[5] Solid-phase immunoassay for human transcobalamin II and detection of the secretory protein in cultured human cells

36

C O B A L A M I N S A N D C O B A M I D E S (B12)

[5]

Comment Recovery of rabbit serum TC-II is - 90%, and purity, as judged from SDS-polyacrylamide gel electrophoresis j8 (Fig. 4) and spectrophotometric criteria, is usually greater than 80% if the stringent wash protocol is performed. Recovery of human IF recovery is consistently ---95% with a purity of >95%. A similar result has been obtained with R-binder from of human saliva. In addition, near-homogeneous preparations of the following Cbl-binding proteins have been obtained: transcobalamin I (chicken serum); granulocyte Cbl-binder (human neutrophils); IF (rat stomachs); IF and non-IF (hog pylorus); and transcobalamins X, Y, and Z (salmon serum). In the above preparations, the Cbl-binding protein is obtained as a complex with AqCbl. The latter can be removed by dialyzing the complex against guanidinium hydrochloride as described by Allen e t al. 4 or by reacting the complex with sodium ascorbate.J9 The apo-binding proteins can then be treated with [57Co]CN-Cbl to form radiolabeled complexes for binding and transport studies. i8 K. Weber, J. R. Pringle, and M. Osborn, this series, Vol. 26, Part C, p. 3. 19 D. W. Jacobsen, Y. D. Montejano, and L. L. Zak, Blood 58, Suppl. 1, 29a.

[5] S o l i d - P h a s e I m m u n o a s s a y for H u m a n T r a n s c o b a l a m i n II a n d D e t e c t i o n o f t h e S e c r e t o r y P r o t e i n in C u l t u r e d H u m a n Cells B y MARIJKE FRATER-SCHRODER

Vitamin B12 (cobalamin, Cbl) in blood is transported by specific carrier proteins, transcobalamin II (TC2) and R-binder.~ The two forms of transport proteins are immunologically and functionally distinct. 2,3 Plasma TC2 (MW 38,000, not glycosylated) is essential for absorption, transport, and promotion of cellular uptake of Cbl in man.~ In contrast with the welldefined role in Cbl distribution, attributed to TC2, the function of the Rbinder type of Cbl-binding proteins, also named haptocorrin, is not clear? Studies on the origin of TC2 have been performed predominantly in animals. Various organs and tissues, including liver and kidney, release or C. A. Hall, ed., "The Cobalamins, Methods in Hematology," Vol. 10. Churchill-Livingstone, Edinburgh and London, 1983. 2 M. Frb.ter-Schr6der, Mol. Cell. Biochem. 56, 5 (1983). 3 E. Nex¢ and H. Olesen, in "BI2"' (D. Dolphin, ed.), Vol. 2, Chapter 3, p. 57. Wiley, New York, 1982.

METHODS IN ENZYMOLOGY, VOL. 123

Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

[5]

RIA FOR HUMANTRANSCOBALAI~41NII

37

produce TC2. 4 D e nooo synthesis has been confirmed in cell culture, using rat hepatocytes, 4 mouse fibroblasts, 4 and mouse mononuclear phagocytes. 5 The actual sites of TC2 synthesis in man are becoming apparent. In vioo investigations suggested that bone marrow 6 and intestine 7 are possible sources of human TC2. Another report indicated that low levels of intracellular TC2 can occur in human fibroblasts. 8 More recently, these findings were supported by the fact that both cultured skin fibroblasts and cultured bone marrow cells synthesize and secrete substantial quantities of TC2. 9 A series of assays based on immunological principles have been reported for the quantification of human TC2 in serum.I°-16 Total, immunoreactive TC2 was determined using various modifications of the classical RIA procedure. ~°,JL13-~5Attempts to quantitate holo-TC2 (approximately 10% of total), and apo-TC2 (a corresponding 90% of total TC2) in serum selectively have also been described. 12,13,~5,16The method for the quantification of total TC2 in human serum presented here uses immobilized antiserum 15 and is preferred by the author, because it is suitable for routine measurements, allowing 100 samples per day to be processed. It requires only 25/zl serum, employs extremely low levels of a stable radioactive label, and can be applied to the detection of low secretory TC2 levels in the medium of cultured human cellsf1,9 A solid-phase R1A for TC2, based on the principles and on the purification schemes presented below, has recently become commercially available.16a 4 p. D. Green and C. A. Hall, this series, Vol. 67, Part F, p. 89. 5 B. Rachmilewitz, M. Rachmilewitz, M. Chaoat, and M. Schlesinger, Blood 52, 1089 (1978). 6 M. Fr~ter-Schr6der, C. Nissen, J. Gmiir, L. Kierat, and W. H. Hitzig, Blood 56, 560 (1980). 7 I. Chanarin, M. Muir, A. Hughes, and A. V. Hoffbrand, Br. Med. J. 1, 1453 (1978). 8 N. Berliner and L. E. Rosenberg, Metab., Clin. Exp. 30, 230 (1981). M. Fr~ter-SchrOder, H. J. Porck, J. Erten, M. R. Miiller, B. Steinmann, L. Kierat, and F. Arwert, Biochim. Biophys. Acta 845, 421 (1985). 10 R. J. Schneider, R. L. Burger, C. S. Mehlman, and R. H. Allen, J. Clin. Invest. 57, 27 (1976). 11 T. A. Morelli, C. R. Savage, J. A. Begley, and C. A. Hall, J. Lab. Clin. Med. 89, 645 (1977). 12 E. Nex¢ and J. Andersen, Scand. J. Lab. Invest. 37, 723 (1977). ~3 M. Frb.ter-SchrOder, P. Vitins, and W. H. Hitzig, in "Vitamin BI2" (B. Zagalak and W. Friedrich, eds.), p. 877. de Gruyter, Berlin, 1979. ~4p. A. Seligman, L. LaDonna, B. S. Steiner, and R. H. Allen, N. Engl. J. Med. 303, 1209 (1980). ~5M. Fr~ter-Schr6der, L. Kierat, R. Y. Andres, and J. ROmer, Anal. Biochem. 124, 92 (1982). 16 j. Lindemans, M. Schoester, and J. van Kapel, Clin Chirn. Acta 132, 53 (1983). ~6, Eurodiagnostics BV, Apeldoorn, The Netherlands.

38

COBALAMINSAND COBAMIDES(BI2)

[5]

Purification of TC2

Preparation 1:TC2 Concentrate, R-Binder-Free for Tracer Preparation The TC2 concentrate described here can be obtained from the Swiss Red Cross Central Laboratory, Berne, Switzerland. The human plasma fraction NB is the starting material. Fraction NB is similar to Cohn fraction III paste in its protein composition, but it is obtained by a slightly different fractionation technique. 17 An ion-exchange procedure, CMSephadex separation 1~-2°was used to concentrate TC2 and also to remove the last trace of R-binder from NB. A recent modification 2° was optimized. At 4°, 6-8 kg of frozen NB (chopped) was stirred overnight and dissolved in 30 liters of 75 mM sodium phosphate, 600 mM sodium chloride, pH 5.2 buffer. Insoluble material was removed by depth filtration through cellulose pads (Filtrox AF9, Switzerland). The filtrate was diluted 3-fold and 30 g dry CM-Sephadex (C50 Pharmacia, Sweden) was added and stirred for 12 hr, in order to absorb TC2. The CM-Sephadex was washed repeatedly with 25 mM sodium phosphate, 200 mM sodium chloride, pH 5.2 buffer. Resuspension in 400 ml elution buffer (50 mM sodium phosphate, ! M sodium chloride, pH 6.0) stirring for 1 hr, and filtration gave the first eluate with 75% of TC2. A second treatment with 200 ml buffer produced the final eluate (25% of TC2). The combined eluates (550 ml) were clarified by filtration through a 3-~m Millipore membrane and stored frozen. The resulting TC2 concentrate contained 40,000-60,000 ng/ liter apo-TC2 measured by its unsaturated vitamin B12 binding capacity (UBBC) 21 or by gel filtration 22 using G-150 Sephadex and 0.1 M phosphate buffer, pH 7.4. The specific protein activity was approximately 0.1 /~g TC2 per mg protein. This preparation was stable at - 2 0 ° for several years. This batch separation can be performed in 4-fold to yield 2200 ml as starting material for the affinity chromatography.

Preparation 2: Affinity Purified TC2 for Antiserum Preparation Thermolabile ligand affinity chromatography, z3 based on the thermolabile immobilization of cyanocobalamin (CN-Cbl) 2° was used for further 17 p. Kistler and H. Nitschmann, Vox Sang. 7, 414 (1962). 18 R. H. Allen and P. W. Majerus, J. Biol. Chem. 247, 7709 (1972). 19 C. R. Savage, A. M. Meehan, and C. A. Hall, Prep. Biochem. 6, 99 (1976). 2o j. van Kapel, B. G. Loef, J. Lindemans, and J. Abels, Biochim. Biophys. Acta 676, 307 (1981). 2~ H. S. Gilbert, J. Lab. Clin. Med. 89, 13 (1977). 22 U° H. Stenman, K. Simons, and R. Grabeck, Scand. J. Clin. Lab. Invest. 21, 202 (1968). 23 E. Nex¢, H. Olesen, D. Bucher, and J. Thomsen, Biochim. Biophys. Acta 379, 189 (1975).

[5]

RIA FOR HUMANTRANSCOBALAMINII

39

purification of TC2. The procedure presented here is preferred over other purification schemes 18a9 because it avoids the relatively drastic exposure to guanidinium chloride to release the binding proteins. A more recent interesting modification of the affinity procedure employs photorelease of Cbl-protein complexes, 24 but this has not yet been put to use for the purification of human TC2. In the thermolabile ligand affinity chromatography procedure, unsaturated Cbl-binding proteins, retained on CN-CblSepharose at 4°, are dissociated together with bound Cbl by increasing the temperature to 37 °. Starting material was either the commercially available concentrate of preparation 1, derived from plasma fraction NB, or the equivalent preparation from Cohn III. 2° The affinity column was prepared with a spacer as described by van Kapel e t a/.2°: 3 g activated CH-Sepharose 4B (Pharmacia, Sweden), washed and swollen according to the instructions of the manufacturer, was coupled to 3,3'-diaminodipropylamine, using 30 ml 10% (v/v) amine in 0.1 M sodium bicarbonate, 0.5 M sodium chloride buffer, pH 8.2, and overnight rotation at room temperature. Excess spacer was removed by washing with coupling buffer. Hydroxocobalamin (OH-Cbl) was added (12 mg in 10 ml coupling buffer) and incubated overnight with the CH-Sepharose conjugate in order to saturate the column with Cbl. The prepared column can be stored at 4°, protected from light, for several days in the presence of excess OH-Cbl. The suspension was transferred to the cold room (4°) and poured into a glass column (2.4 x 7.5 cm), washed with coupling buffer to remove excess OH-Cbl, resuspended in 30 ml of coupling buffer containing 0.2 M KCN, and incubated for 1 hr in order to convert bound OH-Cbl to CN-Cbl. This step is important in order to prevent unspecific absorption of other proteins to the affinity ligand. Unbound CN- was removed by washing with coupling buffer and the column was equilibrated with 0.1 M sodium phosphate, 1 M sodium chloride buffer, pH 6.0, and used directly for affinity chromatography of apoTC2 as described, z° The column contains approximately 2.7 mg cyanocobalamin, enough to bind 70 mg of TC2 protein. Eight 2.2 liter batches of the CM-Sephadex eluate (derived from a 4-fold batch of preparation 1 or Cohn III extract) were thawed at 4° and successively applied to the affinity column (flow rate 150 ml/hr) in a period of 8 days. Between each batch, the column was rigorously washed with 50 ml portions of (1) 50 mM sodium phosphate, 1 M NaC1, pH 6.0, (2) 0.1 M Tris-HC1, 1 M NaCI, pH 8.0, and (3) 25 mM sodium phosphate, 0.2 M NaCI, pH 5.2. The rinses 24 D. W. Jacobsen, Y. D. Montejano, and F. M. H u e n n e k e n s , Anal. Biochem. 113, 164 (1981).

40

COBALAMINS AND COBAMIDES (Bl2)

[5]

were repeated twice in reversing order. After the final washing, the column was resuspended in 15 ml of 0.1 M Tris-HCl, 1 M NaC1, 0.02% NaN3 buffer, pH 8.0, transferred to a clean glass column, and TC2-cobalamin was eluted from the affinity adsorbent with twice 25 ml of warmed Tris-HC1 buffer after 5 and 10 hr of incubation at 37°. The cooled effluents were directly concentrated to about 20 ml in an Amicon ultrafiltration cell (model 52), with a YM10 membrane (Amicon). The concentrate was centrifuged at 105,000 g for 1 hr and the supernatant was chromatographed in two portions on a Sephacryl S-200 column with the pH 8.0 Tris-HCl buffer. The pure TC2-cobalamin fractions were pooled and concentrated as above and stored at - 2 0 °. The purification scheme and yield of TC2 is outlined in Table I.

Immunization Procedure Two rabbit anti-human TC2 antiserum preparations derived from two different sources of purified TC2 were equally suitable in the radioimmunoassay (RIA) described here. Approximately 1 mg of pure holo-TC2 in 2.2 ml, prepared according to Savage e t al., 19 was mixed with an equal volume of Freund's adjuvant. One-half of this preparation was injected via a footpad into each of two male New Zealand White rabbits. A booster injection of the same amount was given to each rabbit 18 days later. The first blood sample was taken 18 days after the booster injection. Subsequent samplings were conducted at 7 day intervals. Whole serum was either frozen at - 2 0 ° or lyophilized for storage. The second anti-human TC2 antiserum 2° was prepared from affinity purified TC2 as described. Rabbits were immunized by intramuscular injections of 0.25 mg pure human holo-TC2 with I ml complete Freund's adjuvant. Immunization was continued every 2 weeks by subcutanous injections of 0.1 mg human holo-TC2. Blood was drawn in one batch by heart puncture, 3 months after the first injection. The second antiserum had similar specificities as the first antiserum, but it could be used at higher dilutions in the assay, indicating that it was four times more active than the first sample.

Preparation of Immobilized Anti-Human TC2 Antiserum (Immunosorbent) A commercial substitute for the solid support used here is named "immunobead reagent" and can be obtained from Bio-Rad Laboratories, Richmond, California. The solid support consisted of copolymer beads of

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42

COBALAMINSAND COBAMIDES(BI2)

[5]

acrylamide and acrylic acid. 25The antiserum was coupled to the - - C O O H groups of the polyacrylamide beads as described for trypsin and ribonuclease A 25 with appropriate modifications presented beforelS: 50 /A of lyophilized reconstituted anti-TC2 antiserum, cleared by centrifugation, was mixed with 100 mg swollen polyacrylamide beads in 30 ml 0.003 M KH2PO4 buffer, pH 6.3, and left for 1 hr at 4°. Ten milligrams carbodiimide [1-ethyl-3-(3-dimethylaminopropyl]carbodiimide.HC1 from Sigma, Germany) was added and the suspension was rotated for 3 hr at 4 °. The insoluble matrix was collected by centrifugation, aspirated, and washed alternatively with phosphate buffer, pH 6.3, phosphate buffers with increasing and decreasing NaC1 content, 4.5 M urea, assay buffer omitting albumin, finishing with assay buffer (0.05 M sodium, potassium phosphate, 0.1% NAN3, 1% Tween 80, and 0.5% human serum albumin, pH 7.4). The final suspension was made up to 10 ml with assay buffer and lyophilized. The lyophilized material could be used for at least 2 years when stored at 4° . Radiolabeling of TC2 (Tracer) The partially purified TC2 concentrate from preparation 1 above (40,000 ng/liter Cbl equivalent apo-TC2) was diluted 20-fold with 0.05 M phosphate, 0.1% NaN3 buffer, pH 7.4 containing radioactive cyanocobalamin (specific activity of 200 mCi/mg from Radiochemical Center, Amersham, England) in order to saturate 80-90% of apo-TC2. After 15 min incubation at 37°, traces of free radioactivity were removed by treatment with albumin-coated charcoal and centrifugation. The final tracer solution was filtered over a 0.45-/zm Millipore sterile filter, and contained approximately 1500 pg/ml labeled TC2. This tracer solution was stored frozen, and was used for up to 6 months. The stability of the TC2[57Co]CN-Cbl complex was tested by gel filtration after incubation for 30 rain at 37°, and 17 hr at 4°. The total radioactivity remained bound to TC2J 1 Preparation of Serum Controls

High control (3000-4000 ng/liter apo-TC2). The partially purified TC2 (UBBC 40,000 ng/liter) from preparation I was diluted 20-fold with normal serum which was also used for the standard curve. The apo-TC2 content of this serum control was thus enhanced 3-fold, without appreciably changing the protein content of the serum. The total TC2 content of 25R. Mosbach, A. C. Koch-Schmidt,and K. Mosbach, this series, Vol. 44, p. 53.

[5]

RIA FOR HUMAN TRANSCOBALAMINII

43

the high control was enhanced 3-fold as well, because both the TC2 concentrate from preparation 1 and the whole serum contain holo-TC2 amounting to 10-12% of total TC2.15 The serum was stored frozen. L o w control (500-600 ng/liter apo-TC2). In vivo parental administration of CN-Cbl causes an increased flux of TC2 into tissues and a corresponding drop of serum level. 26 Blood was withdrawn 30 min after 200/~g CN-Cbl was injected intramuscularly in a healthy person, and it was noticed that the serum level had dropped to 55% of the preinjection TC2 level. The resulting TC2 level (500-600 ng/liter) was in the order of magnitude observed for heterozygous-deficient relatives of TC2-deficient patients. 2 The serum was stored frozen. TC2-free control (<25 ng/liter apo-TC2). TC2-free serum was used to test interference of tracer binding to serum components. It was made by treating normal serum with immobilized anti-TC2 antiserum.~5 One hundred microliters of anti-TC2 antiserum bound to 400 mg polyacrylamide beads was rotated with 5 ml serum from a healthy individual for ! hr at 22 ° and overnight at 4 ° . Centrifugation yielded serum with reduced TC2 which was again incubated with the same amount of regenerated immobilized anti-TC2 antiserum (bound TC2 was removed to regenerate the solid support by washing in 0.2 M glycin, 0.5 M NaCI at pH 2.8, final washing with 0.05 M sodium phosphate, 0.5% human serum albumin buffer, pH 7.4). Subsequent gel filtration (Sephadex G-150 as in Ref. 22) disclosed lack of apo-TC2 (<25 pg/ml), whereas the unsaturated R-binder fraction in the treated serum was retained. The total TC2 content of two " T C 2 - f r e e " serum preparations measured by the RIA presented here was found to be 80-100 pg/ml Cbl equivalent. This indicates that slight interference from serum components does occur, and that this interference is in the order of 5-10% of normal total TC2 levels. Standard Curve and the Determination of Total TC2 in the Reference Sample Fresh serum from whole clotted blood, drawn from 5 healthy donors, was mixed and frozen in aliquots after the addition of 0.1% NAN3; it could be used for at least 6 months for the standard curve described in Table II. Heparin which complexes with TC2 alters the antigen behavior of TC2 in the RIA and should be avoided. The total TC2 level of the mixed serum was calculated by comparison with total TC2 in the TC2 concentrate (preparation 1) as follows. The endogenous Cbl content of the TC2 con26j. A. Begley, T. A. Morelli, and C. A. Hall, N. Engl. J. Med. 297, 614 (1977).

44

COBALAMINS A N D COBAMIDES (BI2)

[5]

T A B L E II ASSAY OF IMMUNOREACTIVE TRANSCOBALAMIN II a

Tube number Total counts Blank Maximal binding Standard curve s Unknown sample

1 2 3 4-11

12, etc.

Serum

Buffer b

CN-Cbl c

Tracer d

Immunosorbent e

---1.56-200 25

-800 300 298.5-100 275

-10 10 10 10

50 50 50 50 50

--500 500 500

a All volumes are in microliters; all tubes in duplicate. Reproduced with permission from

Fr~ter-Schr6der e t al.15 b c d e s

A s s a y b u f f e r (see section on immunosorbent). 300 n g / m l . Labeled T C 2 : 3 3 0 n g / l i t e r [ 5 7 C o ] C N - C b I - T C 2 in assay buffer. Immobilized anti-TC2 (1 : 10,000) in assay buffer. Except for tube 10 (200/zl serum and 100/xl buffer), u s e 100/zl o f appropriately diluted reference serum (1 : 6 4 - 1 : 1 for tubes 3-9) and add 2 0 0 / z l of a s s a y buffer, in order to obtain TC2 levels ranging from 1.87 to 240 pg/tube.

centrate was high (3500 ng/liter Cbl) and could be accurately measured by means of radioisotope dilution technique. 27 This value together with the known apo-TC2 content of the sample gave an accurate estimate of the total TC2 level. Absolute TC2 levels in the standard curve serum were then determined using this known sample as a cross reference. Procedure for Solid Phase RIA A s s a y Conditions 15

Immunoreactive TC2 was measured by competitive inhibition of labeled TC2, using the suspension of immobilized antiserum as a specific binder. Optimal assay conditions for the determination of TC2 in serum are compiled in Table II. The reagents and serum samples were diluted with assay buffer as indicated. Final dilutions were prepared 60 min before use, and kept at room temperature. The 60-min period seemed to be necessary to obtain the optimal stabilizing effect of the nonionic detergent Tween 80 in the assay. Step 1. Eight reference samples for the standard curve, controls, and unknown samples were diluted as shown in Table II. 27 R. P. B r i t t , F. G. B o l t o n , C. A. Cull, a n d G. H. S p r a y , Br. J. Haematol. 16, 457 (1969).

[5]

RIA FOR HUMAN TRANSCOBALAMINII

45

Step 2. An excess amount of 3 ng CN-Cbl in 10/xl was added to each tube, mixed, and incubated for 15 min, to saturate all binding sites to eliminate any interference from traces of free radioactive CN-Cbl. Step 3. T r a c e r (TC2-[57Co]CN-Cbl) was added and mixed well. Step 4. Immunosorbent, kept in suspension by gentle vortexing, was added, mixed once, and incubated overnight at 4 ° . Step 5. The tubes were centrifuged (10 min at 2500 g at 4 °) and the supernatant was immediately removed. The pellet was hardly visible at this point and the supernatant could be aspirated 2 mm from the bottom of the tube. The pellet was counted directly. Step 6. A standard curve was made by plotting the percentage binding against TC2 concentrations of the standard on semilogarithmic paper or logit-log paper (Fig. 1). B0 = cpm max. binding - cpm blank

%B/Bo = cpm standard or sample - cmp blank cpm max. binding - cpm blank The corresponding TC2 value of each unknown sample could be read from the standard curve obtained. Calculation of the unknown sample could also be conducted with the aid of a computer program using logitlog linear regression and iterative optimization of the correlation. Multiplication by the dilution factor gave the concentration per ml of original material. Sensitivity and Specificity of the Assay and Total TC2 Levels Measured in Serum The sensitivity of this assay is 1.87 pg TC2 per tube (Fig. 1). To show that the anti-TC2 antiserum discriminates between human R-binder and TC2, normal serum was treated with excess immobilized anti-TC2 antiserum, as described above in the section on preparation of serum controls. It was then demonstrated by gel filtration that only TC2 is removed, whereas the R-binder activity was unchanged. This confirmed that the antiserum was indeed selective for TC2. The displacement curve for TC2 in a fraction of 107 lysed human granulocytes per ml containing only Rbinder, shown in Fig. 1, confirmed that the anti-TC2 antiserum does not react with R-binder. Species specificity was demonstrated when mouse serum (70,000 ng/ liter mouse transcobalamin II) was tested against the immobilized antihuman TC2 antiserum in this assay. An apparent TC2 level of 176 ng/liter Cbl equivalent was measured. Thus, only 0.3% cross-reactivity between

46

COBALAMINS AND COBAMIDES(B~2)

[5]

B/Bo% lOO _

80_

60_

40_

20_

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3.12

6.25

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pg/tube 1 1.87

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25

50

100

200

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FIG. 1. Solid-phase competitive RIA for TC2. Curve 1: standard displacement calibration curve, based upon the competition between increasing amounts of unlabeled TC2 and its 57Co-labeled analog (TC2-Cbl) for binding to antibody. Curve 2: displacement of labeled TC2 from antiserum by increasing volumes of low control serum containing 534 ng/liter TC2. Curve 3: lack of displacement of labeled TC2 by increasing volumes of a sample prepared from lysed granulocytes with a UBBC of 8470 ng/liter due to R-binder and no detectable TC2. Reproduced with permission from Frhter-Schr6der et al. t5

mouse and man was observed. 15,z8 The same antiserum against human TC2 exhibited further specificities when reacted with TC2 in serum from eight other species of animals. 29 Reactivity of holo-TC2 versus apo-TC2 was tested using the first antibody preparation in this report. The displacement curves were superimposable, indicating that the immunoreactivities of holo-TC2 and apo-TC2 were identical. 11 Reproducibility was tested as follows. Repeat assays of a high control sample on 35 different days yielded 3570 ___260 ng/liter ( - S D ) Cbl equivalent TC2. The coefficient of variation was 7% and the corresponding variation for N = 35 was 1.2%. 15 Both apo- and holo-TC2 are stable in serum (either human or fetal calf serum) for at least 2 weeks at room 28 M. Fr~ter-Schr6der, O. Hailer, R. Gmiir, L. Kierat, and G. Anastasi, Biochem. Genet. 20, 1001 (1982). z9 M. Haus, P. D. Green, and C. A. Hall, Proc. Soc. Exp. Biol. Med. 162, 295 (1979).

[5]

RIA FOR HUMANTRANSCOBALAMINII

47

temperature in a closed tube. ~5The unknown factor responsible for stabilization of apo- and holo-TC2 in serum is partially removed by the purification procedure and purified TC2 is degraded rapidly in the absence of high salt concentrations, but CN-Cbl complexation stabilizes purified TC2 adequately at assay conditions.~5 Normal levels for total TC2 in serum from I00 healthy blood donors, using the assay presented here, have been reported. ~5The mean level and SD were 1140 -+ 260 ng/liter TC2. The levels in males (N = 61, 1150 -+ 240 ng/liter) and females (N = 39, 1130 - 280 ng/liter) did not differ. Three patients with congenital TC2 deficiency had immunoreactive TC2 levels varying from 22 to 39% of the normal mean, and lacked apo-TC2 activity. z,~5 Nine healthy relatives of these three patients, shown by other methods to be heterozygous for the deficiency,z had immunoreactive TC2 levels ranging from 31 to 79% of the normal mean.

Quantification of Secretory TC2 in Human Cell Cultures

Synthesis and Secretion of TC2 by Cultured Skin Fibroblasts The solid-phase RIA presented here has been applied to identification and quantification of TC2 which is secreted by fibroblasts. 9,3° Immunoreactive TC2 levels in the cell culture medium, which contains no free Cbl, were practically the same as apo-TC2 levels measured by G-150 gel filtration, indicating that TC2 is secreted predominantly in its apo form. 9 Fibroblast cultures were derived from skin biopsies obtained from healthy individuals. Confluent cultures (2-5 x l06 cells per flask) were maintained in 15 ml Eagle's minimal essential medium (MEM) supplemented with I0% fetal calf serum (Gibco) and the usual antibiotics plus fungizone. The medium was replaced when the cells were confluent and collected after a fixed number of days (periods from 7 to 14 days were found to be convenient) in order to measure the secretion rate of TC2. Medium sample preparation for investigation of the TC2 content was conducted as follows9: an equal volume of saturated solution of ammonium sulfate (4 M) was added to medium from one culture flask, to concentrate and precipitate TC2 selectively, at 50% saturation. After 16 hr at 4° and subsequent centrifugation (8000 g, 15 rain) the precipitate was reconstituted in RIA assay buffer (omitting albumin as above) equivalent to 10% of the original medium volume, and dialyzed against this buffer for 6 hr at 4 ° with two buffer changes (absence of NH4 + was tested with Nessler's reagent). Samples were stored at -20 ° or assayed directly. 3o M. Sacher, F. Paky, and M. Frhter-Schr6der, Heir. Paediatr. Acta 38, 549 (1983).

48

COBALAMINS AND COBAMIDES (BI2)

[5]

TC2 detection in medium samples: The RIA procedure in Table II was adapted for the investigation of cell culture medium by using larger unknown sample volumes (200 /zl) and correspondingly less assay buffer (100/zl) to detect low TC2 concentrations. It was also possible to analyze more dilute samples using volumes of up to 500 /zl, by increasing the immunosorbent concentration, or by increasing the total assay volume, accordingly. A background control for supplemented medium without cell contact was necessary to determine the residual fetal calf serum-derived cross-reacting material, which had to be subtracted from the TC2 levels obtained in cell culture medium. 9,3°

Synthesis and Secretion of TC2 by Cultured Bone Marrow Liquid bone marrow cultures were derived from spongiosa residues taken from otherwise healthy individuals, undergoing orthopedic surgery and prepared as describedg: 5 × 106 dextran sedimented bone marrow cells were maintained in 6 ml supplemented MEM, containing 20% fetal calf serum and antibiotics. The cell-free medium was investigated after a culture period of approximately 7 to 14 days, in the same way as described for skin fibroblasts, to quantify the secretory capacity of the cultured bone marrow. Both the cultured skin fibroblasts and bone marrow cells (with continuously changing cell populations) were found to secrete increasing quantities of TC2 which accumulates in the medium. The amount of TC2 secreted per flask by 2-5 million cells for both types of cultures ranged from 200 to 2000 pg Cbl equivalent TC2, depending on the culture period and on culture conditions. 9 Applications and General Comments Significant elevations of apo-TC2 concentrations in serum have been observed in Gaucher's disease, 3~ in multiple myeloma, 32 in certain other forms of cancer, 33 after bone marrow transplantation) 4 and in various forms of active autoimmune disease, 35 but not in rheumatoid arthritis. 36 31 H. S. Gilbert and N. Weinreb, N. Engl. J. Med. 295, 1096 (1976). 32 R. Carmel and D. Hollander, Blood 51, 1057 (1978). 33 H. S. Jensen, P. Gimsing, F. Pedersen, and E. Hippe, Cancer 52, 1700 (1983). 34 E. Naparstek, B. Rachmilewitz, M. Rachmilewitz, Z. Fuchs, and S. Slavin, Br. J. Haematol. 55, 229 (1983). 35 M. Fr~tter-Schr6der, W. H. Hitzig, P. J. Grob, and A. B. Kenny, Lancet 2, 238 (1978). 36 M. Fr~ter-Schr6der, A. Fontana, K. Fehr, and L. Kierat, Schweiz. Med. Wochenschr. 114, 1396 (1984).

[5]

RIA FOR HUMAN TRANSCOBALAMINII

49

H o w e v e r , elevated levels of TC2, measured in synovial fluid, can help to discriminate rheumatoid arthritis patients from those with osteoarthrosis. 36'37 Subsequent clinical investigations have confirmed the usefulness of determining total immunoreactive TC2 levels as well, in the clinical course of systemic lupus erythematosus (SLE) and dermatomyositis. 38 Diagnostic criteria of abnormal and absent gene products in TC2dependent inborn errors of Cbl utilization have been defined by measurements of TC2 in serum and in cell culture. Three genetic subgroups have been proposed. 2 Early diagnosis of suspected TC2 deficiency can be performed in a cord serum sample with the RIA for TC2, because it has been shown that the n e w b o r n ' s own genetic TC2 type is already expressed in cord blood. 39 Now that measurement of TC2 biosynthesis by quantification of TC2 in human cell culture medium has become accessible, it may be possible to learn more about the natural dynamics of TC2 in man in the future. It has been reported that several other human cell types, not mentioned here, secrete relatively small quantities of TC2. 9'4° Prenatal diagnosis of TC2 deficiency may be possible by investigating amniotic fluid cell cultures, where TC2 synthesis has been d e m o n s t r a t e d f The rate of TC2 secretion by fibroblasts can be influenced in various ways: CN-Cbl addition to the medium caused a decrease of net secretion (presumably as a consequence of increased uptake of the T C 2 - C b l complex), whereas chloroquine and NHaCI, inhibitors of lysosomal proteolysis, significantly increased the quantity of TC2 recovered in the cell culture medium (presumably as a consequence of inhibition of intracellular degradation). 9 Acknowledgments The author is indebted to Dr. C. A. Hall, Dr. J. Lindemans, and Dr. H. J. Porck for generously supplyingthe antiserum preparations described above, and to Ms. L. Kierat and

Dr. W. H. Hitzig for helpful discussions and support. Supported by research Grants 3.023-81 from the Swiss National Science Foundation, the Kantonal-Ziircher Liga for Krebsbek/~mpfung, and the Prof. Dr. Max Clo6na Foundation, Switzerland. ~7p. A. Christensen, Y. Brynskov. P. Gimsing, and J. Petersen, Stand. J. Rheumatol. 12, 268 (1983). 3~U. L~isser, L. Kierat, P. J. Grob, W. H. Hitzig, and M. Fr8ter-Schr6der, Clin. lmmunol. Immunopathol. 36, 345 (1985). 3,~H. J. Porck, M. Fr'Mer-Schr6der, R. R. Frants, L. Kierat, and A. W. Eriksson, Blood 62, 234 (1983). 4oR. Rabinowitz, B. Rachmilewitz, M. Rachmilewitz, and M. Schlesinger, Isr. J. Med. Sci. 18, 740 (1982). 4~ M. Frater-Schr6der, P. Krieg, L. Kierat, J. Erten, and W. H. Hitzig, Heir. Paediatr. Acta Suppl. 50, Abstr. 115 (1984).