CA1231892A - Immunodiagnostic test for syphilis and other treponemal infections - Google Patents

Abstract

IMMUNODIAGNOSTIC TEST FOR SYPHILIS
AND OTHER TREPONEMAL INFECTIONS
ABSTRACT OF THE DISCLOSURE

A component for the rapid detection of anti-Treponemal antibodies or Treponemal immunogenic proteins adapted for use as a selective foundation material in standard immunoassays is provided. The component com-prises a quantity of fibronectin protein adhered to an insoluble support matrix. The fibronectin component provides a foundation material for the selective binding of Treponemal protein immunogens, either in the form of whole Treponemal organisms or solubilized protein frac-tions thereof. Incorporation of the fibronectin component and either anti-Treponemal antibody or Treponemal protein immungens into a standard immunoassay test-pack provides an immunological assay for the presence of respective complementary Treponemal protein immunogen or anti-Treponemal antibody in a biological sample.

Description

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UTSK:024 IMMUNODIAGNOSTIC TEST FOR SYPHILIS
AND OTHER TREPONEMAL INFECTIONS

The present invention relates to immunological diagnostic assays; and more particularly it relates to immunological diagnostic tests for syphilis and related Treponemal infections.
Syphilis is a unique disease associated with a complex host response which may be accompanied by inter-mittent periods of latency and classical stage develop-ment. Neither protective immunogens nor mechanisms of host resistance have been clearly defined. Also, little information is available concerning the biological-chemical properties of T. pallidum that relate to viru-lence. The limitations of the model system, including the inability to sequentially in vitro passage virulent treponemes, represent serious experimental deficiencies in developing potential diagnostic assays and vaccinogens.
Currently available immunodiagnosis assays for syphilis is inadequate. Routinely a "non-specific" screening test (VDRL, cardiolipin antigen) is used which causes many false-positive reactions. For example numerous infections and autoimmune disorders as well as syphilis cause increases in the detected anti-cardiolipin antibodies.
Also, this test fails to identify significant numbers of ~2~3~

syphilis-positive test samples. Furthermore, the antigen-specific test for conirmation of syphilis is most often the FTA-ABS slide tests which requires whole orga-nisms, fluorescein conjugated reagents and fluorescence microscopy, which involves a time-consuming, expensive and qualltative assay. Therefore, millions of serodiagnostic tests are performed each year in the U.S. alone under clearly suboptimal assay conditions resulting in a tremen-dous economic and emotional burden in our population. In addition, immuno-diagnosis of pinta and yaws (other human treponemal diseases) is unsatisfactory.

The present invention should markedly assist in improved diagnosis of these treponemal infections. The Applicants have evolved a strategy that permits identifi-cation and characterization of T. pallidum virulence determinants which are believed to be present in syphilis and these other pathogenic spirochetes. As an outcome this strategy, Applicants have devised a rational and experimentally effective method for serodiagnosis of syphilis and related Treponemal infections that can be used routinely with grea~~y improved specificity.

This invention provides a component for the rapid detection of anti-Treponemal antibodies or Treponemal immunogenic proteins which component is adapted for use as the selective foundation agent in standard immunodiag-nostic assay techniques.

In accordance with this invention a component for a diagnostic test pack useful for the immunological detec-tion of Treponemal protein immunogens or antibodies specific for Treponemal protein immunogens is provided.
The component comprises a quantity of fibronectin protein adhered to an insoluble support matrix. In some applica-tions of immunological assays techniques such as for the ~3~

detection of antiTreponemal antibodies in a biological sample, it is desirable that the component of fibronectin adhered to the insoluble support matrix further includes Treponemal protein immunogens bound to the fibronectin protein.

The fibronectin component thus provided serves as a selective foundation material adapted for use in a variety of standard immunoassay systems, such as radioimmuno-assays, enzyme linked immunosorbent assays, and fluores-cent tagged immunoassays. By employing the fibronectin component of the present invention in the standard immuno-assays systems, there is provided a rapid, simple, reli-able and sensitive immunoassay for the detection of either anti-Treponemal antibodies or Treponemal immunogenic proteins present in a biological sample. The presence of either anti-Treponemal antibodies or Treponemal immuno-genic proteins in a biological sample, such as blood serum taken from a host, is an indication of Treponemal infec-tion of the host system.

The invention will be described in terms of preferredembodiments which represent the best mode known to the inventors at the time of this application.
The present invention provides immunodiagnostic assay for syphilis and other infections such as yaws and pinta caused by closely related spirochetes. The causative organism of these diseases resides with the bacterial Treponema family, namely Treponema pallidum (syphilis), Treponema pertenue (yaws) and Treponema carateum (pinta).
The present invention encompasses the use of fibronectin protein as a selective receptor for the adhesion of immunogenic Treponemal protelns. Fibronectin protein when adhered to an insoiuble support matrix provides a reactive foundation for immunodiagnostic assays effective to detect ~2~

either Treponemal protein immunogens or anti-Treponemal antibodies present in biological liquid samples.

Fibronectin (I 450,000) is an adhesive glycoprotein, one form of which circulates in the plasma and another form of which is a cell-surface protein mediating cellular adhesive interactions. Fibronectins are important com-ponents of connective tissue where they crosslink to collagen. Further, fibronectin is a protein involved in the aggregation of platelets.

In accordance with this invention fibroneçtin protein is adhered to an insoluble support matrix. An insoluble support matrix provides a biologically inert carrier system for the fibronectin so as to allow the fibronectin to be easily separated from a liquid phase. For conve-nience of separation, it is preferred that the support matrix be a water insoluble solid support matrix.
Examples of suitable insoluble support matrixes include polystyrene beads; latex spheres; agarose ordextran gel heads; glass slides, beads or well containers; filter paper; microtiter plates composed of polymer substrates;
and plastic dip sticks.

Fibronectin protein can be adhered to the insoluble support matrix by a variety of means including surface adhesion or coating to the support matrix, covalent or affinity binding to the support matrix, or interstitial binding within a loosely woven matrix support.
Further in accordance with this invention the fibro-nectin component can be provided in form wherein a quan-tity of Treponemal immunogenic proteins is specifically reacted with the fibronectin. Fibronectin readily and selectively associates with Treponemal immunogPnic pro-teins, particularly Treponemal proteins comprising the ~31~39~

Treponemal organism's outer member. A number of the Treponemal protein immunogens have been identified, specifically termed protein Pl ~i39,500 daltons); P2 (29,500 daltons); P3 (25,500 daltons); P4 (20,000 5 daltons); P5 (59,000 daltons) and P6 (42,500 daltons).
Purification and identification of these proteins are described by Applicants' articles: "Surface-associated Host proteins on Virulent Treponema pallidum," Infect.
Immun. 26: 1048-56 (1979); "Surface Characterization of Virulent Treponema pallidum", Infect. Immun. 30:814-23 (1980)*, and "Molecular Characterization of Receptor Binding Proteins and Immunogens of Virulent TrePonema Pallidum", J. Exp. Med. 151:573-ô6 (1980) . , Further, Applicants have discovered 15 that these purified proteins offer highly sensitive probes for the detection of anti-treponemal antibody, see e.g.
"Enzyme-linked Immunosorbent Assay for the Detection of Serum Antibody to Outer Membrane Proteins of Treponema Pallidum", Fr. J. Vener. Dis. 59:75-9 (1983).
Within the context of this invention, either whole Treponemal organisms or solubilized protein fractions of Treponemes can be bound to the fibronectin component or provided as a separate component for subsequent binding to 25 fibronectin. It is preferred however that the quantity of Treponemal immunogenic proteins be provided in the form of a solubilized protein fraction as opposed to whole Treponemal organisms.

In order that the invention may be more clearly understood, illustrative embodiments will be further described in terms of the following examples, which should not be construed to limit the scope of this invention.
* Appendices A, B, C and D, photocopies placed on file herewith ,~

A. Gelatin-Agarose purification of human plasma fibronectin.

1. A l x 10 cm column was prepared with Gelatin-agarose sigma Chemical Co., St. Louis) and equilibratedwith phosphate-buffered saline (PBS) supplemented with 0.05M cyclohexylaminopropane sulfonic acid (CAPS) prior to addition of fresh plasma as outlined below. After exten-sive washing of the column of unbound materials, fibro-nectin was eluted with l.OM NaBr, 0.05M NaAC, pH 5ØEluted material was then dialyzed overnight against 4 liters of O.lM NaCl, O.OlM CaC12, and 0.05M CAPS, pH 11Ø
The fibronectin was evaluated by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and 99% purification of plasma fibronectin was achieved in this manner. Fibro-nectin wa then aliquoted into small volumes into plastic tubes and frozen at -70C until further use.

2. Fifty ml of freshly drawn citrated human blood was centrifuged at 1000 rpm for 10 minutes. Plasma was decanted into sterile centrifuge tubes and re-centrifuged at 3000 rpm for 10 minutes. Fifteen ml of plasma was combined with an equal volume of 0.05M CAPS for processing in gelatin-agarose as above. The remaining plasma was frozen at -70C until further use.

B. Preparation of fibronectin-coated polyvinvl chloride microtiter wells.

1. Purified fibronectin was diluted no less than 1:5 in carbonate coating buffer (1.6 g/L NaC03, 3.0 g/L
NaHC03, and 0.2 g/L NaN3, pH 9.6), and 50 l giving the appropriate amount of protein was aliquoted into indivi-dual wells of 96 well microtiter plates (Dynatech Labs).
The plates were incubated overnight at 4C followed by washing once in PBS-1% bovine serum albumin (BSA).

lZ3~2 Finally, PBS-BSA was added to individual wells and plates stored at -20C until needed.

2. Fibronectin-coated wells are allowed to thaw at room temperature followed by an additional incubation for 60 minutes at 37C. The wells were then washed at least 3 times with PBS and tapped dry. Varying amounts of Treponema pallidum detergent extract obtained as outlined below were then added and wells allowed to incubate for 60 minutes at room temperature. Fifty microliter volumes of solubilized T. pallidum extract were employed for experi-ments using serum reagents from experimental animals rabbits and mice3. Only 5 l amounts of treponemal extract in 45 l PBS were employed for experiments using sera from normal individuals and humans at various stages of infection with syphilis or other treponematosis.

3. Solubilization of T. pallidum organisms.

T. pallidum organisms (1 x 101 cell number) were obtained from testicular materials of infected rabb ts.
Treponemes were clarified from host materials, washed 3 times with PBS and stored at -70C until use. Pellets were resuspended in 0.5 ml NaCl-EDTA-Tris [NET; 150 mM
NaCl, 5 mM disodium ethylenediaminetetraacetate (EDTA), and 5 mM tris (hydroxymethyl) amino-methane (Tris)]
buffer, pH 7.4 containing 1% Zwittergent 3-12 detergent (Cal Biochem Co., La Jolla, Ca3 and 1 mM phenylmethyl-sulfonyl chloride (PMSF). The suspension was subjected to homogeniz,ation (10 strokes) followed by layering on a 5%
sucrose cushion and centrifugation at 100,000 x g for 30 minutes. The supernatant containing solubilized T.
pallidum proteins was diluted in NET + PMSF to give a 0.1%
Zwittergent 3-12 concentration.

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C. Use of T. pallidum antigens bound to fibronectin on microtiter plate wells for detection of antibody to syphilis and other treponematosis.

l. After addition of 50 Al of a solution containing soluble T. pallidum protein antigens to fibronectin-coated microtiter wells, the wells were incubated at room temper-ature for 60 minutes followed by washing 3 times with PBS.
Control serum or serum from rabbits infected with T.
pallidum was diluted in NET and 50 Al of diluted serum reagent added to wells. After a 60 minute incubation at 37C, the wells were again washed 3 times with PBS fol-lowed by addition of 50 Al of indicator antibody (goat anti-rabbit immunoglobulin G) coupled to alkaline phos-phatase (Miles Labs). Fifty Al p-nitrophenylphosphate was then added to extensively washed (3 times with PBS + 3 times with distilled water) wells incubated 60 minutes at 37C. After a 30 minute incubation at 37C, the wells were examined spectrophotometrically at 405 nm for indica~
tion of enzyme activity. Values using serum from syphi-litic animals were always greater than background values obtained using normal rabbit serum using the same serum dilutions. Results are tabulated in Table I.

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LE I

Reactivity of rabbit syphilitic serum with T. pallidum materials adsorbed to fibronectin coated micro~iter plate wells Absorbance (40 ) _ _ _ US RSS
Experiment 1:101:100 1:10 1:100 1:100 , 1. 50 ng Fibronectin/well a. 50 l Solubilized T. pal/wcll .042+.015 0 1.337+.047 > 1.396 1.314+.08 b. 200 l Solubili7-ed T. pal/well .096*.018 .037+.044 1.329+.06 1.359 ~-1.309 2. 100 ng Fibronectin/w~ll a. 50 l T. pal/well .054+.009 0 1.253+.136 1.347 1.256+.099 NRS, normal rabbit serum; dilutic,n in NET buffer RSS, rabbit syphilitic serun 2. A similar protocol was employed when using sera from normal individuals, individuals having syphilis at different stages of infection or individuals having unrelated infections. Fifty l of undiluted human serum was employed, and the incubation period was increased to 90 minutes at 37C. The incubator antibody consisted of alkaline phosphatase-conjugated goat anti-human lgG.
Serum from a patient with yaws, another syphilis-like infec-tion caused by T. pertenue, was also employed similarly.

3. Substrate (p-nitrophenylphosphate) was prepared in standard diethanolamine buffer consisting of 9.7%
diethanolamine, 0.2% NaN3, 0.01% MgC12.6H20, pH 9.8.
Results are tabulated in Table II.

TABLE II

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Reactivity of Human Syphilitic Serum from Patients at Different Stages of Infection with T. ~allidum Materials Absorbed to Fibronectin Coated Microtiter Well Plates Serum Sample/Stage of Infection Absorbance (405 nm 1. Normal .012 + .002 2. Normal .044 + .006 153. Primary Syphilis .080 + 002

4. Latent Syphilis .091 + .018

5. Latent Syphilis .200 + .017 . Yaws .113 + .005 7. Yaws .289 + .010 8. Group A streptococcus infection .020 + .005 9. Trichomoniasis .030 + .010 D. Results and unique features.

Results indicate that antibody responses by animaIs or humans infected with syphilis and other treponematosis can be detected using T. pallidum antigens adsorbed onto fibronectin-coated microtiter plate wells The specifi-city of the reaction between fibronectin and T pallidum molecules was demonstrated by gel electrophoretic anal-ysis. Three prominent treponemal molecules, Pl, P2 and P3 characterized as those responsible for the adherence properties of virulent spirochetes were found to bind to fibronectin with high affinity .. , ~23~

Thus broadly, the invention contemplates a test component for a diagnostic test pack useful for the i.mmunological detection of anti-Treponema pallidum ant:ibody which comprises insolubilized fibronectin and antigenic Treponema pallidum proteins bound to the insolubilized fibronectin.
In a further embodiment, the invention provides for a diagnostic test pack for the detection of anti-Treponema pallidum antibodies in packaged combination which comprises a first container of insolubilized fibronectin and antigenic Treponema pallidum proteins bound to the insolubilized fibronectin to form a single component, and a second container of a quantity of indicator antibody sufficient for binding with the anti-Treponema pallidum antibody to be detected.
The invention also encompasses an immunoassay for the detection of anti-Treponema pallidum antibody present in a biological sample which comprises the steps of providing insolubilized fibronectin having bound thereto antigenic Treponema pallidum proteins to form an insolubilized fibro-nectin, treponemal protein product, contacting and incubat.ing the insolubilized fibronectin, treponemal protein product for a sufficient time and with a sufficient quantity of the biological sample to permit binding reactions to occur between the insolubilized fibronectin, treponemal protein product and the biological sample, and determining the presence of anti-Treponema pallidum antibody bound to the insolubilized fibronectin, treponemal protein product by contacting the insolubilized fibronectin, treponemal protein product with a quantity of indicator antibody sufficient for binding with the anti-Treponema pallidum antibody.
While the components and methods of this invention have been described in terms of preferred embodiments constituting -the best mode known to Applicants at the time of this invention, it will be apparent to those of skill in the art that various changes may be made in the invention without departing from the scope thereof, which is defined by the following claims.

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Claims (20)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A test component for a diagnostic test pack useful for the immunological detection of anti-Treponema pallidum antibody, the test component comprising:
insolubilized fibronectin and antigenic Treponema pallidum proteins bound to the insolubilized fibronectin.

2. The component according to Claim 1 wherein the fibronectin protein is adhered to a water insoluble, solid support matrix.

3. The component according to Claim 2 wherein the fibronectin is adhered to glass or polymer substrates.

4. The component according to Claim 2 wherein the solid support matrix is a testing vessel, dip stick or insuspensible beads.

5. The component according to Claim 1 wherein the fibronectin is dip coated to glass or a solid polymer support matrix.

6. The test component of Claim 1 wherein the insolubilized fibronectin comprises fibronectin insolubilized to a solid support matrix selected from the group of beads, latex particles, glass slides, well containers, filter paper, microtiter plates, and plastic dip sticks.

7. The test component of Claim 1 wherein the antigenic Treponema pallidum proteins are outer membrane proteins P1, P2, or P3.

8. A diagnostic test pack for syphilis and related Treponemal infections, which test pack provides components for the detection of antibodies specific for proteins of Treponema, the pack comprising:
a quantity of fibronectin protein adhered to an insoluble support matrix;
a quantity of immunogenic Treponemal proteins capable of reacting specifically with both fibronectin protein and the anti-Treponemal antibody to be detected; and a quantity of indicator antibody capable of reacting specifically with the anti-Treponemal antibody to be detected, but which indicator antibody does not react with either the fibronectin protein or the Treponemal proteins.

9. A diagnostic test pack for syphilis and related Treponemal infections which test pack provides components for the detection or immunogenic proteins of Treponema, the pack comprising:
a quantity of fibronectin protein adhered to an insoluble support matrix;
a quantity of anti-Treponemal antibody capable of reacting specifically with the immunogenic proteins of Treponema to be detected; and a quantity of indicator antibody consisting of an antibody portion coupled to an indicator moiety, wherein the antibody portion is either the anti-Treponemal antibody or an antibody capable of reacting with anti-Treponemal antibody.

10. A diagnostic test pack for the detection of anti-Treponema pallidum antibodies, the pack comprising in packaged combination:
(a) a first container of insolubilized fibronectin and antigenic Treponema pallidum proteins bound to the insolubilized fibronectin to form a single component; and (b) a second container of a quantity of indicator antibody sufficient for binding with the anti-Treponema pallidum antibody to be detected.

11. The test pack according to Claim 8, Claim 9 or Claim 10 wherein the indicator antibody is an enzyme linked antibody, a fluorescent tagged antibody, or a radiolabelled antibody.

12. The test pack according to Claim 8, Claim 9 or Claim 10 wherein the indicator antibody is an enzyme linked antibody and said pack further includes a third container of a quantity of substrate sufficient for binding with the enzyme to produce a visually detectable product.

13. The test pack of Claim 8, Claim 9 or Claim 10 wherein the insolubilized fibronectin comprises fibronectin insolubilized to beads, latex particles, glass slides, well containers, filter paper, microtiter plates, or plastic dip sticks.

14. The test pack of Claim 8, Claim 9 or Claim 10 wherein the antigenic Treponema pallidum proteins are outer membrane proteins P1, P2, or P3.

15. A method for the immunological detection of Treponemal protein immunogens present in a biological liquid sample, the method comprising the steps of:
providing a testing vessel containing a quantity of fibronectin protein adhered to an insoluble support matrix;
adding to the testing vessel a quantity of the liquid biological sample;
adding to the testing vessel a quantity of anti-Treponemal antibody capable of reacting specifically with Treponemal protein immunogens;
incubating in the testing vessel the mixture of biological sample and anti-Treponemal antibody for a time sufficient to permit binding reactions to occur;
after incubation, separating and washing the insoluble support matrix from the liquid sample, and determining the presence of antibody bound to the solid support matrix, the detection of antibody indicating the presence of Treponemal protein immunogens in the biological sample. 15

16. A method for the immunological detection of anti-Treponemal antibody present in a liquid biological sample, the method comprising:
providing a testing vessel containing a quantity of fibronectin protein adhered to an insoluble support matrix;
adding to the testing vessel a quantity of Treponemal protein immunogens capable of reacting specifically with anti-Treponemal antibody;
adding to the testing vessel a quantity of the liquid biological sample;
incubating in the testing vessel the mixture of biological sample and Treponemal protein immunogens for a time sufficient to permit binding reactions to occur;
after incubation, separating and washing the insoluble support matrix from the liquid sample; and determining the presence of antibody bound to the solid support matrix, the detection of antibody indicating the presence of anti-Treponemal antibody in the biological sample.

17. An immunoassay for the detection of anti-Treponema pallidum antibody present in a biological sample, the method comprising:
(a) providing insolubilized fibronectin having bound thereto antigenic Treponema pallidum proteins to form an insolubilized fibronectin, treponemal protein product;
(b) contacting and incubating the insolubilized fibronectin, treponemal protein product for a sufficient time and with a sufficient quantity of the biological sample to permit binding reactions to occur between said insolubilized fibronectin, treponemal protein product and said biological sample; and (c) determining the presence of anti-Treponema pallidum antibody bound to the insolubilized fibronectin, treponemal protein product by contacting the insolubilized fibronectin, treponemal protein product with a quantity of indicator antibody sufficient for binding with the anti-Treponema pallidum antibody.

18. The method of Claim 15, Claim 16 or Claim 17 wherein the indicator antibody is an enzyme linked antibody, a fluorescent tagged antibody, or a radiolabelled antibody.

19. The method of Claim 15, Claim 16 or Claim 17 wherein the antigenic Treponema pallidum proteins are outer membrane proteins P1, P2, or P3.

20. The method according to Claim 15, Claim 16 or Claim 17 wherein the amount of antibody bound to the solid support matrix is determined by contacting the insoluble support matrix with a quantity of indicator antibody reagent selected from the group consisting of enzyme linked antibody, fluorescent tagged antibody, or radiolabelled antibody, the antibody portion of which is capable of reacting specifically with the antibody bound to the insoluble support matrix; thereafter determining the enzyme activity, fluorescent activity, or radioactivity of the respective indicator antibody bound to the solid support matrix. 17