UNCLASSIFIED

AD NUMBER

AD819932

NEW LIMITATION CHANGE

TOApproved for public release, distributionunlimited

FROMDistribution authorized to U.S. Gov't.agencies and their contractors;Administrative/Operational Use; JUL 1967.Other requests shall be referred to theArmy Biological Laboratory, Attn:Technical Releases Branch, Fort Detrick,MD 21701.

AUTHORITY

BDRL, per d/a ltr dtd 28 Sep 1971

THIS PAGE IS UNCLASSIFIED

AD

t4

STECHNICAL MANUSCRIPT 390

MICRODIFFUISION IODINATIONOF PURIFIED MICROBIAL ANTIGENS

FOR COPRECIPITATION SEROLOGY

Jack Gruber0 o~rV 0. Wright

*409** C4

JULY 1967 4STT= # UNCLASSU'X.I

this docuiment is subject to special export conztrols and *aftransmittal to foreign~ governmnt or lo ci I w be

COCOCOCCO ado Gal) wih prior approval of

* .. 0.../7

DEPARTMEN OFTH AM

CR*CC2F

Fort DeoddcFrederick, Maryland D 0 C

0SEPO 2 167

Reproduction of this publication in whole or inpart is prohibited except with permission of theCwmmanding Officer, Fort Detrick, ATTN: TechnicalReleases Branch, Technical Information Division,Fort Detrick, Frederick. Maryland, 21701. However,DDC is authorized to reproduce the publication forUnited States Government purposes.

DDC AVAILABILITY NOTICES

Qualified requesters may obtain copies of thispublication from DDC.

Foreign announcement and dissemination of thispublication by DDC is not authorized.

Release or announcement to the public is notauthorized.

DISPOSITION INSTRUCTIONS

Destroy this publication when it is no longerneeded. Do not return it to the originator.

The findings in this publication are not to beconstrued as an official Department of the Armyposition, unless so designated by other authorizeddocuments.

I.Frederick, Maryland 21701

TECOICAL MANUSCRIPT 390

MICROPIFFUSION IODINATION OYF PURIFIED MICROBIAL ANTIGENSFOR COPRECIPITATION SEROLOGY

I George G. Wright

Medical Investigation DivisionMEDICAL SCIENCES LABORATORY

Project 1C01450IB71LA July 1967

1

2

In conducting the research described in this report, the

investigators adhered to 6he "Guide for Laboratory AnimalFacilities and Care," as promulgated by the Committee onthe Guide for Laboratory Animal Facilities and Care of theInstitute of Laboratory Animal Resources, National Academyof Sciences-National Research Council.

ABSTRACT

Conventional methods for introduction of 1131 may modifyor destroy the serological reactivity of labile microbialantigens. A modified microdiffusion iodination method thateliuinates the exposure of antigens to excess oxidizingagent produced satisfactory trace labeling of purifiedprotective antigen of Bacillus anthracis and enterotoxin Bof Staphylococcus aureus without detectable change in pre-cipitating activity. The iodinated antigens proved suit-able for use in the ammonium sulfate coprecipitationtechnique of Farr.

i'I

.: i . . . !i

3

MICRODIFFUSION IODINATION OF PURIFIED MICROBIAL ANTIGENSFOR COPRECIPITATION SEROLOGY

Although combining powu ior antigen is rW-.u&L6meit"1 !-v e--31 oantibody activity, most quantitative serological methods measure onlysecondary effects of such combination. Accordingly, these methods mayprovide inaccurate estimates of antigen combining power and inadequatetests of the significance of specific antibodies in acquired resistancet. infection. The ammonium sulfate coprecipitation technique of Farr,1

using antigen labeled with iodinel 31, provides a quantitative and sensitivemeasure of the capacity of antisera to bind protein antigens. Despite thesensitivity and precision of the method,3 it has been virtually neglectedby those studying resistance to infection, probably in part because ofdifficulty in iodinating relatively labile microbial .ntigens. During astudy of the application nf coprecipitation serology to certain purifiedmicrobial antigens,s a microdiffusion procedure was tmployed for traceiodination. This method provided labeled antigens suitable for serologicaluse and should facilitate the application of Farr's method to other antigen-antibody systems.

Protective antigen of Bacillus anthracis. prepared in our laboratory,'and enterotoxin B of Staphylococcus aureus kindly suiplied by Dr. E.J.

Schantz, were investigated. Attempts to iodinate the urified anthraxprotective antigen by the direct nitrous acid procedure did not yieldsuitably labeled material that retained serological activity. Use ofchloramine-T and other procedures also failed to yield suitable material.Because of this difficulty a modification of the microAiffusion procedureof Banerjee and Ekins5 was investl-ated. This technique involves transferof iodine to the protein solution by gaseous diffusion ind eliminates addi-tion of excess oxidizing agents to the protein.

The modification employed was similar to that described by Seth foriodinaLion of serum globulin. In place of a Conway microdiffusion cell,a modified 50-ml Erlenmeyer flask wab used. A section of glass tubing2.5 cm high and 1 cm in diameter was sealed to the bottom of the flask,thus forming a separate inner compartment. The protein solation to beiodinated, usually 1 ml, was pipetted into the outer chamber of the vessel.Two-tenths milliliter o 130.002 M KI was pipetted into the central compart-ment, 50 to 100 pc of 1131 (as NaI 31 ) were added, and the flask was sealcdwith a skirt-type vaccine stopper. Stock acid dichromate solution (0.27 MNa2Cr2O7 in 36 N h2SO4 ) was diluted 1:20, and 0.2 ml was added to the centralcompartment by a syringe with attached 3- or 4-inch needle. A large excessof oxidizing agent must be a-oided; otherwise the iodine will be furtheroxidized to iodate. The flask was held at room temperature (23 to 25 C)and gently rotated occasionally. After 1 hour the labeled protein solutionwas withdrawn with another syringe and needle. The free iodide was removedby passage through a Sephadex G-25 column or by dialysis.

I4

In studies with the purified anthrax proteLive antigen (1 mg per ml),80% of the radioiodide was c=,/erted to gaseous iodine and approximately20% of the toLal iodine was transferred to the protein solution. Thetransferred iodine bound to antigen ranged from 10 to 35%. Usually, about1 atom of iodine was bound per molecule of antigen. lodination resultingZV -I ' '-!1- ±h L~,l . 4 , Z iodine peL Luolec". of antige-did not affect the precipitating activity of the antigen; labeling abovethe level of 7 atoms per molecule caused appreciable destruction.

In studies with the purified enterotoxin B (2 mg per ml), 70 to 90%of the initial radioiodide was converted to gaseous iodine and approximately25% was transferred to the toxin. Efficiency of labeling averaged approxi-mately 15% of the transferred iodine. Usually 0.5 to 1.5 atoms of iodinewere bound per molecule of toxin. Both an Ouchterlony plate titration anda modified Oudin procedure10 indicated that essentially all of the precipi-tating activity was retained after iodination and dialysis.

With both anLigens, iodination by microdiffusion resulted in labeledmaterial suitable for use in the ammonium sulfate coprecipitation technique.Tests with the labeled anthrax protective antigen revealed marked differ-ences in the combining capacities of various equine sera (Fig. 1). Hyper-immune pony sera at 1:10 dilution precipitated 86 to 98% of the labeledantigen preparation, although only 7% was precipitated by a 1:10 dilutionof normal horse serum, Differences in immune sera were evident azjd werecharacterized by determining the dilution of serum required to precipitate50% of the antigen. The reciprocal of that serum dilution representedthe titer of the serum. Labeled eiLterotoxin B also proved suitable foruse in coprecipitation serology. Using 1.6 M ammonium sulfate forcoprecipitation, differences were found in the combining capacities ofnormal and immune rabbit sera (Fig. 2).

-. ,.

1 100 Lcbeled antigen at 5 Aug/mI concentration

o600

C0

60-

20 o Normal serum0

0 Immune serum P609

X Immune serum P50I 1 -I I IIi1/10 1/20 1/40 1/0-0 1/160 1/320 1/640 1/12801/25601/5120

Final Dilution of Sera

Figure 1. Coprecipitation of Labeled Anthraxc Pro~ective Antigen 4048and Equine $or& bv 1.4 M Amnonium Sua.

6

100 Labeled antigen at 2 ug/nvl concentration

AC

00

4-

Ca

60-

0.

,C 40

4-o 20L A WNormal rabbit serum II!

x Immune rabbit serum (MI-1)of

0 . . . 1 I 1 i1/10 1/20 1/40 1/80 1/160 1/320 1/640 1/12801/25601/5120

Final Dilution of SeraFigre 2. Coprecipittion of Labeled Enterotixmr; B and Rabbit Sera

Using 1.6 H Ammorium Sulfate.

7

SIUI*AY

Conventional methods for introduction of I may miudify or destroythe serological reactivity of labile microbial antigens. A modifiedmicrodiffusion iodination method ':hat eliminates the exposure of antigensto excess oxidizing agent produced satisfactory trace labeling of purifiedprotective antigen of Rac4ljux anthracis and enterotoxin B of Staphylococcusctureus without detectable change in precipitating activity. The iodinatedantigens proved suitable for use in the ammonium sulfate coprecipitationtechnique of Farr.

8

L=A~TUR CIED

1. Farr, R.S. 1958. A quantitative immunochemical measure of the primaryinteraction between I* BSA and antibody. J. Infect. Die. 103:239-262.

2. Minden, P.; Reid, R.T.o; Farr, R.S. 1966. A comparison of somecouneily used methods for detecting antibodies to bovine serum albuminin human serum. J. Immunol. 96:180-187.

3. Gruber, J.; Wright, G.G. 1966. Determination of antibody to purifiedmicrobial antigens by ammonium sulfate coprecipitation (Farr method).Federation Proc. 25:308.

4. Wright, C.G.; Luksas, A.J. 1964. Electrophoretic and serologicalcomrlexity of the protective antigen of Bacillus anthracis.Federation Proc. 23:191.

5. Schantz, E.J.; Roessler, W.G.; Wagman, J.; Spero, L.; Dunn-ry, D.A.;Bergdoll, M.S. 1965. Purification of staphylococcal enterotoxin B.Biochemistry 4:1011-1016.

6. Johnson, A.; Day, E.D.; Pressman, D. 1960. The effect of iodinntionon antibody activity. J. Immunol. 84:213-220.

7. Hunter, W.M.; Greenwood, F.C. 16 2. Preparation of iodine-131labelleu human growth hormone of high specific activity. Nature194:495-496.

8. Banerjee, R.N.; Ekins, R.P. 1961. A simple microdiffusion techniquefor the radiolodination of proteins. Nature 192:746-747.

9. Seth, S.K. 1964. A study of the nechaninm of inactivation of anti-haptene antibody by cobalt-60 gawi.l radiation. Ph.D. Dissertation.Universi~y of Mkchigan, Ann Arbor, Michigan.

10. Weirether, F.J.; Lewis, E.E.; Rosenwald, A.J.: Lincoln, R.E.1966. Rapid quant'tative serolog',-al assay of staphylccoccalenterotoxin S. Appl. Microbiol. 14:284-291.

DOCUMENT CONTROL DATA -R & D(S*CufrE l c.amifleogIii of title, hodir of abstract W4 ind*AM# Annottion mof A bentaWOe *?. IN Me oll ngon is cl.**ifft.6

1. ORIGINATING ACTIVITY (Cotpof edlo) I" RKORT ISCUAITY CLASI~ICATION

Department of the Army UnclassifiedFort Detrick, Frederick, Maryland, 21701 bGRU

V. RMPOPRT TITLE9

KICRODIFFUSION IODINATION OF PURIFIED MICROBIAL AN~TIGENS FOR COPRECIPITATIONSEROLOGY

4. OESCAIPTIVE OEX 2' ftpI ~ilti.118

S. AUTHONISI (jFirst ntwo Ald. WHO1 1n@ n~lI~~)

Gruber, Jack (NWI)Wright, George G.

II. REPORT DATE 7&. TOTAt NO. OF985 PAS Tb. wo. a, us,'.July 1967 10 1 10

4S. CONTRACT ON GRANT NO . ONIOINATOROS REVORT NU611ERIDI(g

h.POJCTN. CO145OIB71A Technical Manuscript 390

0. 30.OTUPIt Rep.OT NO(91 (Any .ocw -. ~ I a" he 0.soetpudVII. ~t)

00. DISTRIBUTION STATEMiENTQualified requesters may obtain copies of this publication from DDC.Foreign announcement and d~ssemination of this publication by DDC is not authorized.Release or announcement to the public is not authorized.

It. SUPPLECMENTARY MOTES I.SONSORING ILITARY ACTIVITY

IL LOTRACFort Detrick, Frederick, Maryland, 21701

Conventional methods for introduction of 1131 may modify or dectroy the serologi-cal reaccivity of labile microbial antigens. A modified microdiffusion iodinationmethod that eliminates the exposure of antigens to excess oxidizing agent producedsatisfactory trace labeling of purified protective antigen of Bacillus anthracis andenterocoxin B of Staphylococcus aureus without detectable change in precipitatingactivity. The iodinated antigens proved suitabla for use in the ammnonium sulfatecoprecipitation technique of Farr.

14. Key Words

*Ant igens*Microdif fusionbacillus anthr cisstapxi2gs.cus lure.Radioactive

DD ems "17 OSI"'s 1I amp1 waJR w"I Unclassified