stipulation that the solute partitions between water and the organic solvent. The differential in vapor pressure between solute and solvent does not seem to be a factor that regulates concentration efficiency. The dipole moment difference between solute and solvent will indicate the extraction efficiency and can be used as an index to select the extraction solvent. For broad spectrum extraction, several solvents can be used either in a series or parallel extractor trains; and with adequate dipole moment
differences between solvents, the extractor train will selectively concentrate on the basis of solute-solvent dipole moment match.
RECEIVED for review May 25, 1972. Accepted July 31, 1972. The USGS does not necessarily endorse any manufacturers product discussed in this text. Approved for publication by the Director of the U.S. Geological Survey.
Factors Affecting Radioimmunoassays: Specifically That of Human Luteinizing Hormone Using Labeled Human Chorionic Gonadotropin Eugene Cerceo Thomas Jefferson University Hospital, Philadelphia, Pa. 19107 Human chorionic gonadotropin (HCG) labeled with iodine425 was investigated as a tracer for the radioimmunoassay of human luteinizing hormone (HLH) using both the double antibody and a solid phase technique. Iodinations were conducted at three specific activities, namely 11.1, 3.93, and 1.57 I*51/HCG. The extent of degradation was followed at -20, 22, and 37 OC by ion exchange and gel filtration chromatography. Standard curves were determined using freshly prepared tracer at the three specified specific activities and utilizing four selected antibody titer values. The results indicated that HCG labeled with iodine-125 to a specific activity of approximately 1.0 lZI/HCG gave the best standard curves. Ideally, the tracer should be freshly prepared for each assay; practically, at the specified specific activity good doseresponse relationships were obtained for a period of approximately two weeks. For radioimmunoassay, the modified solid phase method developed in this investigation gave results comparable to the double antibody technique. I n either case, the parameter most affecting accuracy and precision was the stability of the tracer hormone.
THE RADIOIMMUNOASSAY TECHNIQUE has allowed the estimation of protein hormones in the picomolar range. It has also been applied to steroids, globulins, and viruses. In general, it can be applied to anything for which an antibody can be produced. Essentially, the basic technique involves the measurement of the competitive binding of labeled and unlabeled antigen for antibody, but naturally anything which inhibits this can be measured as apparent antigen if the system in question is not carefully examined. Therefore, as radioimmunoassays are determined over a period of time, the immunological integrity of the system should be followed by noting the position of the standard curve, its point of inflection, and the per cent tracer bound in the “zero” standard. The specificity, sensitivity, and precision of the radioimmunoassay method ( I , 2) is a direct function of the nature and properties of the radioiodinated hormone. In the case of gonadotrophins not only is the identity of the labeled (1) W. D. Odell, G. Abraham, H. R. Raud, R. S. Swerdloff, and D. A. Fisher, Acta E i i d o c r i d . (CopcnAagcn), Suppl., 139-142, 54 ( 1969). (2) S. A. Berson and R. S. Yalow,.J. Cliri. Imesi., 38,1996 (1959).
species at times questioned, but also its immunological activity. The sensitivity and specificity of the assay also depends primarily on the avidity and specificity of the interaction between the antigen and the specific binding sites of the antibody. This becomes the most important single factor in establishing a satisfactory radioimmunoassay, and thus requires close observation. Generally, in the antigen-antibody interaction, the energy of binding is determined by the complementary relationship between the antigenic determinant, those parts of the antigen molecule which combine with the binding sites of the antibody, and the combining sites of the antibody molecule. Here a number of forces are involved, including ionic interactions between charged polar groups, hydrogen bonding, van der Waal‘s forces, hydrophobic interactions, and London forces operating between non-polar groups, It is likely that no single type of bonding predominates, although the relatively long range electrostatic attraction is probably responsible for initial binding between the molecule, followed by secondary interactions when the distance is of the order of a few Angstroms. The close fit of molecular structures which is necessary for a high binding energy may also explain the great specificity of the antigen-antibody reaction. Since the stereochemistry of antibodies assumes such a prominent role, the general structure of these macromolecules merits a closer look. Thus, antibodies are gamma-globulins belonging to one of a number of classes of which the principal members are IgG, IgM, and IgA. All three consist of both light and heavy chains containing some 200 to 450 amino acids, respectively. In most mammalian species, the IgG group is the most important and is the only one involved in the radioimmunoassay reaction. Their molecular weights are in the area of 160,000 and they circulate at a concentration of 10 to 15 mg/ml. The most important factors in antibody production are the ability of the antigen to provoke an antibody response in the chosen animals and its availability in adequate amounts. After it is finally obtained, it is found that the antisera with the greatest avidity usually yields the most sensitive assays, and such an antiserum is produced with the aid of Freunds complete adjuvant.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
93
1,401
45
1.30C 40
1,zoc ’0
-
b
w 35
1,lOC
I
E
>
s
% 3 2
1,ooc
2
30
8
ze’ 8
8
8
900
d ti
*
;
3
E.
2
25
800
i
g
z
700 5
Figure 2. Radioimmunoassay of human luteinizing hormone (HLH.) using the solid phase technique and showing the spread in values of each point ( N = 4)
600 5
10
15
20
25
HUMAN L U T E I N I Z I N G HORMONE (mILI/ml)
A. Equilibration of antibody to hormone for a period of 48 hours at 22 “C B. Equilibration of antibody to hormone for a period of 24 hours at 22 “C
Figure 1. Radioimmunoassay of human luteinizing hormone (HLH) using the solid phase technique
Once the antiserum is obtained, it is best stored in the lyophilized state rather than freezing. Such sera can be kept for relatively long periods of time without loss of specificity or titer. In the general handling of sera, not lyophilized, rapid freezing is to be preferred, since this probably contributes as much to the safety of the material as the absolute temperature at which it is finally stored. Coupling antisera to solid phases (3-6) such as cyanogen bromide-activated Sephadex particles seems to lend added stability to the antibody. The lack of specificity in radioimmunoassays may be attributed to at least three factors: the preparation of the hormone used for immunizations may be impure and may thus stimulate the formation of different populations of antibodies, some of which may be directed against the impurities; impurities present in the labeled hormone preparation; and cross-reactions arising from the fact that some hormones carry common structural portions, either along the polypeptide chains, or in the case of glycoprotein hormones, within the glucidic moiety. Thus, once specificity is obtained, then attention is focused on separation. And the factors that affect the separation of antibody bound from free hormones can be listed as follows: behavior of damaged label, effect of variations in protein concentrations, extent of separation of bound from free hormone, volume of the equilibrating mixture which can be (3) K. E. Kirkham and W. M. Hunter, “Radioimmunoassay Methods,” Churchill tivingston, London, 1971, p 193. (4) P. Cuatrecasas, J. Biol. Chem., 245, 3059 (1970). ( 5 ) J. Porath and P. Flodin, Nature, 183,1657 (1959). (6) J. Porath, ibid., 218,834 (1968). 94
10
HUMAN L U T E I N I Z I N G HORMONE ( m S J / n l )
20
handled, speed of separation, skill of the operator, and effect of non-specific factors present in physiologic fluids. For assays of these fluids, particularly plasma or serum, the cumulative effects of differences between individual plasmas can cause both incubation damage and interference in antigen-antibody reaction. Such damage can best be evaluated through use of affinity chromatography utilizing antibody chemically coupled to sepharose gel (7). Further, what constitutes damage has been a topic of some controversy. Basically, the condition refers to changes tha: may occur in the immunological behavior of a hormone in which its ability to bind with specific antibody may be impaired, This may also be associated with subtle changes ir the physicochemical properties of the hormone, resulting ir some loss of the normal ability to adhere to cellulose columns or become adsorbed to charcoal or silica. When referring to iodination damage, a number of param. eters seem to be involved: namely, the presence of radioiodine substituted for hydrogen in the aromatic ring of tyrosine ma] cause loss of reactivity (8), the reagents used in the iodination procedure may damage the antigen, damage may be caused b j internal radiation during and after iodination, and damage may be caused by noxious substances present in the aqueous solutions of Na1251supplied by the manufacturer. The shelf-life of iodinated preparations is dictated by the slow loss of iodide, the decay catastrophe (9) which refers to a (7) P. Cuatrecasas, J . Biol. Clzem., 245, 3059 (1970). (8) F. C. Greenwood, W. M. Hunter, and J. S. Glover, Biocliem. J . , 89,114 (1963). (9) S. A. Berson and R. S. Yalow, J . Clirz. Incest., 47, 2725 (1968).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
70
50
t
60
E
40
8 50
4c
A B C D
3c
I
I
1
I
0
10
20
30
MlMAN LUTEINIZINO HORMONE
lm’LU/ml)
Figure 4. Radioimmunoassay of HLH using HCG tracer of 11.1 12SI/HCG specific activity Titers
2(
I
,
,
.
,
5
SPECIFIC ACTIVITY
I
t
I
10
3
8
A. B.
1 1 C. 1 D. 1
1 15
0
1 2 5 ~ / ~ ~ ~
Figure 3. Binding to antibody as a function of specific activity for HCG at various titer values Titers A . 1 :20,000 B. 1 :30,000 C. 1 : 40,000 D . 1 :50,000
situation resulting when two radioactive iodine atoms are present at different sites in one molecule of the labeled compound. This causes loss of immunoreactivity when one disintegrates and disrupts the molecule. Changes such as these not only lower both assay precision and sensitivity but also tend to impair specificity because of the unpredictable manner in which the altered labeled hormone may react. In the iodination procedure, a number of factors should be considered. One of these is that the volumes of the reagents should be kept to a minimum since the per cent of incorporation of the iodine is dependent upon the concentration of the protein. This minimum volume should be buffered to pH 7.5, since above pH 8 iodination of groups other than tyrosine can occur (10). Another factor is that the amount of chloramine-T used is dictated by the quantity and the nature of the protein which is being iodinated, since the reagent can react directly with groups on the protein. Moreover, it seems that radioiodine solutions of high specific activity neutralize some of the oxidative capacity of the chloramine-T, probably through degradation, rearrangement of structure, and formation of free radicals as a result of gamma radiation. And the (10) A. E. Freedlender, in “Protein and Polypeptide Hormone,” part 111, M. Margoulies, Ed., International Congress Series, No. 161. Amsterdam: Excerpts Medica Foundation, 1969, pp 608--10.
I 40
:20,000 : 30,000
: 40,000 :50,000
addition of sodium metabisulfite solution in a larger volume than recommended (11, 12) lessened the concentration of possibly damaging substances toward gonadotrophins. In iodinating, two factors are important in affecting the level of substitution of iodine into a peptide macromolecule. These are the following:
(1) If iodination is carried out at a pH of 7.5 to 8.0, then a normal tyrosine will ionize only slightly to the anion and most of the iodination will proceed through the small fraction of anions. Once iodinated, the tendency for a second iodine substitution increases, so that under these conditions, the tendency of the tyrosine is to be disubstituted. (2) The degree of substitution is dependent upon the stereochemistry of the individual tyrosines. Therefore, those tyrosines readily accessible will be iodinated first and these are the tyrosines which tend to have the greatest involvement in antibody binding. An obvious point is that the more tyrosines the molecule has and the larger it is, then the less likely it is that the most critical ones will be affected. In this respect, it should be remembered that if a macromolecule has a number of iodinatable sites, but these cannot be substituted without losing a very large degree of binding, then replacing the iodine with tritium may prove to be advantageous. For if one or two orders of magnitude are lost in the radioactivity, the advantage of preserving molecular (11) W. M. Hunter, Acta. Etidocrirzol. (Copenhagen), Suppl., 142, 134 (1969). (12) F. C.Greenwood, W. M. Hunter, and J. S. Glover, Biochern. J.,89,114(1963).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
95
'k
6i
60
50
t 2o
1
I
0
10
I 20 W J W L U T E I N I Z I N S HOmONE
I 30 IrnlU/V
40
1)
Figure 5. Radioimmunoassay of HLH using HCG tracer of 3.93 1251/HCG specific activity
W J M L U T E I N I Z I N G HORMONE ( P I U h l )
Figure 6. Radioimmunoassay of HLH using HCG tracer of 1.57 I*jI/HCGspecific activity
Titers A . 1 :20,000 B. 1 :30,000 C . 1 :40,000 D. 1 :50,000
identity by using tritium may outweigh the disadvantages of its relatively low specific activity. Therefork, having considered-all of the foregoing, it became the purpose of this investigation to compare two radioimmunoassay methods (specifically for HLH): one utilizing solid phase and the other the double antibody technique. Also the stability of the tracer (labeled HCG) was studied as a function of specific activity. The shape of the corresponding radioimmunoassay curves was observed as a function of parameters affecting the assay along with possible methods to stabilize the system. EXPERIMENTAL
Materials. Sephadex G-25 cross-linked dextran resin was supplied by Pharmacia Fine Chemicals, Inc., Piscataway, N.J. This resin is especially advantageous in hormone separation based on molecular sieve chromatography. AG1 -X8 styrene type quaternary ammonium ion exchange resin, chloride form, 200-400 mesh was obtained from Bio-Rad Laboratories, New York, N.Y. These resins are strongly basic anion exchangers composed of quaternary ammonium groups attached to a styrene-divinylbenzene polymer lattice. The resin was washed with large volumes of deionized water until the effluent yielded a conductivity equivalent to 5 ppm NaC1. The advantage of chosing such a resin is its high affinity for the iodide ion. Human Luteinizing Hormone (immunochemical grade) LER 960, 200 micrograms per ml of buffer solution (0.01M phosphate buffer, 0.14M sodium chloride, pH 7.0) was obtained from the National Pituitary Agency, Endocrinology Study Section, National Institute of Arthritis and Metabolic Diseases, Bethesda, Md. 96
t Titers 1 :20,000 8. 1 :30,000 C. 1 :40,000 D. 1 :50.000 A.
Iodine-125, 98 carrier-free, sodium iodide in sodium hydroxide solution, 2.00 millicuries, specific activity of 1.8 x 104 millicuries per mg in a volume of 10 microliters was supplied in plastic polyvials by Cambridge Nuclear Corporation, Cambridge, Mass. Lyophilized Human Chorionic Gonadotropin antiserum was obtained from ICN Nutritional Biochemicals Corporation, Cleveland, Ohio. Human Chorionic Gonadotrophin from Organon, Inc., West Orange, N.Y .,was purified using carboxymethyl cellulose and DEAE, lyophilized, and diluted in 0.1Mphosphate buffer, pH 7.4 to a concentration of 1.00 mg/ml. All gamma radiation counting was conducted in the Model 4230 Automatic Counting System obtained from Nuclear Chicago Corporation, Des Plaines, Ill. Procedures. The method of iodination has been previously described (13) and basically involved reacting 2 mCi of Nal*&Iwith 2.0 to 25 kg of purified HCG (1 mg/ml> and treating with 10 p1 of an aqueous solution of Chloramine-T (2.5 mgiml). Chloramine-T in aqueous solutions slowly yields hypochlorous acid and, hence, is a mild oxidizing agent. Addition of this reagent to a slightly alkaline aqueous solution of protein and iodide results in the incorporation of the iodine into the tyrosine functions of the protein. Next 50 pl of Sodium Metabisulfite (2.5 mg/ml) was added as a reducing agent to convert excess iodine back to the iodide ion. The reaction mixture was placed on a 20-ml Sephadex G-25 gel column and chromatographed with 0.1M phosphate buffer, pH 7.4. It should be noted that the diluent throughout this procedure was 0.4M phosphate buffer, pH 7.4 except where otherwise specified. (1 3) F. C. Greenwood and W. M. Hunter, Nufure, 194,495 (1962).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
Prior to the radioimmunoassay polystyrene tubes were coated with antibody (14,15)by first etching the inside surfaces with a spray of toluene. The tubes were inverted and allowed to dry at room temperature overnight. The following morning, 1.0 ml of titered antibody in 0.1M phosphate buffer (raised to pH 10.0) was added t o each tube and allowed t o react for at least 5 minutes, after which the antibody solutions were decanted from all the tubes and the tubes washed 3 times with 2 ml of phosphate buffered (pH 7.4) saline. A final wash of 2 ml of 2 . 5 z Bovine Serum Albumin in phosphate buffered saline u a s made t o ensure the absence of nonspecific binding sites. The radioimmunoassay was now performed according to the procedure shown in Table I. RESULTS AND DISCUSSION
For the determination of HLH, two radioimmunoassay techniques were considered; namely, that of double antibody and solid phase employing coated tubes. The former assay method utilizes a second antibody directed against the antigen-antibody complex to cause precipitation of the bound hormone. The latter method involves binding the antibody to a solid support, in this case the walls of a plastic tube, and thus the free hormone is separated from the bound by merely separating the fluid from the tube. The results of extensive work have shown that, in general, the precision and accuracy of the double antibody technique can be Comparable to that of the solid phase; but the advantages of solid phase are speed and economy. The double antibody method has the disadvantage of employing a n extra antibody and a n extra period of equilibration, so that for clinical purposes, and in this investigation, attention was focused primarily on the solid phase technique (Figure 1). In elucidating the parameters affecting the assay, the following factors were found to be significant: affinity and avidity for the labeled and unlabeled hormone, specificity of the antibody, specific activity of the label, the time during the equilibration period that the label is introduced into the assay, both the temperature and time of the equilibration period, and the extent of degradation of the labeled hormone. A closer look a t these factors revealed that if the affinity and avidity were good. satisfactory standard curves were obtained. Note that the antibody cannot be expected to be highly specific since there is cross ieaction to I-ILH (thus the justification for the assay), and some cross reaction with FSH and TSH. Too high a specific activity for labeled H C G yielded high counting rates in the assay. but resulted in accelerated breakdown of the hormone; and too low a specific activity yielded a very low counting rate. but vastly improved hormone stslbility. Thus, in this latter situation, in order to acquire enough counts, the concentration of the trace required a n increase to the point where it was no longer a trace. In essence, this effect decreased the useable concentration range of the standard curve. Thus a n optimum situation between these two extremes wab sought. and this was found by labeling the tracer at a specific activity equal to approximately 1.0 l?jI/HCG. Iodinating 25 pg of HCG with 2 mCi of iodine-125 yielded such a specific activity. Addition of the label at later times during the equilibration process seemed to bring the inflection point of the standard curve to lower concentrations. This had the effect of decreasing the range of the standard curve. This study also revealed that although equilibration of hormone to antibody -.
~
~~___-______
Table I. Radioimmunoassay of HLH by Coated Tube Solid Phase Method' Volume of buffer containing VolConcenHCG-'2'1 Volume of and tration ume of patient's 0.1 stanof stds, plasmas. mIU/ plasma, Sample dards, ml No. P1 Pl Pl 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Ai At A3
Bi B2
B3
100 100 100 100 100 100 100 100 I00 100 100 100 100 100 100 100 100 100 100 100 100 I00 100 100 100 100 100 I00 100 100
0.00 0.00 0.00 0.00 0.25 0.25 0.50 0.50 0.75 0.75 1.0 1.0 1.5 1.5 2.0 2.0 2.5 2.5 3.0 3.0 5.0 5.0 7.5 7.5 10 10 15 15 25 25
... ...
... ...
... ...
...
... ...
... ...
... ... , . .
...
... ... ... ... ...
... ... ...
... ...
900 900 900 900 900 900 900 900 900 900 900 900 9 00 900 900 900 900 9G0 900
...
900
... ...
900 900 900 900 900 900
... ... ... ... ... ... ...
... 100 100 100 100 100 100 100
900
900 900 900 900 900 900 900 900 900 900
Etc. Equilibrate at 22 'C for 48 hours, separate supernatant from tubes, and count tubes for bound hormone.
is fully achieved in 72 hours a t 4 "C, it is not fully completed in 24 hours a t 22 "C. Rather, a period of 48 hours a t 22 "C was needed (Figure 2). The most important of the disrupting effects on the radioimmunoassay was the extent of degradation of the 'ZZI-HCG. This depended on many factors, some of which were the length of time and temperature a t which the radioiodinated hormone was stored, quality of the Na-l?jI, age of the HCG, and the length of time in contact with the iodinating reagents. To help evaluate the extent of degradation, column chromatography was conducted using Sephadex G-25. In the radioimmunoassay, this chromatographic procedure corrects for tracer molecules lost due to deiodination, but does not correct for losses in precision and accuracy resulting from losses in immunological activity. This loss is caused by damage to the protein molecule due to direct self-radiation, and radiolysis resulting in the formation of highly reactive free radicals, and thus a myriad of polymerized molecular species. This loss of precision and accuracy becomes a more important factor in the lower concentration standards.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
e
97
90
r
b Figure 7. Partitioning of HCGJZ5Iand lz5I-in 0.1M phosphate buffer containing 0.1 bovine serum albumin as a function of time on AGl-XS ion exchange resin Specific activity of tracer = 11.1 1251/HCG Bacteriocide = trace of sodium azide Temperatures A . -20 "C,14 freeze-thaw cycles B. 22 "C C. 31 "C
w
I
B P a 2o 10
t
I
0
500
\"C
1 1.003
ELAPSED T I M E (HOURS)
t
4 Figure 8. Partitioning of HCG-l?>Iand l*"I-in 0.1M phosphate buffer containing 0.1 bovine serum albumin as a function of time on AG1-X8 ion exchange resin Specific activity of tracer = 3.93 125I/HCG Bacteriocide = trace of sodium azide Temperatures A . -20 T, 14 freeze-thaw cycles B. 22 "C c. 37 'C
! 401 30
0
1,000
500 ELAPSED TIME (HOURS)
b Figure 9. Partitioning of HCGJ29 and 1251- in 0.1M phosphate buffer containing 0.1 bovine serum albumin as a function of time on AG1-X8 ion exchange resin
c
Specific activity of tracer = 1.57 *SIjHCG Bacteriocide = trace of sodium azide Temperatures A. -20 "C, 13 freeze-thaw cycles B. 22 'C C. 37 "C
I
I 500 ELESEO
98
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
:IVE
1.000 (.!OL'KSI
100
Table 11. Iodination of HCG at Three Different Activities Using the Chloramine -T Method Na 1 2 j l taken HCG taken for Specific for iodination, activity, iodination, Pg 1251,"CG mCi 2.00 2.00 2.00
2.2 11.5 27.5
h
11.1 3.93 1.57
Since the condition of the HCG-lZ4 seemed to be most critical to the performance of a suitable assay, close analytical observations were undertaken. Thus purified, HCG was iodinated at various specific activities as shown in Table 11. Following separation of the iodinated peptide from the reaction products using column chromatography with Sephadex G-25, the labeled protein was immediately used in a standard curve determination at four selected titers (Figures 3-6) and split into three separate fractions and stored at -20 "C: 22 "C, and 37 "C. The extent of deiodination was measured by noting the amount of radioactivity absorbed by AG1-X8 ion exchange resin. Figures 7, 8, and 9 illustrate the stability of the hormone labeled at three specific activities, stored at the three different temperatures, and measured over a period of 1000 hours. In essence, these figures show the partitioning of l * T atoms between the aqueous phase and the resin. Since samples stored at -20 "C were of primary interest, a parallel determination was conducted using column chromatography employing Sephadex G-25. Figure 10 shows the results of this study. The method yields a more accurate indication of the amount of degradation present. Having observed the effect of AG1-X8 resin on free iodide ions, consideration was next given to its use as a means of removing radioiodide ions and possible small radioiodide-containing fragments from the solution of the labeled peptide. Thus the label would be free of any appreciable buildup of radioiodide ions, which could interfere in the assay by producing an apparently lower per cent binding and possible spurious results. Figure 11 shows standard curves using two samples of '*jI-HCG; one in the presence of AG1-X8 resin, and the other purified on Sephadex G-25. Our studies have shown that the resin not only binds the radioiodide ions and possible small molecular fragments, but also a certain amount of the immunoreactive label. Generally, the labeled hormone should be used within approximately two weeks following iodination or repurified by way of affinity chromatography utilizing antibody coupled to sepharose gel. Parallel studies using column chromatography on Sephadex G-100 with samples four weeks old yielded doublets for the protein peak. The first half of the doublet was attributed to damaged, polymerized lZ5I-HCGand the second half to essentially pure, immunoreactive 12jI-HCG. This has been demonstrated through standard curve determinations. Therefore, summarizing and expanding our observations on HCG-l*SI as a radioimmunoassay tracer the following has been concluded: (1) It seems to be more stable than radioiodinated HLH.
(2) Iodinated at a specific activity of approximately 1.0 1*31,"CG, it has shown good stability and yet has yielded enough counts for the trace addition to still be considered a tracer. ( 3 ) Use of ion exchange resin, such as AG1-X8 to remove iodide ion seems to be of doubtful effectiveness due to
\ \
' O B
' A C 40
1
1
1
500
0
*
>
1
1.000
ELAPSED TIME ( H O U R S )
Figure 10. Extent of degradation of HCG-lZ61determined by column chromatography on Sephadex G-25 as a function of time Temperature, -20 'C,6 freeze-thaw cycles Bacteriocide = sodium azide Buffer = 0.1M phosphate containing 0.1% bovine serum albumin Specific activity ( Y / H C G ) A.
1.57
B.
3.93 11.1
c.
simultaneous adsorption of some immunologically reactive label. (4) Use of a protein, such as Bovine Serum Albumin, does not lend sufficient shielding to the label's self-gamma radiation to be effectiveas a stabilizer. ( 5 ) When stored in the radioactive concentration of 1.0 to 1,500 pCiiml at -20 "C, chromatographic purity determinations revealed basically the same deiodination rate. (6) Deiodination values of l*jI-HCG at a given specific activity but stored in different plastic tubes at -20 "C showed different extents of degradation (Figure 12). This implies that deiodination involves complex kinetics, such that the reaction mechanisms are probably dependent upon many parameters, one of which is the specific activity and thus the number of iodines per HCG molecule. When considering the radioimmunoassay of HLH using the aforementioned tracer, the following has been concluded: (1) The double antibody technique yields slightly greater precision and accuracy than the solid phase technique utilizing antibody coated polystyrene tubes. (2) Another solid phase method (26) employing precipitation of polystyrene from a toluene solution onto the walls (16) Robert Ness, Drexel University, Philadelphia Pa., unpublished data, 1972.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
99
"
\ \ 0
I 0
20
30
40
500 ELWSED TW.E
HUMAN L U T Z I N I Z I N G HOWONE ( m I C / m l l
Figure 11. Radioimmunoassay of HLH using ( A ) HCGIz3I purified by colum chromatography on Sephadex G-25 and ( B ) H C G J 4 purified with AGl-XI( ion exchange resin
of glass tubes has yielded comparable results, although there has been difficulty in attaining suitable adherence of the plastic to the walls of the glass tubes. (3) The use of talc tablets as a method of separation has never been satisfactory although success has been reported (17). (4) Parameter most affecting accuracy and precision is the stability of the labeled hormone. ( 5 ) Best curves have been obtained utilizing freshly iodinated and purified hormone from the tail of the chromatographic hormone peak. The leading edge or the peak itself always seems to contain a certain amount of non-immunoreactive product. This is attributed to damaged and polymerized HCG fragments. (6) Utilization of 0.1 plasma in the buffer solution employed for the standards simulated the effects of proteolytic enzymes in the sample tubes. Thus, during equilibration the label in all tubes is affected in the same manner. The absence of this step can lead to false high values of HLH resulting from binding inhibition and breakdown of the tracer in the sample tubes. The alternative is a two-step non-equilibrium method in which the antibody is first allowed to react with standards and plasma samples for a sufficient time followed by reaction with the tracer. In this wsy the proteolytic enzymes and any other harmful components of the plasmas fail to come in contact with the labeled hormone. (7) Minimum equilibration times for both solid phase and double antibody technique involving antigen-antibody interaction is at least 48 hours a t 22 "C or 72 hours at (17) R. A. Levine, R. K . Donabedian, and L. G. Sobrinko, C h i . Chem.. 17,931 (1971). 100
'
'9
I
I
10
A " 0
l,Y""
(I%OURS)
Figure 12. Extent of degradation of HCGJZ5I determined by column chromatography on Sephadex G-25 as a function of time. Each point is a separately frozen sample Temperature -20°C Bacteriocide sodium azide Buffer O.lA4 phosphate Freezer-thaw cycles 1 Sliecific activity (1251/HCG) A . 0.92 B. 1.45
4 "C. Shorter reaction times tend to yield greater scatter in the results of the lower concentration standards. The rate of equilibration is a function of concentration as well as temperature. (8) Recently, enzymatic radioiodination of HCG has been accomplished (18, 19) using lactoperoxidase, hydrogen peroxide, and sodium iodide-125. This milder form of iodination is reported as being gentle, simple, and rapid. Future work should involve comparison of the degradation curves and standard radioimmunoassay curves to those obtained by the chloramine-T method in this investigation. Finally, departing from the specifics of this investigation and taking a general view of radioimmunoassay and its impact on medical science, it should be said that aside from the assay of hormones (peptide and steroid), drugs, vitamins, globulins, etc., the technique i s finding applications in new and exciting areas of immunology. The reason being that medically, it has become apparent that defects in the human immune system probably underlie a number of common disorders, a few (18) Y . Miyachi, J. Vaitukaitis, E. Nieschlag, and M. Lipsett, J . Cliii. Eiidocriirol. Metob., 34, 23 (1972). (19) J. Marchalonis, Biocliem. J . . 113,299 (1969).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
being multiple sclerosis, rheumatoid arthritis, and cancer. The long-range hope of researchers is that a way be found to manipulate the immune system so as to be better able to contend with otherwise incurable diseases. Radioimmunoassay is perfectly suited not only to cope with the analytical problems of these investigations, but also to shed some light on the chemical and biological mechanisms of these diseases.
ACKNOWLEDGMENT The author would like to thank Linda Salvatore for her gracious assistance in the preparation of this paper; also for technical and experimental assistance to thank Sheldon Schlaff and Carl Steinberg.
RECEIVED for review June 23, 1972. Accepted September 8,1972.
Procedure for Quantitative Electron Probe Microanalysis P. W. Wright B. H . P. Melbourne Research Laboratories, P. 0. Box 274, Clayton, 3168, Victoria, Australia Quantitative electron probe microanalysis at the Melbourne Research Laboratories of BHP is applied to many varied investigations. Mineral specimens, sintered ores, refractories, and steel at all stages of production are among the specimens examined. The paper outlines the specimen and standard preparation techniques for electron probe microanalysis. The analysis procedures are discussed; particularly the automatic operation of the X-ray spectrometers and data collection for subsequent computing. A timesharing computer facility is used on-line t o correct raw X-ray data and print-out final analyses. The computer programs, that have been developed in BASIC at MRL, are described. They are compared with programs from other electron probe laboratories. Finally, results of analyses of sulfide minerals and oxides are presented to illustrate the value of quantitative electron probe microanalysis in mineral exploration and steel production.
ELECTRON PROBE MICROANALYSIS has been established as an analytical technique for at least ten years. Any suitably prepared solid specimens can be examined by this technique, which can analyze selected areas as small as a micron in diameter. Regions for analysis are positioned optically under a focused electron beam (
