Vol. 2, No. 5 Printed in U.S.A.
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1975, p. 377-381 Copyright © 1975 American Society for Microbiology
Rapid Separation of Immunoglobulin M from Immunoglobulin G Antibodies for Reliable Diagnosis of Recent Rubella Infections W. FRISCH-NIGGEMEYER Institut fur Virologie der Universitat Wien, A-1095 Wien Kinderspitalgasse 15, Austria
Received for publication 19 May 1975
Chromatography on controlled pore glass was adapted for the separation of immunoglobulin M (IgM) and immunoglobulin G (IgG) rubella antibodies from 0.3-ml samples of human serum. An extremely sharp separation of IgM from IgG antibodies could be obtained within 40 min. Nonspecific inhibitors were removed before chromatography by precipitation with high-molecular-weight dextran sulfate, and the titer of rubella antibodies in the different classes of immunoglobulins were assayed with a modified hemagglutination inhibition technique. The combination of these methods is recommended for routine tests. It permits an accurate diagnosis of recent rubella infection within a few hours.
The clinical diagnosis of rubella is sometimes difficult. A typical rubella-like illness may be caused by viruses other than rubella, e. g., some enteroviruses. In addition, a large proportion of rubella infections take a subclinical course and do not produce any overt signs of disease (16). A critical investigation has demonstrated that the doctor's diagnosis could be verified by serological methods in less than 50% of cases (1). It is well known that infection with this virus may cause congenital malformations of the child, influencing sight, hearing, heart, brain and teeth (26). The danger of the occurrence of such defects is not entirely restricted to the early months of pregnancy (9). Spontaneous abortion and stillbirths can also be caused by rubella virus (29, 31). Therefore, it is important that patients who are exposed to or develop rubella symptoms during pregnancy undergo serological tests to determine whether infection has occurred. As in many other infectious diseases (27), a primary infection with rubella virus causes the antibody titer to develop in a characteristic pattern. A few days after onset of the rash, a steep rise of antibodies occurs in the M-fraction of the immunoglobulins (IgM). Titers reach a maximum after 5 to 14 days, then diminish, and by 8 to 12 weeks later specific IgM generally cannot be detected. There is a similar rise of specific antibodies in the G-class of immunoglobulins (IgG), but hardly any decrease; titers stay constant for many months and are probably measurable for life (3, 18). Antibodies of the immunoglobulin A (IgA) class show a transient
appearance similar to IgM antibodies. Titers increase sharply, fall off gradually, and disappear at about the same time as the IgM titers
(3). This phenomenon permits one to distinguish a recent rubella infection acquired during pregnancy from a long past one, by separate titration of IgM (+IgA) and IgG antibodies, even in the absence of clinical symptoms (7, 15). However, clear-cut separation of immunoglobulin classes by the classical techniques of density gradient centrifugation (14, 20) or gel chromatography (3, 7, 11, 15) is a time consuming procedure, and generally not feasible for routine clinical tests. By using controlled pore glass (CPG) (12), permeation chromatography of serum samples can be performed in 1/io to 1/20 of the time necessary for the above mentioned methods (8, 13). This paper describes a further development of this method for immunoglobulin separation that uses simpler procedures and smaller samples, requires less time, and yields sharper separation. MATERIALS AND METHODS Sera. Sera were examined either fresh or after storage at -20 C. Erythrocytes. Pigeon erythrocytes were prepared by centrifuging the blood for 10 min at 1,000 x g and washing the cells three times with 10-fold volumes of dextrose-gelatin-veronal buffer (DGV) (5). They could be stored concentrated at 4 C for up to 5 days. Dextran sulfate. Dextran sulfate Na-salt (DS) Pharmacia, Uppsala, was used. It was prepared from dextran with a molecular weight of 500,000 and
377
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FRISCH-NIGGEMEYER
contained 17 + 1% sulfur; 0.5 to 2% phosphate was added for stabilization. A 1% solution was stable for about 14 days when stored at 4 C. CPG. Apart from one experimental batch with a 18.5-nm pore diameter, provided by W. Haller, Washington, D. C., CPG was obtained from Electro Nucleonics Inc., Fairfield, N.J., lot no. 372 with a pore diameter of 17.7 nm ± 4.6% and lot no. 0059 with a pore diameter of 19.0 nm ± 6.2%, and the experimental batch all gave excellent results. An earlier lot no. 240 with a 21.5-nm pore diameter yielded slightly broader peaks but was still usable. Lots no. 312 (15.6 nm) and no. 120 (17.1 nm) were not satisfactory. Rubella antigen. A freeze-dried antigen prepared from detergent-treated rubella virus was obtained from Behringwerke AG, Marburg. The contents of 1 ampoule were dissolved in 1 ml of distilled water and then further diluted with DGV purchased from Rohm und Haas, Pharma Ges.m.b.H., Darmstadt, to obtain 4 to 8 hemagglutinating units for the hemagglutination inhibition (HI) test. Treatment of sera for CPG chromatography. To remove nonspecific hemagglutination inhibitors and also natural hemagglutinins, 0.3 to 0.35 ml of serum was mixed with 0.12 ml of 0.1 M CaCl2 and precipitated with 0.03 ml of 1% dextran sulfate. After 15 to 30 min in the refrigerator, the precipitate was sedimented by centrifugation for 5 min at 3,000 x g. Then, without stirring up the sediment, 0.2 ml of a 50% suspension of pigeon erythrocytes in DGV was added. After an additional 15 min at 4 C, the cells were sedimented by centrifugation for 10 min at 1,000 x g and the resulting 0.5 ml of supernatant fluid was aspirated into a 1-ml syringe. Preparation of column. A column of 8-mm diameter and 75-cm length was used. It was fitted on both ends with a closure constructed for a minimum of dead space. It contained a coarse frittered disk of stainless steel and a silicon-rubber septum (Fig. 1). Perforation of the septum v Li a syringe needle connected the columin with th, ibing. Packing with the glass powder was done under intermittent orbital vibration (10), using a small electric motor directly attached to the column. After filling, the column was rinsed with 200 ml of distilled water which was then replaced by the eluting medium. Calibration with Blue Dextran from Pharmacia, Uppsala, and benzyl alcohol demonstrated a void volume of 16.5 ml and a total free volume of 33 ml. Chromatography on CPG. A 0.5-ml amount of the treated serum was injected slowly into the column by means of a 1-ml syringe. For elution, a 0.1 M glycine buffer of pH 9.2 with 0.2 M NaCl was used. It was pumped through the column at a speed of 0.8 to 1.0 ml/min. The eluate was monitored at 280 nm and collected in fractions of 32 drops which corresponded to about 1.8 ml. Radial Immunodiffusion. The fractions to be examined by this technique (23) were dialyzed overnight at 8 C against a 100-fold volume of 0.01 M glycine buffer, pH 9.2. They were then evaporated at room temperature to approximately 0.2 ml, using a vacuum of 15 to 20 Torr. This was followed by lyophi-
J. CLIN. MICROBIOL.
Cap Septum Holder Septum SpacerGasket Space Porous Plate Closure Body
L I1 _
I
Envelope Gasket Envelope FIG. 1. Exploded view of a closure for the CPG column, designed for minimal dead space. lization, and the dry samples were dissolved in 0. 18 ml of distilled water. Diffusion was performed on Immunoplates from Hyland, Costa Mesa. After 16 h,
the plates were photographed and from the diameter of the precipitation zones the concentrations of IgM, IgA, and IgG could be determined. HI test. Serial dilutions of each of the fractions to be examined were made in 0.2-ml volumes with DGV on plastic plates from Rohm and Haas, Pharma Ges. m.b.H., Darmstadt. Four to eight hemagglutination units of rubella antigen were added to each well, and after 1 h at room temperature, 0.2 ml of an 0.2%T suspension of pigeon erythrocytes in DGV was added. Tests were refrigerated to permit sedimentation of the erythrocytes, and the patterns were read after 1 h.
RESULTS 2 Figure shows the ultraviolet absorption and the concentrations of the three classes of immu-
noglobulins as measured by radial immunodiffusion after CPG chromatography of 0.35 ml of serum treated as described. Three peaks could be observed. The first corresponded to proteins of large molecular weight which were not able to penetrate into the pores of the glass matrix. Immunodiffusion demonstrated that practically all of the IgM was contained in this peak. The IgG, which could diffuse easily into the pores, was found in the second peak, which also contained albumin and other immunologically inactive proteins. The third peak consisted mainly of transferrin, albumin, and small amounts of IgG (13). The IgA eluted about 2 ml ahead of the IgG.
379
IGM:IGG SEPARATION FOR DIAGNOSIS OF RUBELLA
VOL. 2, 1975
pg /ml
^/oAbs.
400
80
350L
70
300O
60
250L
50
2001
40
1501
30
10OO0
20
50-
10
00
10
20
50
40
30
ml
FIG. 2. Separation of IgM, IgA, and IgG from 0.35 ml of human serum. Chromatography on CPG with 17.7-nm pore diameter. Elution with 0.1 M glycine buffer, pk 9.2, was at 0.8 ml/min. Absorption of proteins was at 280 nm. Concentration of immunoglobulins was assayed by radial immunodiffusion.
The fact of most practical relevance is that no IgG could be detected in the fractions of the exclusion volume. This is also clearly visible from Fig. 3 which, in addition to the ultraviolet absorption curve, shows the HI titer of rubella antibodies of a patient with a past infection. The single peak of HI antibody practically coincides with the IgG values demonstrated by immunodiffusion. In sharp contrast, HI titers of fractionated serum from patients with recent rubella infections showed two peaks. In addition to the peak corresponding to the IgG, they also showed clearly detectable and sometimes high titers in the fractions of the exclusion volume, where the IgM eluted from the column (Fig. 4). The HI titers were assayed using the fractions without further dilution. In relation to the original serum, these fractions were diluted about 1:5 by the diffusion occurring during the chromatographic procedure. The HI titers proved to be stable at least 3 days when the fractions were stored at 4 C. In an earlier investigation (8) a column of 11-mm diameter and 1-mm length was used for 1- to 5-ml samples of serum for more than 50 runs before it became clogged. No decrease in sharpness of separation could be observed with continued use of the
HI
80
°
Abs.
70 1281 60
50
20
324
_ 10
16 8 4L
0
0
10
20
30
40
50 ml
FIG. 3. Absorption of proteins at 280 nm and HI titer of rubella antibodies from a patient with a long past infection. Chromatography was as in Fig. 2, but eluted at 1.0 ml/min.
column. In the present study it was found that clogging occurred only in the first few millimeters of the layer of porous glass grains. This
380
FRISCH-NIGGEMEYER
J. CLIN. MICROBIOL.
son of chromatography on CPG with separation using Sephadex G-200 was made in an earlier study. The Sephadex chromatography usually 128I -160 gave good separation of IgM, IgA, IgG, and albumin, but had to be run overnight. Ten IgMpositive and 14 IgM-negative samples were \ -~~~~~50 tested with both methods. There were slight 140 differences in the HI titers obtained as is to be expected due to different final dilutions, but in 64r-30 no case could either IgM or IgG be demonstrated by one method and not the other (8). 20 Also, three IgM-positive and three IgM-nega32tive serum samples were centrifuged for 16 h in a sucrose density gradient (4). As with CPG 16-_ chromatography, the IgM was separated clearly from the combined IgA and IgG antibod30 40 ml 50 10 20 0 ies and no discrepancy between CPG chromatogFIG. 4. Absorption of proteins was at 280 nm and raphy and density-gradient centrifugation HI titers of rubella antibodies was from a patient could be observed when the fractions were exwith a recent infection. Chromatography was as in amined by the HI test. Comparing CPG chromaFig. 2 and 3, but eluted at 0.9 ml/min. tography with density-gradient centrifugation, it is again the greater rapidity of the former could be removed with a spatula and replaced which makes it superior to the equally reliable by fresh CPG in a few minutes without having centrifugation method. However it should be to refill the whole column. mentioned that centrifugation does not require removal of nonspecific HI inhibitors. DISCUSSION Nonspecific inhibitors of rubella hemagglutiIn contrast to the organic gels usually used nation which are present in serum belong to the for permeation chromatography, CPG is not class of low-density lipoproteins (14, 28), which compressible, and therefore allows an ex- can be precipitated with sulfurated carbohytremely rapid flow of the eluant. When eluting drates (6, 24). Irreversible precipitation can be with 1 ml/min, the IgM left the column 18 min achieved either with polyanions of rather low after application of the serum. The bulk of the molecular weight and addition of Mn21 ions, or IgG appeared only 9 min later, and 40 min after with high-molecular-weight polyanions tothe start, a new sample could be injected. This gether with Ca2+ ions (2). We choose highextraordinary speed eliminates the necessity to molecular-weight dextran sulfate and Ca2+ use cooled columns or even to work in the cold rather than heparin and Mn2+, because with room. Indeed, it was found that slightly sharper heparin the nonspecific inhibitors are not separations could be obtained at 30 C and at always totally removed (22); the serum had to room temperature than when the column was be diluted about six-fold and a precipitate of cooled with water at 8 C. The use of 0.1 M MnO2 formed during chromatography on the glycine in the eluant provides a pH value at CPG grains in the column. The best conditions which adsorption of the immunoglobulins to for quantitative precipitation of low-density the glass surface is negligible, and also disso- lipoproteins were found to be 0.05% DS and ciates aggregates of IgG (17) which eventually 0.25 M Ca2` (4). form. The reproducibility of the CPG chromaReliable and total removal of inhibiting lowtography is excellent; the IgM appeared with- density lipoproteins enables one to assay the out exception in fractions 10 and 11. The peak of titer of IgM and IgG rubella antibodies by the the IgG was slightly less sharp; most IgG could HI test. This method does not require materials be collected in fractions 14 and 15. for tissue culture, is much simpler to perform CPG chromatography is now used in our labo- than immunofluorescence, and is more sensiratory as a routine procedure. We regard a tive than the complement fixation test (21). We serum as positive for rubella IgM antibodies if used a slight modification of the technique iniinhibition of hemagglutination is observed in tially described (30). Using DGV for preparafractions 10 and 11. This is the case even when tion of serum dilutions (19) and pigeon erythroinhibition is seen only in the first well, since cytes (25) permitted unequivocal reading of the the dilution of IgM antibodies with respect to sedimentation patterns. whole serum is already 1:5 to 1:10. A compariThe combination of dextran sulfate precipitaHI
AAbs. 70
VOL. 2, 1975
IGM:IGG SEPARATION FOR DIAGNOSIS OF RUBELLA
tion of low-density lipoproteins, permeation chromatography on CPG and the HI test with pigeon erythrocytes seems to be the method of choice at present for detection of rubella IgM antibody, from the standpoint of simplicity, speed, sensitivity and reproducibility. A diagnosis of recent rubella infection can be made on the same day the blood sample is taken. AC KNOWLE DGMENTS For valuable advice I am indebted to Ch. Kunz, Vienna, and W. Haller, Washington. I am also grateful to Electro Nucleonics Inc., Fairfield, N.J. for sending free samples of CPG. The immunodiffusion was performed excellently by Beatrix Schadbauer and gradient centrifugation was performed by F. Heinz. This investigation was supported by a grant from Fonds zur Forderung der wissenschaftlichen Forschung, Vienna. LITERATURE CITED 1. Banatvala, J. E., J. M. Best, J. Bertrand, N. A. Bowern, and S. M. Hudson. 1970. Serological assessment of rubella during pregnancy. Br. Med. J. 3:247-250. 2. Bernfeld, P., J. S. Nisselbaum, B. J. Berkeley, and R. W. Hanson. 1960. The influence of chemical and physicochemical nature of macromolecular polyanions and their interaction with human serum j3-lipoproteins. J. Biol. Chem. 235:2852-2859. 3. Burgin-Wolff, A., R. Hernandez, and M. Just. 1971. Separation of rubella IgM, IgA and IgG antibodies by gel filtration on agarose. Lancet p. 1278-1280. 4. Burstein, M., and H. R. Scholnick. 1973. Lipoproteinpolyanion-metal interactions. Adv. Lipid Res. 11:68108. 5. Clarke, D. H., and J. Casals. 1958. Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. Am. J. Trop. Med. Hyg. 7:561-573. 6. Cornwell, D. G., and F. A. Kruger. 1961. Molecular complexes in the isolation and characterization of plasma lipoproteins. J. Lipid Res. 2:110-134. 7. Freymuth, F., and C. Morel. 1972. Mise en evidence des IgM par filtration sur gel Sephadex G 200. Ann. Biol. Clin. (Paris) 30:579-583. 8. Frisch-Niggemeyer, W., F. Heinz, and H. Stemberger. 1974. Eine schnelle und verlaBliche Methode zur Erkennung frischer bzw. alterer Infektionen mit Rubella-Virus. Immun. Infekt. 2:231-237. 9. Fucillo, D. A., and J. L. Sever. 1973. Viral teratology. Bacteriol. Rev. 37:19-31. 10. Gschwender, H., H. W. Haller, and P. H. Hofschneider. 1969. Large scale preparations of viruses by steric chromatography on column of controlled pore glass. Biochim. Biophys. Acta 190:460-469. 11. Gupta, J. D., V. Peterson, M. Stout, and A. M. Murphy. 1971. Single sample diagnosis of recent rubella by fractionation of antibody of Sephadex G 200. J. Clin. Pathol. 24:547-550. 12. Haller, W. 1965. Chromatography on glass of controlled pore size. Nature (London) 206:693-696. 13. Haller, W., K.-D. Tympner, and K. Hannig. 1970. Preparation of immunoglobulin concentrates from human serum by chromatography on controlled pore glass.
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Anal. Biochem. 35:23-31. 14. Haukenes, G., and J. Aasen. 1971. Rubella hemmagglutination inhibitors: their separation from antibodies. Acta Pathol. Microbiol. Scand. Sect. B. 79:679-685. 15. Hofmann, H., and Ch. Kunz. 1973. Zur serologischen Fruhdiagnose von Virusinfektionen. Wien. Klin. Wochenschr. 85:490-493. 16. Horstmann, D. M. 1971. Rubella: the challenge of its control. J. Infect. Dis. 123:640-654. 17. Isliker, H. C. 1957. The chemical nature of antibodies. Adv. Prot. Chem. 12:387-463. 18. Iwakata, S., A. J. Rhodes, and H. A. Labzoffsky. 1972. The significance of specific IgM antibody in the diagnosis of rubella employing the immunofluorescence technique. C. M. A. J. 106:327-330. 19. Kayser, F. H. 1969. Erfahrungen mit dem Robeolen Hamagglutinations-hemmtest. Zentralbl. Bacteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 209: 275-278. 20. Laufs, R., and B. Fleckenstein. 1972. Zur serologischen Diagnostik der Rotelnerkrankung wahrend der Schwangerschaft und die Rotelnembryopathie beim Neugeborenen. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 220:235-241. 21. Lennette, E. H., N. J. Schmidt, and R. L. Magoffin. 1967. The haemagglutination inhibition test for rubella: a comparison of its sensitivity to that of neutralization, complement fixation and fluorescent antibody tests for diagnosis of infection and determination of immunity status. J. Immunol. 99:785-793. 22. Liebhaber, H. 1970. Measurement of rubella antibody by hemagglutination inhibition. II. Characteristics of an improved HAI test employing a new method for the removal of non-immunoglobulin HA inhibitors from serum. J. Immunol. 104:826-834. 23. Mancini, G., A. 0. Carbonara, and J. F. Heremans. 1965. Immunochemical quantitation of antigen by single radial immunodiffusion. Immunochemistry 2:235-254. 24. Oncley, J. L., K. W. Walton, and D. G. Cornwell. 1957. A rapid method for the bulk isolation of lipoproteins from human plasma. J. Am. Chem. Soc. 79:46664669. 25. Petermans, J., and C. Huygelen. 1967. L'emploi d'hematies de pigeons dans le test d'inhibition de l'hemagglutination de la rubeole. Presse Med. 75: 2177-2178. 26. Prinzie, A. 1971. R6telnembryopathie und Rotelnimpfstoff. Dtsch. Med. Wochenschr. 96:1367-1371. 27. Schmitz, H., and R. Haas. 1972. Determination of different cytomegalovirus immunoglobulins (IgG, IgM, IgA) by immunofluorescence. Arch. Gesamte Virusforsch. 37:131-140. 28. Shortridge, K. F., and F. Biddle. 1972. Rubella virus nonspecific haemagglutination inhibitor: evidence for the role of glycolipid bound to low density (/3) lipoprotein. Clin. Chim. Acta 42:285-294. 29. Skinner, C. W. 1961. The rubella problem. Am. J. Dis. Child. 101:78-86. 30. Stewart, G. H., P. D. Parkman, H. E. Hope, R. D. Douglas, J. P. Hamilton, and H. M. Meyer. 1967. Rubella virus haemagglutination-inhibition test. N. Engl. J. Med. 276:554-557. 31. Tartakow, I. J. 1965. The teratogenicity of maternal rubella. J. Pediatr. 66:380-381.
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1975, p. 377-381 Copyright © 1975 American Society for Microbiology
Rapid Separation of Immunoglobulin M from Immunoglobulin G Antibodies for Reliable Diagnosis of Recent Rubella Infections W. FRISCH-NIGGEMEYER Institut fur Virologie der Universitat Wien, A-1095 Wien Kinderspitalgasse 15, Austria
Received for publication 19 May 1975
Chromatography on controlled pore glass was adapted for the separation of immunoglobulin M (IgM) and immunoglobulin G (IgG) rubella antibodies from 0.3-ml samples of human serum. An extremely sharp separation of IgM from IgG antibodies could be obtained within 40 min. Nonspecific inhibitors were removed before chromatography by precipitation with high-molecular-weight dextran sulfate, and the titer of rubella antibodies in the different classes of immunoglobulins were assayed with a modified hemagglutination inhibition technique. The combination of these methods is recommended for routine tests. It permits an accurate diagnosis of recent rubella infection within a few hours.
The clinical diagnosis of rubella is sometimes difficult. A typical rubella-like illness may be caused by viruses other than rubella, e. g., some enteroviruses. In addition, a large proportion of rubella infections take a subclinical course and do not produce any overt signs of disease (16). A critical investigation has demonstrated that the doctor's diagnosis could be verified by serological methods in less than 50% of cases (1). It is well known that infection with this virus may cause congenital malformations of the child, influencing sight, hearing, heart, brain and teeth (26). The danger of the occurrence of such defects is not entirely restricted to the early months of pregnancy (9). Spontaneous abortion and stillbirths can also be caused by rubella virus (29, 31). Therefore, it is important that patients who are exposed to or develop rubella symptoms during pregnancy undergo serological tests to determine whether infection has occurred. As in many other infectious diseases (27), a primary infection with rubella virus causes the antibody titer to develop in a characteristic pattern. A few days after onset of the rash, a steep rise of antibodies occurs in the M-fraction of the immunoglobulins (IgM). Titers reach a maximum after 5 to 14 days, then diminish, and by 8 to 12 weeks later specific IgM generally cannot be detected. There is a similar rise of specific antibodies in the G-class of immunoglobulins (IgG), but hardly any decrease; titers stay constant for many months and are probably measurable for life (3, 18). Antibodies of the immunoglobulin A (IgA) class show a transient
appearance similar to IgM antibodies. Titers increase sharply, fall off gradually, and disappear at about the same time as the IgM titers
(3). This phenomenon permits one to distinguish a recent rubella infection acquired during pregnancy from a long past one, by separate titration of IgM (+IgA) and IgG antibodies, even in the absence of clinical symptoms (7, 15). However, clear-cut separation of immunoglobulin classes by the classical techniques of density gradient centrifugation (14, 20) or gel chromatography (3, 7, 11, 15) is a time consuming procedure, and generally not feasible for routine clinical tests. By using controlled pore glass (CPG) (12), permeation chromatography of serum samples can be performed in 1/io to 1/20 of the time necessary for the above mentioned methods (8, 13). This paper describes a further development of this method for immunoglobulin separation that uses simpler procedures and smaller samples, requires less time, and yields sharper separation. MATERIALS AND METHODS Sera. Sera were examined either fresh or after storage at -20 C. Erythrocytes. Pigeon erythrocytes were prepared by centrifuging the blood for 10 min at 1,000 x g and washing the cells three times with 10-fold volumes of dextrose-gelatin-veronal buffer (DGV) (5). They could be stored concentrated at 4 C for up to 5 days. Dextran sulfate. Dextran sulfate Na-salt (DS) Pharmacia, Uppsala, was used. It was prepared from dextran with a molecular weight of 500,000 and
377
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FRISCH-NIGGEMEYER
contained 17 + 1% sulfur; 0.5 to 2% phosphate was added for stabilization. A 1% solution was stable for about 14 days when stored at 4 C. CPG. Apart from one experimental batch with a 18.5-nm pore diameter, provided by W. Haller, Washington, D. C., CPG was obtained from Electro Nucleonics Inc., Fairfield, N.J., lot no. 372 with a pore diameter of 17.7 nm ± 4.6% and lot no. 0059 with a pore diameter of 19.0 nm ± 6.2%, and the experimental batch all gave excellent results. An earlier lot no. 240 with a 21.5-nm pore diameter yielded slightly broader peaks but was still usable. Lots no. 312 (15.6 nm) and no. 120 (17.1 nm) were not satisfactory. Rubella antigen. A freeze-dried antigen prepared from detergent-treated rubella virus was obtained from Behringwerke AG, Marburg. The contents of 1 ampoule were dissolved in 1 ml of distilled water and then further diluted with DGV purchased from Rohm und Haas, Pharma Ges.m.b.H., Darmstadt, to obtain 4 to 8 hemagglutinating units for the hemagglutination inhibition (HI) test. Treatment of sera for CPG chromatography. To remove nonspecific hemagglutination inhibitors and also natural hemagglutinins, 0.3 to 0.35 ml of serum was mixed with 0.12 ml of 0.1 M CaCl2 and precipitated with 0.03 ml of 1% dextran sulfate. After 15 to 30 min in the refrigerator, the precipitate was sedimented by centrifugation for 5 min at 3,000 x g. Then, without stirring up the sediment, 0.2 ml of a 50% suspension of pigeon erythrocytes in DGV was added. After an additional 15 min at 4 C, the cells were sedimented by centrifugation for 10 min at 1,000 x g and the resulting 0.5 ml of supernatant fluid was aspirated into a 1-ml syringe. Preparation of column. A column of 8-mm diameter and 75-cm length was used. It was fitted on both ends with a closure constructed for a minimum of dead space. It contained a coarse frittered disk of stainless steel and a silicon-rubber septum (Fig. 1). Perforation of the septum v Li a syringe needle connected the columin with th, ibing. Packing with the glass powder was done under intermittent orbital vibration (10), using a small electric motor directly attached to the column. After filling, the column was rinsed with 200 ml of distilled water which was then replaced by the eluting medium. Calibration with Blue Dextran from Pharmacia, Uppsala, and benzyl alcohol demonstrated a void volume of 16.5 ml and a total free volume of 33 ml. Chromatography on CPG. A 0.5-ml amount of the treated serum was injected slowly into the column by means of a 1-ml syringe. For elution, a 0.1 M glycine buffer of pH 9.2 with 0.2 M NaCl was used. It was pumped through the column at a speed of 0.8 to 1.0 ml/min. The eluate was monitored at 280 nm and collected in fractions of 32 drops which corresponded to about 1.8 ml. Radial Immunodiffusion. The fractions to be examined by this technique (23) were dialyzed overnight at 8 C against a 100-fold volume of 0.01 M glycine buffer, pH 9.2. They were then evaporated at room temperature to approximately 0.2 ml, using a vacuum of 15 to 20 Torr. This was followed by lyophi-
J. CLIN. MICROBIOL.
Cap Septum Holder Septum SpacerGasket Space Porous Plate Closure Body
L I1 _
I
Envelope Gasket Envelope FIG. 1. Exploded view of a closure for the CPG column, designed for minimal dead space. lization, and the dry samples were dissolved in 0. 18 ml of distilled water. Diffusion was performed on Immunoplates from Hyland, Costa Mesa. After 16 h,
the plates were photographed and from the diameter of the precipitation zones the concentrations of IgM, IgA, and IgG could be determined. HI test. Serial dilutions of each of the fractions to be examined were made in 0.2-ml volumes with DGV on plastic plates from Rohm and Haas, Pharma Ges. m.b.H., Darmstadt. Four to eight hemagglutination units of rubella antigen were added to each well, and after 1 h at room temperature, 0.2 ml of an 0.2%T suspension of pigeon erythrocytes in DGV was added. Tests were refrigerated to permit sedimentation of the erythrocytes, and the patterns were read after 1 h.
RESULTS 2 Figure shows the ultraviolet absorption and the concentrations of the three classes of immu-
noglobulins as measured by radial immunodiffusion after CPG chromatography of 0.35 ml of serum treated as described. Three peaks could be observed. The first corresponded to proteins of large molecular weight which were not able to penetrate into the pores of the glass matrix. Immunodiffusion demonstrated that practically all of the IgM was contained in this peak. The IgG, which could diffuse easily into the pores, was found in the second peak, which also contained albumin and other immunologically inactive proteins. The third peak consisted mainly of transferrin, albumin, and small amounts of IgG (13). The IgA eluted about 2 ml ahead of the IgG.
379
IGM:IGG SEPARATION FOR DIAGNOSIS OF RUBELLA
VOL. 2, 1975
pg /ml
^/oAbs.
400
80
350L
70
300O
60
250L
50
2001
40
1501
30
10OO0
20
50-
10
00
10
20
50
40
30
ml
FIG. 2. Separation of IgM, IgA, and IgG from 0.35 ml of human serum. Chromatography on CPG with 17.7-nm pore diameter. Elution with 0.1 M glycine buffer, pk 9.2, was at 0.8 ml/min. Absorption of proteins was at 280 nm. Concentration of immunoglobulins was assayed by radial immunodiffusion.
The fact of most practical relevance is that no IgG could be detected in the fractions of the exclusion volume. This is also clearly visible from Fig. 3 which, in addition to the ultraviolet absorption curve, shows the HI titer of rubella antibodies of a patient with a past infection. The single peak of HI antibody practically coincides with the IgG values demonstrated by immunodiffusion. In sharp contrast, HI titers of fractionated serum from patients with recent rubella infections showed two peaks. In addition to the peak corresponding to the IgG, they also showed clearly detectable and sometimes high titers in the fractions of the exclusion volume, where the IgM eluted from the column (Fig. 4). The HI titers were assayed using the fractions without further dilution. In relation to the original serum, these fractions were diluted about 1:5 by the diffusion occurring during the chromatographic procedure. The HI titers proved to be stable at least 3 days when the fractions were stored at 4 C. In an earlier investigation (8) a column of 11-mm diameter and 1-mm length was used for 1- to 5-ml samples of serum for more than 50 runs before it became clogged. No decrease in sharpness of separation could be observed with continued use of the
HI
80
°
Abs.
70 1281 60
50
20
324
_ 10
16 8 4L
0
0
10
20
30
40
50 ml
FIG. 3. Absorption of proteins at 280 nm and HI titer of rubella antibodies from a patient with a long past infection. Chromatography was as in Fig. 2, but eluted at 1.0 ml/min.
column. In the present study it was found that clogging occurred only in the first few millimeters of the layer of porous glass grains. This
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J. CLIN. MICROBIOL.
son of chromatography on CPG with separation using Sephadex G-200 was made in an earlier study. The Sephadex chromatography usually 128I -160 gave good separation of IgM, IgA, IgG, and albumin, but had to be run overnight. Ten IgMpositive and 14 IgM-negative samples were \ -~~~~~50 tested with both methods. There were slight 140 differences in the HI titers obtained as is to be expected due to different final dilutions, but in 64r-30 no case could either IgM or IgG be demonstrated by one method and not the other (8). 20 Also, three IgM-positive and three IgM-nega32tive serum samples were centrifuged for 16 h in a sucrose density gradient (4). As with CPG 16-_ chromatography, the IgM was separated clearly from the combined IgA and IgG antibod30 40 ml 50 10 20 0 ies and no discrepancy between CPG chromatogFIG. 4. Absorption of proteins was at 280 nm and raphy and density-gradient centrifugation HI titers of rubella antibodies was from a patient could be observed when the fractions were exwith a recent infection. Chromatography was as in amined by the HI test. Comparing CPG chromaFig. 2 and 3, but eluted at 0.9 ml/min. tography with density-gradient centrifugation, it is again the greater rapidity of the former could be removed with a spatula and replaced which makes it superior to the equally reliable by fresh CPG in a few minutes without having centrifugation method. However it should be to refill the whole column. mentioned that centrifugation does not require removal of nonspecific HI inhibitors. DISCUSSION Nonspecific inhibitors of rubella hemagglutiIn contrast to the organic gels usually used nation which are present in serum belong to the for permeation chromatography, CPG is not class of low-density lipoproteins (14, 28), which compressible, and therefore allows an ex- can be precipitated with sulfurated carbohytremely rapid flow of the eluant. When eluting drates (6, 24). Irreversible precipitation can be with 1 ml/min, the IgM left the column 18 min achieved either with polyanions of rather low after application of the serum. The bulk of the molecular weight and addition of Mn21 ions, or IgG appeared only 9 min later, and 40 min after with high-molecular-weight polyanions tothe start, a new sample could be injected. This gether with Ca2+ ions (2). We choose highextraordinary speed eliminates the necessity to molecular-weight dextran sulfate and Ca2+ use cooled columns or even to work in the cold rather than heparin and Mn2+, because with room. Indeed, it was found that slightly sharper heparin the nonspecific inhibitors are not separations could be obtained at 30 C and at always totally removed (22); the serum had to room temperature than when the column was be diluted about six-fold and a precipitate of cooled with water at 8 C. The use of 0.1 M MnO2 formed during chromatography on the glycine in the eluant provides a pH value at CPG grains in the column. The best conditions which adsorption of the immunoglobulins to for quantitative precipitation of low-density the glass surface is negligible, and also disso- lipoproteins were found to be 0.05% DS and ciates aggregates of IgG (17) which eventually 0.25 M Ca2` (4). form. The reproducibility of the CPG chromaReliable and total removal of inhibiting lowtography is excellent; the IgM appeared with- density lipoproteins enables one to assay the out exception in fractions 10 and 11. The peak of titer of IgM and IgG rubella antibodies by the the IgG was slightly less sharp; most IgG could HI test. This method does not require materials be collected in fractions 14 and 15. for tissue culture, is much simpler to perform CPG chromatography is now used in our labo- than immunofluorescence, and is more sensiratory as a routine procedure. We regard a tive than the complement fixation test (21). We serum as positive for rubella IgM antibodies if used a slight modification of the technique iniinhibition of hemagglutination is observed in tially described (30). Using DGV for preparafractions 10 and 11. This is the case even when tion of serum dilutions (19) and pigeon erythroinhibition is seen only in the first well, since cytes (25) permitted unequivocal reading of the the dilution of IgM antibodies with respect to sedimentation patterns. whole serum is already 1:5 to 1:10. A compariThe combination of dextran sulfate precipitaHI
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IGM:IGG SEPARATION FOR DIAGNOSIS OF RUBELLA
tion of low-density lipoproteins, permeation chromatography on CPG and the HI test with pigeon erythrocytes seems to be the method of choice at present for detection of rubella IgM antibody, from the standpoint of simplicity, speed, sensitivity and reproducibility. A diagnosis of recent rubella infection can be made on the same day the blood sample is taken. AC KNOWLE DGMENTS For valuable advice I am indebted to Ch. Kunz, Vienna, and W. Haller, Washington. I am also grateful to Electro Nucleonics Inc., Fairfield, N.J. for sending free samples of CPG. The immunodiffusion was performed excellently by Beatrix Schadbauer and gradient centrifugation was performed by F. Heinz. This investigation was supported by a grant from Fonds zur Forderung der wissenschaftlichen Forschung, Vienna. LITERATURE CITED 1. Banatvala, J. E., J. M. Best, J. Bertrand, N. A. Bowern, and S. M. Hudson. 1970. Serological assessment of rubella during pregnancy. Br. Med. J. 3:247-250. 2. Bernfeld, P., J. S. Nisselbaum, B. J. Berkeley, and R. W. Hanson. 1960. The influence of chemical and physicochemical nature of macromolecular polyanions and their interaction with human serum j3-lipoproteins. J. Biol. Chem. 235:2852-2859. 3. Burgin-Wolff, A., R. Hernandez, and M. Just. 1971. Separation of rubella IgM, IgA and IgG antibodies by gel filtration on agarose. Lancet p. 1278-1280. 4. Burstein, M., and H. R. Scholnick. 1973. Lipoproteinpolyanion-metal interactions. Adv. Lipid Res. 11:68108. 5. Clarke, D. H., and J. Casals. 1958. Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. Am. J. Trop. Med. Hyg. 7:561-573. 6. Cornwell, D. G., and F. A. Kruger. 1961. Molecular complexes in the isolation and characterization of plasma lipoproteins. J. Lipid Res. 2:110-134. 7. Freymuth, F., and C. Morel. 1972. Mise en evidence des IgM par filtration sur gel Sephadex G 200. Ann. Biol. Clin. (Paris) 30:579-583. 8. Frisch-Niggemeyer, W., F. Heinz, and H. Stemberger. 1974. Eine schnelle und verlaBliche Methode zur Erkennung frischer bzw. alterer Infektionen mit Rubella-Virus. Immun. Infekt. 2:231-237. 9. Fucillo, D. A., and J. L. Sever. 1973. Viral teratology. Bacteriol. Rev. 37:19-31. 10. Gschwender, H., H. W. Haller, and P. H. Hofschneider. 1969. Large scale preparations of viruses by steric chromatography on column of controlled pore glass. Biochim. Biophys. Acta 190:460-469. 11. Gupta, J. D., V. Peterson, M. Stout, and A. M. Murphy. 1971. Single sample diagnosis of recent rubella by fractionation of antibody of Sephadex G 200. J. Clin. Pathol. 24:547-550. 12. Haller, W. 1965. Chromatography on glass of controlled pore size. Nature (London) 206:693-696. 13. Haller, W., K.-D. Tympner, and K. Hannig. 1970. Preparation of immunoglobulin concentrates from human serum by chromatography on controlled pore glass.
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Anal. Biochem. 35:23-31. 14. Haukenes, G., and J. Aasen. 1971. Rubella hemmagglutination inhibitors: their separation from antibodies. Acta Pathol. Microbiol. Scand. Sect. B. 79:679-685. 15. Hofmann, H., and Ch. Kunz. 1973. Zur serologischen Fruhdiagnose von Virusinfektionen. Wien. Klin. Wochenschr. 85:490-493. 16. Horstmann, D. M. 1971. Rubella: the challenge of its control. J. Infect. Dis. 123:640-654. 17. Isliker, H. C. 1957. The chemical nature of antibodies. Adv. Prot. Chem. 12:387-463. 18. Iwakata, S., A. J. Rhodes, and H. A. Labzoffsky. 1972. The significance of specific IgM antibody in the diagnosis of rubella employing the immunofluorescence technique. C. M. A. J. 106:327-330. 19. Kayser, F. H. 1969. Erfahrungen mit dem Robeolen Hamagglutinations-hemmtest. Zentralbl. Bacteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 209: 275-278. 20. Laufs, R., and B. Fleckenstein. 1972. Zur serologischen Diagnostik der Rotelnerkrankung wahrend der Schwangerschaft und die Rotelnembryopathie beim Neugeborenen. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 220:235-241. 21. Lennette, E. H., N. J. Schmidt, and R. L. Magoffin. 1967. The haemagglutination inhibition test for rubella: a comparison of its sensitivity to that of neutralization, complement fixation and fluorescent antibody tests for diagnosis of infection and determination of immunity status. J. Immunol. 99:785-793. 22. Liebhaber, H. 1970. Measurement of rubella antibody by hemagglutination inhibition. II. Characteristics of an improved HAI test employing a new method for the removal of non-immunoglobulin HA inhibitors from serum. J. Immunol. 104:826-834. 23. Mancini, G., A. 0. Carbonara, and J. F. Heremans. 1965. Immunochemical quantitation of antigen by single radial immunodiffusion. Immunochemistry 2:235-254. 24. Oncley, J. L., K. W. Walton, and D. G. Cornwell. 1957. A rapid method for the bulk isolation of lipoproteins from human plasma. J. Am. Chem. Soc. 79:46664669. 25. Petermans, J., and C. Huygelen. 1967. L'emploi d'hematies de pigeons dans le test d'inhibition de l'hemagglutination de la rubeole. Presse Med. 75: 2177-2178. 26. Prinzie, A. 1971. R6telnembryopathie und Rotelnimpfstoff. Dtsch. Med. Wochenschr. 96:1367-1371. 27. Schmitz, H., and R. Haas. 1972. Determination of different cytomegalovirus immunoglobulins (IgG, IgM, IgA) by immunofluorescence. Arch. Gesamte Virusforsch. 37:131-140. 28. Shortridge, K. F., and F. Biddle. 1972. Rubella virus nonspecific haemagglutination inhibitor: evidence for the role of glycolipid bound to low density (/3) lipoprotein. Clin. Chim. Acta 42:285-294. 29. Skinner, C. W. 1961. The rubella problem. Am. J. Dis. Child. 101:78-86. 30. Stewart, G. H., P. D. Parkman, H. E. Hope, R. D. Douglas, J. P. Hamilton, and H. M. Meyer. 1967. Rubella virus haemagglutination-inhibition test. N. Engl. J. Med. 276:554-557. 31. Tartakow, I. J. 1965. The teratogenicity of maternal rubella. J. Pediatr. 66:380-381.
