WO1990002336A1

WO1990002336A1 - Immunoassay for determining a chlamydial antigen comprising pretreatment of the sample with a chelating agent

Info

Publication number: WO1990002336A1
Application number: PCT/GB1989/000995
Authority: WO
Inventors: David Sherwood; Paul Richard Stirk
Original assignee: Celltech Diagnostics Limited
Priority date: 1988-08-26
Filing date: 1989-08-25
Publication date: 1990-03-08
Also published as: AU4194289A; GB8820375D0

Abstract

In an assay for chlamydial antigens, the sample suspected of containing antigens is contacted with a chelating agent, for example ethylenediaminetetraacetic acid, in the absence of an anzyme such as lysosyme, prior to contact with an anti-Chlamydia antibody.

Description

IMMUNOASSAY FOR DETERMINING A CHLAMYDIAL ANTIGEN COMPRISING PRETREATMENT OF THE SAMPLE WITH A CHELATING AGENT

This invention relates to an immuno-assay

procedure for the detection of chlamydial antigens . in particular, the invention relates to an

immuno-assay procedure for the detection of chlamydial antigens in a cl inical specimen .

Human and animal infection with chlamydial organisms is widespread . Infection with Chlamydia trachomatis , reported to be the most important human chlamydial pathogen, may result in a number of disease syndromes , including , in humans , trachoma and

oculogenital diseases such as conjunctivitis and urethritis . Other Chlamydia induced diseases in humans include psittacosis , the result of infection with Chlamydia psittaci .

A number of methods have been devised for the laboratory detection of chlamydial infections. These generally rely on the use of either cytological techniques, culture procedures, or serological and immunological diagnostic methods. Cytological

techniques and culture procedures are each exacting and time consuming and can only be performed

realistically in large well-equipped laboratories. On the other hand, serological and immunological

diagnostic methods are generally simpler and quicker to operate and offer the potential for more widespread use, such as for example, in doctor's surgeries. Many of the serological or immunological techniques

currently available, however, lack the necessary sensitivity and/or specificity for use as reliable diagnostic tools. This is particularly so with immuno-assays that have been developed in which use is made of antibodies to chlamydial antigens. It is generally recognised that the sensitivity of such methods is lower than might have been predicted, particularly when the source of the chlamydial antigen is a clinical specimen.

We have now developed a simple to operate highly sensitive immuno-assay employing antibodies for the detection of chlamydial antigens. The assay relies on the discovery that pretreatment of a sample containing chlamydial antigens with a metal chelating agent surprisingly leads to an increase in sensitivity. The effect is particularly marked when the sample is a clinical specimen.

Chelating agents have been used previously in connection with various types of immuno-assay, for example:- GB 2032619 discloses a specific antibody mixture where a chelating agent removes the inhibiton of binding of said mixture by removal of heavy metal cations.

GB 1447751 discloses a method for the manufacture of chelates of ^-containing .organic substances.

EP 0220858 discloses the manufacture and use of hybridomas producing monoclonal anti-human breast cancer antibodies.

EP 0210863 discloses a stable liquid form of tetramethylbenzidine for use in immunoassays utilising polyclonal and monoclonal antibodies.

WO 8403698 discloses a novel compound one end of which is a chelating agent.

WO 8707147 discloses a method for the production of monoclonal antibodies to an immunogenic

antibody/antigen conjunction.

None of these methods disclose the particularly advantageous results which can be achieved

specifically by the use of a chelating agent in an assay for chlamydial antigens. US 4683196 discloses an assay for

Chlamydia-derived material, in which a class-specific reagent is reacted with chlamydial antigen.

US 4663291 discloses a Chlamyida assay, in which the membrane protein is solubilised by a detergent at elevated temperatures, in the present of alkali.

US 4497899 discloses an assay for the detection of Chlamydia trachomatis, in which antigen material is coated non-specifically onto a solid phase and

subsequently caused to react with a Chlamydia-specific antibody, in the presence of a chelating agent such as EDTA. The purpose of the EDTA is not specified, but the contact of the EDTA with the Chlamydia-derived material does not take place until after the

immobilisation of the antigen to the solid phase.

US 4617264 discloses a pretreatment method for a sample, prior to carrying out an assay for material derived from certain gram negative bacteria such as, for example, N-gonorrhoeae and Chlamydia. The method disclosed involves the contacting of the sample material with an enzyme capable of hydrolysing bonds between N-acetylglucosamine and N-acetylmuramic acid and a chelating agent.

We have discovered that the pretreatment of a sample containing chlamydial antigens with a chelating agent in the absence of an enzyme of the type required in US 4617264 gives rise to an appreciable increase in sensitivity.

Accordingly, the present invention provides a method of carrying out an immuno-assay for determining a chlamydial antigen in a sample, which method

comprises immobilising chlamydial antigen present in the sample to a solid support contacting the said chlamydial antigens with one or more anti-Chlamydia antibodies, including a labelled anti-Chlamydia antibody, and determining the result of the assay in dependence upon the formation of a complex between the said labelled anti-Chlamydia antibody and any

chlamydial antigen present in the sample,

characterised in that the sample is mixed with a chelating agent prior to the said immobilisation of the said antigen, in the absence of an enzyme capable of hydrolysing bonds between N-acetylglucosamine and N-acetylmuramic acid.

The chelating agent for use in the assay

according to the invention may be any polydentate compound containing two or more donor atoms. Examples of donor atoms include nitrogen, oxygen, sulphur, phosphorus, arsenic, and selenium atoms.

Examples of suitable chelating agents include inorganic chelating agents such as polyphosphates, e.g. sodium tripolyphosphate; and organic chelating agents such as aminocarboxylic acids,

aminoalkylphosphonic acids, hydroxycarboxylic acids, 1,3, diketones, polyamines and aminoalcohols.

Particularly suitable organic chelating agents include hydroxycarboxylic acids such as citric acid, gluconic acid and the salts thereof, polyamines, such as tetra-aza-cyclotetradecane and

tetra-aza-cyclopentadecane, and, especially,

aminocarboxylic acids such as

ethylenediaminetetraacetic acid.

The immunoassay may be of any suitable format for detecting Chlamydia trachomatis or Chlamydia psittaci or both. Thus, the assay may be a solid or liquid phase competitive or non-competitive immuno-assay, for example a two site immunometric assay or a dual competition assay such as a dual analyte assay of the type described in UK Patent Specifications Nos.

2029011 and 2116318. The exact quantity of chelating agent used in the assay will depend on the nature of the sample and the assay format in use, and may be empirically determined by appropriate small-scale tests in accordance with conventional practice. In general, however, we have found that the chelating agent is advantageously present with the sample in a concentration up to about 100mM.

The sample, or an extract thereof, may be mixed with chelating agent at any suitable time, providing it is prior to incubation with the anti-Chlamydia antibody. Thus, for example, the sample, e.g. a urino-genital specimen, may be collected in a suitable medium containing the chelating agent. Alternatively, for some specimens, e.g. urine, the chelating agent may be added direct to the sample, once it has been collected. The time between the mixing of sample and chelating agent and the subsequent incubation with antibody is not critical, but preferably we have found that a minimum time of 5 minutes is desirable to obtain optimum sensitivity.

The sample and chelating agent may be mixed at any suitable temperature, for example in the range 0°-120°C, e.g. at ambient termperature. We have found however that it is possible in some instances to increase the sensitivity of the assay according to the invention by heating the sample in the presence of chelating agent.

The heating of a Chlamydia sample in order to increase sensitivity is disclosed, for example, in EP 0183383

Thus, according to a preferred aspect of the invention we provide an immuno-assay for determining chlamydial antigen in a sample, wherein said sample, or an extract thereof, is contacted with one or more anti-Chlamydia antibodies, including a labelled anti-Chlamydia antibody, and the final assay result is dependent on the formation of a complex between said labelled anti-Chlamydia antibody and any chlamydial antigen present in said sample, characterised in that said sample, or an extract thereof, is mixed with a chelating agent and heated prior to incubating said sample or extract with anti-Chlamydia antibody.

In this aspect of the invention, the sample is preferably a clinical specimen.

The sample and chelating agent are generally heated, for example to a temperature from 80°-120ºC, especially around 100°C, for example for a period of from 5 to 15 minutes.

In order for the chelating agent to be effective, it is important that it is caused to contact the sample prior to or during the immobilisation of the Chlamydial antigen to the solid support. The

immobilisation of the chlamydial antigen may be carried out using a specific binding reaction (e.g, by first binding a Chlamydia specific antibody to the support), or non-specifically (for example, as

disclosed in US 4497899). The sample is preferably contacted with the chelating agent prior to incubation of the sample with anti-Chlamydia antibody,

particularly labelled antibody.

The ability, in accordance with the present invention to carry out an improved Chlamydia assay in the absence of a hydrolytic enzyme is a substantial advantage as compared with US 4617264, particularly in the formulation of assay kits.

The various solutions employed in the assay preferably comprise one or more detergents, for example of the type disclosed in EP 0132948.

A selection of suitable assay systems, which also serves to illustrate the invention, is described in the following Examples.

In Examples 1 to 6 the following basic enzyme immunoassay (EIA) procedure was used, except where indicated otherwise:

STEP

1. Monoclonal antibody (a) passively adsorbed to solid phase of a plastic microtitration plate

(96-well).

2. Laboratory antigen (b) or clinical specimen (100 microlitre) incubated in coated wells at ambient temperature (15°C-30°C) for 60 minutes.

3. Pre-formed complexes of biotinylated

monoclonal antibody (a) streptavidin horseradish

peroxidase (100 microlitre) added and incubated at ambient temperature for 60 minutes.

4. Wells washed with an acetate/detergent based buffer.

5. Tetramethylbenzidine based substrate (100 microlitre) added to washed wells and incubated at ambient temperature for 30 minutes.

6. Dilute sulphuric acid (50 microlitre) added to stop substrate reaction.

7. Colour read photometrically at an absorbance of 450nm.

(a): Laboratory grown Chlamydia trachomatis

serovar LGV2 in McCoy cells was used

throughout,

(b): Monoclonal antibody directed to the genus specific epitope of Chlamydia spp.

Example 1

Use of Chelating agent Ethylenediaminetetraacetic acid

- disodium salt (EDTA) in antigen diluent.

The basic EIA methodology was followed using the following buffers: Antigen diluent: 100mM Tris/HCl+ pH8.5 with or without

EDTA.

Conjugate diluent: 0.1M phosphate buffered saline/

1% bovine serum albumin (BSA)/0.05%Tween 20 detergent, pH7.5

Diluted antigen was either treated by heating to

100ºC for 10 minutes or untreated prior to use in the

EIA. Results of these experiments are shown in

Table 1. Treatment of antigen with EDTA either at ambient temperature or at 100°C prior to assay has a beneficial effect on assay performance (Table 1).

Similar improvements of assay performance using EDTA in the antigen diluent was observed when other antigen and conjugate diluents were used (Table 2).

Example 2

Use of Chelating agent (EDTA) in the presence of swabs The basic EIA methodology was used using the following buffers:

Antigen diluent: Triethanolamine buffer/detergent pH8.5 with or without EDTA.

Conugate diluent: barbital buffer/detergent pH8.5.

The following treatments were prepared for this

Example:

(a) chlamydial antigen was diluted in antigen diluent with or without EDTA

(b) chlamydial antigen was diluted in antigen diluent (with or without EDTA) which had previously been incubated with Exogen

Minitip Swabs (Exogen Limited, Scotland, UK) for 18 hours at ambient temperature.

All dilutions were assayed and the results of the assays are shown in Table 3. The results show that the inclusion of EDTA in antigen diluent has a

beneficial effect on assay performance in both the presence and absence of swabs. Example 3

Effect of temperature on the use of chelating agent

(EDTA) in the presence of swabs.

The basic EIA methodology was used with the following buffers:

Antigen diluent: Triethanolamine buffer/EDTA pH8.5 Conjugate diluent: Triethanolamine buffer/detergent pH8.5

Antigen was diluted in antigen diluent which had not been exposed to Exogen Swabs (see Example 2) or in diluent in which Exogen Swabs had been incubated at ambient temperature for 18 hours. Prior to assay, diluted antigen was either incubated at ambient temperature for 10 minutes or incubated at 100°C for 10 minutes.

Results are shown in Table 4.

The data indicates the beneficial role of EDTA in the assay in the presence of swabs is unaffected by temperature.

Example 4

Beneficial effect of a Chelating agent (EDTA) on the performance of clinical specimens (a) Urine Specimens

The basic EIA methodology was used with the following buffers:

Antigen diluent: Tris/HCl buffer pH8.5 with or without EDTA.

Conjugate diluent: Triethanolamine buffer/detergent pH8.5

The sediment from three Chlamydia positive and three Chlamydia negative urine specimens (collected from patients attending a sexually transmitted disease clinic) were pelleted using centrifugation. The pellets were resuspended in antigen diluent (with or without EDTA) and either treated at ambient temperature or 100°C before assay. The results of these assays are shown in Table 5.

The data shows that the presence of EDTA in antigen diluent improves the detection of Chlamydia in resuspended urine pellets. This improvement is unaffected by increasing treatment temperature prior to assay.

(b) Urino-genital specimens

The basic EIA methodology was used. Antigen diluent and conjugate diluent variation are shown in Table 6. Urino-genital swabs (Exogen Minitip Swab) were taken from the cervix of female patients and the urethra of male patients attending a sexually

transmitted disease clinic. Swabs were collected into antigen diluent and transported to the laboratory for analysis. A duplicate swab was taken for each patient for Chlamydia testing by a reference method (either cell culture or a commercially available enzyme immuno-assay). The swab sequence for patient sampling was randomised between the reference method and the antigen diluent.

The results of these assays are shown in Table 6. The data indicate that the chelating agent is an important component in the antigen diluent to achieve increased assay sensitivity on urino-genital

specimens. Increasing specimen pre-treatment

temperature had no effect on assay sensitivity.

Example 5

Effect of subsequent addition of Chelating Agent

(EDTA) to antigen diluent exposed to swabs.

The basic EIA methodology was used with the following buffers:

Antigen diluent: Triethanolamine buffer/detergent pH8.5

Conjugate diluent: Barbital buffer/detergent pH8.5. The following treatments were used to dilute

Chlamydia antigen:

(a) antigen diluent only.

(b) antigent diluent after exposure to Exogen swabs for 18 hours at ambient temperature.

(c) antigen diluent containing EDTA after

exposure to Exogen swabs for 18 hours at ambient temperature.

(d) chlamydial antigen diluted as in treatment (b) and incubated for 1 hour at ambient temperature before EDTA was added.

Results of the assays are shown in Table 7.

The data indicates that the sequence in which EDTA is added in an assay in the presence of swabs can affect the sensitivity of the assay.

Example 6

Use of various Chelating Agents

The basic EIA methodology was used with the following buffers:

Antigen diluent: Triethanolamine buffer/detergent pH8.5

Conjugate diluent: Barbital buffer/detergent pH8.5

The following teatments were used to dilute chlamydial antigen;

(a) antigen diluent only

(b) antigen diluent after exposure to Exogen

swabs for 18 hours at ambient temperature

(c) antigen diluent containing EDTA

(d) treatment (c) after exposure to Exogen swabs for 18 hours at ambient temperature

(e) antigen diluent containing

Tetra-aza-cyclotetradecane

(f) treatment (e) after exposure to Exogen swabs for 18 hours at ambient temperature (g) antigen diluent containing Tetra-aza-cyclopentadecane

(h) treatment (g) exposed to Exogen swabs for 18 hours at ambient temperature.

The data indicate that both alternative chelating agents behave in a similar manner to EDTA.

Example 7

Effect of Chelating Agents (EDTA) on an assay

incorporating enzyme amplification An amplified enzyme immuno-assay procedure was carried out in a manner similar to that employed in Example 1, with the following modifications:

Alkaline phosphatase (50 microlitres) was

substituted for streptavidin horseradish peroxidase in step 3 of the immuno-assay and the antibody employed was not biotinylated

In step 5, the bound alkaline phosphatase was determined by the method of EP 0132948. NADPH

substrate (100 microlitres), was utilised in place of NADP and incubated at ambient temperature for 40 minutes.

Colour was read spectrophotometrically at an absorbance of 492nm.

The following buffers were employed:- antigen diluent; 100mM Triethanolamine/1%

polyoxyethyleneether detergent (Trade Mark TRITON X-705) with or without EDTA, at pH7.5.

conjugate diluent; 100mM

Triethanolamine/2.0% BSA/0.5% zwitterionic deterge (Trade Mark SB16) /0.1% Triton X-705 at pH7.5.

Washing buffer: 10mM Tris/HCL /60mM Ammonium sulphate /0.025% Triton X-705/0.05% SB14 at pH8.0

Various dilutions of antigen were either

untreated, or heat treated to 100ºC for 15 minutes prior to use in an enzyme immunoassay. The result are shown in Table 9. It can be seen that the treatment of the antigen sample with EDTA at ambient temperature has a beneficial effect on assay

performance.

Example 8

Use of various Chelating Agents in

Amplified Enzyme Immuno-assay Example 7 was repeated using the following alternative chelating agents in place of EDTA:- (a) antigen diluent only (blank)

(b) antigen diluent containing

1, 2-cyclohexanediamine tetraacetic acid (CDTA),

(c) antigen diluent containing

[ethylene-bis (oxyethylenenitrilo)]

tetraacetic acid (EGTA),

(d) antigen diluent containing nitriloacetic acid (NTA) the results are shown in

Table 10.

TABLE 1

A450*

Treatment Dilution of No EDTA in With EDTA in

Chlamydia antigen diluent antigen diluent

10^-2 2.41 2.41

No heat 10^-3 0.48 0.60 treatment 10^-4 0.23 0.25

negative control 0.21 0.24

10^-2 2.43 2.48

Treatment by 10^-3 0.39 0.55 heating at 10^-4 0.24 0.22

100°C for 10 negative control 0.22 0.18

*Absorbance at 450nm - All values are means of two or three replicates.

TABLE 2

A450*

Antigen diluent (AD) Dilution of No EDTA in With EDTA in and conjugate diluent Chlamydia antigen diluent antigen diluent+

AD; Triethanolamine 10^-2 1.86 2.28

buffer/detergent pH8.5 10^-3 0.84 1.37

CD; barbital buffer/ 10^-4 0.20 0.23

detergent pH8.5 negative control 0.06 0.05

AD; Triethanolamine buffer 10^-2 2.71 2.76

pH8.5 10^-3 1.69 1.91

CD; Triethanolamine buffer/ 10^-4 0.39 0.47

detergent pH8.5 negative control 0.18 0.26

AD; Tris/HCl buffer pH8.5 10 ^-2 2.75 2.77

10^-3 2.10 2.33

CD; Triethanolamine buffer/ 10^-4 0.36 0.46

detergent pH8.5 negative control 0.18 0.17

*Absorbance at 450nm - All values are means of two or three replicates.

+Antigen treated with EDTA at ambient temperature.

TABLE 3

A450*

Diluent Dilution of No EDTA in With EDTA in

Used Chlamydia antigen diluent antigen diluent

Antigen diluent only 10^{- 2} 1.68 1.71

10 ^{- 3} 0.95 0.66

10 ^{- 4} 0.26 0.18 negative control 0.16 0.08

Antigen diluent after 10^-2 0.31 1.37 exposure to Exogen 10^-3 0.20 0.42

Swabs 10^-4 0.18 0.10

negative control 0.17 0.07

*Absorbance at 450nm - All values are the means of two or three replicates.

TABLE 4

A450*

Diluent Used Dilution of Ambient temperature 100°C

Chlamydia 10 minutes 10 minutes

Antigen diluent 10^-2 2.50 2.50

10^-3 2.54 2.37

10^-4 0.55 0.45 negative control 0.19 0.18

Antigen diluent 10^-2 2.50 2.48 after exposure to 10^-3 2.06 1.66

Exogen Swabs 10^-4 0.36 0.27 negative control 0.22 0.17

*Absorbance at 450nm - All values are means of two or three replicates.

TABLE 5

A450*

Treatment Urine Specimen Chlamydia No EDTA in With EDTA in

Status+ antigen diluent antigen diluent

ambient 1 positive 0.10 0. 14 temperature 2 positive 0.25 0. 52 for 10 minutes 3 positive 0.18 0. 31

4 negative 0.08 0. 07

5 negative 0.10 0. 10

6 negative 0.08 0. 08

100 °C for 1 positive 0.08 0.09

10 m inu tes 2 positive 0.18 0.37

3 positive 0.16 0.27

4 negative 0.06 0.07

5 negative 0.09 0.09

6 negative 0.07 0.07

+By cell culture or commercially available EIA.

*Absorbance at 450nm - All values are means of duplicates.

TABLE 6

System EDTA in Number of No of specimens No of specimens antigen diluent samples positive by reference positive by

method EIA (%sensitivity)

AD : Triethanolamine

buffer/detergent ρH8.5

CD : Barbital buffer/detergent No 52 8 1 (12.5) pH8.5.

ST : ambient temperature,

10 minutes

AD : as above

CD : as above Yes 95 13 9 (69)

ST : as above

AD : as above

CD : as above YES 91 6 4 (67)

ST : 100ºC, 10 minutes

AD : as above

CD : Triethanolamine buffer/ YES 97 8 6 (75)

detergent pH8.5

ST : 100ºC, 10 minutes

AD-antlgen diluent, CD-conjugate diluent, ST-sample pre-treatment

TABLE 7

Dilution of A450*

Chlamydia Treatment (a) Treatment (b) Treatment (c) Treatment (d)

10^-2 2.09 0.85 1.92 0.49

10^-3 1.33 0.19 0.75 0.14

10 ^-4 0.32 0.10 0.22 0.10 negative control 0.15 0.10 0.11 0.12

*Absorbance at 450nm - All values are means of two or three replicates

TABLE 8

TREATMENTS

A450*

Dilution of (a) (b) (c) (d) (e) (f) (g) (h) Chlamydia

10 ^-2 1.86 0.73 2.28 2.08 2.17 2.17 1.90 1.87

10^-3 0.84 0.19 1.38 1.08 1.01 1.02 0.71 0.72

10 ^-4 0.20 0.10 0.23 0.18 0.11 0.15 0.12 0.12 neσative control 0.06 0.07 0.05 0.05 0.03 0.04 0.05 0.05

*Absorbance at 450nm - All values are means of two or three replicates.

TABLE 9 A*492nm

Treatment Dilution of No. EDTA EDTA in

Chlamydia in Diluent Antigen Diluent

1/4 NT 3.015

1/16 NT 2.310

No treatment 1/64 NT 0.764

negative NT 0.075

Treatment by 1/4 2.921 1.140 heating at 1/16 1.588 0.342

100°C for 15 1/64 0.450 0.126 minutes negative 0.084 0.076

NT = Not tested

*Absorbance at 492nm - All values are means of two or more replicates.

TABLE 10 A492*

Treatment Dilution (a) (b) (c) (d)

of Chlaydia

3/4 NT 2.898 2.932 2.909 No treatment 1/16 NT 1.503 1.342 1.562 (ambient 1/64 NT 0.384 0.404 0.492 temperature) negative NT 0.085 0.091 0.098

Treatment 1/4 NT 0.762 1.813 1.772 by heating 1/16 1.395 0.257 0.555 0.601 at 100°C for 1/64 0.470 0.132 0.204 0.206 15 minutes negative 0.089 0.089 0.091 0,087

NT = Not tested

*Absorbance at 492nm - All values are means of two or more replicates.

Claims

1. A method of carrying out an immuno-assay for determining a chlamydial antigen in a sample, which method comprises immobilising chlamydial antigen present in the sample to a solid support contacting the said chlamydial antigens with one or more

anti-Chlamydia antibodies, including a labelled anti-Chlamydia antibody, and determining the result of the assay in dependence upon the formation of a complex between the said labelled anti-Chlamydia

antibody and any chlamydial antigen present in the sample, characterised in that the sample is mixed with a chelating agent prior to the said immobilisation of the said antigen, in the absence of an enzyme capable of hydrolysing bonds between N-acetylglucosarnine and N-acetylmuramic acid.

2. A method as claimed in Claim 1, wherein the said mixing of the sample with a chelating agent is carried out prior to incubation of the sample with

anti-Chlamydia antibody.

3. A method as claimed in claim 2, wherein the chelating agent is a polydentate compound containing two or more donor atoms selected from nitrogen, oxygen, sulphur, phosphorus, arsenic and selenium.

4. A method as claimed in Claim 3, wherein the chelating agent is a polyphosphate, an aminocarboxylic acid, and aminoalkylphosphonic acid, a

hydroxycarboxylic acid, a 1,3, diketone, a polyamine, or an aminoalcohol.

5. A method as claimed in Claim 4, wherein the chelating agent is citric acid, gluconic acid or a salt thereof, a polyamine, or an aminocarboxylic ac id .

6. A method as claimed in Claim 5 , wherein the chelating agent is tetra-aza-cyclotetradecane, tetra-aza-cyclopentradecane , or

ethylenediaminetetraacetic .

7. A method as claimed in Claim 1, wherein the sample is heated in the presence of the chelating agent , prior to contact with the anti-Chiamydia antibody.