Fluorescent Treponemal Antibody Studies*

FLUORESCENT TREPONEMAL ANTIBODY STUDIES* W. E. DEACON, PH.D. AND ELIZABETH M. FREEMAN, MS. Fluorescent antibody methods were originally introduced by Coons and associates in 1942. Included in this first publication was a procedure for antibody-fluorescein conjugation and a de-

and are then thoroughly washed with saline to remove excess serum. Fluorescein labeled antihuman globulin, the reaction indicator, is then added to the smear. Treponemes which have

scription of fluorescent reactions obtaincd by

coupled with human antibody will, in turn,

means of ultraviolet light microscopy. This communication, and later ones by other authors, indicated that fluorescent antibody procedures were

tion can then be visualized with an ultraviolet

practical for rapid and direct identification of infectious agents, and might be applied in a variety of fields.

couple with the fluoreseein conjugate. The reaclight microscope. A smear treated with a strongly reactive serum will reveal brilliantly fluorescent treponemes. A smear treated with a serum containing smaller amounts of antibody will demon-

Fluorescent antibody reactions may be ob-

strate treponemes with less brilliance or none

tained in either of two ways. The direct reaction is most frequently employed for pathogen iden-

at all. FTA reactions are illustrated in the following photomierographs:

tification and consists of preparing a highly specific antiserum in an appropriate animal and conjugating the gamma globulin with fluorescein. The value of the finished product or conjugate is

Figure 1—Treponema palliduin as seen by

judged by its ability to react with, or stain, a

the usual darkfield using tungsten light. Figure 2—Treponema pallidum, weakly fluorescent as seen by UV light (weakly reactive

serum—treponemes with less brilliance would The indirect reaction in contrast to the direct not be seen) and would constitute a nonreactive procedure is performed in two stages. First, the result. pathogen is treated with a specific unlabeled antiFigure 3—Treponeina pallidum, brilliantly serum (example: rabbit serum); and then after fluorescent as seen with UV light—reactive result. On the basis of extended studies, the antibodies washing thoroughly to remove unreaeted serum, fluoreseein labeled goat antirabbit globulin is ap- detected by the FTA test appear to be similar or plied to detect the presence of rabbit antibody. identical to those identified by the well known The Fluorescent Treponemal Antibody (FTA) TPI test. This conclusion is supported by comtest is an example of the indirect reaction, and as parative findings in the Serology Evaluation and employed, is used to detect human treponemal Research Assembly (SERA) Study conducted in antibody. Since the pathogen (Treponema palli- 1957 by the Venereal Disease Branch, CDC. In dum) is a known quantity, the procedure has this work, six TPI procedures, as performed by been modified to detect the unknown quantity five laboratories, are compared. The following re(human treponemal antibody or human globulin). sults illustrate the close agreement of TPI and The FTA test is performed in the following FTA findings. In Category A, Presumed Normals—346 manner. First, Treponema pallidum is extracted from rabbits in the same manner as is used in the specimens Treponema Pallidum Immobilization (TPI) test. 1.7—5.5% Reactive Range TPI's The intact treponemes are dried on microscope PTA 1 .5% Reactive slides, and after fixing with acetone, are exposed Category B, Diseases Other Than Syphilis— to the human test serums. Slides are gently ro- 83 specimens tated at 37° C. to facilitate antibody coupling, 9—13% Reactive Range TPI's FTA 7% Reactive Presented at the Twentieth Annual Meeting of The Society for Investigative Dermatology, Inc., Category C, Untreated Primary Syphilis—130 Atlantic City, New Jersey, June 7, 1959. specific pathogen and no others.

* From the Venereal Disease Research Laboratory, Venereal Disease Branch, Communicable

Disease Center, U. S. Public Health Service, P.O. Box 1S5, Chamblee, Georgia.

249

specimens

Range TPI's PTA

25—50% Reactive

60% Reactive

250

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Vu;. I

FIG, 2

Category D, Treated Early Syphilis—51 specimens

Range TPI's FTA

as used in these illustrations is based on known syphilis categories consisting of 504 specimens

27—53% Reactive

(early, untreated; early, treated; and late,

38% Reactive Category H, Lcprosy—29 specimens Range TPI's 0—3% Reactive FTA 0% Reactive

treated). Specificity is based on results obtained

with Presumed Normals, and Diseases Other Than Syphilis, consisting of 409 specimens.

Figure 4 shows the distribution of test reThe following slides illustrate the relative sults. Circles indicate Treponema pallidum tests, sensitivity and specificity of various test pro- triangles are lipoidal tests, squares arc Reiter cedures entered in the SERA Study. Sensitivity

treponeme procedures. The large square at the

251

FLUORESCENT TREPONEMAL ANTIBODY

FIG. 3 SEROLOGY EVALUATION AND RESEARCH ASSEMBLY (SERA) SPECIFICITY. PERCENT

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left encloses those tests with the most desirable results in terms of specificity and sensitivity.

Further studies on the mechanism of the FTA test have now been accomplished and may be

Figure 5 is an enlargement of the left hand described briefly as follows: square. Test results are identified. Three TrepLipid antibody produced in rabbits by injeconema pallidtm tests (TPI, U. Michigan, TPI- tion of VDRL antigen, when used in the FTA

200, VDRL), FTA (VDRL), are within the procedure, reveals that surface antigens of square. Of the three procedures, the FTA test in this analysis is the most favorably located.

Treponema pallidum do not react with this ma-

terial. On the other hand, contaminating sub-

252

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stances which are found to be present in all lipids are not a part of the normal antigenic surtreponemal smears obtained from rabbit testes face constituents of intact Treponema patlidum were shown to react with lipid antibody. This as used in the TPI test. The source of this matefinding suggested that normal testicular tissue rial, as previously indicated, is probably normal might serve as a source of nonspecific reactivity. testicular antigen which has been placed on the The removal of reactivity to VDRL antigen from rabbit and human sera by absorption with normal

testicular tissue has proven this antigenie relationship. Absorption of reactive serum with VDRL anti-

gen removes the reactivity with several serological tests for syphilis, but leaves reactivity when tested with the FTA procedure and the TPI. Since Hardy, et al., and other workers have

treponeme surfaces during the extraction process. CONCLUSIONS

The FTA test is basically a simple procedure. Problems arising from nonspecifleity are most likely concerned with the treponemes (lipid antigens), and as a result, carefully tested antigens should be selected. That is, each new batch of antigen should be checked for lipid contamination. This may be done by checking with anti-

described intact Treponema patlidum antigens as being reactive in both lipid and treponemal anti- lipid serums. Also, tests should be made for sensibody systems, an investigation of the Hardy anti- tization of the treponemes by rabbit antibodies.

gen in the PTA procedure was undertaken.

Once an antigen has been found satisfactory, it

Smears were prepared in the usual manner using Hardy's antigen and were exposed to rabbit antiVDRL antigen globulin. Subsequent treatment with fluoreseein labeled goat antirabbit indicator confirmed the lipid antigenie activity of Treponema paltidum prepared by the Hardy method. On the basis of this experiment and those previ-

may be stored in several ways, such as, refrigeration at 50 C., freezing at —4Q° C., and by lyo-

ously described, it would appear that reactive

technies, the need for such expenditure is justified.

philizing. Fluoreseein labeled antihuman and antirabbit globulins are available commercially.

The greatest cost appears to be in purchasing fluorescent equipment. However, with the increasing applications of fluorescent antibody

FLUORESCENT TREPONEMAL ANTIBODY

253

DISCUSSION Da. MARION B. SULZBERGER (New York, students. Bruce Webster of New York gave a N. Y.): I would like to just discuss the point most stimulating talk at the recent Symposium about the absorption of the anti-lipoidal anti- on Venereal Diseases in Baltimore on this subbodies by the testicular material. I would like to ject. He pointed out that the crying need is for ask Dr. Freeman and Dr. Deacon whether they somebody interested in teaching syphilis. Such a have tried the fluorescent antibody technic against man will find plenty of material in any hospital the culture Reiter treponemes, which might not today. In a community hospital in an upper midcarry any of these organ materials since they dle class area of Philadelphia, with which I am have been cultured on thioglycollate medium. If connected, I saw within the past six weeks one the authors have done so, what have the results patient with advanced tabes dorsalis, one with a been? wide open syphilitic aortic regurgitation, one DR. Dt& L. SCHAMBERG (Elkins Park, Pa.): As with a syphilitic aortic aneurysm filling half of his a syphilophile for a good many years now, I am chest as well as, of course, a number of serologic gratified to hear this group of three papers on problems. syphilis. They bespeak our continued interest in I am happy to have heard these papers. I hope syphilology, a field to which our professional for- we will hear more of them as the years go on. I bears contributed much of which we are proud. hope that dermatologists will continue to be I am especially proud to recall my own father's syphilologists.

clinical and laboratory studies in syphilis. But there are signs in recent years that we are losing onr interest in this fascinating disease. Only one of the papers presented today was by a member of our dermatologie fraternity. Fewer and fewer of us have a major interest in syphilis. Hence, the death of Dr. Charles Rein a few years ago was not only a tragedy to those of us who loved him, but left an unfilled void in the field to which he gave so much. We hear the plaint that there are not enough syphilitic patients in teaching hospitals to train

MISS ELIZABETH M. FREEMAN (in closing):

Thank you for the discussion. In answer to Dr.

Sulzberger's question, the use of the Reiter treponeme in the FTA test has not given specific

reactions in our hands. This is probably due to lipid antigen on its surface. The availability of fluorescein isothiocyanate, and the brilliance ob-

tained when using this compound, offers the opportunity to label specific sera to prove the relationship of the Reiter and the Nichol strain. We feel these findings may be very interesting.

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linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.

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