coated charcoal immunoassay of insulin

Coated Charcoal Immunoassay of Insulin

VICTOR HERBERT, KAM-SENG LAU,1 CHESTER W. GOTTLIEB,2

AND SHELDON J. BLEICHER3

Department of Hematology, The Mount Sinai Hospital, New York City, and Metabolic Research Unit,The Jewish Hospital of Brooklyn, Brooklyn, New York

ABSTRACT. Charcoal premixed with dex-tran of average molecular weight 80,000 al-most instantly adsorbs free insulin but re-jects antibody-bound insulin. The use ofsuch dextran-coated charcoal makes simplerand more rapid the immunoassay of insulin in

biologic fluids, using radioisotope dilutionwith 131I-insulin and "biopsy" of the insulinpool by antibody to insulin. The procedurehere described yields a straight line graphwhen insulin added is plotted against insulinrecovered. (J Clin Endocr 25: 1375, 1965)

IT HAS previously been reported thatcharcoal coated with a large molecule

(albumin) almost instantly adsorbs asmall molecule (vitamin B12) but willnot adsorb the small molecule when it isbound to its large molecule carrier (serumvitamin Bi2 binding protein or intrinsicfactor concentrate) (1,2). This method ofseparation of small from large moleculesby "instant dialysis" using coated char-coal has been successfully applied to as-says for vitamin Bi2 and its carriers (2,3), iron and its carrier (iron-bindingglobulin) (4), and for in vitro assay ofthyroid function (5).

In theory, charcoal coated with mole-cules of appropriate molecular size andconfiguration may be used for essentiallyinstant separation of any free agent fromthe same agent bound to a carrier, pro-vided there is a significant difference in

Received March 10, 1965; accepted June 10,1965.

Supported in part by USPHS Grants AM09564, AM 09062, AM 08106 and T4-CA-5126,and the Albert A. List, Frederick Machlin, andAnna Ruth Lowenberg Funds.

1 Research Trainee of the World HealthOrganization.

2 Trainee of the National Cancer Institute(Grant T4-CA-5126).

3 Jewish Hospital of Brooklyn.

molecular size and configuration betweenthe agent alone and the agent when com-plexed with its carrier. The present re-port, an abstract of which has recentlyappeared (6), extends this concept to theseparation of insulin from insulin-anti-body complexes.

MaterialsBuffer. Sodium barbital, 14.714 g, and so-dium acetate, 9.714 g, are made up to 500 mlvolume with distilled water. To 100 ml ofthis buffer base are added 1800 ml of NaCl,0.85 g/100 ml and 100 ml of 0.1N hydro-chloric acid, to yield a final pH of 7.4 (7).

Charcoal Suspension. Norit A neutral phar-maceutical grade decolorizing carbon (char-coal) was purchased from Amend Drug andChemical Company, Inc., New York, N. Y.A 5 g/100 ml suspension of this charcoal isprepared by adding the charcoal to buffer.

Dextran Solution. Dextran 80, of 80,000average molecular weight as determined bylight scattering, was purchased from Phar-macia Inc., New Market, N. J. A 0.5g/100 ml solution of this dextran is preparedby adding the powder to buffer.

Dextran-Coated Charcoal. This is prepared bymixing equal volumes of charcoal suspensionand dextran solution. The mixture is shakenfor 10 sec, labeled "Dextran 80-coated char-coal" and stored at 4 C. Periods of storage upto a month did not appear to affect the

1375

1376 HERBERT, LAU, GOTTLIEB AND BLEICHER Volume 25

TABLE 1. Protocol for standardization of 131I-insulin (sequence of addition of reagents;numbers represent volumes in ml)

Albuminbuffer

350 mgalb./

100 ml

1811.

insulin

0.5 ml

Standardcold

insulin20 nXJ/0.5 ml

Antibody control(with '"I-insulinalone) 3.0 0.5 — Mix

Antibody control(with 13lI-insulin andstd. cold insulin) 2.5 0.5 0.5

Supernatant control 3.5 0.5 —

Antibodyserumdiluted

1:100,000

0.5

0.5

Mix andincubateat 37 Cfor 2 hr

Dextrancoated

charcoal

2.0

2.02.0

Mix and centrifuge at 3000rpm fc;r 15 min. Decantsupernates into countingtubes and count radioac-tivity in a scintillationcounter.

Equation for standardization of l31I-insulin:

'"I-insulin =MU std. cold insulinB'

B - B 'B =cpm of antibody control with 131I-insulin alone minus cpm of supernatant control.B'=cpm of antibody control with 131I-insulin and standard cold insulin minus cpm of supernatant control.

ability of the dextran-charcoal suspension tofunction in assay for insulin.

Human Albumin. Normal serum albumin(human) USP, as Albumisol liquid, was pur-chased from Merck Sharp & Dohme. Thisproduct contains 12.5 g normal human serumalbumin in 250 ml of buffered diluent. Com-mercial salt-poor serum albumin, for un-known reasons, does not function in this as-say system as well as does normal serumalbumin unless the dextran concentration israised or the quantity of albumin in the sys-tem is increased.

Albumin-Buffer. Albumin-buffer is preparedby adding 7 ml of Albumisol to 93 ml ofbuffer to give an albumin concentration of350 mg/100 ml.

Insulin. Unlabeled pork insulin was suppliedby Dr. W. Kirtley of Eli Lilly and Com-pany, Indianapolis, Ind. A stock solution of1 Mg/ml is prepared in albumin-buffer. Fromthis a standard solution of 0.8 ng (20 /IU) per0.5 ml albumin-buffer is made. These solu-tions are stored at 4 C.

131I-insulin (porcine) with specific activityranging from 113 to 298 mc/mg was obtainedfrom Abbott Pharmaceuticals, Inc., throughthe courtesy of Edward E. Grehn, and wasused as supplied. Some pork 131I-insulin wasprepared by one of us by the method ofHunter and Greenwood (8), with specificactivities of 800-1000 mc/mg. The prepared131I-insulin was purified by adsorption ontoWhatman cellulose powder and elution with

iodoacetate-treated plasma (9). The 131I-in-sulin is diluted with albumin-buffer to pro-vide a solution of approximately 0.8 ng (20

131I-insulin per 0.5 ml.

Procedure for Assay of ulI-insulin. Everynewly prepared solution of 131I-insulin is as-sayed against the cold insulin standard (0.8ng/0.5 ml albumin-buffer) using dextran-coated charcoal (Table 1). The amount of131I-insulin present is calculated from theformula:

/ B' \ng 131I-insulin = ng cold insulin I — )

where B =net cpm of supernatant containingantibody and 131I-insulin

B'=net cpm of supernatant containingantibody, 131I-insulin and standardcold insulin.

This equation is derived from equation (v),vide infra.

Insulin Antibody. Antibody to insulin wasprepared in guinea pigs by injection of porkinsulin in complete Freund's adjuvant andshown not to discriminate between humanand pork insulin. A 1:200 dilution of anti-body in buffered-albumin is prepared as astock solution and is stored at — 20 C be-tween usages.

For the assay to work successfully irres-pective of the serum insulin level, it is essen-tial to select a quantity of antibody whichwill have a maximal capacity to bind lessthan the total quantity of insulin present.The quantity of antibody is routinely se-

October 1965 COATED CHARCOAL IMMUNOASSAY OF INSULIN 1377

lected to have a maximal capacity to bindapproximately 60 to 80% of the standardamount of 131I-insulin used in each test. Todetermine the maximal insulin-binding ca-pacity of an antibody, a fixed quantity ofantibody is incubated for 2 hr at 37 C in aseries of tubes containing increasing quanti-ties of labeled insulin. Dextran-coated char-coal is added at the end of incubation toseparate free from bound insulin. The quan-tity of bound insulin is determined by count-ing the radioactivity in the decanted super-natant fluid after centrifugation sedimentsthe charcoal. Fig. 1 shows the insulin-bindingcapacity of 0.5 ml of a 1:100,000 dilution ofguinea pig anti-insulin antibody serum, de-termined in this manner. The graph showsthat the maximum binding capacity of 0.5 mlof a 1:100,000 dilution of this particularantibody is for 14.2 yuU of insulin.

The method can also be applied to deter-mine the quantity of antibody needed toachieve binding of 60 to 80% of a givenamount of labeled insulin. In this reapplica-tion, 20 MU of labeled insulin is incubated at37 C for 2 hr in a series of tubes containingincreasing volumes of antibody serum in1:100,000 dilution. The quantity of insulinbound is determined as before. Fig. 2 showsthe results of such an experiment. From thegraph, the quantity of antibody that bindsapproximately 70 % of 20 /zU can be deter-

10 20 30 40 50 60INSULIN ADDED (.uUnits)

70

FIG. 1. The insulin-binding capacity of 0.5 ml ofa 1:100,000 dilution of guinea pig anti-insulinantibody serum is depicted. The maximumbinding capacity, represented by the plateau ofthe graph, is 14.2 nU of insulin.

0 0.2 0.4 0.6 0.8 1.0QUANTITY OF ANTIBODY ADDED

(ml OF 1:100,000 DILUTION )

FIG. 2. The insulin-binding capacities of in-creasing quantities of antibody after incubationwith a fixed quantity of insulin are depicted.From this graph the quantity of antibodyneeded to bind 60 to 80 % of the labeled insulinis determined.

mined and this quantity is selected for use inthe assay.

MethodTable 2 summarizes the assay procedure,

which is schematically depicted in Fig. 3. Alltests are done in duplicate in 10 ml testtubes. The reagents are added in the volumeand sequence indicated in Table 2. Thetubes are incubated for 2 hr in a 37 C waterbath. After incubation, 2.0 ml of dextran-coated charcoal is added to all tubes. Thetubes are then capped with Parafilm andmixed by repeated inversion for approxi-mately 10 sec. They are then centrifuged for15 min at 3000 rpm. The charcoal forms asolid button at the bottom of the tubes andthe supernatant fluid is decanted into count-ing tubes and counted in a well-type scintilla-tion detector. Variability of results was de-termined using samples of plasma and serumobtained simultaneously from the same sub-ject. Each sample was run 10 times within asingle assay; results were 48.4+5.1 n\J forthe serum and 46.6 + 4.9 juU for the plasmasample. The plasma sample was run in eachof 4 subsequent separate assays and gaveresults of 48, 46, 50 and 43 /zU, for a meanvalue of 46.7 ±1.8 /xU.


TABLE 2. Insulin assay protocol (sequence of addition of reagents;numbers represent volumes in ml)

Antibody controlSupernatant control

(antibody)Unknown serumSupernatant control

(serum)

Albuminbuffer

350 mgalb./

100 ml

1311-

insulin20 /L»U/

0.5 ml

Unknownserum

Antibodyserumdiluted

1:100,000

3.0

3.52.9

3.4

0.5

0.50.5

0.5

0.5

— Mix —0.1

0.1

0.5

Mix andincubateat 37 Cfor 2 hr

Equation for calculation of serum insulin level:

/xU insulin per 0.1 ml serum =//U m I-insulin I 1 I.\B' /

B =cpm of antibody control minus cpm of supernatant control (antibody).B'=cpm of unknown serum minus cpm of supernatant control (serum).

Dextran-coated

charcoal

2.0

2.02.0

2.0

Mix and centrifuge at 3000rpm for 15 min. Decantsupernates into countingtubes and count radioac-tivity in a scintillationcounter.

Calculation of Serum Insulin Level. Net cpmare obtained by subtracting the cpm of theappropriate supernatant controls from theirrespective tubes.

Let B =net cpm of antibody controland B' =net cpm of standard cold insulin con-

trol. The quantity of 131I-insulin present in 0.5ml is given by the equation:

/ B' \MU 131I-insulin = 20 ( — — )

\ B — B /

If B"=net cpm of unknown serum, thequantity of insulin present in 0.1 ml of un-known serum is given by the equation:

insulin/0.1 ml serum

131I-insulin F-0Alternatively, B/B' can be plotted against

the quantity of cold insulin used, and astraight line graph obtained (9) (see Fig. 5);insulin values for unknown sera can be de-termined from this. The quantity of 131I-insulin used in the assay is indicated by thepoint where the graph intersects the abscissaby extrapolation (9).

SERUM INSULINREMAINS IN

SUPERNATANTFLUID

I 1 3 1 - INSULIN

TOTAL INSULININ

TEST TUBEINCUBATE

37°Cx2HR.

FREE INSULININSULIN BOUNDTO ANTIBODY

("INSULIN BIOPSY")'

ANTIBODY

REMOVEDBY

DEXTRAN-COATED

CHARCOAL

FIG. 3. The principle ofradioisotope dilution asapplied to charcoal im-munoassay of insulin isschematically depicted.


Derivation of Formula [see Hales and Randle(10)]Let M = mass of 131I-insulin added and R its radio-

activity (cpm)Let m =mass of 131I-insulin bound by antibody

and B its radioactivity (cpm)

2.5

R BSpecific activity of mI-insulin = — = —

M m(i)

If B" = radioactivity (cpm) of mI-insulin boundby antibody after dilution by a mass m" ofcold insulin, then the new specific activityafter radiodilution =

R B"M + m" mSubstituting for R from equation (ii)B X M/m _ B"M + m" m

B X M _ B"(M + m")m m

B X M = B"(M + m")

- ~ X M = M + m"

B

(iii)

(iv)

, B /(v )

Using this formula, the preparation of astandard curve using varying known quanti-ties of insulin becomes unnecessary, and only2 reference quantities are needed: the quan-tity of 131I-insulin used, and the cpm of theantibody control. As pointed out by Halesand Randle (10), this formula only holdswhen the affinity of antibody for labeled andunlabeled antigen is the same and when theamount of antigen bound by antibody isfixed.

ResultsLinear Relation. Fig. 4 shows a radiodilu-tion curve similar to that obtained withimmunoassay for insulin as originallydescribed by Berson and Yalow (9).Known quantities of cold insulin wereadded to 22.5 /iU of 131I-insulin and totalinsulin was assayed by the charcoalmethod.

Fig. 5 shows the same data replotted,using on the ordinate the ratio of B/B'in place of B/F, where B =cpm of 131I-

10 20 30 40 50ADDED COLD INSULIN (>i Units)

FIG. 4. Radiodilution curve obtained by char-coal immunoassay, in which the bound to freeratio of 131I-insulin is plotted against the addedcold insulin.

o C •o o.c

•a a S cE E.o

i " ' 5

30 20 10 20 30 40 50

-Added Cold Insulin {p Units)

60

FIG. 5. The data of Fig. 4 are replotted to illus-trate that charcoal immunoassay of insulin canbe represented by a linear graph.


10 20 30 40 50ADDED COLD INSULIN ( p Units )

FIG. 6. The data of Fig. 4 are plotted to showthe correlation between added and recoveredcold insulin.

insulin bound by antibody and B ' = cpmof diluted 13lI-insulin bound by the samequantity of antibody. The straight linegraph obtained supports the evidencepresented by Hales and Randle (10),who showed by antibody precipitationthat radioimmunoassay of insulin can berepresented by a linear graph.

Recovery of Known Amounts of AddedCold Insulin. Fig. 6 shows the good cor-

relation between added cold insulin andrecovered insulin in the charcoal assay.In Fig. 6 the recovered insulin is ob-tained by using the same data as for Fig.1 and 2 to solve for equation (v), givenearlier.

Measurement of Plasma Insulin. Fig. 7and 8 show plasma insulin levels in thecourse of oral glucose tolerance testsperformed on different days on the samepatient. They indicate that plasma insu-lin levels obtained by the charcoal pro-cedure are essentially similar to those ofthe chromatographic method of im-munoassay of Berson and Yalow (9).This would be expected since the char-coal assay separates free from bound in-sulin, as does the chromatographicmethod of Berson and Yalow (9).

Fig. 9 shows plasma insulin levels inthe course of an intravenous glucosetolerance test. The values obtained bythe charcoal method are similar to thosefor the chromatography method. Thesmall rise in insulin level between the 30-and 40-minute samples is faithfully re-corded by both methods. However, inthe 60- and 90-minute samples the char-coal method recorded higher insulin val-ues by 26 and 23 yuU, respectively. The

200 200

!nsulin(Chromatogrophy-4 da j Incubation )

50 100 150 200

TIME ( minutes)

250 300

FIG. 7. Comparison ofinsulin levels by thecharcoal and chromato-graphic immunoassaysduring the course of anoral glucose tolerancetest.


175

150

Et 125Q.

FIG. 8. Comparison of ••§insulin levels by the 3 100charcoal and chromato- 3graphic immunoassays zduring the course of an ^ 7 5

oral glucose tolerance w

test. - _ ..< 50 •/ ;

••. Insulin (Charcool-2hr. Incubation)

75

150

125 §

100 6>

ui75 oo

3

50

25

In»uli

. •ORAL GLUCOSE 100 yn.--:::: i•<doy Incubation) |

25 m

50

insulin levels in the fasting sample (28/*U) and the 90-minute sample (30 fAJ)were almost identical in the charcoalmethod, but in the chromatographymethod they were wide apart (21 and 7JUU, respectively).

Fig. 10 shows the plasma insulin val-ues obtained in the course of threetolbutamide tolerance tests performedon the same patient over a period of twomonths.

These results show a good over-all cor-relation between the charcoal and thechromatography methods. The shapes ofthe insulin curves in these tests are essen-tially similar in both methods.

No significant difference between insu-lin values obtained from serum or hepa-rinized plasma has been observed. How-ever, use of serum samples is preferredas they store better (i.e., clots do notform in stored serum, but do form instored heparinized plasma).

DiscussionA number of immunologic micro-

methods have been described for the as-say of insulin in serum (9-12). All ofthem depend on the addition of a stan-dard or unknown solution of unlabeled

100 150 200

TIME (Minutes)

250 300

insulin to a fixed amount of 131I-insulin,followed by "biopsy" of the total pool ofinsulin with a fixed amount of antibodyto insulin, and then separation of the freeinsulin from that bound to antibody.The reduction in quantity of radioactiv-ity bound to antibody is directly propor-tional to the quantity of unlabeled in-sulin present in the pool. The methoddescribed in the present paper appears

120

-• 100

80

60

3! n< 40?

20'

-•IV. GLUCOSE 25 gm.

Insulin (Charcoal)

Insulin (Chromatagrophy)

250

200 -g.

150 e

100!

50

20 40 60 80TIME (Minutes)

100 120

FIG. 9. Insulin levels obtained in the course of anintravenous glucose tolerance test.


~ 140

I5; 120a.

| 100

3 802^ 60enS 40

PATIENT D 910 64

--..JnsulinJChromalography)

IV. TOLBUTAMIDE I git

120 Q

100 £

f

60S

40 »QO

20 q

0 10 20 30 40 50 60 70 80 90 100 110 120TIME (Minutes )

— 140EZ 120a.

I 100

5 802o 60in

J 4 0 :

PATIENT D 1019-64

v ^ Jnsuhn ( Chromalography)

PATIENT D II 12 64 EO

1202u

100 ^6

80 m

o20 S

TOLBUTAMIDE Igm. icn D. J I.v. TOLBUTAMIDE Ign

10 20 30 40 50 60 70 80 90 100 110 120TIME (Minutes)

? Tpsuiin(Chon;pol|

10 20 30 40 50 60 70 80 90TIME (Minutes)

100 110 120

FIG. 10. Plasma insulin levels in the course of 3 tolbutamide tolerance tests performed on thesame patient over a period of 2 months. A reduced insulin response in this patient is demonstratedby both methods.

to have advantages of greater simplicityand rapidity over those previously de-scribed.

As indicated in Fig. 1 and 2 of thepresent paper, and in Fig. 8 of the paperby Hales and Randle (10), a given quan-tity of antibody has a fixed maximalcapacity to bind insulin. Hales andRandle used a quantity of antibody solarge in relation to the quantity of 131I-insulin used that the theoretical straightline relationship for isotope dilution didnot hold, and they were therefore forcedto use the relationship of added insulin toB/B'. In the present study, the quantityof antibody chosen was closer to thesmall amount theoretically necessary sothat the theoretical relationship for iso-tope dilution will hold (an amount belowthe plateau of Fig. 2). By using such anamount, a true linear relationship be-tween cold insulin added and insulin re-covered can be obtained, such as was ob-

tained with even a slightly larger quan-tity of antibody, as illustrated in Fig. 6.It should be noted that the fact that agiven quantity of antibody has a fixedmaximal binding capacity tends to sup-port the concept (9) that insulin is a uni-valent antigen.

In the charcoal studies, most of theantigen bound was bound within twohours at 37 C; hence the routine use ofthis time and temperature, which alsoreduces incubation damage to labeledinsulin. As in prior methods, it is notnecessary for the antigen-antibody reac-tion to go to completion; all that isnecessary is that time and temperatureof incubation be the same for all sam-ples. When it is desired to use microquantities of serum, or short incubationtimes, as the quantity of antigen (insu-lin) is decreased, the weight of antibodyper unit weight of antigen should be in-creased.


Volumes of the various solutions areunimportant in the charcoal assay, andmay be altered as desired. However, theratio of antigen to antibody and ofalbumin to dextran to charcoal shouldnot be significantly altered. The markedsensitivity of charcoal assay for insulinmay be further increased by using moreplasma, since there is theoretically nolimit to the amount of plasma whosetotal insulin content may be assayed.Use of charcoal also makes unnecessarythe need to screen batches of chromatog-raphy paper in order to find paper withfavorable insulin-binding affinity in thepresence of plasma.

For convenience in assaying the entirerange of expected plasma insulin levels,0.8 ng (20 iAJ) of 131I-insulin is used per0.1 ml of unknown plasma. Of this quan-tity of 131I-insulin, 7 1 % is bound by 0.5ml of a 1:100,000 dilution (i.e., 2xlO~5

ml) of our guinea pig antibody serum,using a two-hour incubation at 37 C(represented in Fig. 4 by the initial B/Fof 2.46). Using this quantity of 131I-insu-lin, insulin levels ranging from 0 to 60/xU/0.1 ml of sample can be accuratelyand reproducibly measured (Fig. 4-6).For concentrations of insulin above 60JUU/0.1 ml, either decreased quantities ofsample or increased quantities of tracermay be used.

The amount of radioactive insulinbound by a fixed quantity of antibody isdetermined by the duration of incuba-tion. As duration of incubation increases,the amount of bound insulin increases;the same is true at 4 C (Fig. 11). Notethat sufficient bound insulin has formedafter one hour of incubation to permitperformance of insulin assay. Only slightincrements in bound insulin occur in thefinal 44 hours of a 48-hour incubationperiod.

Norit A pharmaceutical grade char-coal, 50 mg, adsorbs, essentially in-stantly, more than 99% of 0.8 ng of 131I-

- No incubation= I hr. incubation at 37° C= 2hrs.incubation at 37°C= 4 hrs. incubation at 37° C= 48hrs. incubation at 4° C= !37hrs. incubation at 4°C

10 20 30 40 50 60ADDED COLD INSULIN (p Units)

FIG. 11. The amount of radioactive insulinbound by a fixed quantity of antibody increaseswith the duration of incubation. Radiodilutioncurve b shows that sufficient insulin binding hasoccurred after 1 hr at 37 C to permit perfor-mance of the charcoal immunoassay.

insulin. Dextran-coated charcoal ad-sorbs, essentially instantly, 90 to 98%of 131I-insulin. Using chromatoelectro-phoresis, it was found that, of the ap-proximately 5% average of the totaladded radioactivity in the supernatantcontrol (i.e., not adsorbed by dextran-coated charcoal), approximately threefourths was due to degraded insulin andone fourth to undegraded insulin. Thus,it appears that dextran-coated charcoaladsorbs approximately 99% of free butvery little degraded insulin.

Theoretically, the proper molecularsieve coat for the charcoal would be amolecule with a molecular weight andconfiguration such that the insulin


polypeptide dimer could pass betweenthe adjacent molecules into the char-coal, but the complex of insulin withantibody would be excluded. Relativelysmall molecules, such as albumin, hemo-globin and dextrans of average molecu-lar weights 10,000, 20,000 and 40,000blocked free insulin from being adsorbedto charcoal. Materials which allowedfree insulin to pass but rejected boundinsulin, in addition to dextran 80 (aver-age molecular weight 80,000), were dex-trans of average molecular weight110,000, 150,000, 250,000, 500,000 and2,000,000, as well as gamma globulin,fibrinogen and Ficoll, a nonionic, highpolymer alcohol of average molecularweight approximately 400,000 (Phar-macia, Inc.). The pores between theadjacent molecules of Ficoll appear to besufficiently small to block passage of in-sulin-antibody complexes.

To minimize insulin degradation andsticking to glassware, insulin and anti-body solutions were made in buffer con-taining 0.35 g/100 ml albumin. Forunknown reasons, when these solutionswere prepared with bovine rather thanhuman serum albumin, insulin-antibodycomplexes were not excluded from dex-tran-coated charcoal as well as whenhuman albumin was used. This wasprobably not due to a blocking effect ofhuman albumin, since human albumin,when used in place of dextran as themolecular sieve, did not adequately ex-clude insulin-antibody complexes fromthe charcoal.

Preliminary studies suggest the assayhere described for insulin and antibodyto insulin in guinea pig serum will be use-ful for assay of anti-insulin-antibodytiters in human serum, for assay of gluca-gon and antibody to glucagon, and, inpreliminary studies with Grumbach,Kaplan and Wolter, for assay of growthhormone and chorionic "growth hor-

mone-prolactin" (placental lactogen)and their antibodies. In all assays forhormones using the "instant dialysis"concept, it must be borne in mind thatthe effective conformation, and there-fore the diffusion behavior of polypep-tides, is strongly influenced by smallalterations in the solvent, particularlyionic strength and temperature (13).Preliminary studies suggest circulating"free" and "bound" insulin of Antoni-ades et at. (14) may be separated byappropriately coated charcoal. Coatedcharcoal appears to be a simple alterna-tive to ultracentrifugation and gel filtra-tion (15) for separating free from boundinsulin at physiologic pH and ionicstrength.

AcknowledgmentWe are indebted to Mr. John Farrelly and

Mrs. Katherine Kellett for technical assistance.

References1. Herbert, V., C. Gottlieb, K.-S. Lau, and

L. R. Wasserman, Lancet 2:1017, 1964.2. Gottlieb, C, K.-S. Lau, L. R. Wasserman,

and V. Herbert, Blood 25: 875, 1965.3. Lau, K.-S., C. Gottlieb, L. R. Wasserman,

and V. Herbert, Blood 26: 202, 1965.4. Herbert, V., M. Fisher, K.-S. Lau, C. Gott-

lieb, N. Gevirtz, and L. R. Wasserman,Amer J Clin Nutr 16: 385, 1965 (Abstract).

5. Herbert, V., C. Gottlieb, K.-S. Lau, and S.Silver, Fed Proc 24: 254, 1965 (Abstract);J Lab Clin Med (in press).

6. Herbert, V., K.-S. Lau, C. W. Gottlieb,R. A. Arky, and S. J. Bleicher, J Clin In-vest 44: 1059, 1965 (Abstract).

7. Michaelis, L., Biochem Z 234: 139, 1931.8. Hunter, W. M., and F. C. Greenwood, Na-

ture (London) 194: 495, 1962.9. Yalow, R. S., and S. A. Berson, J Clin In-

vest 39: 1157, 1960.10. Hales, C. N., and P. J. Randle, Biochem J

88: 137, 1963.11. Grodsky, G. M., and P. H. Forsham, J

Clin Invest 39:1070,1960.12. Morgan, C. R., and A. Lazarow, Diabetes

12: 115, 1963.13. Craig, L. C., J. D. Fisher, and T. P. King,

Biochemistry (Wash) 4: 311, 1965.14. Antoniades, H. N., A. M. Huber, B. R.

Boshell C. A. Saravis, and S. N. Gershoff,Endocrinology 76: 709, 1965.

15. Chao, P. Y., J. H. Karam, and G. M.Grodsky, Diabetes 14: 27, 1965.

coated charcoal immunoassay of insulin

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