72 BIOCHEMICAL EDUCATION October 1976 Vol. 4 No. 4

A LABORATORY-CLASSROOM EXPERIMENT ILLUSTRATING AFFINITY CHROMATOGRAPHY AND RADIOIMMUNOASSAY: PURIFICATION AND ASSAY OF INSULIN

BONNIE LEE,* JEAN-CLAUDE DILLON, & GEORGE WOLFT D e p a r t m e n t of Nutr i t ion and Food Science Massachuse t t s Inst i tute of Technology Cambr idge Massachuse t t s 02139, U.S.A.

In teaching a course on methods of biochemical analysis, we have found the following experiment very useful; it is designed to demonstrate the techniques and principles of affinity chroma- tography and radioimmunoassay as methods of purification and assay for insulin, and can be performed within two or three 3-hr laboratory periods.

The technique of affinity chromatography has become widespread in biochemical research because of its technical simplicity and its high degree of biological specificity in the purification of macromolecuies and complex biological structures. The basic principle behind this technique is the immobilization of the ligand to an insoluble, porous support, which can then be used to bind specifically (but reversibly) the component that is to be isolated. This component can then be eluted by appropriate means, either specifically by an eluant resembling the ligand or by a "deforming" system, such as a change in pH. A convenient affinity column for isolation of insulin from serum was described by Akanuma and HayashiX: the guinea pig antibovine antibody is attached to a Sepharose 4B gel activated by cyanogen bromide; the insulin is then bound to the column and can be eluted with dilute acetic acid.

Radioimmunoassay provides a simple and sensitive method for measuring low insulin concentrations in plasma or serum, z'5 An unlabeiled antigen (insulin) competes with a radioactively labelled antigen ([12sI]insulin) for the binding sites of the antibody (insulin antibody). The unlabelled antigen therefore diminishes the binding of the labelled antigen. The quantity of antigen in unknown samples can be determined, then, by comparing the decrease in antibody binding of labelled antigen in unknown solutions with that in known standard solutions.

The unbound antigen can be separated from the antibody- antigen complex, and the degree of binding of radioactive antigen can be determined by liquid scintillation counting. An easy and convenient way to obtain all the materials necessary for this assay is by purchasing the "Insulin Radioimmunoassay Kit (IM 78)" (Amersham/Searle Corp., Arlington Heights, Ill., U.S.A.) at a modest price of about $100; it supplies sufficient material for 400 assays. In order to separate the unbound antigen effectively, one adds a second antibody to the antigen-antibody complex, causing precipitation of the complex. The second antibody is then directed against the one that had originally reacted with the antigen.

The Insulin Radioimmunoessay Kit employs the assay method of Hales and Randle6'7: here the second antibody is reacted with the first before the first one is used. The first antibody (a guinea pig antibody against insulin) reacts with the second antibody (a rabbit anti-guinea pig antibody) to form an insoluble complex. The insulin (either unlabelled, from serum, or SaSl-labelled) can still combine with the first antibody, adding to the insoluble complex in a relatively short time. The bulk of the double antibody precipitate can be increased with additional second antibody and then separated from unbound insulin by filtration.

Materials The experiment requires the following materials (with suggested

sources): cyanogen bromide-activated Sepharme 4B, stabilized by dextran and lactose (Pharmacia Fine Chemicals, Piscataway, N.J., U.S.A.); bovine insulin antibody (raised in guinea pigs) and rabbit

guinea pig antherum (Miles-Yeda Ltd., Rehoroth, Israel); "RIA- fluor" (New England Nuclear, Boston, Mass., U,A.A.), a liquid scintillation fluor solution adapted for t2Sl counting; filtration apparatus and fibre filters (pore size: 10.8tun) (Millipore Corp., Bedford, Mass., U.S.A.); horse serum (Grand Island Biological Co., Grand Island, N.Y., U.S.A.); a mixer for rapid mixing of contents of test tubes; and standard human insulin (Eli Lilly, Indianapolis, Ind., U.S.A.), made to 2410~U/ml in Tris-BSA buffer. C['ris-BSA buffer consists of 0.14 NaC1 and 10 mM Tris chloride, containing 0.25% bovine serum albumin (BSA); #U is microunit).

Buffer A was made of 40 mM isotonic phosphate buffer (contain- ing, per liter, 9.0g of NaCI and 6.2 8 of NaH, PO,.2H,O, pH adjusted to 7.4), 0.5% BSA, and 6 mM sodium mercurithio- salicylate. This buffer is used for dilution of standards and iodinated insulin and for the preparation of "Buffer B."

Buffer B is a high-protein buffer used for washing antibody precipitates. It consists of 500ml each of horse serum and Buffer A, filtered carefully through diatomaceous earth to give a bright, clear wash fluid. It should be stored at 2-4°C.

The Insulin Radioimmunoassay Kit provides: insulin binding reagent (antihuman insulin serum from guinea pig reacted with anti-guinea pig serum from rabbit) with phosphate buffer, preservative, EDTA, and BSA as stabilizer; ['aSl]insulin (about 20~Ci); and human insulin standard solution (160~U/ml in phosphate buffer). When reconstituted, 100~1 of the insulin binding reagent will bind approximately 40% of 450pg of [S2Sl]insuiin. When reconstituted with Buffer A, 100~1 of the [12el]insulin solution will contain 250pg of [t~sI]insulin (or about 0.05 ~Ci of 12si)"

Expaqmemsl Preparation o f column, s Swell and wash 2 8 of the cyanogen

bromide-Sepharose 4B gel with 200ml of a I0 "s M HCI solution for 15 rain on a glass filter. Mix the gel in a test tube with babout 25 mg of the substance to be coupled - - antibovine insulin from guinea pig, dissolved in a 0.1 M NaHCO~ buffer solution containing $ ml of 0.5 M NaC1; this mixture should then be rotated end over end for 2 hr at room temperature or overnight at 4°C. Other gentle stirring methods may be employed, but magnetic stirrers should be used with great care to avoid fragmentation of the gel beads. Wash away unbound material with coupling buffer; any remaining active groups are then reacted with 1 M ethanolamine at pH 8.0 over a 1- 2hr period. Use three washing cycles to remove noncovalently absorbed protein, each cycle consisting of a wash at pH 4.0 (0.1 M acetate buffer, containing 1 M NaCI) followed by a wash at pH 8.0 (0.1 M borate buffer containing 1 M NaCI).

Binding and elution o f insulin on affinity chromatography column. After equilibrating the column with Tris-BSA buffer, charge it with 1 ml of the insulin solution to be purified at a flow rate of 60ml/hr. Wash the column with 60ml of Tris-BSA buffer (tubes 1-10, with about 6ml being collected for each tube). The insulin is absorbed to the column at pH 7.8. Next wash the column with 24ml of Tris buffer (no BSA) (tubes 11-14), in order to eliminate any BSA that may interfere with the protein assay of the einted insulin. Without BSA in the buffer, the pH increases

*Present address: Department of English, University of Virginia, Charlottesville. Virginia 22093, U.S.A. tTo whom all inquiries concerning this report are to be sent.

BIOCHEMICAL EDUCATION October 1976 Vol. 4 No. 4 73

slightly, and small amounts of insulin dissociate from the column, but not enough to spoil the results. Finally, wash the column with 30ml of 1M acetic acid (tubes 15-20), in order to elute the bound insulin. The pH of tubes 16-19 rises immediately from pH 2.5 to 7.8 when approximately 4 ml of 2.5 N NaOH are added to each; check the pH with a pH meter. The column can be reused after equilibration of the matrix with Tris-BSA buffer.

Preparation of standard curve and radioimmunoassay for insulin. Each group of two students receives 2ml of standard solution (160/~U/ml). The dilutions shown in Table 1 are made by each group. Number all reaction tubes and follow the scheme for the assay* (see Table 2). The antigen-antibody microprecipitate, which forms in tubes 3-20, can be separated by the following filtration procedureS:

1. Float sufficient Millipore filter discs for a day's use, one at a time, on the surface of some Buffer B in a beaker and leave them in the beaker until required.

2. Assemble the microanalysis filter holder on the vacuum filtration flask, fit the first filter disc, and rinse with Buffer B.

3. At the end of the 2t-hour incubation period, remove the reaction tubes from the refrigerator one at a time, mix with a test tube mixer, and deliver the solution onto the filter using a disposable pipette; apply suction and wash the tube with two successive portions (approx. 1 ml each) of ice-cold buffer, transferring the washings to the filter. Washing of the filtration apparatus between individual samples is not necessary. It is convenient to use two disposable pipettes for a batch of filtrations, one for transferring the assay samples and washes from the reaction tubes to the filter and another for transferring Buffer B to the reaction tubes. After transferring the second wash, this pipette is clean enough to be used for the next sample without further treatment. When filtration of a sample is complete and while the filter is still under vacuum, remove the glass reservoir from

V o l u m e o f V o l u m e o f Fbtal insalla standard Buffer A concentration

(ed) (ml) (~U/ml) 0.2 0.2 80 0.2 0.6 40 0.2 1.4 20 0.2 3.0 10

Table 1. Dilutions to be made of standard insulin

:E ~L 0

:5,000 -~e

\

2,000 -

t,ooo -

0 I I I I 20 40 80 160

Insulin, FU/m l

Figure 1. Standard curve: radioactivity of precipitate against insulin concentration.

the filter and lift off the filter disc with the aid of a scalpel blade and forceps.

4. Place each filter in a clean counting tube and count the radioactivity in all samples and in an empty background tube. The filters should be in the same position in all tubes to minimize geometric effects on count rate. Count the radio- activity of the 2 total count tubes containing only 100/A of It =Sl] insulin working solution at the same time.

Count the filters in vials containing 15 ml of RIA-fluor in a liquid scintillation counter. Tubes 1 and 2 give total counts of [t=Sl] insulin added. Tubes 3 and 4 give zero insulin (i.e., total [l=sI] insulin bound). Values in tubes 3 and 4 should be 25-40% of those in tubes 1 and 2. s Plot the results as counts/min versus the concentration of insulin in standard. The concentration of the unknowns can be read in terms of the concentration in the standards. Figure 1 depicts a typical standard curve.

Next, for protein assay* and radioimmunoassay, take 100-~1 aliquots of the tubes from the eluate of the affinity column (indicated in Table 3). In addition, perform an insulin radio- immunoassay and protein assay on a 100-~1 aliquot of the original

Standard curve (/~U/ml)

Unknown Unknown 10 20 40 80 160 Blank sampk 1 mmpk 2

Tube numbers 1-2" 3-4 5-6 7-8 9-10

Buffer A (/~1) - 100 - - -

Appiopriate human insulin standard dilution (/~l)t - - 100 100 100

Unknown ( ~ 1 ) . . . . .

Binding reagent (td) - 100 100 100 100

11-12 13-14 15-16 17-18 19-20

- - 200 - -

1 0 0 100 - - -

- - - 1 0 0 1 0 0

1 0 0 1 0 0 - 1 0 O 1 0 0

Mix. Incubate in refrigerator at 2-4 degrees centigrade for 45 minutes. Add 100 1 of 1:400 dilution of guinea pig antiserum to increase bulk. Add:

[ 1251] insulin, 250 pg (~1) I00 100 100 100 100 100 100 100 100 100

Mix. Incubate in refrigerator for 2V4 hr (or between classroom sessions). Filter.

*Do not filter. tSee Table 1.

Table 2. Scheme for assay of insulin (standard and unknown) adapted from "Insulin Radioimmunoassay ''s

74 BIOCHEMICAL EDUCATION October 1976 Vol. 4 No. 4

Tube Step number Imuifm Vo/ume Insulin Pro~b,

(~u/aa) (ad) (~u) (~s/~) Fixation 5 0 5 0 not determined

Washing 13 0 5 0 1.0

Acetic acid elution 15 5 6 0 undetectable

Acetic acid elution 16 180 6 1080 undetectable

Acetic acid elution 17 160 6 960 undetectable

Acetic acid elution 18 20 8 120 undetectable

Acetic acid elution 19 10 8 80 undetectable

Acetic acid elution 20 5 5 25 undetectable

Table 3. Purification of insulin on an affinity column (placed on column: 2410t~U insulin, in 1 ml Tds-BSA buffer [0.96~U inlmlinl/~g protein] )

insulin sample (before being placed on the column). Table 3 tabulates the results.

D ~ l ~ i M d o n As Table 3 shows, the recovery of insulin averages 94%, with the

bulk eiuting in two tubes shortly after the acetic acid wash. In the tubes containing the insulin, protein eiution is so small that it is undetectable (at least under these assay conditions, with 100-td samples).

This insulin purification experiment can be completed in two or three laboratory periods of 3hr each, the difference depending upon whether the students prepare the affinity columns themselves or not. The first session can be used for preparation of the column; the second semion for the insulin binding, the washing of insulin through the column, the preparation of the standard curve, and the incubation of unknown samples. The samples can be left to incubate in the refrigerator between the second and third sessions, and the third session can consist of filtration and liquid scintillation counting.

The insulin solution used in our classroom experiments is made artificially from pure standard human insulin and BSA; in preparing it, we attempt to simulate serum but also to find a concentration high enough (about 2500t~U/ml) for a convenient assay to be performed directly on the eluate from the column. Akanuma et al. le described the purification of insulin from pancreatic vein se~um containing about 1350~U/ml. Their method could, of course, be adapted to purify insulin from such a source, or even from serum, if the eluate from the affinity column can be concentrated.

It should he pointed out that the (commercially obtainable) antibody used for the affinity column acts against bovine insulin, whereas the purified insulin is from the human. Cross reaction is sufficient, however, to allow complete binding of the human insulin.

ACKNOWLEDGMENT The authors are grateful to Amersham/Searle Corp., Arlington

Heights, IU., for permission to quote from the Insulin Radio- immunoassay Kit instruction manual.

REFERENCES I Akanuma, Y. and Hayashi, M., in MethodJ/n Enr#mo/oLv, VOl. 34 (Jacoby, W. B. and Wllcheck, M., eds.), Academic Press, New York, 1974, p. 93.

z Berson, S. A., Yalow, R. S., Bauman, A., Rothschild, M. A., and Newerly, K., ./. Clin. Invest. 35:170 (1956).

3 Berson, S. A. and Yalow, R. S.,J. Clin. Invest. 38:1996 (1959).

4 Yalow, R. S. and Berson, S. A., Nature 184:1648 (1959). Yalow, R. S. and Berson, S. A. ,~ Clin. Invest. 39: 1157(1960).

e Hales, C. N. and Randle, P. J., Lancet !: 200 (1963).

7 Hales, C. N. and Randle, P. J., Biochem. 3. 88:137 (1963).

s "Insulin Radioimmunoassay", brochure accompanying Insulin Radioimmunoassay Kit (IM 78), Amersham/Searle Corp., Arlington Heights, Ill., 1976.

9 Lowry, O. H., Rosebrough, N. S., Farr, A. L., and Randall, R. J.,f. Biol. Chem. 193: 265(1951).

10 Akanuma, Y., Kyzuya, T., Hayashi, M., Ide, T., and Kuzuya, N., Biochem. Biophys. Res. Commun. 38:947 (1970).

AIBS Directory of Blesei~ee ~ a n t s and Faculties in the United States and Canada

By Peter Gray. Second edi t ion, 1975. Dowden, Hutch inson and Ross, Inc. , a n d d is t r ibuted by Hals tead Press, a division of John Wiley and Sons, Inc. Pp. 660. £14.00 or $27.75.

The information in this directory of the American Institute of Biological Sciences was collected by means of questionnaires sent out to 2,529 departments, of which 92% replied including 2% who requested the entry "No information available." The 90% response has resulted in this impressive compilation - - the Faculty Index, for instance, listing the names of about 30,000 individuals. Information about a department includes the name of the Chairman, his telephone number, names of the professors, the number of other staff such as research assistants, the degree programmes, graduate and undergraduate degrees ~nd their requirements and a list of the graduate and undergraduate courses available for graduate credit.

The compiler admits that there are difficulties in defining a biosclence department and has taken the granting of an academic degree as the necessary condition for inclusion. Thus while all biochemistry departments would get in, a distinguished department of neurosurgery doing fine research, for instance, would be

excluded. Just as biological taxonomy is bedevilled by name changes and the activities of "splitters" and "lumpers", so bioscience departments have been afflicted by the same troubles. For instance, during the 1950s and 1960s, "Anatomy" fell into disfavour and departments were quick to change either their names or their habits or both. Biological Structure or Cell Biotosy became in-names so one must not be surprised nowadays to find a Department o f C'~tobiolosy offering instruction in human gross anatomy. While most of the older universities in the United Kingdom have separate departments of Botany and Zoology, it is interesting to note that such are rare in the New WoHd and Departments of Biology are more usual, an integration which seems logical to those of us who believe in the biochemical unity of life.

The first edition was published in 1967 and the present second edition in 1975. There does not appear to he any indication as to when the questionnaires were sent out and some of the information could be out-of-date if the gestation period of the book has been long. It would have been useful if the date of receipt of the completed questionnaire had been added to the other departmental information provided.

The reviewer has found the directory to be a useful book to have available, particularly in tracing the current (o/le hopes) addresses of bioscientists in the U.S.A. and Canada and would recommend Librarians to purchase the directory for the reference shelves of their Departmental or University Libraries.

B. A. Kilhy