124
`
`F. Chiodi, A. Siden and E. &by
`
`Francesca Chiodi'
`Ake Siden'
`Eva Osby2
`
`'Department of Neurology, Huddinge
`University Hospital, Huddinge
`'Department of Haematology,
`Karolinska Hospital, Stockholm
`
`Electrophoresis 1985.6, 124-128
`Isoelectric focusing of monoclonal immunoglobulin G, A
`and M followed by detection with the avidin-biotin system
`
`The sera of49 subjects with IgG, IgA or IgM benign monoclonal gammopathy-com-
`ponents were examined by agarose gel isoelectric focusing followed by Coomassie
`Brilliant Blue R-250 staining as well as avidin-biotin amplified double-antibody
`peroxidase labelling after nitrocellulose blotting. IgG-components gave 2- 10 distinct
`bands within the isoelectric point (pl)-range of pH 6.5-9.5, IgA-components were
`focused into 10-15 bands with pl-values of pH 4.5-6.5 and the IgM-components
`gave 1-2 fractions within the pl-range of pH 4.5-6.5. The avidin-biotin peroxidase
`complex (ABC) technique exhibited some tendency to non-specific labeling related to
`the avidin-biotin component and depending on the relative amounts of the individual
`proteins. However, the method has a definite potential for the visualization of sub-
`microgram quantities of immunoglobulins blotted to nitrocellulose membranes after
`isoelectric focusing.
`
`1 Introduction
`
`Immunoglobulin M-components are relatively often observed
`when patient sera are subjected to electrophoretic separation.
`Such changes are most frequently caused by a plasma cell
`dyscrasia such as benign monoclonal gammopathy (BMG),
`multiple myeloma, Waldenstrom disease, and light or heavy
`chain disease. The incidence of BMG in a population is about
`0.5 % and increases with age [ 11 while the other conditions are
`rare. During studies of possible associations between BMG
`and peripheral neuropathy [2, 31, an investigation of the ab-
`normal immunoglobulin components from subjects with
`BMG was initiated. Agarose gel isoelectric focusing was used
`for the analytical studies and the components were detected by
`use of the avidin-biotin system. Isoelectric focusing is a high-
`resolution electromigration technique which has proved
`useful for studies of proteins in biological fluids [41. This
`method separates the sample molecules on the basis of their
`respective pl. Separations by use of isoelectric focusing give a
`complex band pattern when mixtures of proteins such as sera
`are examined. Thus, when individual proteins are studied, this
`technique should be combined with a method for detection of
`the protein of interest. The avidin-biotin system has, since its
`introduction IS], found many applications for mainly im-
`munological investigations [6J. The ABC technique [71, em-
`ploying a primary antibody, a biotinylated secondary an-
`tibody and an avidin-biotin peroxidase complex, was used for
`the detection of immunoglobulins which had been blotted to
`nitrocellulose membranes. This report states our experiences
`with examinations of IgG, IgA and IgM BMG-components.
`
`2 Materials and methods
`
`2.1 Materials
`
`The serum samples were obtained from 49 subjects with a
`mean age of 62 years (range 39-81 years) in whom the
`diagnosis of BMG was based on a thorough haematological
`
`Correspondence: Dr. Ake Siden, Department of Neurology, Huddinge
`University Hospital, S-141 86 Huddinge, Sweden
`
`Abbreviations: M-component, monoclonal component; BMG, benign
`monoclonal gammopathy; PI, isoelectric point; ABC, avidin-biotin per-
`oxidase complex: PBS, phosphate buffered saline
`
`investigation and follow-up, performed as previously describ-
`ed [ 31. The monoclonal component had been detected by agar
`gel electrophoresis and was identified by immunofixation 181.
`The immunoglobulin levels had been determined by rocket im-
`munoelectrophoresis and turbidimetric immunoassay 19,101.
`Among the samples, 34 showed IgG-components (29 K and
`5 il. light chain types), 10 IgA-components (4 K and 6 il. light
`chain types) and 5 IgM-components (4 K and 1 h light chain
`types). The total IgG concentration ofthe sera with IgG-com-
`ponents was 8-32 g/liter (mean value 18 g/liter), theIgA con-
`centration of the samples with IgA-components was 4-22 g/
`liter (mean value 14 g/liter) and the IgM concentration of
`those with such components was 3-18 g/liter (mean value
`11 g/liter). Prior to isoelectric focusing the samples were
`diluted with phosphate buffered saline (PBS) to a concentra-
`tion of the relevant immunoglobulin class of 1 g/liter (Coo-
`massie staining) and 20 mg/liter (avidin-biotin system).
`
`2.2 Isoelectric focusing
`
`Agarose gel plates (approximately 2 mm thick) of 0.8 o/o w/v
`agarose (IsoGel; FMC Corporation, Marine Colloids Div.,
`Rockland, USA) and with 5.4 % v/v Ampholine carrier
`ampholytes, pH 3.5-10 (LKB, Stockholm, Sweden), were
`cast on a support medium (GelBond film; FMC Corporation,
`Marine Colloids Div.) of the dimensions 110 x 205 mm.
`Isoelectric focusing was performed by use of the 2 1 17 Mul-
`tiphor, 2197 Power Supply and 2209 MultiTemp (LKB) at a
`constant wattage of 15 W with an initial voltage of 300 V
`(50 mA) and a final voltage of 800 V (1 8 mA). The electrode
`distance was 100 mm and the electrode solutions were 0.5 M
`acetic acid (anode) and 0.5 M sodium hydroxide(cathode); the
`temperature of the MultiTemp was set at + 5 O C . For Coo-
`massie Brilliant Blue R-250 staining, 20 pg (20 pl, l g/liter) of
`the relevant immunoglobulin was applied by 5 x 10 mm
`sample application pieces (LKB) 40 mm (IgG) or 50 mm (IgA
`and IgM) from the anode; the separation was performed for
`60 min (IgG), 75 min (IgA) or 90 min (IgM). Some samples
`found to give an unsatisfactory separation were subjected to
`chloroform extraction for lipid elimination which corrected
`the problem. Also included on each plate were normal samples
`as well as a mixture of p l marker proteins (Isoelectric Focus-
`ing p l Calibration Kit pH 3-10; Pharmacia, Uppsala,
`Sweden). Isoelectric focusing prior to detection by the avidin-
`biotin system was performed at the same basic conditions but
`with the application of 0.3 pg (15 pl, 20 mg/liter) of the rele-
`
`0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1985
`
`01 73-0835/85/0303-0124 $02.50/0
`
`Ex. 1021 - Page 1 of 6
`
`AMGEN INC.
`Exhibit 1021
`
`

`

`124
`
`F. Chiodi, A. Siden and E. &by
`
`Francesca Chiodi'
`Ake Siden'
`Eva Osby2
`
`'Department of Neurology, Huddinge
`University Hospital, Huddinge
`'Department of Haematology,
`Karolinska Hospital, Stockholm
`
`Electrophoresis 1985.6, 124-128
`Isoelectric focusing of monoclonal immunoglobulin G, A
`and M followed by detection with the avidin-biotin system
`
`The sera of49 subjects with IgG, IgA or IgM benign monoclonal gammopathy-com-
`ponents were examined by agarose gel isoelectric focusing followed by Coomassie
`Brilliant Blue R-250 staining as well as avidin-biotin amplified double-antibody
`peroxidase labelling after nitrocellulose blotting. IgG-components gave 2- 10 distinct
`bands within the isoelectric point (pl)-range of pH 6.5-9.5, IgA-components were
`focused into 10-15 bands with pl-values of pH 4.5-6.5 and the IgM-components
`gave 1-2 fractions within the pl-range of pH 4.5-6.5. The avidin-biotin peroxidase
`complex (ABC) technique exhibited some tendency to non-specific labeling related to
`the avidin-biotin component and depending on the relative amounts of the individual
`proteins. However, the method has a definite potential for the visualization of sub-
`microgram quantities of immunoglobulins blotted to nitrocellulose membranes after
`isoelectric focusing.
`
`1 Introduction
`
`Immunoglobulin M-components are relatively often observed
`when patient sera are subjected to electrophoretic separation.
`Such changes are most frequently caused by a plasma cell
`dyscrasia such as benign monoclonal gammopathy (BMG),
`multiple myeloma, Waldenstrom disease, and light or heavy
`chain disease. The incidence of BMG in a population is about
`0.5 % and increases with age [ 11 while the other conditions are
`rare. During studies of possible associations between BMG
`and peripheral neuropathy [2, 31, an investigation of the ab-
`normal immunoglobulin components from subjects with
`BMG was initiated. Agarose gel isoelectric focusing was used
`for the analytical studies and the components were detected by
`use of the avidin-biotin system. Isoelectric focusing is a high-
`resolution electromigration technique which has proved
`useful for studies of proteins in biological fluids [41. This
`method separates the sample molecules on the basis of their
`respective pl. Separations by use of isoelectric focusing give a
`complex band pattern when mixtures of proteins such as sera
`are examined. Thus, when individual proteins are studied, this
`technique should be combined with a method for detection of
`the protein of interest. The avidin-biotin system has, since its
`introduction IS], found many applications for mainly im-
`munological investigations [6J. The ABC technique [71, em-
`ploying a primary antibody, a biotinylated secondary an-
`tibody and an avidin-biotin peroxidase complex, was used for
`the detection of immunoglobulins which had been blotted to
`nitrocellulose membranes. This report states our experiences
`with examinations of IgG, IgA and IgM BMG-components.
`
`2 Materials and methods
`
`2.1 Materials
`
`The serum samples were obtained from 49 subjects with a
`mean age of 62 years (range 39-81 years) in whom the
`diagnosis of BMG was based on a thorough haematological
`
`Correspondence: Dr. Ake Siden, Department of Neurology, Huddinge
`University Hospital, S-141 86 Huddinge, Sweden
`
`Abbreviations: M-component, monoclonal component; BMG, benign
`monoclonal gammopathy; PI, isoelectric point; ABC, avidin-biotin per-
`oxidase complex: PBS, phosphate buffered saline
`
`investigation and follow-up, performed as previously describ-
`ed [ 31. The monoclonal component had been detected by agar
`gel electrophoresis and was identified by immunofixation 181.
`The immunoglobulin levels had been determined by rocket im-
`munoelectrophoresis and turbidimetric immunoassay 19,101.
`Among the samples, 34 showed IgG-components (29 K and
`5 il. light chain types), 10 IgA-components (4 K and 6 il. light
`chain types) and 5 IgM-components (4 K and 1 h light chain
`types). The total IgG concentration ofthe sera with IgG-com-
`ponents was 8-32 g/liter (mean value 18 g/liter), theIgA con-
`centration of the samples with IgA-components was 4-22 g/
`liter (mean value 14 g/liter) and the IgM concentration of
`those with such components was 3-18 g/liter (mean value
`11 g/liter). Prior to isoelectric focusing the samples were
`diluted with phosphate buffered saline (PBS) to a concentra-
`tion of the relevant immunoglobulin class of 1 g/liter (Coo-
`massie staining) and 20 mg/liter (avidin-biotin system).
`
`2.2 Isoelectric focusing
`
`Agarose gel plates (approximately 2 mm thick) of 0.8 o/o w/v
`agarose (IsoGel; FMC Corporation, Marine Colloids Div.,
`Rockland, USA) and with 5.4 % v/v Ampholine carrier
`ampholytes, pH 3.5-10 (LKB, Stockholm, Sweden), were
`cast on a support medium (GelBond film; FMC Corporation,
`Marine Colloids Div.) of the dimensions 110 x 205 mm.
`Isoelectric focusing was performed by use of the 2 1 17 Mul-
`tiphor, 2197 Power Supply and 2209 MultiTemp (LKB) at a
`constant wattage of 15 W with an initial voltage of 300 V
`(50 mA) and a final voltage of 800 V (1 8 mA). The electrode
`distance was 100 mm and the electrode solutions were 0.5 M
`acetic acid (anode) and 0.5 M sodium hydroxide(cathode); the
`temperature of the MultiTemp was set at + 5 O C . For Coo-
`massie Brilliant Blue R-250 staining, 20 pg (20 pl, l g/liter) of
`the relevant immunoglobulin was applied by 5 x 10 mm
`sample application pieces (LKB) 40 mm (IgG) or 50 mm (IgA
`and IgM) from the anode; the separation was performed for
`60 min (IgG), 75 min (IgA) or 90 min (IgM). Some samples
`found to give an unsatisfactory separation were subjected to
`chloroform extraction for lipid elimination which corrected
`the problem. Also included on each plate were normal samples
`as well as a mixture of p l marker proteins (Isoelectric Focus-
`ing p l Calibration Kit pH 3-10; Pharmacia, Uppsala,
`Sweden). Isoelectric focusing prior to detection by the avidin-
`biotin system was performed at the same basic conditions but
`with the application of 0.3 pg (15 pl, 20 mg/liter) of the rele-
`
`0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1985
`
`01 73-0835/85/0303-0124 $02.50/0
`
`Ex. 1021 - Page 2 of 6
`
`

`

`Electrophoresis 1985,6, 124-128
`
`Isoelectric focusing of monoclonal immunoglobulin
`
`125
`
`vant immunoglobulin in preformed slits at the positions men- Table 1. Isoelectric focusing followed by staining with Coomassie Brilliant
`tioned above.
`Blue R-250
`
`BMG type
`
`Number
`of samples
`
`Isoelectric focusing findings
`
`IgG
`
`IgA
`IgM
`
`34
`
`10
`5
`
`All samples exhibited distinct abnor-
`mal bands within the pf range of pH
`6.5-9.5;
`the number of
`individual
`in 17 seraand 6-10 in
`bands was 2-5
`17 sera; probably IgG M-components
`No abnormal bands within the pf range
`of pH 6.5-9.5; complex band pattern
`in regions corresponding to more acidic
`pZ values; obvious need of a method
`for identification of individual proteins
`
`changes (Fig. 1) are clearly different from the polyclonal
`appearance of norma IgG and make the occurrence of an IgG
`M-component probable. However, the final conclusion con-
`sidering the nature of such findings has to be based on methods
`for specific identification. None of the samples with IgA or
`IgM BMG-components exhibited abnormal bands within the
`p l range of pH 6.5-9.5. Evaluation of the regions correspond-
`ing to more acidic p l values offers considerable difficulties
`because of the large number of proteins focused within that
`range. This fact, as well as the obvious need of amethod for the
`identification of IgG, A and M in a study of these immuno-
`globulins, was our reasons for applying the avidin-biotin
`amplified double-antibody peroxidase labelling.
`
`3.2 Detection by avidin-biotin amplified double-antibody
`peroxidase labelling
`
`Table 2 gives the results of isoelectric focusing followed by
`avidin-biotin amplified double-antibody peroxidase labelling.
`The abnormal band spectra covering a region of about
`0.1-1.5 pH unitswithin theplrangeofpH6.5-9Sinserawith
`IgG BMG-components were identified as IgG with a homo-
`
`2.3 Protein detection
`
`For Coomassie staining the gels were first immersed for
`10 rnin in a fixative solution (32 1 g oftrichloroacetic acid and
`103.8 g sulphosalicylic acid diluted to 3000 ml in distilled
`water), washed in ethanol (950 ml/liter) in 20 min,pressed un-
`der ethanol-soaked filter paper and dried in warm air. The gels
`were then stained in a 0.5 % w/v solution of Coomassie
`Brilliant Blue R-250 (Sigma Chemical Co., St. Louis, MO,
`USA) in acetic acid/ethanol/distilled water (10/36/54 by
`volume) for 10 rnin at +20 "C, followed by destaining with the
`samesolution without the dye and finally drying in warm air.
`
`Protein transfer to nitrocellulose membranes and avidin-
`biotin amplified double-antibody peroxidase labelling was
`performed at room temperature essentially as described by
`Olsson et al. 11 11. The gel surface was briefly touched with a
`filter paper (1577; No. 134473; Schleicher & Schull, Dassel,
`FRG) moistened in 0.1 M PBS, pH 7.3, followed by the ap-
`plication of a nitrocellulose membrane (BA 85, No. 401 198;
`Schleicher & Schull) prewetted in PBS which was overlaid
`with a PBS-moistened filter paper, several layers of dry filter
`papers and a glass plate and pressed against the gel surface for
`1 h. The nitrocellulose membrane was then soaked in 3 %
`bovine serum albumin (Albumin, Bovine Fraction V; Sigma
`Chemical Co.) in PBS for 30 rnin in order to block the empty
`protein-binding spaces. After blocking, the membrane was in-
`cubated with the primary antiserum (diluted 1 5 0 0 in PBS)for
`2 h. The following primary antisera were used: rabbit anti-
`human sera specific for a-chains and secretory component,
`IgG Fc-fragments, y-chains, K and h light chains (Dakopatts,
`Glostrup, Denmark). After three washings for 10 rnin each
`with PBS the membrane was incubated for 2 h with bio-
`tinylated goat antiserum to rabbit IgG (Vectastain ABC Kit,
`Rabbit IgG; Vector Laboratories., Burlingame, USA) diluted
`1:400 in PBS. The membrane was washed three times for
`10 rnin each with PBS and then incubated for 1 h with ABC
`(Vectastain ABC Kit) diluted 1:200. After three final
`washings in PBS for 10 rnin each, staining was performed by
`incubation of the membrane in a solution of40 mg of 3-amino-
`9-ethylcarbazole and 2.5 ml of dimethylformamid (Sigma
`Chemical Co.) in 47.5 ml of 0.05 M sodium acetate, pH 4.5,
`followed by the addition of 50 p1 of 0.3 M hydrogen peroxide.
`When an optimal color development was achieved the mem-
`brane was washed in distilled water and dried. The evaluation
`of the protein patterns after Coomassie staining and avidin-
`biotin amplified immunological detection was performed
`visually and based on comparisons with normal samples as
`well as experiences from previous studies of abnormal im-
`munoglobulins [2, 12, 131.
`
`3 Results
`
`3.1 Staining with Coomassie Brilliant Blue R-250
`
`The findings on isoelectric focusing followed by staining with
`Coomassie Brilliant Blue R-250 are summarized in Table 1.
`All samples with IgG BMG-components exhibited a spectra
`of2-lodistinct bands within theplrangeofpH 6.5-9.5. Such
`
`Figure 1. Isoelectric focusing followed by staining with Coomassie Brilliant
`Blue R-250 of (a) normal serum and (b) two sera with IgG BMG-compo-
`nents. The anode in this and the following figures was at the bottom and the
`cathode at the top. The position corresponding to a pf value ofabout pH 6.5
`is indicated by an arrow-head.
`
`Ex. 1021 - Page 3 of 6
`
`

`

`126
`
`F. Chiodi, A. Siden and E. &by
`
`Electrophoresis 1985.6. 124- 128
`
`Table 2. Isoelectric focusing followed by avidin-biotin amplified double-antibody
`peroxidase labelling
`
`Number
`BMG type of samples
`
`Isoelectric focusing findings (the frequencies are
`given as number of samples)
`
`Heavy and light chain identities based o n the
`x
`ABC technique
`Y
`a
`P
`K
`
`pZ range
`pH4.S-6.5 pH6.5-9.5
`
`Number of distinct bands
`1 2-5
`6-10
`11-15
`
`a) 10 bands
`b) 2bands
`
`geneous light chain type (Fig. 2). A few samples exhibited
`minimal staining for the alternative light chain, probably a
`non-specific labelling (vide infru). There was complete agree-
`ment between the light chain identification with the present
`method and that based on immunofixation after standard
`electrophoresis. The number of individual bands detected
`within the IgG BMG-components by the avidin-biotin system
`did not deviate significantly from that shown by Coomassie
`staining. Samples with IgA and IgM BMG-components were
`found to have this immunoglobulin focused within the PI
`range of pH 4.5-6.5 and there were no distinct fractions with
`more alkaline p l values. The ranges stained by primary an-
`tisera against a or y heavy chains exhibitedone dominant light
`chain type which was identical to that shown by immunofixa-
`tion after standard electrophoresis. IgA of sera with BMG-
`components was focused into 10- 15 individual bands cover-
`ing a region of about 1.5-2.0 pH units within the p l range of
`pH 4.5-6.5. Two samples exhibited an anodal or cathodal
`predominance but otherwise the a-chain findings were similar
`to those of normal IgA (Fig. 3). The sera with IgM BMG-com-
`ponents exhibited 1-2 relatively broad fractions which were
`distinctly positive for y-chains (Fig. 4). These components
`
`Figure3. Isoelectricfocusing withdetectinnofa-chains by useofthe avidin-
`biotin system performedon serum with(a)normalIgA and(b)two sera with
`IgA BMG-components.
`
`covered a region of about 0.5 pH units within the anodal,
`middle or cathodal part of the p l range of pH 4.5-6.5. Sera
`with normal IgM did not exhibit such prominent fractions
`stained by antisera against y-chains.
`
`Figure 2. Isoelectric focusing followed by avidin-biotin amplified double-
`antibody peroxidase labelling of (a) IgG Fc-fragments and (b) A-chains.
`Sample with normal IgC to the right and sample with an IgG BMG-compo-
`nent of h-chain identity to the left.
`
`Figure 4. Tsoelectric focusing of three sera with IgM BMG-components
`followed by detection of p-chains with the avidin-biotin system. The broad
`prominent fractions correspond to IgM. A partial non-specific labelling of
`IgA can also be observed, especially in the sample to the left.
`
`Ex. 1021 - Page 4 of 6
`
`

`

`Electrophoresis 1985,6, 124-128
`
`Isoelectric focusing of monoclonal immunoglobulin
`
`127
`
`4 Discussion
`Suitable methods for protein separation and detection are
`prerequisites for the diagnosis ofplasma cell dyscrasias as well
`as for the investigations of monoclonal immunoglobulins.
`Agar or agarosegel electrophoresis combined with techniques
`for protein staining and immunological detection, e. g. im-
`munofixation, have mostly been used for these purposes 1 141.
`However, the excellent resolution of isoelectric focusing made
`this method a promising tool for investigations of monoclonal
`immunoglobulins [ 151. Isoelectric focusing performed in an
`agarose gel offers high resolution combined with low molec-
`ular sieving and gives flexibility in the choice of the protein
`detection method. This technique is well suited for examina-
`tions not only of IgG but also IgA and IgM [ 16,171. The ABC
`technique [7l,combined with nitrocellulose blotting, has prov-
`ed useful for studies of oligoclonal IgG in unconcentrated
`cerebrospinal fluid [ 1 11.
`
`The present study was restricted to examinations of benign
`IgG, IgA and IgM hl-components and comparisons with the
`corresponding normal immunoglobulins. Our primary in-
`terest was to investigate the applicability of nitrocellulose blot-
`ting and the ABC technique for visualization of BMG-compo-
`nents after agarose gel isoelectric focusing of sera. All three
`types of BMG-components were well detected by the method.
`We did not observe any definite qualitative differences be-
`tween the benign IgA M-components included in this study
`and normal IgA. Further studies are needed before definite
`conclusions can be drawn about the possible spectrotype
`differences of normal and monoclonal IgA [ 171. The band pat-
`terns of IgG and IgM M-components were in all cases
`qualitatively different from those of the normal immuno-
`globulin.
`
`The application of the avidin-biotin system for detection of
`nitrocellulose blotted proteins holds promising potentials for
`studies of proteins in biological fluids. However, the advan-
`tages and limitations of this method have still to be evaluated.
`During these investigations of normal and monoclonal IgG,
`IgA and IgM, we have found evidence of non-specific labelling
`depending on the relative amounts of protein; similar observa-
`tions have been made with studies of cerebrospinal fluid IgM
`I 181. This non-specific labelling is related to the avidin-biotin
`component and not to the primary antiserum. Morris and
`Saelinger [ 191, on the basis of experience with ultra-structural
`localization of antigens, have also discussed the problem of
`non-specific binding. They have emphasized the capacity of
`avidin, carrying a positive net charge at physiological pH
`values, to bind electrostatically to negatively charged proteins
`and the potential binding of the vitamin biotin to serum pro-
`teins.
`
`According to our experience the present method has, in spite
`of the limitations mentioned above, a definite potential for in-
`vestigations of immunoglobulin M-components separated by
`isoelectric focusing. The immunoglobulin component occur-
`ing in such conditions generally has a high relative concentra-
`tion which significantly reduces the risk of non-specific label-
`ing of other proteins. However, the system is less suitable for
`investigations of a possible biclonality of the component.
`Definite advantages of the technique are its high sensitivity,
`relatively simple performance and low antiserum consump-
`tion. The low sample load of the isoelectric focusing run will
`also reduce the risk of artefacts such as skewed bands,
`isoelectric precipitation and aggregation phenomena.
`
`Figure 5. Isoelectric focusing of a serum with a monoclonal IgG-compo-
`nent. After nitrocellulose blotting labelling was performed by the avidin-
`biotin system with a primary antiserum against (a) IgG Fc-fragments, (b)a-
`chains and (c) p-chains. The non-specific labelling of the IgG-component is
`indicated by arrow-heads.
`
`3.3 Non-specific labelling
`
`Indications for non-specific labelling were observed when nor-
`mal sera were examined for IgA (non-specific staining of IgG)
`or IgM (non-specific staining of IgA and IgG) by the ABC
`technique. Light chain typing of BMG-components in a few
`cases exhibited a similar but less pronounced tendency. These
`findings, as well as experiments with heavy chain labelling of
`sera with monoclonal immunoglobulins (Fig. 51, have in-
`dicated that the problem is related to a high relative concentra-
`tion of the immunoglobulin(s) which exhibit non-specific
`staining. Subsequent investigations have shown that this type
`of labelling is referable to the avidin-biotin component and not
`to the type of primary antibody. Absorption against human
`immunoglobulins of the second antibody did not prove effec-
`tive in eliminating the problem; neither did trials with different
`lots of avidin or other reagents. Several physico-chemical
`modifications of the procedure were also tried, with negative
`results: changing the blocking agent, heating of
`the
`nitrocellulose membrane after blotting, changing the pH
`values or ionic strengths of the buffers, chloroform extraction
`of the samples prior to isoelectric focusing. The really signifi-
`cant problem caused by this non-specific labelling was ob-
`served when normal sera were examined for IgM. For that
`reason a modification based on an initial immunofixation and
`omission of the nitrocellulose blotting was tried. Briefly, after
`isoelectric focusing (0.8 pg of IgM) the gel was immunofixed
`with the primary antibody, thoroughly washed and finally
`processed (secondary antibody, etc.) in a similar way as after
`nitrocellulose blotting. The primary and secondary antisera as
`well as the ABC were all diluted 1 : 100 in PBS. Several samples
`with normal IgM have been examined by this method and IgM
`was found to be focused in the p l range of pH 4.5-6.5. The
`staining was relatively homogeneous and no prominent in-
`dividual bands were observed.
`
`Ex. 1021 - Page 5 of 6
`
`

`

`128
`
`D. Waiterova el a[.
`
`Electrophoresis 1985.6. 128- 132
`
`This work was supported by grants from the Swedish Society
`of Medical Sciences. The skilful technical assistance of M s
`Carina Gudmundsson is gratefully acknowledged.
`
`Received September 13, 1984
`
`5 References
`
`I 1 I Axelsson, U., Bachmann, R. and Hallen, J., Acta med..Scand. 1966,
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`
`1 1
`1 .
`
`Daniela Walterova'
`ZdenEk Stransky2
`Vladimir Preininger
`Vilim Simanek'
`
`Capillary isotachophoresis of some isoquinoline
`alkaloids*
`
`'Institute of Medical Chemistry,
`Medical Faculty
`21nstitute of Analytical and Organic
`Chemistry, Faculty of Natural
`Sciences, University Palacky,
`0 I o m o u c
`
`The isotachophoretic behavior of quaternary benzo/c/phenanthridine, protober-
`berine and aporphine alkaloids in different electrolyte systems is described. The con-
`centration of the leading ion and the pH value of the leading electrolyte affect the
`relative effective mobilities of the alkaloids. The system of pH 4.7, containing the
`leading ion K' (0.005 M) counter ion acetate, and the terminating ion p-alanine
`(0.02 M), has been selected for the quantitative determination ofthe studied alkaloids
`in model mixtures and plant extracts.
`
`1 Introduction
`
`Quaternary isoquinoline alkaloids are ubiquitous in the plant
`family Papaveraceae 11 I. A suitable analytical method for a
`rapid quantitative determination of quaternary isoquinoline
`alkaloids is still lacking. The methods used so far have been
`basedonthin-layer chromatography [2,31. High performance
`liquid chromatography was used to determine sanguinarine
`(Fig. la) in saliva and in dental plaques (41. Isotachophoresis
`has been used for analyses of many cations [5-81 including
`quaternary quinoline and isoquinoline ions [9]. In alkaloid
`chemistry, this method has been applied to determine, for
`example, theophylline [ 101 and atropine [ 111 in plasma or
`some tertiary bases in pharmaceutical preparations [ 121. We
`employed capillary isotachophoresis for the determination of
`
`Correspondence: Prof. Dr. Vilim Simanek, Institute of Medical Chemistry,
`Palacky 1 University,S. Allende 3, CS-775 15 Olomouc,Czechoslovakia
`* Part XCVI in the series: Isolation, Chemistry and Biology of Alkaloids
`from Plants of Papaveraceae. For part XCV see Acta Univ. Palacki
`Olomuc. (Olomouc) 1985,110, in press
`
`sanguinarine (Fig. la) and chelerythrine (Fig. lb) in quater-
`nary benzophenanthridine fractions from Chelidonium majus
`L. [ 13 1. The present study describes the isotachophoretic
`behavior of some quaternary benzo/c/phenanthridine (Fig.
`la-d), protoberberine (Fig. le-1) and aporphine alkaloids
`(Fig. Im-p) as well as the quantitative determination of
`sanguinarine (Fig. 1 a), chelerythrine (Fig. 1 b), berberine
`(Fig. If), and coptisine (Fig. li) in model mixtures and in plant
`extracts.
`
`2 Materials and methods
`
`The alkaloids sanguinarine (Fig. la) and chelerythrine
`(Fig. Ib) were isolated from roots of the plant Chelidonium
`majus L. [14]; berberine (Fig. If), coptisine (Fig. li) and
`magnoflorine (Fig. lp) from aerial parts of Corydalis ophio-
`carpa Hook et Thorns. [ 151. Coralyne (Fig. 11) was synthetiz-
`ed from papaverine [ 161; bulbocapnine methiodide (Fig. lm)
`and isothebaine methiodide (Fig. In) were prepared by
`methylation of tertiary bases with methyl iodide.
`
`0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1985
`
`01 73-0835/85/0303-0 128 $02.50/0
`
`Ex. 1021 - Page 6 of 6
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