- Email: [email protected]
Radioimmunoassay, a goal or a tool? The setup of a reliable, fast, and cheap radioimmunoassay for vasopressin in biological samples
Journal of ControlledRelease, 2 1 ( 1992 ) 23-36 0 1992 Elsevier Science Publishers B.V. All rights reserved
23 016%3659/92/$05.00
COREL 00736
Radioimmunoassay, a goal or a tool? The setup of a reliable, fast, and cheap radioimmunoassay for vasopressin in biological samples Jeroen A. Ten Haaf”, Dirk Terwel”, Bert J.M. Van de Heijningb and Tjeerd B. Van Wimersma Greidanusb aDepartment ofNeuropsychology
and Psychobiology, University ofLimburg, Maastricht, Netherlands; bRudolfMagnus for Pharmacology, Utrecht, Netherlands
(Received
Key words: Vasopressin; standard
17 September
Institute
199 I ; accepted in revised form 4 February 1992 )
Radioimmunoassay;
Plasma determination;
Introduction The introduction of the radioimmunoassay (RIA) by Yalow and Berson [ 1 ] has been of great importance to almost the whole field of clinical and biological sciences. In principle it enabled researchers to determine the level of every biological substance against which an antibody can be raised. Basically RIA is the comparison of the competition between a known amount of radio-iodinated ligand (so-called tracer) and an unknown amount of unlabeled ligand on the one hand with the competition between standard amounts of unlabeled and labeled ligand on the other hand for a known amount of antibody. Binding is plotted against known amount of ligand and unknown amounts are determined by interpolation (Fig. 2). The highest sensitivity of the assay is obtained at a concentration of antiserum at which 50% of the tracer is bound. This concentration is deterCorrespondence to: J.A. ten Haaf, Dept. of Neuropsychology and Psychobiology, P.O. Box 616, 6200 MD Maastricht, Netherlands.
Tissue determination;
Internal
mined by serial dilution of antiserum (Fig. 2). As a source of antibodies can be used either serum of immunized rabbits or larger animals, or the culture medium of hybridomas of lymphocytes of immunized mice and myeloma cells. If other methods of assay are already available RIA is generally more efficient and sensitive. However, together with improvement in terms of efficiency and sensitivity, the RIA, since it is an indirect method, confronted researchers with the problem of interference by specific and nonspecific matrix factors present in biological samples. The level of nonspecific interferences is generally reduced by extraction of the substance of interest prior to RIA. However, since the RIA usually is performed on large numbers of samples on a routine basis, extraction procedures should be quick and simple and consequently sample extracts still contain (a great deal of) impurities. In order to anticipate this pitfall, Yalow and Berson state that the effect of interfering substances should be tested and that it must be assured that the milieu of the unknown sample is identical with that of the known standards. With respect to the other problem, the presence
24
in the matrix of substances resembling the ligand of interest (specific interference), the use of antibodies with a high affinity for the specific ligand and low cross-reactivity with resembling substances is a prerequisite for reliable RIA. The present article will describe our experience with the setup and the routine use of a RIA for arginine-vasopressin (VP), see Fig. 1. This neurohypophyseal peptide, besides its classical peripheral vasopressor and antidiuretic functions, is thought to play a role in learning and memory processes in the brain [ 2,3 1. Since these functions are within the scope of our research, a reliable and efficient protocol for determination of VP levels in biological samples is a fundamental analytical tool. RIAs for VP are considered difficult for a number of reasons: ( 1) VP is a small peptide (9 amino-acids) and therefore a poor antigen, (2) its level in the circulation and some brain regions is extremely low (in plasma of the Wistar rat about l-2 fmol VP/ml, in humans sometimes even lower [ 4]), so its determination is relatively sensitive to matrix-interference, and (3) it is sensitive to endogenous peptidase activity. The purpose of this study was to raise in rabbits sensitive and specific antisera to VP, to develop reliable protocols for the sampling of (brain) tissue and blood plasma from rats or humans, to extract VP from these matrices and to determine VP by RIA in a reproducible way. In addition, we will discuss some of the experiences of others with the determination of VP.
cys----s-s--1
Cys-Pro-Arg-Gly-NH2 ‘ 8 7
9
Fig. 1. The 9-amino acid arginine-vasopressin molecule: position I and 6 are interconnected by a disulphide bridge to form the characteristic ring structure. The tyrosine-residue, which is [ ‘*‘I ] iodinated during the radiolabeling procedure, is indicated by an asterisk. Theoretically, monoclonal antibodies will recognize one specific site, the polyclonal antibody subspecies are directed towards more sites of the molecule (see also Fig. 3 ).
Production of antisera against vasopressin The antigenicity of small peptides can be enhanced by conjugation to a protein carrier [ 5 1. Several investigators have raised antibodies against a conjugate of VP and some carrier, either coupled with glutaraldehyde or carbodiimide [ 671. Rabbits that produce a high affinity antibody against VP become polyuric [ 8 1. Some of the antibodies raised are susceptible to proteins and salts [ 9- 111 . This warns against the direct measurement of VP in plasma or tissue samples. To date direct measurement has only been attempted with success once [ 121. When applying direct measurement special measures have to be taken to prevent slow degradation of VP. Fyhrquist et al. [ 12 ] used s-aminocaproic acid for this purpose. We raised antisera in six male white New Zealand rabbits (code W l-W6). VP (arginine vasopressin, pressor activity: 500 I.U./mg, obtained from Organon, Oss, The Netherlands) was conjugated to bovine thyroglobulin (Type I, Sigma? St, Louis, U.S.A.) with carbodiimide (Sigma), generally according to Skowsky and Fisher [6]. Prior to the injection the conjugate was freshly emulsified 3 : 4 in complete Freund’s adjuvant (Difco Laboratories, Detroit, U.S.A. ). Six injections of 0.5 ml emulsion, containing 0.25 mg VP, were given intramuscularly with a two week interval. From then, every two months a booster injection was given. Small portions of blood (circa 3 ml) of each animal were harvested one week after each injection by puncture of the lateral ear vein, allowed to clot for 2 h at 4’ C in polypropylene tubes, spun down (2000 g, 30 min, 4”C), and stored in 100 ~1 portions at - 20°C. A fresh portion was used to test binding capacity and titre (see Fig. 2 ) , for details see Radioimmunoassay for vasopressin. If an antiserum met the test criteria (maximal binding > 85% and a titre < 1:40,000), the animal was anaesthetized and all the blood collected. The specificities of the two antisera that met the test criteria, W 1 and W4, were tested by determining the displacement of tracer from the antibody by a variety of substances (peptides), more or less resembling VP (e.g. oxytocin, VP fragments, and
25 TABLE 1 Protocol for the radio-iodination 1.
2. 3. 4. Fig. 2. Left panel: Dilution curves for two antisera (W 1 and W4). Abscissa: dilution factor (X 10d3); Ordinate: percentage binding of tracer to serum. Right panel: Displacement curves of antisera W 1 and W4. Abscissa: fmol VP/tube added to incubation; ordinate: percentage binding of tracer.
of VP
Add to a 12 X 75 mm glass tube: 50 ~1 of 0.5 M phosphate buffer, pH 7.5, 10 c(g peptide dissolved in 10 ~10.2 M acetic acid, 1 mCi NarZS1dissolved in 10 ~1 dilute NaOH solution (Amersham, UK), 40 ,ug chloramine-T dissolved in 10 ~1 50 mM phosphate, pH 7.5. Mix the contents of the tube with a few finger flicks. After 60 s add 1 ml of a suspension of 150 mg Dowex 1 X8 (Cl- form). Transfer the supernatant to a Sephadex G-25 column (45 cm~0.9 cm) equilibrated with a solution containing 0.01 M acetic acid and 1.25 mg BSA/ml. Elute with the same solution at a rate of 12 ml/h.
be expressed as “VP-immunoreactivity (VPir ) levels”, rather than “VP levels”. Most of the antibodies reported in the literature have a K, near 10” M-l. Few antibodies have been obtained with a K, higher than lOI M-’ [7,13]. Wl has aK,of1.55x10”M-‘;W4hasoneof10”M-’. Antibodies Glick-1, Skowsky-7 1 and W 1 have been used in numerous studies.
Iodination of vasopressin t fmol .; ;
Dmol 10
1001000
1
I 10
pmol 1
lfmol .;
;
lb
loolob
;
lb
Fig. 3. Cross reactivities of antisera Wl (left panel) and W4 (right panel) with vasopressin fragments and oxytocin. Abscissa: fmol (or pmol) added peptide/tube in RIA. Ordinate: Logit log transformation of percentage antibody-tracer displacement. Numbers refer to vasopressin fragments (e.g. l9: VP-( l-9); pGlu4-9: [pyroGlu4, Cyt6]VP-(4-9); OT: oxytocin). VP-fragments were kindly provided by Organon Oss, the Netherlands. Crossreactivity on molar basis (as percentage of EDSo of VP-( l-9) ) of the fragments are as follows: Wl: VP-(2-9): 56; VP-(3-9): 25; VP-(4-9): 20; VP-(5-9): 13; VP-(7-9): 0.05; OT: 0.004; VP-( l-8): 0.004; W4: VP(3-9):
other peptides, see Fig. 3 ). W 1 is directed against the C-terminus of the vasopressin molecule, W4 to the C- as well as the N-terminus. Although we obtained highly specific antibodies, one has to keep in mind that data obtained by RIA should
In the radio-iodination of VP generally chloramine-T is used as the oxidizing agent of iodine [ 141. Despite the fact that Iodogen has been reported to be a milder oxidant than chloramine-T [ 151, which of the two is used does not result in differences in immunogenicity of the tracer. Iodination conditions should be chosen such as to prevent formation of the diiodinated species, since this species is unstable [ 161. The iodination reaction is stopped by scavenging the iodide with Dowex 1 x 8 ( 100-200 mesh in Cl- form) or another anion-exchange resin [ 17- 191 or by the addition of serum albumin [ 201. Metabisulphite can not be used to this end, since it will reduce the disulphide bridge of the VP molecule. After iodination free iodide, labeled and unlabeled components in the reaction mixture can readily be separated by high performance liquid chromatography [ 2 11, although good separation can also be obtained by chromatography on a
26
Sephadex G-25 column when eluting with low concentrations acetic acid containing 1.25 mg serum albumin per ml to prevent adsorption of the peptide to the column [ 19 1. Table 1 gives a detailed protocol for the iodination of VP. Instead of going through these quite laborious procedures, to date we obtain iodinated VP of high specific activity commercially ( Amersham) .
Collection/dissection and extraction of biological samples Plasma Plasma preparation Plasma of rats is obtained according to the protocol described in Table 2. Blood from human subjects is collected into ice-cold Greiner Vacuette (Greiner, Alphen a/d Rijn, The Netherlands) tubes to which we added 5 mg heparin dissolved in 100 ~1 water by injection in advance. Subsequently, the tubes are placed on ice. No further precautions are needed to prevent degradation of VP, except when blood of pregnant women is collected. Pregnancy plasma contains high levels of cystine-aminopeptidase activity [ 221. This activity can be inhibited TABLE 2 Plasma sample preparation; all steps should be carried out at 4°C unless indicated otherwise 1.
2.
3.
4.
5. 6.
For reasons of reproducibility, transport animals at least 30 min before decapitation to the (quiet) room where they will be decapitated. Experiments should always take place around the same time of day. Add 100 ,ul Hz0 to which 5 mg heparin (Leo, Ballerup, Denmark) is added, into ice-cold tubes (polypropylene or polyethylene). Place plastic funnels in the tubes. Take the animal from its cage quietly, decapitate it quickly, and collect the trunk blood (8- 10 ml) in the tube. Close tube and rotate it immediately by hand to mix the blood with the heparin. Rotate gently in order to prevent hemolysis. Place the tubes immediately on ice until centrifugation within 1 h. Centrifuge tubes (2000 xg, 30 min). Collect plasma in tubes and store at - 20°C until use.
sufficiently by addition of 0.1 ml of a solution of 60 mg/ml 1, lo-phenanthroline [ 231 to the tubes. This inhibitor does not interfere with the radioimmunoassay. Plasma sample extraction VP has been extracted from plasma in various ways. Originally, gel filtration was used to separate VP from other plasma components [ lo]. This laborious method was soon replaced by organic solvents extraction using acetone and petroleum ether [ 10,161 or extraction by adsorption to a solid phase, such as glass powder [4,13,17,24] or diatomaceous earth [ 181. Recently, extraction on C 18- or C8 cartridges has come into use [ 25-28 1. At our laboratories we use either extraction with Vycor glass powder, principally according to Dogterom et al. [ 41, or extraction on Bond Elut C8 cartridges (LCR series, Analytichem Int., Sopar Biochem) [28]. The recovery of extraction with Vycor is 75%; extraction on Bond Elut C8 cartridges is 96% (see Table 6 ) . Tables 3 and 4 provide protocols for both extraction techniques. Plasma internal standard preparation More or less dependent on the type of matrix and adsorbent used for this purpose, an additional but important phenomenon is the co-extraction of matrix factors, that will interfere in most cases with antibody-ligand binding characteristics, leading to erroneous data if one does not anticipate [ 10,11,29 1. Therefore, in the development of an extraction protocol special attention must be given to account for this. In our opinion, the only right approach is to include a so-called internal standard at the start of the extraction procedure that is subjected to the same technical manipulations as the unknown samples. A concomitant advantage of this strategy is the absence of the need to correct final data for recovery (e.g. due to volume losses or efficiency of the adsorbent) once the procedure is implemented. Nevertheless it will always be necessary to determine the recovery percentage (by comparing the internal standard displacement curve
27 TABLE 3
TABLE 4
Procedure for the Vycor-extraction of vasopressin from plasma; all steps should be carried out at 4”C, unless indicated otherwise; use polystyrene tubes resistant to 60°h acetone
Extraction of vasopressin from plasma samples by solid phase Bond Elut C&silica columns
1.
2.
3.
4.
5.
6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17.
Vycor glass powder (mesh 140, Corning Glass Works, New York, U.S.A.), should be activated at 700°C for at least 24 h in advance of the experiment. Vycor can be kept indefinitely at 700°C. Thaw the frozen stocks as described in Table 2 at 4°C. Centrifuge stocks (2000 X g, 10 min) to spin coagulated proteins down. Prepare from stocks 0.5-2 ml plasma volumes (standard medium and unknowns) in tubes (preferably in triplicate). Standards and unknowns should have the same volume. Add to the standard medium tubes 50 ~1 Verona1 buffer (see section Incubation), containing no - for blank and nonspecific determination - or known amounts of VP (0.125-64 fmol). Add to each tube 100 ~1 from a constantly stirred Vycar suspension (2 g Vycor per 10 ml H20, 4°C). Vortex. Let the tubes tumble in a rotary tumbler for 30 min. Centrifuge tubes (2OOOxg, 10 min). Discard supernatants of standards by suction. Decant and pool supernatants of unknowns, and store these at -20°C. This material can be used as internal standard medium in a next extraction procedure. Resuspend the Vycor pellets in 0.5 ml HZ0 by Vortex. Centrifuge at 2000 xg for 10 min. Remove the supernatant by suction. Resuspend the Vycor pellets in 0.5 ml 2 N HCl by Vortex. Centrifuge at 2000~gfor 10 min. Remove the supernatant by suction. Resuspend the Vycor pellets in 0.5 ml acetone/H,0 (60: 40, v/v) by Vortex. Let the tubes tumble in a rotary tumbler for 30 min. Centrifuge at 2000 Xg for 10 min. Decant the acetone/H,0 fractions into plastic tubes. Vacuum evaporate until dryness in a Speed Vat.
with the external standard curve, see Table 6) as a check for overall performance of the extraction procedure and subsequent RIA. The ideal internal standard medium should contain exactly the same matrix, devoid of any trace of the specific l&and. With some exceptions (e.g. plasma of the Brattleboro rat), plasma or animals must be pretreated in order to get rid of the ligand to obtain the internal standard medium: either “pre-ex-
I.
6. 7.
8.
Connect C8 columns to a vacuum manifold column processor and activate columns by passing 4 ml methanol. Wash with ml H20. Do not let columns run dry. Add 4 ml 0.1 M HCl to 1 ml acidified (HAc) plasma (standards and unknowns). Apply this mixture to the column. Allow at least 5 min for the mixture to run through. Apply 4 ml 10% (vol/vol) acetonitrile in 0.1% trifluoro-acetic acid (TFA) to wash impurities from the column. Elute with 1.5 ml 60% acetonitrile in 0.1% TFA. Preserve the eluate for step 8. Cleanse the column with 4 ml 1OOI acetonitrile, 4 ml 8 M urea, and 4 ml HzO. Columns can be reused up to 20 times. Eluates are dried in a Speed Vat Concentrator (Savant). Residues are diluted and subjected to RIA (section Incubation).
traction” of plasma or over-hydration of animals can be a tool for this purpose. Note that each time a plasma extraction is carried out, the Vycor supematants of the unknown samples can be used as internal plasma standard medium in a next extraction procedure (see Table 3). From Fig. 4 it is clear that the displacement curve obtained by dilution of 24 h water-deprived normal rat plasma exactly parallels the internal standard curve prepared in Brattleboro plasma. The standard curves prepared from plasma of overhydrated normal rats and from Vycor pre-extracted normal plasma also perfectly parallel the curve prepared from Brattleboro plasma in Fig. 4 (data not shown). Furthermore, there is no difference in the respective blank and nonspecific binding values between the internal standard series prepared from Brattleboro plasma, “hydrated” normal plasma, and Vycor pre-extracted normal plasma. Therefore, Vycor pre-extracted normal plasma, plasma from overhydrated animals, as well as Brattleboro plasma can serve as internal standard medium. However, since a single standard tube prepared from Brattleboro plasma or “hydrated” normal plasma accounts for approximately one rat, this approach
28 50 ,
I
J
oi----
0
----I
01
““““I
“.““‘I
1
““‘.‘I
10
100
“...-I
1000
Fig. 4. Displacement of tracer from antibody by internal standard extracts (A ) and extracts of serial dilutions of plasma from a 24 h waterdeprived rat (A ). Brattleboro plasma was used as standard medium and as diluent.
is both not ethical and economically not affordable on a routine scale if one includes enough internal standards to obtain reliable results. For the setup of an internal standard series we add known amounts of vasopressin to pre-extracted plasma, obtained as described in Table 3. The internal standard is then subjected, together with the unknowns, to the same extraction protocol. see Table 3.
after weighing and before homogenization the tissue samples are kept frozen by keeping the Eppendorf vials afloat in a vessel with liquid nitrogen. Microdissection is performed by a punch technique. The punch technique was originally developed by Palkovits [ 3 11, but we introduced some practical improvements. Palkovits takes punches from frozen brain slices, whereas we use lyophilized brain slices. The way lyophilized brain slices are prepared is especially important, since in a conventional freeze-drier, brain slices become distorted and cracked. Therefore we use a - 20°C freezer that has a device to trap water. Lyophilizing this way does not change the shape or volume of the brain slices. The slices obtained are soft and elastic and punches can be easily made from them. We both used the conventional and the new punch technique on bilateral halves of the same coupes and found identical levels of VPir in the punched brain nuclei. Punch needles can be readily made of stainless hypodermic needles (Delvo, Switzerland). Needles are first made blunt end and then sharpened. As a shaft a short piece of a lo-ml syringe is used. Punches are removed from the lumen of the needle with a stainless steel rod. Depending on the size of the nucleus needles of different internal diameters can be chosen.
Brain tissue TABLE 5
Tissue is dissected either at the macrolevel or at the microlevel. Macrodissection is performed with a pair of sharp forceps (Inox No. 7 ). The brain is removed from the skull and dissected at 4°C generally according to Gispen et al. [ 30 ] into a number of different structures, such as the septum, hypothalamus, hippocampus, and medulla oblongata. For the dissection we make use of external landmarks, such as the sulcus rhinalis and chiasma opticum. Intern landmarks, such as the anterior commissure and corpus callosum are made visible by a transversal cut at the optic chiasm. Dissected brain structures are put in an Eppendorf vial and stored at - 80’ C. Before and
Extraction of vasopressin from brain tissue
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Homogenize brain tissue in 33 or more volumes 1 M acetic acid. Pipet I-ml portions of the homogenate into Eppendorfvials. Boil the homogenates for 5 min. Centrifuge at SOX lo3 Xg for 30 min. Pipet 0.85 ml of the supernatants into LP3 tubes. Vacuum evaporate until dryness in a Speed Vat Concentrator. Wash residues in I.3 ml ethanol/water (50: 50, v/v). Repeat 4. Decant ethanol/water fractions into polystyrene tubes. Repeat 6.
29 TABLE 6 Characteristics
of standard curves for different
Extraction
Tissue amount or volume of plasma
&, (%)
(amounts of) biological samples detection limit fmol/tube
NS (Oh1
r2
Apparent recovery (O/O)
Assay coefficient of variation (%) intra
no ethanol (1 1, 1, Vycor C8
buffer 1 mg tissue 2mg 4mg 8mg 2 ml plasma 1 ml plasma
48.1 47.4 46.0 44.3 41.8 42.2 20.0
3.1 3.8 3.8 4.3 5.7 5.1 6.2
0.19 0.24 0.25 0.29 0.41 0.36 0.50
1.7 1.8 3.3 3.0 3.3 0.5 2.0
0.999 0.996 0.997 0.995 0.998 0.998 0.999
100 82 82 72 54 61 96
inter
9.5 8.9 5.7 10.6 8.6 10.0
12.1 8.6 16.0
Diluent buffer used in the RIA consisted of the Verona1 buffer as described in section Incubation. Binding= (counts-NS)/TNS ), in which counts = non-adsorbed counts, NS = nonspecifically bound counts and T= total counts. Binding data were transformed according to Rodbard’s logit-log equation: In (B/ [&-B] ) = - n*lnVP + b, and the parameters of this equation were estimated with the least squares method. From these parameters EDSo was calculated according to ED,,= n/b. The limit of detection is defined as the amount of VP at which B=&-3*SD. Coefftcients of determination ( r2) were calculated for the logit-log equations. Apparent recovery: EDSo of internal standard curve as percentage of EDSo of external standard curve. Intra- and inter assay coefficients of variation (SD/mean) are based on 12 determinations on pools of plasma or tissue homogenate containing 4 fmol*ml-’ synthetic VP.
Tissue sample extraction
since the peptide is degraded dases only [ 35 1.
Vasopressin has mostly been extracted from tissue by the boiling acid method, [ 32,331. In the literature it is reported that these residues are soluble. It is our experience, however, that residues obtained this way are largely insoluble and a great deal of the peptide material is trapped. Extracted this way recovery depends on the tissue concentration of the homogenate. Some have used Vycor extraction after the boiling acid method [ 34,35 1, probably in an attempt to get rid of insoluble material and improve recovery. When we used Vycor extraction on brain tissue as described by Dogterom et al. [ 341, recovery still was very poor. We developed a simple additional step following acid extraction (see Table 5). Insoluble residues are washed in 50% ethanol/water. Recoveries of radio-iodinated VP after this two step extraction procedure are 85% and are independent of the tissue concentration in the range of 1 to 8 mg/ml homogenate. The use of acetic acid in the extraction procedure makes it unnecessary to use enzyme inhibitors to prevent degradation of VP during extraction,
Tissue internal standard preparation
by neutral pepti-
As tissue internal standard solutions we use a VPir free tissue homogenate to which known amounts of VP have been added. VPir free tissue homogenate is obtained by incubation of the homogenate for 3 h at 37°C prior to acidification to 1 M acetic acid. During incubation endogenous VP is degraded by amino- and endopeptidases [ 361. It must be assured that the tissue concentration in the unknown samples is the same as in the internal standards. From the internal standards VPir is extracted the same way as VPir is extracted from unknown samples. Table 6 gives the characteristics of some tissue- and plasma internal standard curves obtained with our methods. From these characteristics it is clear that internal and external standard curves were not superimposable, which strongly indicates that it is necessary to use internal standardization. Increasing the amount of tissue lowered the detection limit per mg tissue, but from Table 6 it can be concluded that it is not much use to mea-
30
sure in more than 8 mg tissue when the RIA is carried out in a volume of 100 ~1.
Radioimmunoassay for vasopressin To enhance the sensitivity of the radioimmunoassay we carry out the incubation of extract with label and antibody in a small volume. Especially when the K, of the antibody is low in comparison to the tracer concentration sensitivity can be improved by volume reduction. This increase in sensitivity is - as discussed before accompanied by concentration of interfering substances which extraction did not completely eliminate. To correct for nonspecific interference with the displacement of tracer from the antibody we routinely use internal standardization (see sections Plasma internal standard preparation and Tissue internal standard preparation).
Incubation Veronal/HSA buffer, pH 8.0, is used as a diluent and contains 20 mM Verona1 (5,5-diethylbarbituric acid, Merck, Germany), 10 mM Na,EDTA (Sigma), 0.14 M NaCl, 0.067 mM Lcystine (Sigma) and 4 mg HSA/ml (Sigma); all chemicals should be of analytical grade. To small plastic tubes is added 25 ~1 tracer solution containing 1 fmol [ lz51-Tyr2]VP (circa 2 nCi), 25 ~1 diluted antiserum Wl (final dilution 1: 512 X 103) and 50 ~1 appropriately diluted plasma- or tissue extract (standard- and unknown sample extracts). The tubes are incubated for 72 h. The whole procedure is carried out at 4” C. Alternatively, tracer is added 24 h after the addition of antiserum to the plasma- or tissue extract. This delayed tracer addition increases sensitivity 2-fold (data not shown),
Separation Separation of bound and free VP has been achieved by precipitation of the antibody with second antibody or polyethylene glycol, binding of antibody to second antibody coupled to a solid phase ( Sac-Cel) and adsorption of the free pep-
tide to coated charcoal. The use of coated charcoal and Sac-Cel is the simplest and both are used at our laboratories; precipitation by second antibody requires another 24 h before separation can be completed, polyethylene glycol precipitation involves the addition of y-globulin in order to assure precipitation. Since the use of dextran as a coating material of charcoal has been criticized [ 3 7 1, we tested Ficoll400 as a coating material. When Ficoll was used as a coating material instead of dextran binding was apparently 80% higher, whereas variation of the dextran- or Ficoll concentration hardly had any effect (Table 7 ) . This indicates that the separation of the free and bound fraction was incomplete with dextran-coated charcoal. Doubts could be raised as to whether coating is a real phenomenon. An alternative explanation could be that Ficoll merely reduces the surface area available for binding. This question was addressed by the determination of the binding of tracer and antibody to charcoal as a function of the charcoal concentration in the absence of, or at a fixed concentration of Ficoll (see Fig. 5 ). It appeared that Ficoll reduces the binding to charcoal of the free as well as the bound fraction, but much more so that of the bound. It can be concluded that coating is a real phenomenon and that Ficoll is a better coating material than dextran. The best TABLE I Characteristics conditions
of standard
curves for different
separation
Separation conditions
B, (%) EDSo (fmol/tube)
Detection limit (fmol/tube)
NS (%)
rz
1D” 6D 1F 3F 0.5D, 0.5F
25.1 26.6 46.2 49.8 48.1
0.32 0.34 0.23 0.20 0.19
0.6 1.7 1.9 1.4 1.7
0.973 0.983 0.998 0.991 0.999
2.7 3.0 3.6 3.4 3.1
“Codes refer to type and amount of coating material in the separation medium. ID= 7.5 mg dextran/ml, 6D=45 mg dextran/ml, lF=7.5 mg Ficoll/ml, 3F=22.5 mg/ml, 0.5D=3.75 mg dextran/ml, 0.5F= 3.75 mg Ficoll/ml. Characteristics were derived as mentioned in the legend of Table 6.
31
‘,t 60 t
-
40 200 ;
antibody
tracer
t
/
,,,,,
I
/
1
LJJe,,,i
1
10
100
1000
10000
Fig. 5. Effect of Ficoll coating of charcoal on binding of iodinated VP and antibody to charcoal as a function of the charcoal concentration. Abscissa: charcoal @g/tube). Ordinate: percentage binding of tracer or antibody. The incubation medium contained 0.1 mg HSA/ml and the separation medium contained no (0 ) or 3.75 mg Ficoll/ml (0 ).
overall results were obtained with Ficoll-dextran-coated charcoal (375 pg Ficoll 400 (Pharmacia, Uppsala, Sweden), 375 pg dextran T 70 (Sigma) and 4 mg charcoal in 100,~l of a 50 mM phosphate buffer, pH 7.4), see Table 7. Validation
The reliability of RIAs has been tested in a number of ways. None of these tests are sufficient. Therefore, as many as possible should be performed. 1. A classical criterion for demonstrating immunochemical identity of standard and unknown is represented by superimposibility of their dilution curves. The plasma internal standard dilution curve and the plasma extract dilution curve illustrate that this criterion was met for the measurement in plasma (Fig. 4 ). Likewise, this requirement was fulfilled for the measurement in brain tissue (results not shown). 2. Synthetic peptide and immunoreactive material should elute in the same fractions during high performance liquid chromatography and account for all the immunoreactivity measured in the RIA. Indeed, immunoreactive material in plasma extracts showed identical behavior with synthetic VP [ 28 1. However, up to 40% of the
immunoreactive material in extracts of extrahypothalamic brain structures did not elute with the intact molecule, but with VP fragments [ 381. 3. Physiological or pharmacological manipulations known to affect the level of the hormone should also affect the level of immunoreactive material. We tested the effects of a number of manipulations on the plasma VPir level in rats: dehydration, salt infusion, hemorrhage, i.p. ethanol, and i.c.v. histamine administration. As expected dehydration resulted in an increase of the plasma VPir level, see Fig. 6 [ 291. Moreover, no difference in VPir was detected by antiserum W 1 and W4 under normal (not deprived) and under water-deprived conditions, indicating that the majority of VPir in the plasma under both conditions is identical with the native VP( 1-9) molecule [ 29 1. Salt infusion resulted in a linear increase in the plasma VPir level with osmolality and hemorrhage resulted in an exponential increase with volume change (Fig. 7). Intraperitoneal ethanol administration had a biphasic effect on plasma VPir levels, see Fig. 8 [ 391, and intracerebroventricular as well as intraperitoneal injection of histamine stimulated plasma VPir dose-dependently, see Fig. 9 [ 40,4 11, respectively. It is not well established yet which physiological manipulations affect 15
10
5
0 Wl
w4
not deprived
Wl
w4
24 h water deprivatm
Fig. 6. Plasma VPir levels (fmol/ml+SEM) of non-deprived and waterdeprived animals, as measured using antisera W 1 and W4. Plasmas of the two groups were pooled each before extraction. Each bar represents 5 determinations.
32 60
r t abc
50
ab
Fig. 7. Responses of Lewis rats to graded blood volume reduction (7% reduction each 5 min), left panel, and salt infusion (i.v. infusion of a 9% salt solution at a rate of 30 pl/ min), right panel. Abscissa: left panel: percentage volume reduction; right panel: osmolality in mOsm. Ordinate: fmol VPir per ml plasma.
1
12,
Fig. 9. Plasma VPir levels (fmol/ml I? SEM) 5 min after intracerebroventricular injection of graded doses histamine. Abscissa: pg histamine. Each group consisted of 7 to 10 animals. One-way ANOVA (F(5,47)=5.9, p
n ETHANOL 0
q n
SALINE SHAM
TABLE 8 INJ
NOINJ /
”
Effect of castration on levels of VP ( fmol/mg protein f SEM) in extrahypothalamic brain regions of male Brown-Norway rats Region
Control
Castration
Septum Amygdala Hippocampus
121.5k5.6 37.8 + 3.0 7.5 I? 0.6
10.7t 1.3’ 8.9?2.9* 1.7kO.3’
*P
0
5
60
Fig. 8. Mean ( ?SEM) vasopressin levels (fmol/ml) in plasma from rats decapitated 5 or 6 min after i.p. ethanol (2 g/kg ethanol ( 15% v/v, absolute ethanol in 0.9% saline)), saline, or sham injection. Other rats (0 min after treatment) were neither injected nor disturbed until decapitation. *pcp
levels of VPir in the brain. A physiological manipulation that unequivocally affects VPir levels in some brain areas is castration [ 42 1. As assessed with our assay castration reduced the level of VPir 75-90% in the septum, amygdala and the hippocampus of male rats (Table 8 ). 4. Levels of VPir determined with antisera that
recognize different epitopes should be identical, if it is assumed that the VPir material consists mainly of the native VP- ( 1-9 ). In plasma extracts identical levels of N- and C-terminal immunoreactivity were detected before and after osmotic stimulation (see Fig. 6 ) . In the septum and amygdala of the brain a 40% higher C-terminal immunoreactivity was measured than Nterminal immunoreactivity (data not shown). Both immunoreactivities correlated highly (r2> 0.9, P
33
age of a fixed amount of ligand recovered, should be reproducible and ideally quantitative. However, a simple, cheap, and reproducible technique with a recovery of > 50% is preferable to a complex procedure with a recovery of, say, 95%. For plasma two techniques used at our laboratories are described. The Bond Elut C8 column extraction of VP gives a 96% recovery, but satisfactory results are obtained with Vycor extraction (6 1% recovery, see Table 6) with the important additional advantage of the concomitant production of plasma internal standard medium (SC.the supematant of Vycor extracted plasma). For tissue VPir determination a simple technique, including a centrifugation step at relatively high g-force and ethanol extraction gives a recovery of 52-82%, depending on the amount of tissue extracted (see Table 6 ) . 6. The coefficient of variation (CV) of repeated determinations of the same sample within one assay and between different assays, is an important parameter for determining the reproducibility of the procedure. As can be seen in Table 6, CVs are well within the generally accepted range.
Discussion Radioimmunoassay of VP is difficult, especially if one wishes to measure basal plasma levels. Antibodies with a K, much higher than 10” M-’ can hardly be raised, whereas basal plasma levels are as low as IO- ” M. Rather large volumes have to be concentrated to obtain enough VP for determination. When working with rats plasma volumes cannot exceed a few milliliters if one wishes to carry out determination in triplicate. To obtain a sensitive enough assay we optimized sample preparation, incubation and separation conditions, and only use tracer with a high specific activity and a small incubation volume. Thus, we were able to measure quantities of VPir as small as 0.2 fmol/ml plasma or 0.4 fmol VPir/mg protein or even lower when delayed tracer addition is used. Our assay is as sensitive as the most sensitive assays reported [ lo,13 1, though we use an antibody that is much less sensitive than these assays relied on. Our an-
tibody is of a sensitivity normally obtained for anti-VP antibodies. Therefore, the methods described herein are of general importance to the determination of VPir. Our assay differs from all but one assay in that it is internally standardized. Skowsky et al. [ 18 ] also used internal standardization and concluded that they did not need it. They extracted VP from 1 ml plasma and dissolved the residue in a volume of 1.5 ml for RIA. Others concentrated 5-20 ml - e.g. [ 17,24,43] - in a volume of 0.5-l .5 ml. Their assays probably suffered from nonspecific interferences, indicated by the fact that they measured detectable levels of VPir in overhydrated subjects, also stressed by Rooke and Baylis [ 13 1. Although a number of methods for the determination of VPir in brain structures have been described, these methods have been validated very poorly - e.g. [ 32,33,35]. The possible effects of matrix factors have not been tested; extraction recoveries have not been adequately assessed. By contrast, our assays described in this article correct for nonspecific interferences and we use simple extraction techniques with a high recovery. Material costs of the determination of one sample can be roughly estimated at twenty-live dollar cents. An analyst can determine VPir levels in 120 tissue samples or 200 plasma samples in two days. Thus we developed reliable, fast and cheap extraction techniques combined with a specific and sensitive RIA for the determination of vasopressin immunoreactivity. Several steps in the described procedures may be applicable in the field of immunoassays for (small) peptides. Although many criteria can be met, as stated in section Validation no assay is perfect. Different RIAs (using different antibodies and/or different extraction techniques) for the same ligand will reflect different aspects of the same material. Even if results are not exactly ‘right’ according to some (hypothetical) absolute reference the RIA is still immensely valuable if it serves to distinguish, on a routine base, between normal and stimulated or decreased levels of the substance under study. Therefore, although RIA should be treated as a goal it is a tool.
34
Acknowledgements
12
The authors wish to thank Mrs. E. AndringaBakker, MS C. Maigret, Mrs I. Koekkoek-Van den Herik, Mrs. M. Markerink-Van Ittersum, Dr. M. Van Lookeren Campagne, Drs. P. Bijlsma, and Drs. T. Van Teunenbroek for their valuable contributions to this work. Parts of the studies were subsidized by the Dutch FUNGO project no. 1335-042 and b ‘Y NIAAA-National Research Service Award-05 196.
13
References
16
I
2
3
4
5
6
7
8
9
10
11
R.S. Yalow alnd S.A. Berson, Assay of plasma insulin in human subjects by immunological methods, Nature, 184 (1959) 1648-1649. T.B. Van Wimersma Greidanus and J.P.H. Burbach, Vasopressin and oxytocin their presence in the central nervous system and their functional significance in brain processes related to behaviour and memory, Acta Endocrinol. Suppl. 276 (1986) 85-94. T.B. Van Wimersma Greidanus and J.M. Van Ree, Behavioral effects of vasopressin, Curr. Top. Neuroendocrinol. 10 (1990) 61-79. J. Dogterom, T.B. Van Wimersma Greidanus and D. De Wied, Vasopressin in cerebrospinal fluid and plasma of man, dog and rat, Am. J. Physiol. 234 (1978) E463E467. T.L. Goodfriend, L. Levine and G.D. Fasman, Antibodies to bradykinin and angiotensin: a use of carbodiimides in immunology, Science 144 ( 1964) 1344-l 346. W.R. Skowsky and D.A. Fisher, The use of thyroglobulin to induce antigenicity to small molecules, J. Lab. Clin. Med. 80 (1972) 134-144. S.N. Oyama, A. Kagan and S.M. Click, Radioimmunoassay of vasopressin: application to unextracted human urine, J. Clin. Endocrinol. Metab. 33 ( 197 1) 739744. J.J. Morton and M.A. Waite, The possible relationship between the afftnity of arginine vasopressin antibodies and the degree of polyuria and polydipsia in actively immunized rabbits, J. Endocrinol. 54 ( 1972) 523-524. G.L. Robertson, L.A. Klein, J. Roth and P. Gorden, Immunoassay of plasma vasopressin in man, Proc. Natl. Acad. Sci. USA 66 (1970) 1298-1305. G.L. Robertson, E.A. Mahr, S. Athar and T. Sinha, Development and clinical application of a new method for the radioimmunoassay of arginine vasopressin in human plasma, J. Clin. Invest. 52 ( 1973) 2340-2352. P. Rooke and P.H. Baylis, Effect of plasma interference on different vasopressin antisera, J. Immunoassay 3 (1983) 133-143.
14
IS
17
18
19
20
21
22
23
24
25
26
F. Fyhrquist, M. Wallenius and H.J.G. Hollemans, Radioimmunoassay of vasopressin in unextracted plasma, Stand. J. Clin. Lab. Invest. 36 (1976) 841-847. P. Rooke and P.H. Baylis, A new sensitive radioimmunoassay for plasma arginine vasopressin, J. Immunoassay3 (1982) 115-131. F.C. Greenwood, W.M. Hunter and J.S. Glover, The preparation of ‘3’I-labelled human growth hormone with a high specific radioactivity, Biochem. J. 89 ( 1963) 114123. P.R.P. Salacinski, C. McLean, J.E.C. Sykes, V.V. Clemont-Jones and P.J. Lowry, Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3~u,6c~,-diphenyl glycoluril, Anal. Biochem. 117 (1981) 136-146. M.K. Husain, N. Fernando, M. Shapiro, A. Kagan and S.M. Glick, Radioimmunoassay of arginine vasopressin in human plasma, J. Clin. Endocrinol. Metab. 37 ( 1973) 616-625. C.G. Beardwell, Radioimmunoassay of arginine vasopressin in human plasma, J. Clin. Endocrinol. Metab. 33 (1971)254-260. W.R. Skowsky, A.A. Rosenbloom and D.A. Fisher, Radioimmunoassay measurement of arginine vasopressin in serum: development and application, J. Clin. Endocrinol. Metab. 38 (1974) 278-287. P. Czernichow, U. Merkelbach and M.B. Valloton, Radioimmunoassay of [ %arginine]-vasopressin. Acta Endocrinol. 80 (1975) 440-452. J. Roth, S.M. Glick, L.A. Klein and M.J. Peterson, Specific antibodies to vasopressin in man, J. Clin. Endocrinol. Metab. 25 (1966) 671-675. N.G. Seidah, M. Dennis, P. Corvol, J. Rochemont and M. Chretien, A rapid high-performance liquid chromatography purification method of iodinated polypeptide hormones, Anal. Biochem. 109 (1980) 185-191. H. Tuppy and A. Nesvadba, Uber die Aminopeptidaseaktivitit des Schwangerenserums und ihre Beziehung zu dessen Vermogen, Oxytocin zu inaktivieren, Md. Chem. 88 (1957) 977-988. J.M. Davison, E.A. Gilmore, J. Durr, G.L. Robertson and M.D. Lindheimer, Altered osmotic thresholds for vasopressin secretion and thirst in human pregnancy, Am. J. Physiol. 246 (1984) FlOS-F109. J.J. Morton, P.L. Padlield and M.L. Forsling, A radioimmunoassay for plasma-vasopressin in man and dog: application to physiological and pathological states, J. Endocrinol. 65 (1975) 41 l-424. F.T. La Rochelle Jr, W.G. North and P. Stern, A new extraction of arginine vasopressin from blood: the use of octadecasyl-silica, Pfltigers Arch. 387 ( 1980) 79-8 1. M. Bevilacqua, R. Meroni, R. Dagani, E. Renesto, C. Baruto and G. Norbiato, Role ofblood osmolality in the regulation of vasopressin secretion in man: application of a new radioimmunoassay method for vasopressin, J. Endocrinol. Invest. 8 (1985) 97-101.
35 27
28
29
30
31 32 33
34
35
MS. Brownfield, J. Greathouse, S.A. Lorens, J. Armstrong, J.H. Urban and L.D. Van de Kar, Neuropharmacological characterization of serotonergic stimulation of vasopressin secretion in conscious rats, Neuroendocrinology 47 (1988) 277-283. B.J.M. Van de Heijning, I. Koekkoek-Van de Herik, T. Ivany and T.B. Van Wimersma Greidanus, Solid phase extraction of plasma vasopressin: evaluation, validation and application, J. Chromatogr. 565 ( 199 1) 159171. J.A. Ten Haaf, Studies on Hormone Release from the Neurointermediate Lobe of the Rat Pituitary Gland, Thesis, University of Utrecht, The Netherlands, 1990. W.H. Gispen, P. Schotman and E.R. De Kloet, Brain RNA and hypophysectomy; a topographical study, Ncuroendocrinology 9 ( 1972) 285-296. M. Palkovits, Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Res. 59 (1973) 449-450. S.M. Glick and M.J. Brownstein, Vasopressin content ofrat brain, Life Sci. 27 (1980) 1103-l 110. R.E. Brinton, P.P. Desmukh, A. Chan, T.P. Davis, S. Hsiao and HI. Yamamura, A non-equilibrium 24 hour vasopressin radioimmunoassay: development and basal levels in the rat brain, Brain Res. 266 (1983) 344-347. J. Dogterom, F.G.M. Snijdewint and R.M. Buijs, The distribution of vasopressin and oxytocin in the rat brain, Neurosci. Lett. 9 (1978) 341-346. J. Hawthorn, V.T.Y. Ang and J.S. Jenkins, Localization of vasopressin in rat brain, Brain Res. 197 ( 1980) 7.581.
36
37
38
39
40
41
42
43
J.P.H. Burbach, D. Terwel and J.L.M. Lebouille, Measurement and distribution of vasopressin-converting aminopeptidase-activity in rat brain, Biochem. Biophys. Res. Commun. 144 ( 1987) 726-73 1. M.A. Binoux and W.D. Odell, Use of dextran-coated charcoal to separate antibody-bound from free hormone: a critique, J. Clin. Endocrinol. Metab. 36 ( 1973) 303-310. J.P.H. Burbach, X.-C. Wang, J.A. Ten Haaf and D. De Wied, Substances resembling C-terminal vasopressin fragments are present in the brain but not in the pituitarygland, Brain Res. 306 ( 1984) 384-387. D.H. Colbern, J.A. Ten Haaf, B. Tabakoff and T.B. Van Wimersma Greidanus, Ethanol increases plasma vasopressin shortly after intraperitoneal injection in rats, Life Sci. 37 (1985) 1029-1032. J.A. Ten Haaf, T.B. Van Wimersma Greidanus, C. Maigret and D. De Wied, Effect of the opioid peptide @-endorphin on the in vivo release of vasopressin in rats under various conditions, Neuroendocrinology 44 ( 1986) 102-107. J. Dogterom, T.B. Van Wimersma Greidanus and D. De Wied, Histamine is an extremely potent releaser of vasopressin in the rat, Experientia 32 ( 1976) 659. G.J. De Vries, R.M. Buijs, F.W. Van Leeuwen, A.R. Gaffe and D.F. Swaab, The vasopressinergic innervation of the brain in normal and castrated rats, J. Camp. Neurol. 233 ( 1985) 236-254. C.I. Johnston, Radioimmunoassay for plasma antidiuretic hormone, J. Endocrinol. 52 (1972) 69-78.
23 016%3659/92/$05.00
COREL 00736
Radioimmunoassay, a goal or a tool? The setup of a reliable, fast, and cheap radioimmunoassay for vasopressin in biological samples Jeroen A. Ten Haaf”, Dirk Terwel”, Bert J.M. Van de Heijningb and Tjeerd B. Van Wimersma Greidanusb aDepartment ofNeuropsychology
and Psychobiology, University ofLimburg, Maastricht, Netherlands; bRudolfMagnus for Pharmacology, Utrecht, Netherlands
(Received
Key words: Vasopressin; standard
17 September
Institute
199 I ; accepted in revised form 4 February 1992 )
Radioimmunoassay;
Plasma determination;
Introduction The introduction of the radioimmunoassay (RIA) by Yalow and Berson [ 1 ] has been of great importance to almost the whole field of clinical and biological sciences. In principle it enabled researchers to determine the level of every biological substance against which an antibody can be raised. Basically RIA is the comparison of the competition between a known amount of radio-iodinated ligand (so-called tracer) and an unknown amount of unlabeled ligand on the one hand with the competition between standard amounts of unlabeled and labeled ligand on the other hand for a known amount of antibody. Binding is plotted against known amount of ligand and unknown amounts are determined by interpolation (Fig. 2). The highest sensitivity of the assay is obtained at a concentration of antiserum at which 50% of the tracer is bound. This concentration is deterCorrespondence to: J.A. ten Haaf, Dept. of Neuropsychology and Psychobiology, P.O. Box 616, 6200 MD Maastricht, Netherlands.
Tissue determination;
Internal
mined by serial dilution of antiserum (Fig. 2). As a source of antibodies can be used either serum of immunized rabbits or larger animals, or the culture medium of hybridomas of lymphocytes of immunized mice and myeloma cells. If other methods of assay are already available RIA is generally more efficient and sensitive. However, together with improvement in terms of efficiency and sensitivity, the RIA, since it is an indirect method, confronted researchers with the problem of interference by specific and nonspecific matrix factors present in biological samples. The level of nonspecific interferences is generally reduced by extraction of the substance of interest prior to RIA. However, since the RIA usually is performed on large numbers of samples on a routine basis, extraction procedures should be quick and simple and consequently sample extracts still contain (a great deal of) impurities. In order to anticipate this pitfall, Yalow and Berson state that the effect of interfering substances should be tested and that it must be assured that the milieu of the unknown sample is identical with that of the known standards. With respect to the other problem, the presence
24
in the matrix of substances resembling the ligand of interest (specific interference), the use of antibodies with a high affinity for the specific ligand and low cross-reactivity with resembling substances is a prerequisite for reliable RIA. The present article will describe our experience with the setup and the routine use of a RIA for arginine-vasopressin (VP), see Fig. 1. This neurohypophyseal peptide, besides its classical peripheral vasopressor and antidiuretic functions, is thought to play a role in learning and memory processes in the brain [ 2,3 1. Since these functions are within the scope of our research, a reliable and efficient protocol for determination of VP levels in biological samples is a fundamental analytical tool. RIAs for VP are considered difficult for a number of reasons: ( 1) VP is a small peptide (9 amino-acids) and therefore a poor antigen, (2) its level in the circulation and some brain regions is extremely low (in plasma of the Wistar rat about l-2 fmol VP/ml, in humans sometimes even lower [ 4]), so its determination is relatively sensitive to matrix-interference, and (3) it is sensitive to endogenous peptidase activity. The purpose of this study was to raise in rabbits sensitive and specific antisera to VP, to develop reliable protocols for the sampling of (brain) tissue and blood plasma from rats or humans, to extract VP from these matrices and to determine VP by RIA in a reproducible way. In addition, we will discuss some of the experiences of others with the determination of VP.
cys----s-s--1
Cys-Pro-Arg-Gly-NH2 ‘ 8 7
9
Fig. 1. The 9-amino acid arginine-vasopressin molecule: position I and 6 are interconnected by a disulphide bridge to form the characteristic ring structure. The tyrosine-residue, which is [ ‘*‘I ] iodinated during the radiolabeling procedure, is indicated by an asterisk. Theoretically, monoclonal antibodies will recognize one specific site, the polyclonal antibody subspecies are directed towards more sites of the molecule (see also Fig. 3 ).
Production of antisera against vasopressin The antigenicity of small peptides can be enhanced by conjugation to a protein carrier [ 5 1. Several investigators have raised antibodies against a conjugate of VP and some carrier, either coupled with glutaraldehyde or carbodiimide [ 671. Rabbits that produce a high affinity antibody against VP become polyuric [ 8 1. Some of the antibodies raised are susceptible to proteins and salts [ 9- 111 . This warns against the direct measurement of VP in plasma or tissue samples. To date direct measurement has only been attempted with success once [ 121. When applying direct measurement special measures have to be taken to prevent slow degradation of VP. Fyhrquist et al. [ 12 ] used s-aminocaproic acid for this purpose. We raised antisera in six male white New Zealand rabbits (code W l-W6). VP (arginine vasopressin, pressor activity: 500 I.U./mg, obtained from Organon, Oss, The Netherlands) was conjugated to bovine thyroglobulin (Type I, Sigma? St, Louis, U.S.A.) with carbodiimide (Sigma), generally according to Skowsky and Fisher [6]. Prior to the injection the conjugate was freshly emulsified 3 : 4 in complete Freund’s adjuvant (Difco Laboratories, Detroit, U.S.A. ). Six injections of 0.5 ml emulsion, containing 0.25 mg VP, were given intramuscularly with a two week interval. From then, every two months a booster injection was given. Small portions of blood (circa 3 ml) of each animal were harvested one week after each injection by puncture of the lateral ear vein, allowed to clot for 2 h at 4’ C in polypropylene tubes, spun down (2000 g, 30 min, 4”C), and stored in 100 ~1 portions at - 20°C. A fresh portion was used to test binding capacity and titre (see Fig. 2 ) , for details see Radioimmunoassay for vasopressin. If an antiserum met the test criteria (maximal binding > 85% and a titre < 1:40,000), the animal was anaesthetized and all the blood collected. The specificities of the two antisera that met the test criteria, W 1 and W4, were tested by determining the displacement of tracer from the antibody by a variety of substances (peptides), more or less resembling VP (e.g. oxytocin, VP fragments, and
25 TABLE 1 Protocol for the radio-iodination 1.
2. 3. 4. Fig. 2. Left panel: Dilution curves for two antisera (W 1 and W4). Abscissa: dilution factor (X 10d3); Ordinate: percentage binding of tracer to serum. Right panel: Displacement curves of antisera W 1 and W4. Abscissa: fmol VP/tube added to incubation; ordinate: percentage binding of tracer.
of VP
Add to a 12 X 75 mm glass tube: 50 ~1 of 0.5 M phosphate buffer, pH 7.5, 10 c(g peptide dissolved in 10 ~10.2 M acetic acid, 1 mCi NarZS1dissolved in 10 ~1 dilute NaOH solution (Amersham, UK), 40 ,ug chloramine-T dissolved in 10 ~1 50 mM phosphate, pH 7.5. Mix the contents of the tube with a few finger flicks. After 60 s add 1 ml of a suspension of 150 mg Dowex 1 X8 (Cl- form). Transfer the supernatant to a Sephadex G-25 column (45 cm~0.9 cm) equilibrated with a solution containing 0.01 M acetic acid and 1.25 mg BSA/ml. Elute with the same solution at a rate of 12 ml/h.
be expressed as “VP-immunoreactivity (VPir ) levels”, rather than “VP levels”. Most of the antibodies reported in the literature have a K, near 10” M-l. Few antibodies have been obtained with a K, higher than lOI M-’ [7,13]. Wl has aK,of1.55x10”M-‘;W4hasoneof10”M-’. Antibodies Glick-1, Skowsky-7 1 and W 1 have been used in numerous studies.
Iodination of vasopressin t fmol .; ;
Dmol 10
1001000
1
I 10
pmol 1
lfmol .;
;
lb
loolob
;
lb
Fig. 3. Cross reactivities of antisera Wl (left panel) and W4 (right panel) with vasopressin fragments and oxytocin. Abscissa: fmol (or pmol) added peptide/tube in RIA. Ordinate: Logit log transformation of percentage antibody-tracer displacement. Numbers refer to vasopressin fragments (e.g. l9: VP-( l-9); pGlu4-9: [pyroGlu4, Cyt6]VP-(4-9); OT: oxytocin). VP-fragments were kindly provided by Organon Oss, the Netherlands. Crossreactivity on molar basis (as percentage of EDSo of VP-( l-9) ) of the fragments are as follows: Wl: VP-(2-9): 56; VP-(3-9): 25; VP-(4-9): 20; VP-(5-9): 13; VP-(7-9): 0.05; OT: 0.004; VP-( l-8): 0.004; W4: VP(3-9):
other peptides, see Fig. 3 ). W 1 is directed against the C-terminus of the vasopressin molecule, W4 to the C- as well as the N-terminus. Although we obtained highly specific antibodies, one has to keep in mind that data obtained by RIA should
In the radio-iodination of VP generally chloramine-T is used as the oxidizing agent of iodine [ 141. Despite the fact that Iodogen has been reported to be a milder oxidant than chloramine-T [ 151, which of the two is used does not result in differences in immunogenicity of the tracer. Iodination conditions should be chosen such as to prevent formation of the diiodinated species, since this species is unstable [ 161. The iodination reaction is stopped by scavenging the iodide with Dowex 1 x 8 ( 100-200 mesh in Cl- form) or another anion-exchange resin [ 17- 191 or by the addition of serum albumin [ 201. Metabisulphite can not be used to this end, since it will reduce the disulphide bridge of the VP molecule. After iodination free iodide, labeled and unlabeled components in the reaction mixture can readily be separated by high performance liquid chromatography [ 2 11, although good separation can also be obtained by chromatography on a
26
Sephadex G-25 column when eluting with low concentrations acetic acid containing 1.25 mg serum albumin per ml to prevent adsorption of the peptide to the column [ 19 1. Table 1 gives a detailed protocol for the iodination of VP. Instead of going through these quite laborious procedures, to date we obtain iodinated VP of high specific activity commercially ( Amersham) .
Collection/dissection and extraction of biological samples Plasma Plasma preparation Plasma of rats is obtained according to the protocol described in Table 2. Blood from human subjects is collected into ice-cold Greiner Vacuette (Greiner, Alphen a/d Rijn, The Netherlands) tubes to which we added 5 mg heparin dissolved in 100 ~1 water by injection in advance. Subsequently, the tubes are placed on ice. No further precautions are needed to prevent degradation of VP, except when blood of pregnant women is collected. Pregnancy plasma contains high levels of cystine-aminopeptidase activity [ 221. This activity can be inhibited TABLE 2 Plasma sample preparation; all steps should be carried out at 4°C unless indicated otherwise 1.
2.
3.
4.
5. 6.
For reasons of reproducibility, transport animals at least 30 min before decapitation to the (quiet) room where they will be decapitated. Experiments should always take place around the same time of day. Add 100 ,ul Hz0 to which 5 mg heparin (Leo, Ballerup, Denmark) is added, into ice-cold tubes (polypropylene or polyethylene). Place plastic funnels in the tubes. Take the animal from its cage quietly, decapitate it quickly, and collect the trunk blood (8- 10 ml) in the tube. Close tube and rotate it immediately by hand to mix the blood with the heparin. Rotate gently in order to prevent hemolysis. Place the tubes immediately on ice until centrifugation within 1 h. Centrifuge tubes (2000 xg, 30 min). Collect plasma in tubes and store at - 20°C until use.
sufficiently by addition of 0.1 ml of a solution of 60 mg/ml 1, lo-phenanthroline [ 231 to the tubes. This inhibitor does not interfere with the radioimmunoassay. Plasma sample extraction VP has been extracted from plasma in various ways. Originally, gel filtration was used to separate VP from other plasma components [ lo]. This laborious method was soon replaced by organic solvents extraction using acetone and petroleum ether [ 10,161 or extraction by adsorption to a solid phase, such as glass powder [4,13,17,24] or diatomaceous earth [ 181. Recently, extraction on C 18- or C8 cartridges has come into use [ 25-28 1. At our laboratories we use either extraction with Vycor glass powder, principally according to Dogterom et al. [ 41, or extraction on Bond Elut C8 cartridges (LCR series, Analytichem Int., Sopar Biochem) [28]. The recovery of extraction with Vycor is 75%; extraction on Bond Elut C8 cartridges is 96% (see Table 6 ) . Tables 3 and 4 provide protocols for both extraction techniques. Plasma internal standard preparation More or less dependent on the type of matrix and adsorbent used for this purpose, an additional but important phenomenon is the co-extraction of matrix factors, that will interfere in most cases with antibody-ligand binding characteristics, leading to erroneous data if one does not anticipate [ 10,11,29 1. Therefore, in the development of an extraction protocol special attention must be given to account for this. In our opinion, the only right approach is to include a so-called internal standard at the start of the extraction procedure that is subjected to the same technical manipulations as the unknown samples. A concomitant advantage of this strategy is the absence of the need to correct final data for recovery (e.g. due to volume losses or efficiency of the adsorbent) once the procedure is implemented. Nevertheless it will always be necessary to determine the recovery percentage (by comparing the internal standard displacement curve
27 TABLE 3
TABLE 4
Procedure for the Vycor-extraction of vasopressin from plasma; all steps should be carried out at 4”C, unless indicated otherwise; use polystyrene tubes resistant to 60°h acetone
Extraction of vasopressin from plasma samples by solid phase Bond Elut C&silica columns
1.
2.
3.
4.
5.
6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17.
Vycor glass powder (mesh 140, Corning Glass Works, New York, U.S.A.), should be activated at 700°C for at least 24 h in advance of the experiment. Vycor can be kept indefinitely at 700°C. Thaw the frozen stocks as described in Table 2 at 4°C. Centrifuge stocks (2000 X g, 10 min) to spin coagulated proteins down. Prepare from stocks 0.5-2 ml plasma volumes (standard medium and unknowns) in tubes (preferably in triplicate). Standards and unknowns should have the same volume. Add to the standard medium tubes 50 ~1 Verona1 buffer (see section Incubation), containing no - for blank and nonspecific determination - or known amounts of VP (0.125-64 fmol). Add to each tube 100 ~1 from a constantly stirred Vycar suspension (2 g Vycor per 10 ml H20, 4°C). Vortex. Let the tubes tumble in a rotary tumbler for 30 min. Centrifuge tubes (2OOOxg, 10 min). Discard supernatants of standards by suction. Decant and pool supernatants of unknowns, and store these at -20°C. This material can be used as internal standard medium in a next extraction procedure. Resuspend the Vycor pellets in 0.5 ml HZ0 by Vortex. Centrifuge at 2000 xg for 10 min. Remove the supernatant by suction. Resuspend the Vycor pellets in 0.5 ml 2 N HCl by Vortex. Centrifuge at 2000~gfor 10 min. Remove the supernatant by suction. Resuspend the Vycor pellets in 0.5 ml acetone/H,0 (60: 40, v/v) by Vortex. Let the tubes tumble in a rotary tumbler for 30 min. Centrifuge at 2000 Xg for 10 min. Decant the acetone/H,0 fractions into plastic tubes. Vacuum evaporate until dryness in a Speed Vat.
with the external standard curve, see Table 6) as a check for overall performance of the extraction procedure and subsequent RIA. The ideal internal standard medium should contain exactly the same matrix, devoid of any trace of the specific l&and. With some exceptions (e.g. plasma of the Brattleboro rat), plasma or animals must be pretreated in order to get rid of the ligand to obtain the internal standard medium: either “pre-ex-
I.
6. 7.
8.
Connect C8 columns to a vacuum manifold column processor and activate columns by passing 4 ml methanol. Wash with ml H20. Do not let columns run dry. Add 4 ml 0.1 M HCl to 1 ml acidified (HAc) plasma (standards and unknowns). Apply this mixture to the column. Allow at least 5 min for the mixture to run through. Apply 4 ml 10% (vol/vol) acetonitrile in 0.1% trifluoro-acetic acid (TFA) to wash impurities from the column. Elute with 1.5 ml 60% acetonitrile in 0.1% TFA. Preserve the eluate for step 8. Cleanse the column with 4 ml 1OOI acetonitrile, 4 ml 8 M urea, and 4 ml HzO. Columns can be reused up to 20 times. Eluates are dried in a Speed Vat Concentrator (Savant). Residues are diluted and subjected to RIA (section Incubation).
traction” of plasma or over-hydration of animals can be a tool for this purpose. Note that each time a plasma extraction is carried out, the Vycor supematants of the unknown samples can be used as internal plasma standard medium in a next extraction procedure (see Table 3). From Fig. 4 it is clear that the displacement curve obtained by dilution of 24 h water-deprived normal rat plasma exactly parallels the internal standard curve prepared in Brattleboro plasma. The standard curves prepared from plasma of overhydrated normal rats and from Vycor pre-extracted normal plasma also perfectly parallel the curve prepared from Brattleboro plasma in Fig. 4 (data not shown). Furthermore, there is no difference in the respective blank and nonspecific binding values between the internal standard series prepared from Brattleboro plasma, “hydrated” normal plasma, and Vycor pre-extracted normal plasma. Therefore, Vycor pre-extracted normal plasma, plasma from overhydrated animals, as well as Brattleboro plasma can serve as internal standard medium. However, since a single standard tube prepared from Brattleboro plasma or “hydrated” normal plasma accounts for approximately one rat, this approach
28 50 ,
I
J
oi----
0
----I
01
““““I
“.““‘I
1
““‘.‘I
10
100
“...-I
1000
Fig. 4. Displacement of tracer from antibody by internal standard extracts (A ) and extracts of serial dilutions of plasma from a 24 h waterdeprived rat (A ). Brattleboro plasma was used as standard medium and as diluent.
is both not ethical and economically not affordable on a routine scale if one includes enough internal standards to obtain reliable results. For the setup of an internal standard series we add known amounts of vasopressin to pre-extracted plasma, obtained as described in Table 3. The internal standard is then subjected, together with the unknowns, to the same extraction protocol. see Table 3.
after weighing and before homogenization the tissue samples are kept frozen by keeping the Eppendorf vials afloat in a vessel with liquid nitrogen. Microdissection is performed by a punch technique. The punch technique was originally developed by Palkovits [ 3 11, but we introduced some practical improvements. Palkovits takes punches from frozen brain slices, whereas we use lyophilized brain slices. The way lyophilized brain slices are prepared is especially important, since in a conventional freeze-drier, brain slices become distorted and cracked. Therefore we use a - 20°C freezer that has a device to trap water. Lyophilizing this way does not change the shape or volume of the brain slices. The slices obtained are soft and elastic and punches can be easily made from them. We both used the conventional and the new punch technique on bilateral halves of the same coupes and found identical levels of VPir in the punched brain nuclei. Punch needles can be readily made of stainless hypodermic needles (Delvo, Switzerland). Needles are first made blunt end and then sharpened. As a shaft a short piece of a lo-ml syringe is used. Punches are removed from the lumen of the needle with a stainless steel rod. Depending on the size of the nucleus needles of different internal diameters can be chosen.
Brain tissue TABLE 5
Tissue is dissected either at the macrolevel or at the microlevel. Macrodissection is performed with a pair of sharp forceps (Inox No. 7 ). The brain is removed from the skull and dissected at 4°C generally according to Gispen et al. [ 30 ] into a number of different structures, such as the septum, hypothalamus, hippocampus, and medulla oblongata. For the dissection we make use of external landmarks, such as the sulcus rhinalis and chiasma opticum. Intern landmarks, such as the anterior commissure and corpus callosum are made visible by a transversal cut at the optic chiasm. Dissected brain structures are put in an Eppendorf vial and stored at - 80’ C. Before and
Extraction of vasopressin from brain tissue
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Homogenize brain tissue in 33 or more volumes 1 M acetic acid. Pipet I-ml portions of the homogenate into Eppendorfvials. Boil the homogenates for 5 min. Centrifuge at SOX lo3 Xg for 30 min. Pipet 0.85 ml of the supernatants into LP3 tubes. Vacuum evaporate until dryness in a Speed Vat Concentrator. Wash residues in I.3 ml ethanol/water (50: 50, v/v). Repeat 4. Decant ethanol/water fractions into polystyrene tubes. Repeat 6.
29 TABLE 6 Characteristics
of standard curves for different
Extraction
Tissue amount or volume of plasma
&, (%)
(amounts of) biological samples detection limit fmol/tube
NS (Oh1
r2
Apparent recovery (O/O)
Assay coefficient of variation (%) intra
no ethanol (1 1, 1, Vycor C8
buffer 1 mg tissue 2mg 4mg 8mg 2 ml plasma 1 ml plasma
48.1 47.4 46.0 44.3 41.8 42.2 20.0
3.1 3.8 3.8 4.3 5.7 5.1 6.2
0.19 0.24 0.25 0.29 0.41 0.36 0.50
1.7 1.8 3.3 3.0 3.3 0.5 2.0
0.999 0.996 0.997 0.995 0.998 0.998 0.999
100 82 82 72 54 61 96
inter
9.5 8.9 5.7 10.6 8.6 10.0
12.1 8.6 16.0
Diluent buffer used in the RIA consisted of the Verona1 buffer as described in section Incubation. Binding= (counts-NS)/TNS ), in which counts = non-adsorbed counts, NS = nonspecifically bound counts and T= total counts. Binding data were transformed according to Rodbard’s logit-log equation: In (B/ [&-B] ) = - n*lnVP + b, and the parameters of this equation were estimated with the least squares method. From these parameters EDSo was calculated according to ED,,= n/b. The limit of detection is defined as the amount of VP at which B=&-3*SD. Coefftcients of determination ( r2) were calculated for the logit-log equations. Apparent recovery: EDSo of internal standard curve as percentage of EDSo of external standard curve. Intra- and inter assay coefficients of variation (SD/mean) are based on 12 determinations on pools of plasma or tissue homogenate containing 4 fmol*ml-’ synthetic VP.
Tissue sample extraction
since the peptide is degraded dases only [ 35 1.
Vasopressin has mostly been extracted from tissue by the boiling acid method, [ 32,331. In the literature it is reported that these residues are soluble. It is our experience, however, that residues obtained this way are largely insoluble and a great deal of the peptide material is trapped. Extracted this way recovery depends on the tissue concentration of the homogenate. Some have used Vycor extraction after the boiling acid method [ 34,35 1, probably in an attempt to get rid of insoluble material and improve recovery. When we used Vycor extraction on brain tissue as described by Dogterom et al. [ 341, recovery still was very poor. We developed a simple additional step following acid extraction (see Table 5). Insoluble residues are washed in 50% ethanol/water. Recoveries of radio-iodinated VP after this two step extraction procedure are 85% and are independent of the tissue concentration in the range of 1 to 8 mg/ml homogenate. The use of acetic acid in the extraction procedure makes it unnecessary to use enzyme inhibitors to prevent degradation of VP during extraction,
Tissue internal standard preparation
by neutral pepti-
As tissue internal standard solutions we use a VPir free tissue homogenate to which known amounts of VP have been added. VPir free tissue homogenate is obtained by incubation of the homogenate for 3 h at 37°C prior to acidification to 1 M acetic acid. During incubation endogenous VP is degraded by amino- and endopeptidases [ 361. It must be assured that the tissue concentration in the unknown samples is the same as in the internal standards. From the internal standards VPir is extracted the same way as VPir is extracted from unknown samples. Table 6 gives the characteristics of some tissue- and plasma internal standard curves obtained with our methods. From these characteristics it is clear that internal and external standard curves were not superimposable, which strongly indicates that it is necessary to use internal standardization. Increasing the amount of tissue lowered the detection limit per mg tissue, but from Table 6 it can be concluded that it is not much use to mea-
30
sure in more than 8 mg tissue when the RIA is carried out in a volume of 100 ~1.
Radioimmunoassay for vasopressin To enhance the sensitivity of the radioimmunoassay we carry out the incubation of extract with label and antibody in a small volume. Especially when the K, of the antibody is low in comparison to the tracer concentration sensitivity can be improved by volume reduction. This increase in sensitivity is - as discussed before accompanied by concentration of interfering substances which extraction did not completely eliminate. To correct for nonspecific interference with the displacement of tracer from the antibody we routinely use internal standardization (see sections Plasma internal standard preparation and Tissue internal standard preparation).
Incubation Veronal/HSA buffer, pH 8.0, is used as a diluent and contains 20 mM Verona1 (5,5-diethylbarbituric acid, Merck, Germany), 10 mM Na,EDTA (Sigma), 0.14 M NaCl, 0.067 mM Lcystine (Sigma) and 4 mg HSA/ml (Sigma); all chemicals should be of analytical grade. To small plastic tubes is added 25 ~1 tracer solution containing 1 fmol [ lz51-Tyr2]VP (circa 2 nCi), 25 ~1 diluted antiserum Wl (final dilution 1: 512 X 103) and 50 ~1 appropriately diluted plasma- or tissue extract (standard- and unknown sample extracts). The tubes are incubated for 72 h. The whole procedure is carried out at 4” C. Alternatively, tracer is added 24 h after the addition of antiserum to the plasma- or tissue extract. This delayed tracer addition increases sensitivity 2-fold (data not shown),
Separation Separation of bound and free VP has been achieved by precipitation of the antibody with second antibody or polyethylene glycol, binding of antibody to second antibody coupled to a solid phase ( Sac-Cel) and adsorption of the free pep-
tide to coated charcoal. The use of coated charcoal and Sac-Cel is the simplest and both are used at our laboratories; precipitation by second antibody requires another 24 h before separation can be completed, polyethylene glycol precipitation involves the addition of y-globulin in order to assure precipitation. Since the use of dextran as a coating material of charcoal has been criticized [ 3 7 1, we tested Ficoll400 as a coating material. When Ficoll was used as a coating material instead of dextran binding was apparently 80% higher, whereas variation of the dextran- or Ficoll concentration hardly had any effect (Table 7 ) . This indicates that the separation of the free and bound fraction was incomplete with dextran-coated charcoal. Doubts could be raised as to whether coating is a real phenomenon. An alternative explanation could be that Ficoll merely reduces the surface area available for binding. This question was addressed by the determination of the binding of tracer and antibody to charcoal as a function of the charcoal concentration in the absence of, or at a fixed concentration of Ficoll (see Fig. 5 ). It appeared that Ficoll reduces the binding to charcoal of the free as well as the bound fraction, but much more so that of the bound. It can be concluded that coating is a real phenomenon and that Ficoll is a better coating material than dextran. The best TABLE I Characteristics conditions
of standard
curves for different
separation
Separation conditions
B, (%) EDSo (fmol/tube)
Detection limit (fmol/tube)
NS (%)
rz
1D” 6D 1F 3F 0.5D, 0.5F
25.1 26.6 46.2 49.8 48.1
0.32 0.34 0.23 0.20 0.19
0.6 1.7 1.9 1.4 1.7
0.973 0.983 0.998 0.991 0.999
2.7 3.0 3.6 3.4 3.1
“Codes refer to type and amount of coating material in the separation medium. ID= 7.5 mg dextran/ml, 6D=45 mg dextran/ml, lF=7.5 mg Ficoll/ml, 3F=22.5 mg/ml, 0.5D=3.75 mg dextran/ml, 0.5F= 3.75 mg Ficoll/ml. Characteristics were derived as mentioned in the legend of Table 6.
31
‘,t 60 t
-
40 200 ;
antibody
tracer
t
/
,,,,,
I
/
1
LJJe,,,i
1
10
100
1000
10000
Fig. 5. Effect of Ficoll coating of charcoal on binding of iodinated VP and antibody to charcoal as a function of the charcoal concentration. Abscissa: charcoal @g/tube). Ordinate: percentage binding of tracer or antibody. The incubation medium contained 0.1 mg HSA/ml and the separation medium contained no (0 ) or 3.75 mg Ficoll/ml (0 ).
overall results were obtained with Ficoll-dextran-coated charcoal (375 pg Ficoll 400 (Pharmacia, Uppsala, Sweden), 375 pg dextran T 70 (Sigma) and 4 mg charcoal in 100,~l of a 50 mM phosphate buffer, pH 7.4), see Table 7. Validation
The reliability of RIAs has been tested in a number of ways. None of these tests are sufficient. Therefore, as many as possible should be performed. 1. A classical criterion for demonstrating immunochemical identity of standard and unknown is represented by superimposibility of their dilution curves. The plasma internal standard dilution curve and the plasma extract dilution curve illustrate that this criterion was met for the measurement in plasma (Fig. 4 ). Likewise, this requirement was fulfilled for the measurement in brain tissue (results not shown). 2. Synthetic peptide and immunoreactive material should elute in the same fractions during high performance liquid chromatography and account for all the immunoreactivity measured in the RIA. Indeed, immunoreactive material in plasma extracts showed identical behavior with synthetic VP [ 28 1. However, up to 40% of the
immunoreactive material in extracts of extrahypothalamic brain structures did not elute with the intact molecule, but with VP fragments [ 381. 3. Physiological or pharmacological manipulations known to affect the level of the hormone should also affect the level of immunoreactive material. We tested the effects of a number of manipulations on the plasma VPir level in rats: dehydration, salt infusion, hemorrhage, i.p. ethanol, and i.c.v. histamine administration. As expected dehydration resulted in an increase of the plasma VPir level, see Fig. 6 [ 291. Moreover, no difference in VPir was detected by antiserum W 1 and W4 under normal (not deprived) and under water-deprived conditions, indicating that the majority of VPir in the plasma under both conditions is identical with the native VP( 1-9) molecule [ 29 1. Salt infusion resulted in a linear increase in the plasma VPir level with osmolality and hemorrhage resulted in an exponential increase with volume change (Fig. 7). Intraperitoneal ethanol administration had a biphasic effect on plasma VPir levels, see Fig. 8 [ 391, and intracerebroventricular as well as intraperitoneal injection of histamine stimulated plasma VPir dose-dependently, see Fig. 9 [ 40,4 11, respectively. It is not well established yet which physiological manipulations affect 15
10
5
0 Wl
w4
not deprived
Wl
w4
24 h water deprivatm
Fig. 6. Plasma VPir levels (fmol/ml+SEM) of non-deprived and waterdeprived animals, as measured using antisera W 1 and W4. Plasmas of the two groups were pooled each before extraction. Each bar represents 5 determinations.
32 60
r t abc
50
ab
Fig. 7. Responses of Lewis rats to graded blood volume reduction (7% reduction each 5 min), left panel, and salt infusion (i.v. infusion of a 9% salt solution at a rate of 30 pl/ min), right panel. Abscissa: left panel: percentage volume reduction; right panel: osmolality in mOsm. Ordinate: fmol VPir per ml plasma.
1
12,
Fig. 9. Plasma VPir levels (fmol/ml I? SEM) 5 min after intracerebroventricular injection of graded doses histamine. Abscissa: pg histamine. Each group consisted of 7 to 10 animals. One-way ANOVA (F(5,47)=5.9, p
n ETHANOL 0
q n
SALINE SHAM
TABLE 8 INJ
NOINJ /
”
Effect of castration on levels of VP ( fmol/mg protein f SEM) in extrahypothalamic brain regions of male Brown-Norway rats Region
Control
Castration
Septum Amygdala Hippocampus
121.5k5.6 37.8 + 3.0 7.5 I? 0.6
10.7t 1.3’ 8.9?2.9* 1.7kO.3’
*P
0
5
60
Fig. 8. Mean ( ?SEM) vasopressin levels (fmol/ml) in plasma from rats decapitated 5 or 6 min after i.p. ethanol (2 g/kg ethanol ( 15% v/v, absolute ethanol in 0.9% saline)), saline, or sham injection. Other rats (0 min after treatment) were neither injected nor disturbed until decapitation. *pcp
levels of VPir in the brain. A physiological manipulation that unequivocally affects VPir levels in some brain areas is castration [ 42 1. As assessed with our assay castration reduced the level of VPir 75-90% in the septum, amygdala and the hippocampus of male rats (Table 8 ). 4. Levels of VPir determined with antisera that
recognize different epitopes should be identical, if it is assumed that the VPir material consists mainly of the native VP- ( 1-9 ). In plasma extracts identical levels of N- and C-terminal immunoreactivity were detected before and after osmotic stimulation (see Fig. 6 ) . In the septum and amygdala of the brain a 40% higher C-terminal immunoreactivity was measured than Nterminal immunoreactivity (data not shown). Both immunoreactivities correlated highly (r2> 0.9, P
33
age of a fixed amount of ligand recovered, should be reproducible and ideally quantitative. However, a simple, cheap, and reproducible technique with a recovery of > 50% is preferable to a complex procedure with a recovery of, say, 95%. For plasma two techniques used at our laboratories are described. The Bond Elut C8 column extraction of VP gives a 96% recovery, but satisfactory results are obtained with Vycor extraction (6 1% recovery, see Table 6) with the important additional advantage of the concomitant production of plasma internal standard medium (SC.the supematant of Vycor extracted plasma). For tissue VPir determination a simple technique, including a centrifugation step at relatively high g-force and ethanol extraction gives a recovery of 52-82%, depending on the amount of tissue extracted (see Table 6 ) . 6. The coefficient of variation (CV) of repeated determinations of the same sample within one assay and between different assays, is an important parameter for determining the reproducibility of the procedure. As can be seen in Table 6, CVs are well within the generally accepted range.
Discussion Radioimmunoassay of VP is difficult, especially if one wishes to measure basal plasma levels. Antibodies with a K, much higher than 10” M-’ can hardly be raised, whereas basal plasma levels are as low as IO- ” M. Rather large volumes have to be concentrated to obtain enough VP for determination. When working with rats plasma volumes cannot exceed a few milliliters if one wishes to carry out determination in triplicate. To obtain a sensitive enough assay we optimized sample preparation, incubation and separation conditions, and only use tracer with a high specific activity and a small incubation volume. Thus, we were able to measure quantities of VPir as small as 0.2 fmol/ml plasma or 0.4 fmol VPir/mg protein or even lower when delayed tracer addition is used. Our assay is as sensitive as the most sensitive assays reported [ lo,13 1, though we use an antibody that is much less sensitive than these assays relied on. Our an-
tibody is of a sensitivity normally obtained for anti-VP antibodies. Therefore, the methods described herein are of general importance to the determination of VPir. Our assay differs from all but one assay in that it is internally standardized. Skowsky et al. [ 18 ] also used internal standardization and concluded that they did not need it. They extracted VP from 1 ml plasma and dissolved the residue in a volume of 1.5 ml for RIA. Others concentrated 5-20 ml - e.g. [ 17,24,43] - in a volume of 0.5-l .5 ml. Their assays probably suffered from nonspecific interferences, indicated by the fact that they measured detectable levels of VPir in overhydrated subjects, also stressed by Rooke and Baylis [ 13 1. Although a number of methods for the determination of VPir in brain structures have been described, these methods have been validated very poorly - e.g. [ 32,33,35]. The possible effects of matrix factors have not been tested; extraction recoveries have not been adequately assessed. By contrast, our assays described in this article correct for nonspecific interferences and we use simple extraction techniques with a high recovery. Material costs of the determination of one sample can be roughly estimated at twenty-live dollar cents. An analyst can determine VPir levels in 120 tissue samples or 200 plasma samples in two days. Thus we developed reliable, fast and cheap extraction techniques combined with a specific and sensitive RIA for the determination of vasopressin immunoreactivity. Several steps in the described procedures may be applicable in the field of immunoassays for (small) peptides. Although many criteria can be met, as stated in section Validation no assay is perfect. Different RIAs (using different antibodies and/or different extraction techniques) for the same ligand will reflect different aspects of the same material. Even if results are not exactly ‘right’ according to some (hypothetical) absolute reference the RIA is still immensely valuable if it serves to distinguish, on a routine base, between normal and stimulated or decreased levels of the substance under study. Therefore, although RIA should be treated as a goal it is a tool.
34
Acknowledgements
12
The authors wish to thank Mrs. E. AndringaBakker, MS C. Maigret, Mrs I. Koekkoek-Van den Herik, Mrs. M. Markerink-Van Ittersum, Dr. M. Van Lookeren Campagne, Drs. P. Bijlsma, and Drs. T. Van Teunenbroek for their valuable contributions to this work. Parts of the studies were subsidized by the Dutch FUNGO project no. 1335-042 and b ‘Y NIAAA-National Research Service Award-05 196.
13
References
16
I
2
3
4
5
6
7
8
9
10
11
R.S. Yalow alnd S.A. Berson, Assay of plasma insulin in human subjects by immunological methods, Nature, 184 (1959) 1648-1649. T.B. Van Wimersma Greidanus and J.P.H. Burbach, Vasopressin and oxytocin their presence in the central nervous system and their functional significance in brain processes related to behaviour and memory, Acta Endocrinol. Suppl. 276 (1986) 85-94. T.B. Van Wimersma Greidanus and J.M. Van Ree, Behavioral effects of vasopressin, Curr. Top. Neuroendocrinol. 10 (1990) 61-79. J. Dogterom, T.B. Van Wimersma Greidanus and D. De Wied, Vasopressin in cerebrospinal fluid and plasma of man, dog and rat, Am. J. Physiol. 234 (1978) E463E467. T.L. Goodfriend, L. Levine and G.D. Fasman, Antibodies to bradykinin and angiotensin: a use of carbodiimides in immunology, Science 144 ( 1964) 1344-l 346. W.R. Skowsky and D.A. Fisher, The use of thyroglobulin to induce antigenicity to small molecules, J. Lab. Clin. Med. 80 (1972) 134-144. S.N. Oyama, A. Kagan and S.M. Click, Radioimmunoassay of vasopressin: application to unextracted human urine, J. Clin. Endocrinol. Metab. 33 ( 197 1) 739744. J.J. Morton and M.A. Waite, The possible relationship between the afftnity of arginine vasopressin antibodies and the degree of polyuria and polydipsia in actively immunized rabbits, J. Endocrinol. 54 ( 1972) 523-524. G.L. Robertson, L.A. Klein, J. Roth and P. Gorden, Immunoassay of plasma vasopressin in man, Proc. Natl. Acad. Sci. USA 66 (1970) 1298-1305. G.L. Robertson, E.A. Mahr, S. Athar and T. Sinha, Development and clinical application of a new method for the radioimmunoassay of arginine vasopressin in human plasma, J. Clin. Invest. 52 ( 1973) 2340-2352. P. Rooke and P.H. Baylis, Effect of plasma interference on different vasopressin antisera, J. Immunoassay 3 (1983) 133-143.
14
IS
17
18
19
20
21
22
23
24
25
26
F. Fyhrquist, M. Wallenius and H.J.G. Hollemans, Radioimmunoassay of vasopressin in unextracted plasma, Stand. J. Clin. Lab. Invest. 36 (1976) 841-847. P. Rooke and P.H. Baylis, A new sensitive radioimmunoassay for plasma arginine vasopressin, J. Immunoassay3 (1982) 115-131. F.C. Greenwood, W.M. Hunter and J.S. Glover, The preparation of ‘3’I-labelled human growth hormone with a high specific radioactivity, Biochem. J. 89 ( 1963) 114123. P.R.P. Salacinski, C. McLean, J.E.C. Sykes, V.V. Clemont-Jones and P.J. Lowry, Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3~u,6c~,-diphenyl glycoluril, Anal. Biochem. 117 (1981) 136-146. M.K. Husain, N. Fernando, M. Shapiro, A. Kagan and S.M. Glick, Radioimmunoassay of arginine vasopressin in human plasma, J. Clin. Endocrinol. Metab. 37 ( 1973) 616-625. C.G. Beardwell, Radioimmunoassay of arginine vasopressin in human plasma, J. Clin. Endocrinol. Metab. 33 (1971)254-260. W.R. Skowsky, A.A. Rosenbloom and D.A. Fisher, Radioimmunoassay measurement of arginine vasopressin in serum: development and application, J. Clin. Endocrinol. Metab. 38 (1974) 278-287. P. Czernichow, U. Merkelbach and M.B. Valloton, Radioimmunoassay of [ %arginine]-vasopressin. Acta Endocrinol. 80 (1975) 440-452. J. Roth, S.M. Glick, L.A. Klein and M.J. Peterson, Specific antibodies to vasopressin in man, J. Clin. Endocrinol. Metab. 25 (1966) 671-675. N.G. Seidah, M. Dennis, P. Corvol, J. Rochemont and M. Chretien, A rapid high-performance liquid chromatography purification method of iodinated polypeptide hormones, Anal. Biochem. 109 (1980) 185-191. H. Tuppy and A. Nesvadba, Uber die Aminopeptidaseaktivitit des Schwangerenserums und ihre Beziehung zu dessen Vermogen, Oxytocin zu inaktivieren, Md. Chem. 88 (1957) 977-988. J.M. Davison, E.A. Gilmore, J. Durr, G.L. Robertson and M.D. Lindheimer, Altered osmotic thresholds for vasopressin secretion and thirst in human pregnancy, Am. J. Physiol. 246 (1984) FlOS-F109. J.J. Morton, P.L. Padlield and M.L. Forsling, A radioimmunoassay for plasma-vasopressin in man and dog: application to physiological and pathological states, J. Endocrinol. 65 (1975) 41 l-424. F.T. La Rochelle Jr, W.G. North and P. Stern, A new extraction of arginine vasopressin from blood: the use of octadecasyl-silica, Pfltigers Arch. 387 ( 1980) 79-8 1. M. Bevilacqua, R. Meroni, R. Dagani, E. Renesto, C. Baruto and G. Norbiato, Role ofblood osmolality in the regulation of vasopressin secretion in man: application of a new radioimmunoassay method for vasopressin, J. Endocrinol. Invest. 8 (1985) 97-101.
35 27
28
29
30
31 32 33
34
35
MS. Brownfield, J. Greathouse, S.A. Lorens, J. Armstrong, J.H. Urban and L.D. Van de Kar, Neuropharmacological characterization of serotonergic stimulation of vasopressin secretion in conscious rats, Neuroendocrinology 47 (1988) 277-283. B.J.M. Van de Heijning, I. Koekkoek-Van de Herik, T. Ivany and T.B. Van Wimersma Greidanus, Solid phase extraction of plasma vasopressin: evaluation, validation and application, J. Chromatogr. 565 ( 199 1) 159171. J.A. Ten Haaf, Studies on Hormone Release from the Neurointermediate Lobe of the Rat Pituitary Gland, Thesis, University of Utrecht, The Netherlands, 1990. W.H. Gispen, P. Schotman and E.R. De Kloet, Brain RNA and hypophysectomy; a topographical study, Ncuroendocrinology 9 ( 1972) 285-296. M. Palkovits, Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Res. 59 (1973) 449-450. S.M. Glick and M.J. Brownstein, Vasopressin content ofrat brain, Life Sci. 27 (1980) 1103-l 110. R.E. Brinton, P.P. Desmukh, A. Chan, T.P. Davis, S. Hsiao and HI. Yamamura, A non-equilibrium 24 hour vasopressin radioimmunoassay: development and basal levels in the rat brain, Brain Res. 266 (1983) 344-347. J. Dogterom, F.G.M. Snijdewint and R.M. Buijs, The distribution of vasopressin and oxytocin in the rat brain, Neurosci. Lett. 9 (1978) 341-346. J. Hawthorn, V.T.Y. Ang and J.S. Jenkins, Localization of vasopressin in rat brain, Brain Res. 197 ( 1980) 7.581.
36
37
38
39
40
41
42
43
J.P.H. Burbach, D. Terwel and J.L.M. Lebouille, Measurement and distribution of vasopressin-converting aminopeptidase-activity in rat brain, Biochem. Biophys. Res. Commun. 144 ( 1987) 726-73 1. M.A. Binoux and W.D. Odell, Use of dextran-coated charcoal to separate antibody-bound from free hormone: a critique, J. Clin. Endocrinol. Metab. 36 ( 1973) 303-310. J.P.H. Burbach, X.-C. Wang, J.A. Ten Haaf and D. De Wied, Substances resembling C-terminal vasopressin fragments are present in the brain but not in the pituitarygland, Brain Res. 306 ( 1984) 384-387. D.H. Colbern, J.A. Ten Haaf, B. Tabakoff and T.B. Van Wimersma Greidanus, Ethanol increases plasma vasopressin shortly after intraperitoneal injection in rats, Life Sci. 37 (1985) 1029-1032. J.A. Ten Haaf, T.B. Van Wimersma Greidanus, C. Maigret and D. De Wied, Effect of the opioid peptide @-endorphin on the in vivo release of vasopressin in rats under various conditions, Neuroendocrinology 44 ( 1986) 102-107. J. Dogterom, T.B. Van Wimersma Greidanus and D. De Wied, Histamine is an extremely potent releaser of vasopressin in the rat, Experientia 32 ( 1976) 659. G.J. De Vries, R.M. Buijs, F.W. Van Leeuwen, A.R. Gaffe and D.F. Swaab, The vasopressinergic innervation of the brain in normal and castrated rats, J. Camp. Neurol. 233 ( 1985) 236-254. C.I. Johnston, Radioimmunoassay for plasma antidiuretic hormone, J. Endocrinol. 52 (1972) 69-78.