Scand. J. elin. Lab. Invest. 39, 685-688, 1979.
A rapid polyethylene glycol assay for gastric intrinsic factor
Scand J Clin Lab Invest Downloaded from informahealthcare.com by Cornell University on 12/29/14 For personal use only.
KNUT-JAN ANDERSEN Section for Clinical Research and Molecular Medicine, University of Bergen, Medical Department A, 5016 Haukeland Sykehus, Bergen, Norway
Andersen, K.-J. A rapid polyethylene glycol assay for gastric intrinsic factor. Scand. J. din. Lab. Invest. 39, 685-688, 1979. The use of polyethylene glycol (mol. wt 4000) in a rapid radioassay for intrinsic factor is reported. The assay is based on the observation that polyethylene glycol precipitates the intrinsic factor-vitamin BIZ complex in the presence of serum from patients with pernicious anaemia having auto-antibodies against intrinsic factor. The assay was found highly reproducible (CV 1.8”/,) and well correlated to the classical method using coated charcoal in the separation step.
Key-words: auto-antibodies; gastric juice; y-globulin; pernicious anaemia; radioassay; vitamin Blz Knut-Jan Andersen, Ph. D., Section for Clinical Research and Molecular Medicine, University of Bergen, Medical Departrrient A, 5016 Haukeland Sykehus, Bergen, Norway
The findings by Taylor [18] and Schwartz [15, 161 that the sera from pernicious anaemia (PA) patients inhibited the gastrointestinal absorption of vitamin B12 (Biz) led to the dernonstration of two types of intrinsic factor (IF) antibodies [12]. These are the ‘blocking’ antibody (AbI), which prevents the formation of the intrinsic factor-vitamin B,z complex (IF-BI2), and the ‘precipitating’ or ‘binding’ antibody (AbII), which combines with the IF-B12 complex [13]. These antibodies have made possible the development of a vast number of radioimmunoassays (RIA) for IF [7] based on the principle that the difference in B12 binding to gastric juice with and without AbI, or the competition between I F and IF-B,, for the 0036-5513/79/ 1 l00-0685$02.00 0 1979 Medisinsk Fysiologisk Forenings Forlag
binding sites of AbII, is a measure of the IF content of the sample. The separation step is the main difference in these methods, of which the coated charcoal assay described by Gottlieb ef at. [8] is the most widely used. Polson et al. [lo] and Iverius & Laurent [9], have reported that high molecular weight linear polymers like polyethylene glycol (PEG) caused a nearly quantitative precipitation of y-globulin. This precipitation method has later been successfully used to separate free and bound hormones in RIA’S [4, 61. Furthermore, PEG was found not to disturb the equilibrium of the ligand-antibody interaction [ 5 ] . The use of PEG in precipitation of the intrinsic factor antibody complex has therefore been studied, and a rapid assay for the direct determination of intrinsic factor is reported. 685
686
G u t - J a n Andersen
MATERIAL A N D METHODS
Scand J Clin Lab Invest Downloaded from informahealthcare.com by Cornell University on 12/29/14 For personal use only.
Materials ["Co]Vitamin B12 (cyanocobalamin; 7.5 Ci/g) was obtained from The Radiochemical Centre, Amersham, U.K. ; Polyethylene glycol (PEG, Carbowax 4000) was obtained from Fluka AG, Buchs., Switzerland; Charcoal Carbo Medicinalis from Norit, N.V., Amsterdam, The Netherlands; and Sephadex G-100 from Pharmacia Fine Chemicals AB, Uppsala, Sweden. Human gastric juice was obtained from patients with peptic ulcer, and processed as described elsewhere [3], while saliva was collected as described by Simons [17]. Serum was obtained from normal adults fasted overnight, and from patients with pernicious anaemia (PA) with antibody to IF. Methods
Charcoul assay. The coated charcoal assay described by Gottlieb et ul. [8] was performed as described elsewhere [3] using serum coated charcoal. Polyethylene glycol (PEG) assays. The PEGassay for IF was performed in small conical polycarbonate tubes as summarised in Table 1, unless otherwise indicated. The effect of p H on the assay was studied using a series of barbital buffers (0.05 molil of sodium barbital) in the pH range 7.6-8.6. The effect of time and TABLE I . Assay protocol for the determination of intrinsic factor using polyethylene glycol Intrinsic factor assay Step of additions and incubations I 2 3 4 5 6
7 8
Blank
[57Co]Vitaniin B,, (PI; 100 /@/I) 30 100 Buffer ( ~ 1 ) Gastric juice (PI) 20 Incubation at 25°C (min) 10 Incubation at 4°C (niin) 10 PA-serum (PI) Normal serum (pi) 50 Incubation at 4°C (niin) 30 Cold PEG (PI; 300 g/l) 200 Precipitate collected by centrifugation (30,000 g . min, 25°C) Radioactivity determined in 100 141 of supernatant
Sample
temperature o n the binding of IF-B,, to antibody was studied at 4,25 and 37°C. Incubations (step 5 , Table I) were performed for 5-120 min. The effect of the amount of antiserum in the PEG assay for IF was studied using the following modification of Table 1: step 4, NaCl (0.15 mol/l; 100--30 pl), PA serum (0-70 PI); step 6, PEG (250 pl). AiralyriJ of PEG-precipitate. To verify that PEG (final concentration 150 g/l) completely separates bound and free B12, the following was done. Volumes five times the standard assay for I F (Table 1) were used. Precipitate remaining after decantation was dissolved in 2 ml of Krebs Ringer Tris (KRT) buffer, pH 7.4, and applied on a Sephadex G-100 column (2.6 x 70 cm) previously equilibrated with KRT. The flow rate was 18 ml/h and 4.5 ml fractions were collected. Radioactivity containing fractions were assayed for I F activity [ 141 using purified guinea-pig intestinal brush borders [2]. Culciilatioris. Using the definition of one IFunit being thespecific IF-binding of 1 ngvitamin B L 2[ll], the concentration of IF can be calculated as follows: IF concentration (unitsil) = (cpm blank -cpm sample). (final assay volume)/(cpm/ng[57Col B I 2 ) . (volume counted). (1 gastric juice in assay) = (cpm blank -cpm sample). 2 . 105/(cpm/ng [57ColB~2). where the simplified formula is the one arrived a t using the volumes given in Table I. Results obtained with the PEG and the charcoal assay were compared by the student t test for paired observations. The slope was determined by a linear regression model. RESULTS A N D DISCUSSION
30 100
20 10 10
50 ~
30 200
The buffer hydrogen concentration (pH 7.6-8.6 did not have any marked effect o n the PEG precipitation of the IF-BIZ-antibody complex (data not shown). However, less seemed to be precipitated a t increasing pH, which is in agreement with previous reports demonstrating a decrease in the precipitation of serum proteins by PEG above pH 8 [5,101. A p H of 7.8 was therefore chosen as optimal and used in the assay.
Scand J Clin Lab Invest Downloaded from informahealthcare.com by Cornell University on 12/29/14 For personal use only.
Determination of intrinsic factor The temperature as well as the incubation time were found to have a marked effect on the subsequent PEG precipitation of the IF-B,,antibody coinplex (Fig. 1). The amount precipitated was found to decrease when the incubation time and temperature were increased. However, the effect at 4'C was negligible (decreased about 3% after 120 min). A 30 min incubation at 4°C was therefore used for the formation of the IF-B, ,-antibody complex, to reduce a possible inhibitory effect of the gastric juice; an effect that might be due to some residual peptic activity o n the antibody. The effect of the amount of antiserum i n the assay was also tested using increasing volumes of PA-serum (Fig. 2 ) . Moderately increased serum concentrations gave increased IF concentrations, demonstrating that an actual complex
20
40
60
80
100
687
had been formed between IF and the antibody. A 'plateau' concentration of PA-serum was used in the assay to ensure complete precipitation of IF-BI2. The contribution of serum bound [ 5 7 C ~ ] B 1in2 the assay was negligible since no further increase in apparent IF concentration was demonstrated above the 'saturation level' (Fig. 2). Only two out of five PA-sera, all demonstrated by the charcoal assay to have high concentrations of Abl, were found to promote the precipitation of IF-B,r. This implies that the actual complex being precipitated by PEG is IF-B,,-AbII. The assay may therefore also be used as a n assay for AbII, since a linear relationship between small amounts of PA-serum and the amount of IF-R, I precipitated was observed (Fig. 2).
120
Time ( m i n )
FIG. I . Time and temperature effect on the PEG assay for intrinsic factor. Incubations were done at 4°C -c'), 25'C (,'>-A), and 37 'C (7 --Y).
( 8 ;
Charcoal m e t h o d Intrinsic factor concentration i u n i t s / ~ ) x 1 0 - ~
~
f
0
-
FIG.3 . Comparison of intrinsic factor (IF) concentrations determined by the charcoal method and the PEG method. IF was determined i i i twelve preparations of human gastric juice and two preparations of human saliva.
-
J
I
Antiserum
in
assay ( p L )
FIG. 2 . Effect of antiserum concentration in the PEG assay for intrinsic factor.
To verify the complete separation of IF-B,, from B l z and B 1 , complexed to other binders in gastric juice, the complex precipitated by PEG was redissolved and subjected to gelfiltration on Sephadex G-100. More than 90"L of the radioactivity was recovered from the column in the void volume, no radioactivity was eluted at volumes corresponding to the non-IF binder o r IF-BI2, and only 3 % was eluted as free B1,. The radioactivity containing fractions were further assayed for possible IF-B, binding to intestinal brush borders, but no such uptake was shown, which clearly
Scand J Clin Lab Invest Downloaded from informahealthcare.com by Cornell University on 12/29/14 For personal use only.
688
Knut-Jan Andersen
demonstrated that all IF-BI, precipitated was antibody bound. The I F concentration was determined in twelve different preparations of human gastric juice and two preparations of human saliva (containing B I 2binder different from IF) to compare (Fig. 3) the PEG-assay (one step method) with the charcoal assay (two step method). The two methods were found t o be highly correlated (correlation coefficient = 0.991), and the salivary B I Zbinder was not detected in the PEG-assay. The reproducibility of the assay was also tested by doing ten simultaneous determinations of IF, and the coefficient of variance was found to be 1.8. The radioassay described here differs significantly from those previously published [7] in the separation procedure as well as sensitivity. Most methods published so far for the determination of IF require at least 200 rtl of gastric juice and 500 pI of PA-serum. The PEG-assay reported here requires only one tenth of these volumes which make the assay of a greater number of IF-preparations possible using the same batch of PA-serum. Furthermore, being a n immunoassay, the PEG-assay may be used not only for the determination of IF, but also for the detection of possible alterations in the immunological properties of IF after exposure to various proteolytic enzymes and subcellular fractions
111. ACKNOWLEDGMENTS This work has been supported by research grants from The Norwegian Research Council for Sciences and the Humanities to whom I a m sincerely grateful. The skillful technical assistance of Ms A.S. Eriksen and Ms. K . Thunold is also acknowledged. REFERENCES
& Schjmsby, H. Purification and characterization of guinea-pig intestinal brush borders. Eiochrr77. J . 152, 157, 1975. 3 Andersen, K . - J . . von der Lippe, G . & Schjunsby, H. Bile and detergent interaction with the radioassay for vitamin B,, hinders using protein and dextrancovered charcoal. Ann. Biochem. 74, 488, 1976. 4 Barrett, M.J. & Cohen, P.S. Radioimmunoassay of serum renin activity and digoxin concentrations with use of polyethykne glycol to separate free and antibody-bound ligand. Clin. Chem. 18, 1339, 1972. 5 Cheung, M.C. & Slaunwhite, W.R., Jr Use of polyethylene glycol in separating bound from unbound ligand in radioimmunoassay of thyroxine. Clin. Chem. 22, 299, 1976. 6 Desbuquois, B. & Aurhach, G . D . Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays. J . elin. Endocr. M e t a b . 33, 732, 1971. 7 Glass, G.B.J. Gastric Intrinsic ractor rind Other Vitamin E l Binders: Biot,hrmistry, Phvsinlogy and Relation to Vitamin B I M(,taholisnr. Georg Thiemc Publishers, Stuttgart, 1974. 8 Gottlieb, C., Lau, K.-S., Wassemian, L.R. & Herbert, V. Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated B,, binding capacity, antibody to IF, and serum unsaturated B I Z binding capacity, Blood, 25, 875, 1965. 9 Iverius, P.H. & Laurent, T.C. Precipitation o r some plasma proteins by the addition of dextran or polyethylene glycol. Biochim. bitiphys. Actn 133, 371. 1967. 10 Polson, A., Potgieter, G.M., Largier, J.F., Mears G.E.F. & Jourbert, F.J. The fractionation of protein mixtures by linear polymers of high molecular weight. Eiochim. hiophgs. Acto 82, 463. 1964. 1 1 Rudbro, P., Christiansen. P.M. & Schwarlr, M . Intrinsic factor secretion in stomach diseases. Lancet ii, 1200, 1965. 12 Roitt, I.M., Doniach, D . .& Shapland, C. Intrinsic factor autoantibodies. Lancet ii, 469, 1964. 13 Schade, S.G., Feick, P.L.,Imrie, M.H. & Schilling R.F. I n i\itro studies on antibodies to intrinsic factor Clin. expl. Irnmun. 2, 399, 1967. 14 Schjenshy, H. & Peters, T.J. The estimation of intrinsic factor using guinea-pig intestinal brush borders. Scand. J . Gastroenterol. 6, 441, 1971. 15 Schwartz, M. Intrinsic factor-inhibiting substance in serum of orally treated patients with pernicious anaemia. Lancet i, 61, 1958. 16 Schwartz, M. Intrinsic factor antibody in serum from patients with pernicious anaemia. Lancet ii, 1263, 1960. 17 Simons, K. Vitamin B I Z binders in human body fluids and blood cells. Sociefy Scientarum Fennica, Comment. Eiol. 27, Fasc. 5, 1964. 18 Taylor, K.B. Inhibition of intrinsic factor by pernicious anaemia sera. Lancer ii, 106, 1959.
,
1 Andersen, K.-J. & von der Lippe. G. The effect of
proteolytic enzymes on the vitamin BI1-binding proteins of human gastric juice and saliva. Scand. J . Ga.rtroenterol. 14, 1979 (In press). 2 Andersen, K.-J., von der Lippe, G., Merkrid, L.
Received 22 March 1979 Accepted 1 J u n e 1979
A rapid polyethylene glycol assay for gastric intrinsic factor
Scand J Clin Lab Invest Downloaded from informahealthcare.com by Cornell University on 12/29/14 For personal use only.
KNUT-JAN ANDERSEN Section for Clinical Research and Molecular Medicine, University of Bergen, Medical Department A, 5016 Haukeland Sykehus, Bergen, Norway
Andersen, K.-J. A rapid polyethylene glycol assay for gastric intrinsic factor. Scand. J. din. Lab. Invest. 39, 685-688, 1979. The use of polyethylene glycol (mol. wt 4000) in a rapid radioassay for intrinsic factor is reported. The assay is based on the observation that polyethylene glycol precipitates the intrinsic factor-vitamin BIZ complex in the presence of serum from patients with pernicious anaemia having auto-antibodies against intrinsic factor. The assay was found highly reproducible (CV 1.8”/,) and well correlated to the classical method using coated charcoal in the separation step.
Key-words: auto-antibodies; gastric juice; y-globulin; pernicious anaemia; radioassay; vitamin Blz Knut-Jan Andersen, Ph. D., Section for Clinical Research and Molecular Medicine, University of Bergen, Medical Departrrient A, 5016 Haukeland Sykehus, Bergen, Norway
The findings by Taylor [18] and Schwartz [15, 161 that the sera from pernicious anaemia (PA) patients inhibited the gastrointestinal absorption of vitamin B12 (Biz) led to the dernonstration of two types of intrinsic factor (IF) antibodies [12]. These are the ‘blocking’ antibody (AbI), which prevents the formation of the intrinsic factor-vitamin B,z complex (IF-BI2), and the ‘precipitating’ or ‘binding’ antibody (AbII), which combines with the IF-B12 complex [13]. These antibodies have made possible the development of a vast number of radioimmunoassays (RIA) for IF [7] based on the principle that the difference in B12 binding to gastric juice with and without AbI, or the competition between I F and IF-B,, for the 0036-5513/79/ 1 l00-0685$02.00 0 1979 Medisinsk Fysiologisk Forenings Forlag
binding sites of AbII, is a measure of the IF content of the sample. The separation step is the main difference in these methods, of which the coated charcoal assay described by Gottlieb ef at. [8] is the most widely used. Polson et al. [lo] and Iverius & Laurent [9], have reported that high molecular weight linear polymers like polyethylene glycol (PEG) caused a nearly quantitative precipitation of y-globulin. This precipitation method has later been successfully used to separate free and bound hormones in RIA’S [4, 61. Furthermore, PEG was found not to disturb the equilibrium of the ligand-antibody interaction [ 5 ] . The use of PEG in precipitation of the intrinsic factor antibody complex has therefore been studied, and a rapid assay for the direct determination of intrinsic factor is reported. 685
686
G u t - J a n Andersen
MATERIAL A N D METHODS
Scand J Clin Lab Invest Downloaded from informahealthcare.com by Cornell University on 12/29/14 For personal use only.
Materials ["Co]Vitamin B12 (cyanocobalamin; 7.5 Ci/g) was obtained from The Radiochemical Centre, Amersham, U.K. ; Polyethylene glycol (PEG, Carbowax 4000) was obtained from Fluka AG, Buchs., Switzerland; Charcoal Carbo Medicinalis from Norit, N.V., Amsterdam, The Netherlands; and Sephadex G-100 from Pharmacia Fine Chemicals AB, Uppsala, Sweden. Human gastric juice was obtained from patients with peptic ulcer, and processed as described elsewhere [3], while saliva was collected as described by Simons [17]. Serum was obtained from normal adults fasted overnight, and from patients with pernicious anaemia (PA) with antibody to IF. Methods
Charcoul assay. The coated charcoal assay described by Gottlieb et ul. [8] was performed as described elsewhere [3] using serum coated charcoal. Polyethylene glycol (PEG) assays. The PEGassay for IF was performed in small conical polycarbonate tubes as summarised in Table 1, unless otherwise indicated. The effect of p H on the assay was studied using a series of barbital buffers (0.05 molil of sodium barbital) in the pH range 7.6-8.6. The effect of time and TABLE I . Assay protocol for the determination of intrinsic factor using polyethylene glycol Intrinsic factor assay Step of additions and incubations I 2 3 4 5 6
7 8
Blank
[57Co]Vitaniin B,, (PI; 100 /@/I) 30 100 Buffer ( ~ 1 ) Gastric juice (PI) 20 Incubation at 25°C (min) 10 Incubation at 4°C (niin) 10 PA-serum (PI) Normal serum (pi) 50 Incubation at 4°C (niin) 30 Cold PEG (PI; 300 g/l) 200 Precipitate collected by centrifugation (30,000 g . min, 25°C) Radioactivity determined in 100 141 of supernatant
Sample
temperature o n the binding of IF-B,, to antibody was studied at 4,25 and 37°C. Incubations (step 5 , Table I) were performed for 5-120 min. The effect of the amount of antiserum in the PEG assay for IF was studied using the following modification of Table 1: step 4, NaCl (0.15 mol/l; 100--30 pl), PA serum (0-70 PI); step 6, PEG (250 pl). AiralyriJ of PEG-precipitate. To verify that PEG (final concentration 150 g/l) completely separates bound and free B12, the following was done. Volumes five times the standard assay for I F (Table 1) were used. Precipitate remaining after decantation was dissolved in 2 ml of Krebs Ringer Tris (KRT) buffer, pH 7.4, and applied on a Sephadex G-100 column (2.6 x 70 cm) previously equilibrated with KRT. The flow rate was 18 ml/h and 4.5 ml fractions were collected. Radioactivity containing fractions were assayed for I F activity [ 141 using purified guinea-pig intestinal brush borders [2]. Culciilatioris. Using the definition of one IFunit being thespecific IF-binding of 1 ngvitamin B L 2[ll], the concentration of IF can be calculated as follows: IF concentration (unitsil) = (cpm blank -cpm sample). (final assay volume)/(cpm/ng[57Col B I 2 ) . (volume counted). (1 gastric juice in assay) = (cpm blank -cpm sample). 2 . 105/(cpm/ng [57ColB~2). where the simplified formula is the one arrived a t using the volumes given in Table I. Results obtained with the PEG and the charcoal assay were compared by the student t test for paired observations. The slope was determined by a linear regression model. RESULTS A N D DISCUSSION
30 100
20 10 10
50 ~
30 200
The buffer hydrogen concentration (pH 7.6-8.6 did not have any marked effect o n the PEG precipitation of the IF-BIZ-antibody complex (data not shown). However, less seemed to be precipitated a t increasing pH, which is in agreement with previous reports demonstrating a decrease in the precipitation of serum proteins by PEG above pH 8 [5,101. A p H of 7.8 was therefore chosen as optimal and used in the assay.
Scand J Clin Lab Invest Downloaded from informahealthcare.com by Cornell University on 12/29/14 For personal use only.
Determination of intrinsic factor The temperature as well as the incubation time were found to have a marked effect on the subsequent PEG precipitation of the IF-B,,antibody coinplex (Fig. 1). The amount precipitated was found to decrease when the incubation time and temperature were increased. However, the effect at 4'C was negligible (decreased about 3% after 120 min). A 30 min incubation at 4°C was therefore used for the formation of the IF-B, ,-antibody complex, to reduce a possible inhibitory effect of the gastric juice; an effect that might be due to some residual peptic activity o n the antibody. The effect of the amount of antiserum i n the assay was also tested using increasing volumes of PA-serum (Fig. 2 ) . Moderately increased serum concentrations gave increased IF concentrations, demonstrating that an actual complex
20
40
60
80
100
687
had been formed between IF and the antibody. A 'plateau' concentration of PA-serum was used in the assay to ensure complete precipitation of IF-BI2. The contribution of serum bound [ 5 7 C ~ ] B 1in2 the assay was negligible since no further increase in apparent IF concentration was demonstrated above the 'saturation level' (Fig. 2). Only two out of five PA-sera, all demonstrated by the charcoal assay to have high concentrations of Abl, were found to promote the precipitation of IF-B,r. This implies that the actual complex being precipitated by PEG is IF-B,,-AbII. The assay may therefore also be used as a n assay for AbII, since a linear relationship between small amounts of PA-serum and the amount of IF-R, I precipitated was observed (Fig. 2).
120
Time ( m i n )
FIG. I . Time and temperature effect on the PEG assay for intrinsic factor. Incubations were done at 4°C -c'), 25'C (,'>-A), and 37 'C (7 --Y).
( 8 ;
Charcoal m e t h o d Intrinsic factor concentration i u n i t s / ~ ) x 1 0 - ~
~
f
0
-
FIG.3 . Comparison of intrinsic factor (IF) concentrations determined by the charcoal method and the PEG method. IF was determined i i i twelve preparations of human gastric juice and two preparations of human saliva.
-
J
I
Antiserum
in
assay ( p L )
FIG. 2 . Effect of antiserum concentration in the PEG assay for intrinsic factor.
To verify the complete separation of IF-B,, from B l z and B 1 , complexed to other binders in gastric juice, the complex precipitated by PEG was redissolved and subjected to gelfiltration on Sephadex G-100. More than 90"L of the radioactivity was recovered from the column in the void volume, no radioactivity was eluted at volumes corresponding to the non-IF binder o r IF-BI2, and only 3 % was eluted as free B1,. The radioactivity containing fractions were further assayed for possible IF-B, binding to intestinal brush borders, but no such uptake was shown, which clearly
Scand J Clin Lab Invest Downloaded from informahealthcare.com by Cornell University on 12/29/14 For personal use only.
688
Knut-Jan Andersen
demonstrated that all IF-BI, precipitated was antibody bound. The I F concentration was determined in twelve different preparations of human gastric juice and two preparations of human saliva (containing B I 2binder different from IF) to compare (Fig. 3) the PEG-assay (one step method) with the charcoal assay (two step method). The two methods were found t o be highly correlated (correlation coefficient = 0.991), and the salivary B I Zbinder was not detected in the PEG-assay. The reproducibility of the assay was also tested by doing ten simultaneous determinations of IF, and the coefficient of variance was found to be 1.8. The radioassay described here differs significantly from those previously published [7] in the separation procedure as well as sensitivity. Most methods published so far for the determination of IF require at least 200 rtl of gastric juice and 500 pI of PA-serum. The PEG-assay reported here requires only one tenth of these volumes which make the assay of a greater number of IF-preparations possible using the same batch of PA-serum. Furthermore, being a n immunoassay, the PEG-assay may be used not only for the determination of IF, but also for the detection of possible alterations in the immunological properties of IF after exposure to various proteolytic enzymes and subcellular fractions
111. ACKNOWLEDGMENTS This work has been supported by research grants from The Norwegian Research Council for Sciences and the Humanities to whom I a m sincerely grateful. The skillful technical assistance of Ms A.S. Eriksen and Ms. K . Thunold is also acknowledged. REFERENCES
& Schjmsby, H. Purification and characterization of guinea-pig intestinal brush borders. Eiochrr77. J . 152, 157, 1975. 3 Andersen, K . - J . . von der Lippe, G . & Schjunsby, H. Bile and detergent interaction with the radioassay for vitamin B,, hinders using protein and dextrancovered charcoal. Ann. Biochem. 74, 488, 1976. 4 Barrett, M.J. & Cohen, P.S. Radioimmunoassay of serum renin activity and digoxin concentrations with use of polyethykne glycol to separate free and antibody-bound ligand. Clin. Chem. 18, 1339, 1972. 5 Cheung, M.C. & Slaunwhite, W.R., Jr Use of polyethylene glycol in separating bound from unbound ligand in radioimmunoassay of thyroxine. Clin. Chem. 22, 299, 1976. 6 Desbuquois, B. & Aurhach, G . D . Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays. J . elin. Endocr. M e t a b . 33, 732, 1971. 7 Glass, G.B.J. Gastric Intrinsic ractor rind Other Vitamin E l Binders: Biot,hrmistry, Phvsinlogy and Relation to Vitamin B I M(,taholisnr. Georg Thiemc Publishers, Stuttgart, 1974. 8 Gottlieb, C., Lau, K.-S., Wassemian, L.R. & Herbert, V. Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated B,, binding capacity, antibody to IF, and serum unsaturated B I Z binding capacity, Blood, 25, 875, 1965. 9 Iverius, P.H. & Laurent, T.C. Precipitation o r some plasma proteins by the addition of dextran or polyethylene glycol. Biochim. bitiphys. Actn 133, 371. 1967. 10 Polson, A., Potgieter, G.M., Largier, J.F., Mears G.E.F. & Jourbert, F.J. The fractionation of protein mixtures by linear polymers of high molecular weight. Eiochim. hiophgs. Acto 82, 463. 1964. 1 1 Rudbro, P., Christiansen. P.M. & Schwarlr, M . Intrinsic factor secretion in stomach diseases. Lancet ii, 1200, 1965. 12 Roitt, I.M., Doniach, D . .& Shapland, C. Intrinsic factor autoantibodies. Lancet ii, 469, 1964. 13 Schade, S.G., Feick, P.L.,Imrie, M.H. & Schilling R.F. I n i\itro studies on antibodies to intrinsic factor Clin. expl. Irnmun. 2, 399, 1967. 14 Schjenshy, H. & Peters, T.J. The estimation of intrinsic factor using guinea-pig intestinal brush borders. Scand. J . Gastroenterol. 6, 441, 1971. 15 Schwartz, M. Intrinsic factor-inhibiting substance in serum of orally treated patients with pernicious anaemia. Lancet i, 61, 1958. 16 Schwartz, M. Intrinsic factor antibody in serum from patients with pernicious anaemia. Lancet ii, 1263, 1960. 17 Simons, K. Vitamin B I Z binders in human body fluids and blood cells. Sociefy Scientarum Fennica, Comment. Eiol. 27, Fasc. 5, 1964. 18 Taylor, K.B. Inhibition of intrinsic factor by pernicious anaemia sera. Lancer ii, 106, 1959.
,
1 Andersen, K.-J. & von der Lippe. G. The effect of
proteolytic enzymes on the vitamin BI1-binding proteins of human gastric juice and saliva. Scand. J . Ga.rtroenterol. 14, 1979 (In press). 2 Andersen, K.-J., von der Lippe, G., Merkrid, L.
Received 22 March 1979 Accepted 1 J u n e 1979
