Proc. Nat. Acad. Sci. USAVol. 71, No. 11, pp. 4533-4535, November 1974

Visual Detection of Hepatitis B Antigen( unoassay/protein adsorption)

IVAR GIAEVER* AND ROBERT J. LAFFINt* General Electric Corporate Research & Development, Schenectady, New York 12301; and t Albany Medical College, Albany,New York 12208

Contributed by Ivar Giaever, August 26, 1974

ABSTRACT When the hepatitis B antigen is firstadsorbed on a specially prepared metal surface, the sub-sequent reaction with its specific antibody producescomplexes that are visible to the unaided eye. Applicationof this principle has led to the development of tests forboth antigen and antibody that compare favorably withthe sensitivity obtained by radioimmunoassay.

While measuring the thickness of films adsorbed on metalsurfaces, Langmuir and Schaefer (1) studied the effect ofapplying antitoxin to a previously adsorbed layer of diphtheriatoxin. These studies were extended to several additional anti-gen-antibody systems by other investigators (2-4). Rothen (5)has continued to study adsorption of protein on metal sur-faces, using an ellipsometer. Vroman (6) and coworkers haveused the ellipsometer to study blood clotting at interfaces and,in addition (7), have described a simple method for visuallydetecting antigen-antibody complexes on anodized tantalumfilms using reflected light. One of us (8) has recently describeda visual method using indium-coated glass slides.This report describes an effort to adapt some of the simple

visual techniques of detecting the antigen-antibody reactionto a clinically important system. Specifically, we have appliedthese methods to detect hepatitis B antigen (HBAg) and itsspecific antibody (HBAb). We are very encouraged by ourresults, as we find that we can detect both HBAg and HBAbwith at least the same sensitivity as in standard radioimmuno-assays. However, it is still too early to state whether the testswill find clinical use.The basic principle of the reactions on metal surfaces is very

simple. First, a monolayer of antigen is adsorbed onto aspecially prepared metal surface. This surface is then exposedto a test serum. If the serum contains anitibody, complexes areformed at the surface and, as a result, the total protein layerbecomes thicker. If the serum does not contain antibody, theprotein layer does not increase significantly in thickness.When the surface is examined visually, it is possible to dis-tinguish between the monolayer of protein where no reactionhas occurred and the double layer that forms if the antigen-antibody reaction has taken place.

In our initial experimentation we found that neither indium-coated slides nor anodized tantalum slides were very satis-factory. Since the HBAg$ is a large protein, the added thick-

Abbreviations: HBAg, hepatitis B antigen; HBAb, hepatitisantibody.t HBAg used in these experiments was purchased from Electro-Nucleonics Lab., Bethesda, Md. The preparation contained2.2 X 1014 particles per ml; protein concentration was 0.692mg/ml.

ness of the antibody layet did not change the contrast enoughfor a sensitive test. This led to the development of a differentsubstrate and the performance of additional steps to enhancesensitivity. The new substrate was an alloy of indium and gold,deposited onto an ordinary cover glass slide 25 X 25mm by vac-uum evaporation techniques. These slides were heated in air to4000 to slightly oxidize the metals. When the color of theslides appeared light brown or gold, they were removed fromthe furnace and cut into four approximately equal pieces. Adetailed description of the preparation of these slides will begiven elsewhere. The additional steps used to increase sensi-tivity were the addition of antiglobulin specific for the anti-body complexes with the HBAg. This second antibody im-proves the contrast of the first antibody layer. Further en-hancement can be achieved by building up multilayers usingthe same principle. Good visual contrast between single andmultiple layers of protein is crucial to the success of the test.

In all our tests described here we first diluted the HBAg1:10 with 0.85% (w/v) NaCl, and then applied two smalldrops about 2 mm in diameter to a metal-coated slide. Afterthe slides were left in a moist chamber for 15-30 min, theywere rinsed with distilled water and gently blown dry withcompressed air. The HBAg is adsorbed from the drops andappears as two small spots on the metal slide. These spots arebarely visible, if at all, to the unaided eye.The first experiment was designed to detect HBAb in

rabbit antiserum§. The antiserum was diluted with normalhuman serum to obtain dilutions from 1: 100 through 1: 12,800.The purpose of using normal human serum as a diluentthroughout these experiments, other than in preparing orig-inal antigen spots, was to duplicate the conditions that wouldexist during an actual test. Slides with antigen spots wereplaced in individual plastic vials containing 3 ml of sample. Inaddition, one vial contained only the human serum diluent as anegative control. To prevent protein denaturation at theliquid-air interface, a disc of Parafilm was floated on the sur-face of each sample. The vials were then placed in a shakerand stirred vigorously for 16 hr.We now removed the Parafilm and added one drop of un-

diluted goat antirabbit globulin directly to the 3-ml sample ineach vial. After the slides were shaken for an additional 30min, they were rinsed with distilled water, placed in a moistchamber, and covered with a 1: 5 dilution of rabbit antigoatglobulin for 30 min.

§ The rabbit antiserum used in these experiments was obtainedfrom the Ortho Diagnostic Electrophoresis Kit.

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4534 Immunology: Giaever and Laffin

FIG. 1. Titration of rabbit HBAb. Slides 1 through 8 represent dilutions of 1:100 through 1: 12,800; slide 9 is a negative control; slide10 has only the original antigen spots.

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FIG. 2. Inhibition test for HBAg, using rabbit HBAb and a pool of HBAg-positive human sera. Slides 1 through 7 represent virusdilutions of 1: 100 through 1: 6400; slide 8 is a positive control for antibody; slide 9 is a negative control; slide 10 has only the originalantigen spots.

Finally the slides were rinsed, blown gently dry with com-

pressed air, and examined. The results are shown in Fig. 1.Slides 1 through 8 have been exposed to the dilutions 1:100through 1:12,800 respectively, slide 9 is the negative control,and slide 10 has only the original antigen spots on the surface.As seen, the spots get progressively lighter as the antibodyconcentration gets more dilute, but they are still visible at a

titer of 1:12,800.The next experiment tested for the presence of HBAb in

human serum. A specimen of human serums that had a titerof 1:32 by counterelectrophoresis was diluted 1:10 through1:160,000. These diluted sera were tested against individualslides with HBAg spots as before. The testing for humanHBAb required one essential change in the procedure. Thefirst antiglobulin, in this case goat antihuman globulin, could

New York State Reference Antibody 149-1, kindly supplied bythe New York State Blood Resources Program.

not be added directly to the vials containing 3 ml of humanserum, since it would be neutralized before it reached theHBAb-HBAg complexes on the metal surface. Therefore, wediscarded the diluent after the 16-hr incubation, and replacedit with 2 ml of 0.85% NaCl. The slides were rinsed with dis-tilled water and placed in the vials with the new diluent. Onedrop of undiluted goat antihuman globulin was added to eachvial. The tests were carried to completion as before.

Inspections of the slides showed visible spots of decreasingcontrast through the 1:80,000 dilution. Only the 1:160,000slide and the negative control slide had no visible spots. Thistiter compares favorably with a 1: 100,000 titer in humanserum determined by radioimmunoassay (9).Having demonstrated the ability of the method to detect

antibody, we carried out an inhibition test for the detection ofHBAg. The principle of such a test is that HBAg, if added insufficient quantity to a serum containing HBAb, will neutral-ize antibodies and prevent them from forming observable

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Visual Detection of Hepatitis B Antigen 4535

complexes with the antigen spots on the slide surface. Com-plete inhibition should occur in antigen excess and at equiv-alence, while partial inhibition should occur in the region ofslight antibody excess.The source of HBAg was a pool of ten human sera positive

by radioimmunoassay 11 at a titer of 1: 800. Using this pool, weprepared seven vials with final dilutions of 1:100 through1: 6400. Each of these vials contained a 1: 6000 dilution of therabbit antiserum for which we had previously obtained a titerof 1: 12,800. Controls included a 1: 6000 dilution of the rabbitantiserum but no HBAg and a vial containing neither HBAgnor HBAb. As in the antibody titration test, each vial con-tained 3 ml of sample, and the test was carried out in exactlythe same manner.

Fig. 2 shows the results of such a test. Slides 1 through 7have been exposed to the dilutions 1:100 through 1:6400,respectively, slide 8 has been exposed only to HBAb, slide 9 toneither HBAg nor HBAb, and slide 10 has only the originalantigen spots on the surface. The first three slides are franklynegative, the absence of spots indicating that HBAg was pres-ent in sufficient quantity to neutralize antibody completely.The noticeable decrease in color intensity on slides 4 and 5,compared to slide 8, the antibody control, is interpreted as beingdue to partial antibody neutralization. Thus, the endpoint wasread at slide 5, the 1:1600 dilution. While this present methodof reading the slides is subjective, objective instrument read-outs can easily be developed.The main limitation to the sensitivity of our test is the

time it takes the antibodies to diffuse to the surface of themetal slide. Thus, it is possible within wide limits to increasesensitivity by sacrificing time if the antibody concentration isadjusted properly. We tested this concept in 1-hr and 4-hr in-cubations, where we used an antibody concentration of 1: 400and 1:1600, respectively, instead of the 1: 6000 used for the

16-hr test. As expected, the HBAg titration came out 1:100and 1:400, which is '/16 and '/4 as sensitive as the 16-hr test.A final experiment tested the effectiveness of human anti-

body in neutralizing HBAg. In this case, we diluted the puri-fied HBAg in human serum in concentrations ranging from692 ng/ml through 5.5 ng/ml. These solutions were testedusing the previously assayed human antiserum diluted1: 20,000. We obtained an endpoint of 22 ng/ml in this case,while the radioimmunoassay of the same samples gave anendpoint of 86 ng/ml.In summary, we have described methods that are capable of

detecting both HBAg and HBAb in a concentration rangecomparable to radioimmunoassay.We are grateful to Dr. D. C. Golibersuch for suggesting the

use of antiglobulins to improve the contrast in our experiment.

1. Langmuir, I. & Schaefer, V. J. (1937) "Optical measurementof the thickness of a film," J. Amer. Chem. Soc. 59, 1406.

2. Shaffer, M. F. & Dingle, J. H. (1938) "A study of antigensand antibodies by the monolayer film technique of Langmuir,"Proc. Soc. Exp. Biol. Med. 38, 528- 530.

3. Porter, E. F. & Pappenheimer, A. M., Jr. (1939) "Antigen-antibody reactions between layers adsorbed on built upstearate films," J. Exp. Med. 69, 755- 765.

4. Rothen, A. & Landsteiner, K. (1939) "Adsorption of anti-bodies by egg albumin films," Science 90, 65- 66.

5. Rothen, A. (1973) "Immunologic and enzymatic reactionscarried out at a solid- liquid interface," Physiol. Chem. Phy8.5, 243-258.

6. Vroman, L., Adams, A. & Klings, M. (1971) "Interactionsamong human blood proteins at interfaces," Fed. Proc. 30,1494-1502.

7. Adams, A. L., Klings, M., Fischer, G. C. & Vroman, L.(1973) "Three simple ways to detect antibody- antigencomplex on flat surfaces," J. Immunol. Methods 3, 227-232.

8. Giaever, I. (1973) "The antibody- antigen reaction: A visualobservation," J. Immunol. 110, 1424-1426.

9. Ginsberg, A. L., Conrad, M. E., Bancroft, W. H., Ling,C. M. & Overby, L. R. (1973) "Antibody to Australiaantigen: Detection with a simple radioimmune assay, inci-dence in military populations, and role in the prevention ofhepatitis B with gamma globulin," J. Lab. Clin. Med. 82,317-325.

1I We determined the titer, using the Abbott radioimmunoassaytest.

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