifugation studies were made on the Spinco Model E, using the analytical Rotor A with a I 2 - m m cell. U.V. and visible spectra were determined on a Beckman recording spectrophotometer. As a control, a mixture of ferritin, immune serum, phosphate buffer and saline was prepared in the same proportions as for conjugation. AIb.
Fern
_
Glob.
Fig. I. Normal r a b b i t serum.
Fig. 2. F e r r i t i n - i m m u n e sera mixture. Alb. + Fern
Alb. + Fern
Fig. 3. I m m u n e c o n j u g a t e d sera (conj. I).
Fig. 4. I m m u n e c o n j u g a t e d sera (conj. II).
Biochim. Biophys. Acta, 47 (1961) 587-588
588
SHORT COMMUNICATIONS
Spectrophotometry of the conjugates and ferritin-serum mixtures failed to reveal a difference in the absorption patterns in either the u.v. or visible range. When the electrophoretic pattern of a normal rabbit serum (Fig. I) was compared with the ferritin-immune serum mixture (Fig. 2), a ferritin-apoferritin peak in the e2-globulin position is evident. A comparison of the normal rabbit serum with the conj. I (Fig. 3) shows an increase in the albumin and globulin peaks, without a distinguishable ferritin peak. When the conj. I I (Fig. 4) is then compared with conj. I, a quantitative increase can be seen in the protein fractions. Ultracentrifugation studies of the ferritin solution revealed a broad colored band which separated rapidly from a slower moving boundary of apoferritin which had a sedimentation coefficient Szo,w = 17 as found b y ROTHEN4. The sedimentation coefficient of ferritin in the ferritin-serum mixture was S2o,w-- 66 and in conj. I it w a s s20,w ~
60.
Although u.v. and visible spectrophotometry could not differentiate free and conjugated ferritin, paper eleetrophoresis presented a method in which differentiation could be obtained. ROTHEN4 states that ferritin consists of a homogeneous protein portion, apoferritin, with the associated micelles of iron complex but he found that both have the same electrophoretic mobility. This is confirmed by the distinct peak found by paper electrophoresis. Conj. I has a pattern in which unbound ferritin is negligible as indicated by the absence of a ferritin peak. The pattern of conj. I I indicates that the area of the peak is directly proportional to the amount of ferritin molecules attached. The specificity of both conjugates have not been altered as determined by immune electron microscopy and immunofluoreseenee. Ultracentrifugation indicates the absence of free ferritin in conj. I. The decrease in the sedimentation coefficient is due, most probably, to an increase in resistance of the ferritin-protein complex. These observations indicate that ferritin is conjugated to the immune serum and that detectable amounts of free ferritin are not present. Paper electrophoresis presents a reliable and reproducible method to confirm the labelling of an immune serum with ferritin. Unattached ferritin presents a specific peak, while a comparison of an unconjugated with a conjugated serum will demonstrate the attachment of ferritin by a linear increase in the albumin and y-globulin peaks. Antibody can be labelled with a variable amount of ferritin as indicated by a quantitative relationship in the protein peaks. This will alter the number of ferritin molecules discernible in an immune reaction by electron microscopy.
Bacteriology, Immunology and Infectious Disease Branch, Armed Forces Institute of Pathology, Washington 25, D.C. (U.S.A.)
(~,HAUNCEY W . SMITH .JOSEPH 1:i'. METZGER
1 C. W. SMITH, J. F. I~IETZGER, S. I. ZACKS AND A. KASE, Pro& Soc, Exptl. Biol. Med., Io 4 (196o) 336. a j . D. MARSHALL, W. C. EVELAND ANn C. W'. SMITH, ProP. Soc. Exptl. Biol. 2lied., io2 (1958) 898. 3 S. GRANICK AND g. MICHAELIS, J . Biol. Chem., 147 (1943) 91. 4 A. ROTHEN, J. Biol. Chem., 152 0944) 679.
Received October I7th, 196o Biochim. Biophys. Acla, 47 (1961) 58"7 588