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Immunofluorescence determination og IgG in cerebrospinal fluid following high performance liquid gel permeation chromatography
Journal of Immunological Methods, 98 (1987) 1-4 Elsevier
1
JIM 04258
Immunofluorescence determination of IgG in cerebrospinal fluid following high performance liquid gel permeation chromatography Hideo H o s o t s u b o 1, K a y o k o Arai 1 and Jun-Ichi Iwamura 2 1 Central Laboratory for Cinical Inoestigation, Osaka Unioersity Hospital, Fukushima, Osaka, and 2 Research Institute of Food Science, Kinki University, Kowakae, Higashi-osaka, Japan
(Received 13 October 1986, accepted 20 November 1986)
A simple technique using high performance liquid gel permeation chromatography for determination of IgG in cerebrolspinal fluid (CSF) is described. Accurate determination of IgG was achieved by precolumn derivatization with fluorescein isothiocyanate-conjugated anti-human IgG Fab, a substance that readily yields a fluorescent immune complex. Immune complexes separated as a well-delineated peak in the column void volume, and were measured by the fluorescence of the column eluates (Ex = 490 nm, Em = 520 nm). The results were reproducible with a deviation of less than 2% and a good linear relationship over the range of 2.0-24.5 mg/dl. The IgG concentration assayed by the column method correlated well with values obtained by laser nephelometry. Key words: Chromatography, high performance liquid gel permeation; Cerebrospinal fluid; IgG; FITC-conjugated anti-human IgG Fab; Fluorescent immune complex
Introduction
High performance liquid gel permeation chromatography (HPL-GPC) has become the analytical method of choice for the rapi d separation of proteins (Sann et al., 1983). Eluted proteins can be directly quantified by UV detection, usually at 280 nm. Although selective for aromatic residues in proteins, detection by UV absorbance at this wavelength is not specific for proteins since a host of other molecules may also absorb to some extent. In order to confirm the presence of a particular protein, a selective detection system has been developed which employs precolumn reaction with antibody. Correspondence to: H. Hosotsubo, Central Laboratory for Clinical Investigation, Osaka University Hospital, Fukushima, Osaka, Japan.
The measurement of immunoglobulins in CSF has always posed a problem due to extremely low levels being present. An important methodological aspect of the analysis of proteins in the CSF is the influence of the concentration of a particular protein in the serum on its concentration in the CSF (Felgenhauer, 1974). For several proteins the CSF:serum ratio correlates with the hydrodynamic radius. In addition, the permeability of the blood-CSF barrier affects the concentration of CSF proteins and this is taken into account by the determination of the ratio. For IgG the quotient of the IgG ratio and the albumin ratio (the IgG index) provides a convenient parameter for the interpretation of IgG in CSF (Tibbling et al., 1977). The major immunoglobulin i n CSF, IgG, is usually measured by standard immunol0gical techniques such as radial immunodiffusion
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
(Mancini et al., 1965), electroimmunodiffusion (Tourtellotte et al., 1971) and laser nephelometry (Steruberg, 1977). Affinity chromatography has been applied in many different low pressure chromatography systems (Phillips et al., 1984) but rarely as a rapid determination system. Our primary aim was to establish a method for the characterization and quantification of IgG (Hosotsubo et al., 1985), but the method developed is applicable to any human CSF protein against which antibody can be raised. This system employs precolumn reaction with fluorescein isothiocyanate (FITC)-conjugated monovalent antibody by HPL-GPC. Precolumn reaction can be easily coupled with a fluorescence detector to provide selective and specific detection. In this report we have studied the separation of immune complexes and free antibody by gel permeation chromatography and determined the optimal conditions for their separation. The immune complex and free antibody pass through a column as two well-defined peaks. The immune complex is eluted in the void volume and is .easily measured distinct from the fluorescence of the bound FITC.
the SPD-6A (Shimadzu) UV detector. The high performance gel permeation chromatography column used was the Shimadzu Shim-pack DIOL-300 (25 cm x 7.9 mm ID, 5/~m diameter, 30 nm pore size). For quantitative data processing, the chromatopac C-R3A was used and the concentration of IgG was calculated from the peak area of the fluorescent immune complex.
CSF samples CSF specimens were obtained from patients with different infectious diseases attending the Hospital of Osaka University. Following the removal of cells by centrifugation at 1200 × g for 10 min, CSF was stored at 4°C and was diluted 10-fold with PBS just before use.
Preparation of fluorescent immune complex for HPL-GPC analysis 20/~1 of CSF (diluted 10-fold with PBS) or IgG standard samples and an equal volume of the diluted FITC anti-IgG Fab (diluted 10-fold with PBS) were mixed in a small tube and directly analyzed by HPL-GPC.
Chromatography Materials and methods
Antigen (lgG standard) Chromatographically purified human IgG was obtained from Kabi (Stockholm, Sweden).
Antisera Anti-human IgG Fab conjugated with FITC (H and L chain specific; lot no. 2275; F / P : 4.26 mg/g; MW: 50000) was purchased from Cappel Laboratories (Downington, PA).
Chromatography apparatus Separations were performed under isocratic conditions using a Shimadzu LC-6A system (Shimadzu, Kyoto, Japan). Samples were applied with a SIL-1A (Shimadzu) valve equipped with a 200 /xl loop and injected with a microsyringe. Fluorescence of antibody and immune complex were measured using 490 mn (excitation wavelength) and 520 nm (emission wavelength) with a RF-530 (Shimadzu) fluorescence spectrometer. Other proteins were monitored at 280 nm using
The void volume of the column contains proteins having a molecular weight greater than 300000. Sample sizes of 10 /~1 proved best for application. During and after injection the pressure ranged between 10 and 20 bar at a constant flow rate of 1.2 ml/min. All analyses were performed at this constant flow rate using phosphatebuffered saline (PBS: 0.07 M phosphate containing 0.14 M NaCI, pH 7.4) as the mobile phase.
Determination of lgG in CSF samples IgG was identified by the retention time of the main fluorescence peak defined by chromatography of a standard fluorescent immune complex. The IgG content of each CSF sample was estimated from the relative peak area using known amounts of authentic IgG as a standard.
Laser nephelometric assay For laser nephelometer (Behringwerke, (Schliep et al.,
nephelometry a Behring laser full-automatic system was used D-3550 Marburg/Lahn, F.R.G.) 1978). Samples, standard and anti-
sera were diluted with a sterile isotonic saline solution and 100 /xl of sample (diluted two-fold) were mixed in a microcuvette with 40 ~1 of antiIgG (LN serum anti-IgG SAW, Behringwerke, diluted five-fold). The mixtures in the cuvettes were mixed and allowed to stand for 30 rain at room temperature. After incubation, the relative light scatter was measured and, using a 9825A calculator (Yokogawa-Hewlett-Packard, Tokyo, Japan) and a third-order polynomial curve-fitting program, standard curves were constructed. A calibration curve was prepared using the 1050 m g / d l standard solution of IgG, diluted to give concentrations of 10.5, 5.25, 2.60, 1.30, 0.65 and 0.33 mg/dl.
Results
Identification of IgG Fig. 1 shows the absorbance and fluorescence elution profiles observed when chromatography was performed with 5 /~1 of the diluted CSF, diluted fluorescent antibody or a mixture containing 5/xl each. The mixture was incubated for 10 min at 37°C before the chromatography. Formation of a fluorescent immune complex in the mixture was indicated by the appearance of a new peak at 4.8 min, the time at which molecules or molecular complexes exceeding the exclusion of the column were eluted.
Stability of fluorescent immune complexes There was no detectable change in the time of elution % for samples stored at 4 ° C for up to 2 weeks.
Linearity of IgG assay The IgG assay was linear for measurements in the final reaction mixture over the concentration range 2.0-24.5 m g / d l (see Fig. 2). This is equivalent to 0.100-1.225 /~g of IgG actually injected on the column.
(A)
~L l 0 retention time(min)
i
/
150-
i
5 I0 retention time (min)
A
120
/ "
.
oo
90.
~
60,
o
o
/ /
30
5'
~'o
retention time (;.in)
Fig. i. Typical H P L - G P C chromatograms. A: Separation of diluted human CSF. B: Pattern produced by a diluted FITCconjugated anti-human IgO Fab. C: Chromatograrn produced by A + B. Chromatography performed on a 250×7.9 m m Shim-pack DIOL-300 column at the flow rate of 1.2 m l / m i n using PBS. UV detector: 280 n m (0.04 AUFS). Fluorescence detector: Ex = 490 nm, Em = 520 nm. Injection volume: 10/~1.
0
/
/ °
/ 7.0
14.0 IgG concentration
21.0
28.0
(mg/dl)
Fig. 2. Standard curve for IgG concentrations over the range 3.5-24.5 m g / d l . 20 ~1 of each diluted IgG standard were added to 20 /~1 of FITC-conjugated anti-IgG Fab and the fluorescence peak area was determined. Each data point indicates a single measured value.
36
33
3O 27
24
? 12
9 6 3
3
6
9
12
15
18
Nephelometric
21 method
24
27
30
33
36
(mg/dl)
Fig. 3. Correlation between CSF IgG measured by laser nephelometric method and the HPL-GPC method. The regression equation and correlation coefficient were y = 0:966x -0.228 andr = 0.997, respectively.
Accuracy of HPL-GPC determination of lgG The I g G levels of CSF samples measured by the H P L - G P C method described here were compared with those obtained by nephelometry. As shown in Fig. 3 the values obtained by the two methods were comparable. G o o d correlation was observed with the nephelometric method ( r = 0.997, n = 40). The recovery of a known amount of purified I g G added to CSF was over 99.6%
Reproducibility test The concentrations of I g G in CSF samples were analyzed up to ten times to determine the reproducibility of the assay system. The H P L - G P C method was highly reproducible and accurate with intra-assay coefficients of variation of 1.343, 0.984 and 0.636% at 4.0, 10.0 and 16.0 m g / d l , respectively.
Discussion We have previously reported that H P L - G P C provides a rapid method for analyzing IgG, and in
this report conclude that H P L - G P C can offer a much more rapid method for analyzing I g G in CSF than nephelometry. Determinations requiring 30 min by nephelometry can be obtained in 10 min by H P L - G P C . Because the separations achieved on the gel permeation column are highly reproducible, the methodology has potential as a reference procedure for characterizing IgG. In this present report, the use of H P L - G P C for quantitative I g G analysis has been examined. Estimates of I g G concentrations in CSF, obtained by H P L - G P C were in agreement with those obtained by nephelometry. The level of accuracy was acceptable, and the effect of other CSF components was insignificant. While the correlation between nephelometry and H P L - G P C for the measurement of I g G in CSF was good ( r = 0.997), this does not imply that all CSF samples can be examined by this method using a single standard curve. It should be emphasized that the present method: (i) measures the concentration of available I g G specifically, (ii) is as accurate as the standard immunological assays in most clinical laboratories, (iii) is faster and yields result on a 5/~1 sample of CSF and (iv) is an inexpensive improvement for diagnostic work, if H P L C equipment is already available.
References Felgenhauer, K. (1974) Klin. Wochenschr. 52, 1158. Hosotsubo, H., Arai, K. and Iwamura, J.-I. (1985) J. Immunol. Methods 85, 115. Mancini, G., Carbonara, A.O. and Heremans, F.F. (1965) Immunochemistry2, 235. Phillips, T.M., More, N.S., Queen, W.D., Holohan, T.V., Kramer, N.C. and Thompson, A.M. (1984) J. Chromatogr. 317, 173. Satan, G., Schneider, G., Loeke, S. and Doerr, H.W. (1983) J. Immunol. Methods 5, 121. Schliep, G. and Felgenhaner, K. (1978) J. Clin. Chem. Clin. Biochem. 16, 631. Steruberg, J.C. (1977) Clin. Chem. 23, 1456. Tibbling, G., Link, H. and Ohman, S. (1977) Scand. J. Ciin. Lab. Invest. 37, 385. Tourtenotte, W.W., Tovolato, B., Parker, J.A. and Comiso, P. (1971) Arch. Neurol. 25, 345.
1
JIM 04258
Immunofluorescence determination of IgG in cerebrospinal fluid following high performance liquid gel permeation chromatography Hideo H o s o t s u b o 1, K a y o k o Arai 1 and Jun-Ichi Iwamura 2 1 Central Laboratory for Cinical Inoestigation, Osaka Unioersity Hospital, Fukushima, Osaka, and 2 Research Institute of Food Science, Kinki University, Kowakae, Higashi-osaka, Japan
(Received 13 October 1986, accepted 20 November 1986)
A simple technique using high performance liquid gel permeation chromatography for determination of IgG in cerebrolspinal fluid (CSF) is described. Accurate determination of IgG was achieved by precolumn derivatization with fluorescein isothiocyanate-conjugated anti-human IgG Fab, a substance that readily yields a fluorescent immune complex. Immune complexes separated as a well-delineated peak in the column void volume, and were measured by the fluorescence of the column eluates (Ex = 490 nm, Em = 520 nm). The results were reproducible with a deviation of less than 2% and a good linear relationship over the range of 2.0-24.5 mg/dl. The IgG concentration assayed by the column method correlated well with values obtained by laser nephelometry. Key words: Chromatography, high performance liquid gel permeation; Cerebrospinal fluid; IgG; FITC-conjugated anti-human IgG Fab; Fluorescent immune complex
Introduction
High performance liquid gel permeation chromatography (HPL-GPC) has become the analytical method of choice for the rapi d separation of proteins (Sann et al., 1983). Eluted proteins can be directly quantified by UV detection, usually at 280 nm. Although selective for aromatic residues in proteins, detection by UV absorbance at this wavelength is not specific for proteins since a host of other molecules may also absorb to some extent. In order to confirm the presence of a particular protein, a selective detection system has been developed which employs precolumn reaction with antibody. Correspondence to: H. Hosotsubo, Central Laboratory for Clinical Investigation, Osaka University Hospital, Fukushima, Osaka, Japan.
The measurement of immunoglobulins in CSF has always posed a problem due to extremely low levels being present. An important methodological aspect of the analysis of proteins in the CSF is the influence of the concentration of a particular protein in the serum on its concentration in the CSF (Felgenhauer, 1974). For several proteins the CSF:serum ratio correlates with the hydrodynamic radius. In addition, the permeability of the blood-CSF barrier affects the concentration of CSF proteins and this is taken into account by the determination of the ratio. For IgG the quotient of the IgG ratio and the albumin ratio (the IgG index) provides a convenient parameter for the interpretation of IgG in CSF (Tibbling et al., 1977). The major immunoglobulin i n CSF, IgG, is usually measured by standard immunol0gical techniques such as radial immunodiffusion
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
(Mancini et al., 1965), electroimmunodiffusion (Tourtellotte et al., 1971) and laser nephelometry (Steruberg, 1977). Affinity chromatography has been applied in many different low pressure chromatography systems (Phillips et al., 1984) but rarely as a rapid determination system. Our primary aim was to establish a method for the characterization and quantification of IgG (Hosotsubo et al., 1985), but the method developed is applicable to any human CSF protein against which antibody can be raised. This system employs precolumn reaction with fluorescein isothiocyanate (FITC)-conjugated monovalent antibody by HPL-GPC. Precolumn reaction can be easily coupled with a fluorescence detector to provide selective and specific detection. In this report we have studied the separation of immune complexes and free antibody by gel permeation chromatography and determined the optimal conditions for their separation. The immune complex and free antibody pass through a column as two well-defined peaks. The immune complex is eluted in the void volume and is .easily measured distinct from the fluorescence of the bound FITC.
the SPD-6A (Shimadzu) UV detector. The high performance gel permeation chromatography column used was the Shimadzu Shim-pack DIOL-300 (25 cm x 7.9 mm ID, 5/~m diameter, 30 nm pore size). For quantitative data processing, the chromatopac C-R3A was used and the concentration of IgG was calculated from the peak area of the fluorescent immune complex.
CSF samples CSF specimens were obtained from patients with different infectious diseases attending the Hospital of Osaka University. Following the removal of cells by centrifugation at 1200 × g for 10 min, CSF was stored at 4°C and was diluted 10-fold with PBS just before use.
Preparation of fluorescent immune complex for HPL-GPC analysis 20/~1 of CSF (diluted 10-fold with PBS) or IgG standard samples and an equal volume of the diluted FITC anti-IgG Fab (diluted 10-fold with PBS) were mixed in a small tube and directly analyzed by HPL-GPC.
Chromatography Materials and methods
Antigen (lgG standard) Chromatographically purified human IgG was obtained from Kabi (Stockholm, Sweden).
Antisera Anti-human IgG Fab conjugated with FITC (H and L chain specific; lot no. 2275; F / P : 4.26 mg/g; MW: 50000) was purchased from Cappel Laboratories (Downington, PA).
Chromatography apparatus Separations were performed under isocratic conditions using a Shimadzu LC-6A system (Shimadzu, Kyoto, Japan). Samples were applied with a SIL-1A (Shimadzu) valve equipped with a 200 /xl loop and injected with a microsyringe. Fluorescence of antibody and immune complex were measured using 490 mn (excitation wavelength) and 520 nm (emission wavelength) with a RF-530 (Shimadzu) fluorescence spectrometer. Other proteins were monitored at 280 nm using
The void volume of the column contains proteins having a molecular weight greater than 300000. Sample sizes of 10 /~1 proved best for application. During and after injection the pressure ranged between 10 and 20 bar at a constant flow rate of 1.2 ml/min. All analyses were performed at this constant flow rate using phosphatebuffered saline (PBS: 0.07 M phosphate containing 0.14 M NaCI, pH 7.4) as the mobile phase.
Determination of lgG in CSF samples IgG was identified by the retention time of the main fluorescence peak defined by chromatography of a standard fluorescent immune complex. The IgG content of each CSF sample was estimated from the relative peak area using known amounts of authentic IgG as a standard.
Laser nephelometric assay For laser nephelometer (Behringwerke, (Schliep et al.,
nephelometry a Behring laser full-automatic system was used D-3550 Marburg/Lahn, F.R.G.) 1978). Samples, standard and anti-
sera were diluted with a sterile isotonic saline solution and 100 /xl of sample (diluted two-fold) were mixed in a microcuvette with 40 ~1 of antiIgG (LN serum anti-IgG SAW, Behringwerke, diluted five-fold). The mixtures in the cuvettes were mixed and allowed to stand for 30 rain at room temperature. After incubation, the relative light scatter was measured and, using a 9825A calculator (Yokogawa-Hewlett-Packard, Tokyo, Japan) and a third-order polynomial curve-fitting program, standard curves were constructed. A calibration curve was prepared using the 1050 m g / d l standard solution of IgG, diluted to give concentrations of 10.5, 5.25, 2.60, 1.30, 0.65 and 0.33 mg/dl.
Results
Identification of IgG Fig. 1 shows the absorbance and fluorescence elution profiles observed when chromatography was performed with 5 /~1 of the diluted CSF, diluted fluorescent antibody or a mixture containing 5/xl each. The mixture was incubated for 10 min at 37°C before the chromatography. Formation of a fluorescent immune complex in the mixture was indicated by the appearance of a new peak at 4.8 min, the time at which molecules or molecular complexes exceeding the exclusion of the column were eluted.
Stability of fluorescent immune complexes There was no detectable change in the time of elution % for samples stored at 4 ° C for up to 2 weeks.
Linearity of IgG assay The IgG assay was linear for measurements in the final reaction mixture over the concentration range 2.0-24.5 m g / d l (see Fig. 2). This is equivalent to 0.100-1.225 /~g of IgG actually injected on the column.
(A)
~L l 0 retention time(min)
i
/
150-
i
5 I0 retention time (min)
A
120
/ "
.
oo
90.
~
60,
o
o
/ /
30
5'
~'o
retention time (;.in)
Fig. i. Typical H P L - G P C chromatograms. A: Separation of diluted human CSF. B: Pattern produced by a diluted FITCconjugated anti-human IgO Fab. C: Chromatograrn produced by A + B. Chromatography performed on a 250×7.9 m m Shim-pack DIOL-300 column at the flow rate of 1.2 m l / m i n using PBS. UV detector: 280 n m (0.04 AUFS). Fluorescence detector: Ex = 490 nm, Em = 520 nm. Injection volume: 10/~1.
0
/
/ °
/ 7.0
14.0 IgG concentration
21.0
28.0
(mg/dl)
Fig. 2. Standard curve for IgG concentrations over the range 3.5-24.5 m g / d l . 20 ~1 of each diluted IgG standard were added to 20 /~1 of FITC-conjugated anti-IgG Fab and the fluorescence peak area was determined. Each data point indicates a single measured value.
36
33
3O 27
24
? 12
9 6 3
3
6
9
12
15
18
Nephelometric
21 method
24
27
30
33
36
(mg/dl)
Fig. 3. Correlation between CSF IgG measured by laser nephelometric method and the HPL-GPC method. The regression equation and correlation coefficient were y = 0:966x -0.228 andr = 0.997, respectively.
Accuracy of HPL-GPC determination of lgG The I g G levels of CSF samples measured by the H P L - G P C method described here were compared with those obtained by nephelometry. As shown in Fig. 3 the values obtained by the two methods were comparable. G o o d correlation was observed with the nephelometric method ( r = 0.997, n = 40). The recovery of a known amount of purified I g G added to CSF was over 99.6%
Reproducibility test The concentrations of I g G in CSF samples were analyzed up to ten times to determine the reproducibility of the assay system. The H P L - G P C method was highly reproducible and accurate with intra-assay coefficients of variation of 1.343, 0.984 and 0.636% at 4.0, 10.0 and 16.0 m g / d l , respectively.
Discussion We have previously reported that H P L - G P C provides a rapid method for analyzing IgG, and in
this report conclude that H P L - G P C can offer a much more rapid method for analyzing I g G in CSF than nephelometry. Determinations requiring 30 min by nephelometry can be obtained in 10 min by H P L - G P C . Because the separations achieved on the gel permeation column are highly reproducible, the methodology has potential as a reference procedure for characterizing IgG. In this present report, the use of H P L - G P C for quantitative I g G analysis has been examined. Estimates of I g G concentrations in CSF, obtained by H P L - G P C were in agreement with those obtained by nephelometry. The level of accuracy was acceptable, and the effect of other CSF components was insignificant. While the correlation between nephelometry and H P L - G P C for the measurement of I g G in CSF was good ( r = 0.997), this does not imply that all CSF samples can be examined by this method using a single standard curve. It should be emphasized that the present method: (i) measures the concentration of available I g G specifically, (ii) is as accurate as the standard immunological assays in most clinical laboratories, (iii) is faster and yields result on a 5/~1 sample of CSF and (iv) is an inexpensive improvement for diagnostic work, if H P L C equipment is already available.
References Felgenhauer, K. (1974) Klin. Wochenschr. 52, 1158. Hosotsubo, H., Arai, K. and Iwamura, J.-I. (1985) J. Immunol. Methods 85, 115. Mancini, G., Carbonara, A.O. and Heremans, F.F. (1965) Immunochemistry2, 235. Phillips, T.M., More, N.S., Queen, W.D., Holohan, T.V., Kramer, N.C. and Thompson, A.M. (1984) J. Chromatogr. 317, 173. Satan, G., Schneider, G., Loeke, S. and Doerr, H.W. (1983) J. Immunol. Methods 5, 121. Schliep, G. and Felgenhaner, K. (1978) J. Clin. Chem. Clin. Biochem. 16, 631. Steruberg, J.C. (1977) Clin. Chem. 23, 1456. Tibbling, G., Link, H. and Ohman, S. (1977) Scand. J. Ciin. Lab. Invest. 37, 385. Tourtenotte, W.W., Tovolato, B., Parker, J.A. and Comiso, P. (1971) Arch. Neurol. 25, 345.