277
Clinica Chimica Acta, 98 (1979) 277-279 0 Elsevier/North-Holland Biomedical Press
BRIEF TECHNICAL NOTE CCA
1132
RAPID MEASUREMENT OF TOTAL PROTEIN, AT,BUMIN AND IgG IN CEREBROSPINAL FLUID
ANDREW
Department (Received
MUIR
* and WILLIAM
of Biochemistry, April
16th,
J. HENSLEY
Royal Prince Alfred Hospital, Sydney,
N.S. W. 2050 (Australi6)
1979)
Introduction The more generally accepted methods for quantitative measurement of albumin and IgG in cerebrospinal fluid, are electroimmunodiffusion (E.I.D.) and radial immunodiffusion (R.I.D.) both of which are relatively labour intensive. The method proposed here consists of measurement of total protein by a method previously described by us f 11 for the measurement of total protein in urine by a trichloracetic acid turbidometric technique; albumin and IgG are measured by an immunological turbidometric technique based on work previously described by Blom and Hjorne [ 2,3] and by Finley et al. [4]. Material and methods Instrumentation The instrument used was a Centrifichem series 300 centrifugal analyser {Union Carbide, Tarrytown, NY). Me~uremen~ were carried out at 340 nm and 37°C. Total protein The method has been previously described [ 11. Measurement of IgG and albumin All antisera and protein standards used were from Hoechst Australia (Behring). Suitable dilution of antisera for both IgG and albumin was found to be 1 in 10 in polyethylene glycol reagent (P.E.G.).
* To whom correspondence
should be addressed.
278
Cerebrospinal
fluid samples
For albumin determination, CSF was diluted 1 in 50 with sodium chloride solution (9 g/l). For IgG determination, 50 ~1 of undiluted CSF were used. Polyethylene glycol reagent 21 g sodium fluoride (0.5 mol/l);
40 g polyethylene glycol (mol. wt. 6000) made up to 1 1 with 0.5 mol/l sodium chloride solution. The P.E.G. was filtered through a 0.22 micron “Millex” (Millipore) membrane before use. Loading of transfer discs Total protein
For this method [l], 10 ~1 of sample and 55 ~1 of diluent, to which 250 ~1 of trichloracetic acid reagent (50 g/l) were added, was dispensed using the standard Centrifichem pipettor. Albumin
All samples with total protein values less than 1 g/l were diluted 1 in 50 with sodium chloride solution (9 g/l), and samples having total protein values greater than 1 g/l were diluted 1 in 100. 100 ~1 of the diluted samples were placed in the transfer discs using Lancer pipettors (Sherwood Medical Industries, St. Louis, MO). 200 ,ul of diluted antisera/P.E.G. reagent were placed in the reagent wells using Lancer pipettors.
50 ~1 of undiluted CSF were used for the estimation of IgG levels. Transfer discs were loaded as for the albumin analysis. Reaction was measured at 37°C the absorbance being measured after 4 min at 340 nm. Under these conditions the absorbance measured at 4 min is proportional to protein concentration. Antigen
excess
Under conditions of antigen excess (antigen concentration being in excess of the amount of antibody available) absorbance is not proportional to concentration, and the protein concentration of the sample must be diluted further. Conditions of antigen excess can readily be recognised if the absorbance readings are measured at 0.5 min intervals for total protein estimation and at 1 min intervals for IgG and albumin estimations, where antigen excess is shown by an immediately high absorbance followed by a rapid decrease in absorbance with time. Results Precision
The results of precision Correlation
studies for albumin and IgG are shown in Table I.
studies
Correlation studies were carried out on 60 samples of CSF with IgG values ranging from 11 to 118 mg/l, and albumin values ranging from 100 to 840 mg/l.
279
TABLE
I
Albumin
I@
Within
Within
batch
n =
25
mean
n =
=
157
mg/1
mean
batch
n =
25 =
572
12
mean
w&/l
n =
=
36
mg/l
12
mean
=
91
S.D.
=
4.08
S.D.
=
3.69
S.D.
=
0.63
S.D.
=
4.86
C.V.
=
2.6%
C.V.
=
0.65%
C.V.
=
1.74%
C.V.
=
5.33%
n =
36
Batch
to
IZ =
batch
(12
on
3 consecutive
36
mean
n = =
158
mg/l
Batch
days)
mean
to
n =
36 =
591
batch
36
mea*
mgjl
mg/l
=
37
mg/l
mean
=
93
S.D.
=
5.16
S.D.
=
18.7
S.D.
=
0.93
S.D.
=
5.03
C.V.
=
3.3%
C.V.
=
3.2%
C.V.
=
2.5%
C.V.
=
5.4%
mg/l
Comparison of the results obtained by the Centrifichem and radial immunodiffusion using Partigen plates (Hoechst, Australia) gave a correlation coefficient of 0.93 for albumin and 0.97 for IgG. Reference ranges The reference ranges obtained from analysis of 60 apparently normal CSF samples, i.e. with normal values for glucose, chloride and with total protein values less than 400 mg/l (range 160 to 400) were 115 to 370 mg/l for albumin (mean 277, SD. 95.5) and 3 to 59 mg/l for IgG (mean 29.1, SD. 11.8). Ganrot and Laurel], using eIectroimmunodiffusion 151, obtained a mean value for albumin of 304 mg/l (S.D. 126) and for IgG of 20.6 mg/l (S.D. 14). The CSF IgG/albumin ratio (expressed as a percentage), in our control group was less than 30%. The method described required 70 ~1 of CSF and has been shown to be as precise as the R.I.D. method. It has the advantage over other methods in that it is less labour intensive, with lower reagent cost and is more rapid than either the R.I.D. or E.I.D. methods. Results for total protein, albumin and IgG may be obtained on 26 CSF sampfes in approximately 45 min. References Muir,
A.
and
Hensley,
Blom,
M. and
Hiorne,
Blow
M. and
Hjorne,
Finley, Ganrot,
P., Williams, K. and
WJ. N.
(1978)
Clin.
N. (1976)
Clin.
J. and
LaurelI,
CIin.
(1975) Byers,
C. (1974)
M.
Chim.
Chem. Chem. (1976)
CIin.
Chem.
Acta
90.
231-239
21.195-198 22.657-662 Clin. 20,
Chem. 571-573
22,1037-1041
Clinica Chimica Acta, 98 (1979) 277-279 0 Elsevier/North-Holland Biomedical Press
BRIEF TECHNICAL NOTE CCA
1132
RAPID MEASUREMENT OF TOTAL PROTEIN, AT,BUMIN AND IgG IN CEREBROSPINAL FLUID
ANDREW
Department (Received
MUIR
* and WILLIAM
of Biochemistry, April
16th,
J. HENSLEY
Royal Prince Alfred Hospital, Sydney,
N.S. W. 2050 (Australi6)
1979)
Introduction The more generally accepted methods for quantitative measurement of albumin and IgG in cerebrospinal fluid, are electroimmunodiffusion (E.I.D.) and radial immunodiffusion (R.I.D.) both of which are relatively labour intensive. The method proposed here consists of measurement of total protein by a method previously described by us f 11 for the measurement of total protein in urine by a trichloracetic acid turbidometric technique; albumin and IgG are measured by an immunological turbidometric technique based on work previously described by Blom and Hjorne [ 2,3] and by Finley et al. [4]. Material and methods Instrumentation The instrument used was a Centrifichem series 300 centrifugal analyser {Union Carbide, Tarrytown, NY). Me~uremen~ were carried out at 340 nm and 37°C. Total protein The method has been previously described [ 11. Measurement of IgG and albumin All antisera and protein standards used were from Hoechst Australia (Behring). Suitable dilution of antisera for both IgG and albumin was found to be 1 in 10 in polyethylene glycol reagent (P.E.G.).
* To whom correspondence
should be addressed.
278
Cerebrospinal
fluid samples
For albumin determination, CSF was diluted 1 in 50 with sodium chloride solution (9 g/l). For IgG determination, 50 ~1 of undiluted CSF were used. Polyethylene glycol reagent 21 g sodium fluoride (0.5 mol/l);
40 g polyethylene glycol (mol. wt. 6000) made up to 1 1 with 0.5 mol/l sodium chloride solution. The P.E.G. was filtered through a 0.22 micron “Millex” (Millipore) membrane before use. Loading of transfer discs Total protein
For this method [l], 10 ~1 of sample and 55 ~1 of diluent, to which 250 ~1 of trichloracetic acid reagent (50 g/l) were added, was dispensed using the standard Centrifichem pipettor. Albumin
All samples with total protein values less than 1 g/l were diluted 1 in 50 with sodium chloride solution (9 g/l), and samples having total protein values greater than 1 g/l were diluted 1 in 100. 100 ~1 of the diluted samples were placed in the transfer discs using Lancer pipettors (Sherwood Medical Industries, St. Louis, MO). 200 ,ul of diluted antisera/P.E.G. reagent were placed in the reagent wells using Lancer pipettors.
50 ~1 of undiluted CSF were used for the estimation of IgG levels. Transfer discs were loaded as for the albumin analysis. Reaction was measured at 37°C the absorbance being measured after 4 min at 340 nm. Under these conditions the absorbance measured at 4 min is proportional to protein concentration. Antigen
excess
Under conditions of antigen excess (antigen concentration being in excess of the amount of antibody available) absorbance is not proportional to concentration, and the protein concentration of the sample must be diluted further. Conditions of antigen excess can readily be recognised if the absorbance readings are measured at 0.5 min intervals for total protein estimation and at 1 min intervals for IgG and albumin estimations, where antigen excess is shown by an immediately high absorbance followed by a rapid decrease in absorbance with time. Results Precision
The results of precision Correlation
studies for albumin and IgG are shown in Table I.
studies
Correlation studies were carried out on 60 samples of CSF with IgG values ranging from 11 to 118 mg/l, and albumin values ranging from 100 to 840 mg/l.
279
TABLE
I
Albumin
I@
Within
Within
batch
n =
25
mean
n =
=
157
mg/1
mean
batch
n =
25 =
572
12
mean
w&/l
n =
=
36
mg/l
12
mean
=
91
S.D.
=
4.08
S.D.
=
3.69
S.D.
=
0.63
S.D.
=
4.86
C.V.
=
2.6%
C.V.
=
0.65%
C.V.
=
1.74%
C.V.
=
5.33%
n =
36
Batch
to
IZ =
batch
(12
on
3 consecutive
36
mean
n = =
158
mg/l
Batch
days)
mean
to
n =
36 =
591
batch
36
mea*
mgjl
mg/l
=
37
mg/l
mean
=
93
S.D.
=
5.16
S.D.
=
18.7
S.D.
=
0.93
S.D.
=
5.03
C.V.
=
3.3%
C.V.
=
3.2%
C.V.
=
2.5%
C.V.
=
5.4%
mg/l
Comparison of the results obtained by the Centrifichem and radial immunodiffusion using Partigen plates (Hoechst, Australia) gave a correlation coefficient of 0.93 for albumin and 0.97 for IgG. Reference ranges The reference ranges obtained from analysis of 60 apparently normal CSF samples, i.e. with normal values for glucose, chloride and with total protein values less than 400 mg/l (range 160 to 400) were 115 to 370 mg/l for albumin (mean 277, SD. 95.5) and 3 to 59 mg/l for IgG (mean 29.1, SD. 11.8). Ganrot and Laurel], using eIectroimmunodiffusion 151, obtained a mean value for albumin of 304 mg/l (S.D. 126) and for IgG of 20.6 mg/l (S.D. 14). The CSF IgG/albumin ratio (expressed as a percentage), in our control group was less than 30%. The method described required 70 ~1 of CSF and has been shown to be as precise as the R.I.D. method. It has the advantage over other methods in that it is less labour intensive, with lower reagent cost and is more rapid than either the R.I.D. or E.I.D. methods. Results for total protein, albumin and IgG may be obtained on 26 CSF sampfes in approximately 45 min. References Muir,
A.
and
Hensley,
Blom,
M. and
Hiorne,
Blow
M. and
Hjorne,
Finley, Ganrot,
P., Williams, K. and
WJ. N.
(1978)
Clin.
N. (1976)
Clin.
J. and
LaurelI,
CIin.
(1975) Byers,
C. (1974)
M.
Chim.
Chem. Chem. (1976)
CIin.
Chem.
Acta
90.
231-239
21.195-198 22.657-662 Clin. 20,
Chem. 571-573
22,1037-1041
