WO1998058259A1

WO1998058259A1 - Stabilization of polypeptides for use in immunoassay procedures

Info

Publication number: WO1998058259A1
Application number: PCT/IL1998/000278
Authority: WO
Inventors: Bella Ohana
Original assignee: Savyon Diagnostics Ltd.
Priority date: 1997-06-19
Filing date: 1998-06-15
Publication date: 1998-12-23
Also published as: IL121116A; IL121116A0; EP0995119A1; JP2002508843A; AU7786398A

Abstract

A method for improving the stability of reagents for detection of antibodies in a sample, comprising: (a) providing a first buffer for the stabilization of polypeptides or proteins attached to a solid support, said buffer containing about 1-20 % sucrose; (b) providing a second buffer for the dilution of the antibody-containing sample and for reacting said immobilized polypeptides with the antibodies present in said sample, said second buffer comprising about 0.05 % (v/v) detergent other than Tween-20.

Description

STABILIZATION OF POLYPEPTIDES FOR USE IN IMMUNOASSAY PROCEDURES

Field of the invention

The present invention relates to the stabilization of immobilized polypeptides, to the improvement of immunoassay procedures.

Background of the invention

Immunosorbent assays have largely replaced the use of agglutination- based assays in detection of immunologically active compounds, e.g. antigens of pathogens or antibodies against them. Due to the fact that in most infections, antibodies occur in much higher concentration than the antigen, and are easily obtained in non-invasive procedures such as sampling body fluids, most diagnostic methods determine the amount of antibody to a given antigen in order to find out whether an infection with the agent that carries the antigen is ongoing. Such assays necessitate the use of antigen to capture the antibody. The antigen can be prepared in three major ways: a) by modifying intact pathogenic organisms, e.g. extracting certain proteins; b) by the use of recombinantly produced proteins of said organism, and c), by the use of synthetic peptides derived from the sequence of antigenic proteins of said organism. The use of defined proteins or fragments thereof has an advantage over the use of whole pathogen extracts; namely the possibility to avoid structures which cross-react with antibodies formed against other organisms or strains, biovars or serovars of the said organism. In many cases it is desired, however, to distinguish between reactive antibodies to one or another strain of a certain microorganism, in some cases it desired to distinguish between biovars or serovars of a certain pathogen. One example of such a situation are the various genotypes of hepatitis B virus, some of which respond well to interferon treatment while others do not. Another example are the two bacterial species Chlamydia pneumoniae and Chlamydia trachomatis. Prevalence for the former is high, with about 60% of the population having been infected at one time, but most infections cured, while the prevalence for the latter is less than 10%, with the possibihty of silent infections going on that may develop into chronic infections. In both situations it is desirable to find out which organism caused a present symptom before starting treatment.

Chlamydia are gram-negative intracellular parasitic bacteria that cause acute and chronic disease in mammalian and avian species. The genus Chlamydia is comprised of four species: C. trachomatis, C. pneumoniae, C. precorum and C. psittaci. C. trachomatis is divided into 15 serovars, which may cause different diseases such as trachoma, lymphogranuloma venereum or sexually transmitted disease. The three different species of Chlamydia are serologically highly cross-reactive. Most of the serological diagnostic assays for Chlamydia use either purified elementary bodies (microimmunofluorescence, MIF and ELISA tests), lipopolysaccharide, LPS or purified major outer membrane protein, MOMP (ELISA tests) as antigens. Genus-specific epitopes are present in all the above antigens, resulting in low species specificity of the available tests. Moreover, a large proportion of the population has been exposed to C. pneumoniae (with no clinical signs), so that the prevalence of anti-Chlamydia antibodies is very high. Therefore, the differentiation between C. pneumoniae and C. trachomatis specific antibodies using conventional serological screening tests (MIF, ELISA, EIA etc.) is deficient.

Similarly to the above considerations concerning specific detection, for the purpose of vaccination, it may be desired to elicit an immunoreaction against certain epitopes only so as to develop immunity against certain organisms or strains or biovars or serovars of organisms preferentially the ones that are most pathogenic, and at the same time not to develop antibodies against e.g. non-pathogenic or less virulent variants of said organism. The set-up of diagnostic kits that allow rapid, reproducible and cost- effective testing of a large number of clinical samples depends, among other parameters, upon the stabihty of its components. Some proteins and peptides are highly unstable, with half-lives ranging from hours to days only. The art is therefore constantly seeking to improve the stability and preservation of the antibody-binding properties of such polypeptides for the use in immunoassays and vaccines.

The Prior Art

Peptide- and protein-based antibody-capture assays are well known in the art and have been described in many publications. Similarly, methods for the stabilization of proteins have been described. A number of such publications uses sucrose as a stabilizing agent, e.g. to preserve growth- enhancing properties of serum (WO9418310), enzyme activity (JP06113847), or drug activity of interferon-beta (EP 551535). However, the use of sucrose has not been reported for immobilized proteins or in peptide-based immunoassays.

It is an object of the present invention to provide improved immunoassays, in which stability of the immobilized polypeptide is enhanced.

It is another object of the invention to provide immunoassays and reagents therefor, which provide improved results.

It is yet another object of the invention to provide reagents and kit components which are useful to improve said immunoassays.

Other objects and advantages of the invention will become apparent as the description proceeds. The term "body fluid", as used herein, comprises fluids sampled from within the body, such as blood or lymph, local secretions, such as tears, semen, urine, sweat, sputum etc., samples obtained by washing (e.g. bronchiolar lavage) or swabbing, including cervical smears, and the like.

The term "peptide", as used herein, comprises peptides obtained by chemical synthesis, or by cleavage, either by chemical means or by using proteolytic enzymes, of a larger peptide or protein.

Summary of the Invention

In one aspect, the invention is directed to a method for improving the stability of reagents for detection of antibodies in a sample, comprising:

(a) providing a first buffer for the stabilization of polypeptides or proteins attached to a solid support, said buffer containing about 1-20% sucrose,

(b) providing a second buffer for the dilution of the antibody- containing sample and for reacting said immobilized polypeptides with the antibodies present in said sample, said second buffer comprising about 0.05% (v/v) detergent.

Said detergent is preferably a detergent other than Tween-20.

According to a preferred embodiment of the invention, the first buffer is a blocking solution in an immunoassay, said blocking solution containing about 0.05 to 4 % (w/v) gelatin, about 1 to 30 % (v/v) normal rabbit serum, 1 to 20% (w/v) sucrose in PBS; and wherein the second buffer is a serum diluent containing about 0.05 to 4% (w/v) gelatin, about 1 to 30% (v/v) normal rabbit serum, and about 0.01 to 2 % (v/v) Triton-X 100 in PBS.

The invention is further directed to a stabilizing solution for the stabilization of a polypeptide or protein attached to a solid support, comprising about 1-20% sucrose in a buffer. An illustrative and non- limitative example of such a solution is a solution containing about 0.03% (w/v) gelatin, about 10% (v/v) normal rabbit serum, and about 10% (w/v) sucrose in PBS.

The invention is also directed to a buffer solution for the dilution of an antibody-containing sample to be reacted with an immobilized polypeptide, comprising about 0.05% (w/v) of a detergent other than Tween-20. An illustrative example of a suitable solution comprises about 0.03% (w/v) gelatin, about 10% (v/v) normal rabbit serum, and about 0.05% Triton-X 100, in PBS. The invention further encompasses kits comprising the stabilization solution and/or the dilution solution of the invention. Such kits can be useful for a variety of tests, e.g. for ELISA. Practically important pathogens to be tested by the invention are Chlamydia trachomatis, or Chlamydia pneumoniae, although the invention is of course in no way limited to the testing of any specific pathogen.

The invention also encompasses a method for detecting an infection in a mammal, said method comprising:

(a) obtaining a serum or other body fluid sample from the person or animal, and diluting said sample using a dilution buffer comprising about 0.05% (w/v) of a detergent other than Tween- 20,

(b) contacting said sample with said immobilized polypeptides, which have been with a stabilization solution containing about 1-20% sucrose in a buffer; and (c) determining the extent of reaction between antibodies present in said sample and said immobilized polypeptides using conventional immunoassay technology.

Detailed Description of the Invention

The present invention provides improvements over the prior art of immunoassay using immobilized antigen. Such assays are carried out by

1) immobilizing a suitable antigen on a sohd support;

2) diluting the sample that should be assayed for the presence of antibodies against the immobilized antigen in a suitable dilution buffer, this buffer being optimized for maximum specific binding of the antibody while minimizing background resulting from non-specific binding of other sample components, e.g. antibodies with different specificities, to the antigen or the sohd support or other components, e.g. the substance used to block binding to the sohd support once the antigen is bound; and

3) washing off unbound antibody and determining the extent of reaction by e.g. the use of a labeled (by enzyme or other means, e.g. radioactively) antibody that recognizes a constant domain of the antibodies that are bound to the antigen.

The invention, as illustrated by the following description, provides substantial improvements in such procedures.

The present invention will now be described in more detail in the following non-limiting examples and their accompanying figures

General Procedures

In the following experiments Chlamydia species-specific human sera were tested for their ability to bind Chlamydia species-specific peptides. The peptides are the subject of two copending patent apphcations of the same applicant herein, filed on the same day as the present application and designated as Attorney's docket 4195/96 and 4326/97, the description of which is incorporated herein by reference. Peptides were synthesized by standard organic chemistry.

Peptides specific for C. trachomatis:

IFDTTTLNPTIASGAGDVK herein designated as Ct4A (SEQ. ID NO. 1)

VDITTLNPTIAGCGSVA herein designated as Ct4B (SEQ. ID NO. 2)

VFDVTTLNPTIAGAGDVK herein designated as Ct4C (SEQ. ID NO. 3)

LAEAILDVTTLNPTITGKGAWS herein designated as Ct4D (SEQ. ID

NO. 4).

CDNENQSTVKTNSVPNMSLDQSK herein designated as Ct2A(SEQ. ID

NO. 5)

LDVTTNATIAGKGTW herein designated as CtVDIV (SEQ. ID NO. 6)

As a specificity control for the immunoassay, the following peptides designed according to the sequence of homologous MOMP domains of the related bacteria Chlamydia pneumoniae were synthesized and used in the immunoassay:

CFSMGAKPTGSAAANYTTAVDRPNPAYNK herein designated as CplA

(SEQ. ID NO. 7)

AFPLPTDAGVATATGTKS herein designated as CpVDIII (SEQ. ID NO.

8)

VKGTTVNANELPNVSLSNGK herein designated as Cp2A (SEQ. ID NO.

9)

LNLTAWNPSLLGNATALSTTDSFK herein designated as Cp4A (SEQ. ID

NO. 10) When testing the reactivity of several peptides for various sera that were positive for Chlamydia trachomatis (by MIF assay), it was found that each serum reacts with a certain subpopulation of peptides. In the following study, peptides Ct2A, Ct4A, Ct4B, Ct4C and Ct4D were tested on IVF (in- vitro-fertilization) sera for IgG reactivity. Twenty-seven sera reacted positively with at least one C. trachomatis peptide. Out of the twenty- seven, only four were positive to Ct2A, seventeen were positive to Ct4A, twenty-three positive to Ct4B, eighteen positive to Ct4C, and twenty were positive to Ct4D. Interestingly, 4 sera were positive exclusively to Ct4D and 5 sera were positive exclusively to Ct4B. In order to detect all C. trachomatis serovars, it is therefore necessary to include all of the above peptides in the assay, i.e. C.t 4A, 4B, 4C and 4D.

Table 1: The sensitivity of C. trachomatis peptides on IVF sera

Stabilization of Peptides

ELISA plates (Maxisorb, Nunc) were coated with peptides (0.3 μg/well) dissolved in 0.05M carbonate buffer pH 9.5 for 1 h at 37°C. Alternatively, coating was done overnight at room temperature or at 4°C. The peptide solutions were removed, the wells filled with blocking solution (0.03% gelatin, 10% normal rabbit serum (NRS) in 0.15 M NaCl, 0.05 M NaP0₄, pH 7.5 [PBS]). After incubation for lh at 37°C the plates were either used immediately or after drying and incubation for various times at 37 °C as indicated in Table 1. Sera were diluted in 1/21 in diluent (0.03% gelatin, 10% NRS, 0.05% Triton-X 100), added to the coated wells and incubated for 1 h at 37°C. After rinsing six times, with PBS/0.05% Tween-20 (PBS/Tween), anti-human IgG-conjugated HRP (diluted 1:15,000 in blocking solution) was added and incubated for 1 h at 37 °C. After rinsing as before, substrate was added and the reaction quantified by measuring OD 450.

Table 2 shows an example of such a stabihty test. For some of the peptides, incubation of the dried coated plates at 37°C led to a drastic reduction in antibody binding (see e.g. Ct2A, Ct4A). In contrast, other peptides, like Ct4B, CplA, or Cp2A, were relatively stable even after 6 days incubation at 37 °C.

Table 2 Stability of various coated peptides after incubation at 37°C

In further experiments, peptide Ct4C was found to be highly unstable when Ct4C-coated and dried plates were incubated at 37 °C. Since this peptide contains a cysteine residue at its N-terminal end, and cysteine is known to be sensitive to e.g. oxidizing conditions, a modified version of peptide Ct4C lacking the cysteine residue was tested for stability and for binding to different sera. Although the modified peptide had much improved stabihty, it reacted much weaker than the original Ct4C peptide (55% less signal in immunoassay) with certain sera. It was therefore desirable to include the cysteine-containing peptide Ct4C in the immunoassay in order to maximize sensitivity.

Example 1

In order to enhance stabihty of the immobilized peptides, sucrose was added and Tween-20 omitted from the blocking solution so that the composition was 0.03% gelatin, 10% NRS, 20% sucrose in PBS. All other reagents were as in Table 2.

The table shows the results of ELISA tests (OD 450) using the indicated peptides for coating and the indicated sera as samples. Serum #11 was a negative control, serum #92 is positive for both C. trachomatis and C. pneumoniae. The serum mix contained 4 sera of different patients that cover most of the serovar reactivities for C. trachomatis.

The results show that all tested peptides are now stable for up to one week at 37°C. Even peptide Ct4C, which is highly unstable when blocking solution without sucrose was used, now shows only a decline of 8 % (from OD 2.9 to 2.67) in reactivity after 37 °C incubation of the peptide-coated plate. The same conclusion can be drawn for e.g. peptide Ct2A, whose reactivity declined from 3 to 0.4 after 6 days at 37 °C (Table 2), whereas when using sucrose-containing blocking solution the reactivity of the peptide is stable (2.47 versus 2.5 after one week 37 °C, Table 3). Example 2 Effect of various sucrose concentrations in the blocking solution on peptide stability

In order to evaluate the optimal sucrose concentration for stabihzation of peptides, 1%, 10% and 20% sucrose was added to the blocking solution (as described for Table 2). The plates were coated with a mixture of C. trachomatis peptides (Ct4A, Ct4B, Ct4C, Ct4D) and blocked. After drying and incubation for 4 weeks at 4°C, room temperature (RT) or 37°C, the plates were tested for their binding activity using 5 sera, 2 negative (F40, and F235) and three positive (#92, F50 and BBI 13) as described above. Only slight differences were observed when 1, 10 or 20% sucrose were used, even after 4 weeks of storage at 37°C. However, the background of the negative serum (F40) was somewhat higher when 1% or 20% sucrose was used (Table 4, row 1, 37°C), therefore 10% was chosen as optimal concentration.

Table 4 Effect of sucrose concentration on stabilization (4 weeks) of immobilized C. trachomatis mix peptides

Example 3 The influence of sucrose and Tween on the stability of distinct peptides

In order to test the influence of sucrose and Tween in the blocking solution on the stability of various peptides, four peptides derived from C. trachomatis and four peptides as well as a mixture of these derived from C. pneumoniae were tested independently. Testing was carried out by incubation of the immobihzed peptides at 37°C for three to ten days and the results compared to peptides which were incubated at 4°C. The following Tables show on their left part ELISA results obtained with peptides incubated at the indicated temperatures and for the indicated times, while at the right side they give the ratio of the results obtained at 37°C incubation versus the results obtained by 4°C incubation. A ratio of about 1 indicates that the peptide is stable when incubated at 37°C, while smaller ratios (<0.9) indicate instability of the immobihzed peptide. ELISA tests were carried out as described for Table 2.

A) Results obtained for C. trachomatis peptides - Tables 5-16 show the results of ELISA assays carried out with immobihzed peptides stored dry for the indicated times and at the indicated temperatures. The assay was carried out as described for Table 2, with 10% sucrose and/or 0.05% Tween-20 ("PBS-T") added to the blocking buffer where indicated.

All of the tested C. trachomatis peptides were essentially stable when blocking buffer containing sucrose was used (see Tables 5, 8, 11, 14).

When sucrose was omitted from the blocking buffer, a significant decrease in reactivity to most sera was observed for peptide Ct4A and Ct4C (Tables 6 and 12), a decrease to three of the sera for Ct4B (Table 9), and a slight decrease to four sera for Ct4D (Table 15). These data show clearly that each peptide displays several epitopes, and that the reactivity of these epitopes to the antisera is influenced differentially storage of the dried immobihzed peptides, but that all epitopes can be preserved when sucrose- containing buffer is used for blocking. When sucrose was omitted from the blocking solution and Tween-20 was added (as is customary in ELISA procedures), the stabihty of some of the peptides was decreased by much, e.g. Ct4A (compare Table 7 to Table 6), and Ct4B (compare Table 10 to Table 9), while the other two peptides were less affected, but still exhibited slightly lower reactivities when Tween-20 was present in the blocking buffer (compare Table 13 to Table 12 and table 16 to Table 15).

Table 5 peptide Ct4A, blocker with sucrose

37°C/4°C serum 4°C 3 day 7 dav 10 dav 3 day 7 dav 10 dav

H 156 1.246 1.437 1.277 1.333 1.153 1.024 1.069

H 171 0.719 0.791 0.709 0.742 1.100 0.985 ! 1.032

H 130 0.627 0.679 0.635 0.558 1.083 1.014 ι 0.890

H 186 0.687 0.712 0.678 0.689 1.036 0.987 ! 1.003

H 203 0.820 0.822 0.862 0.740 1.002 1.051 ^! 0.902

M92+H 163 0.767 0.737 0.825 0.745 0.962 1.076 i 0.971

F 111 0.483 0.495 0.466 0.477 1.025 0.965 ^! 0.989

F 210 0.402 0.302 0.245 0.328 0.752 0.609 < 0.816

Table 6

Table 7

Table 8 peptide Ct4B, blocker with sucrose

37°C/4°C serum 4°C 3 day 7 day 10 day 3 day 7 day 10 day

H 156 0.298 0.321 0.319 0.330 1.077 1.072 1.109

H 171 0.481 0.481 0.496 0.483 1.000 1.031 1.004

H 130 0.673 0.683 0.659 0.599 1.015 0.979 0.890

H 186 0.642 0.623 0.609 0.567 0.971 0.949 ; 0.884

H 203 0.526 0.528 0.538 0.507 1.004 1.022 0.963 92+H 163 0.308 0.291 0.278 0.262 0.946 0.902 ^■ 0.852

F 111 0.218 0.265 0.258 0.254 1.213 1.181 ^! 1.163

F 210 0.266 0.260 0.260 0.276 0.979 0.979 1.040

Table 9

Table 10

Tablell

Table 12

Table 13

Table 14 peptide Ct4D, blocker with sucrose

37°C/4°C serum 4°C 3 day 7 day 10 day ref (605) 3 day 7 day 10 day

H 156 0.568 0.618 0.520 0.577 1.530 1.088 0.915 1.016

H 171 0.542 0.543 0.536 0.544 | 0.429 1.003 0.989 1.004

H 130 0.439 0.406 0.403 0.391 ' 0.802 0.926 0.919 0.891

H 186 0.712 0.726 0.726 0.713 ¹ 0.769 1.019 1.020 1.001

H 203 0.292 0.285 0.267 0.280 ¹ 0.766 0.974 0.913 0.959

M92+H16 0.372 0.351 0.368 0.345 ¹ 1.248 0.943 0.991 0.927

F 111 0.366 0.327 0.309 0.318 ι 0.210 0.893 0.843 0.867

F 210 0 352 0.333 0.322 0.335 I 0.221 0.945 0.915 0.950

Table 15

Table 16

B Results obtained with C. pneumoniae peptides

In order to determine whether the presence of sucrose in the blocking buffer only affects C. trachomatis peptides (all of the tested peptides were from the VDIV region of the MOMP protein and therefore homologous in their sequence), or also affects the stabihty of other peptides, various peptides derived from the MOMP protein sequence of C. pneumoniae were tested for stability as described above for C. trachomatis peptides.

When sucrose was present in the blocking buffer, peptides C.pVDIII, CplA were essentially stable (see Tables 17, 23). Also a mixture of peptides (CplA, C.p2A, C.pVDIII. C.p4A) was stable (Table 20). Peptide Cp2A was somewhat less stable under these conditions, as indicated by ratios (37°C/4°C) ranging between 0.8 and 0.9 with four of the sera tested (Table 26, M92, H171+H226, H171, H247 after ten days storage).

When sucrose was omitted from the blocking solution, however, the stability of the reactivity of the peptides was reduced for two sera in the case of C.p VDIII (Table 18), for five sera in the case of the peptide mixture (Table 21), and for four sera in the case of C.p 1A (Table 24). The slight instability of peptide C.p 2A observed with sucrose-containing buffer (Table 26, ratios between 0.8 and 0.9 for M92, H171+H226, H171 and H156) was significantly increased when sucrose was omitted from the buffer (Table 27, ratios between 0.5 and 0.8 for the same sera).

The addition of Tween-20 to the blocking solution as customary in the art of ELISA further reduced the stabihty of the C. pneumoniae peptides, as can be seen for C.p VDIII (compare Table 19 to Table 14), and the peptide mixture (compare Table 22 to Table 21). On the other hand, the stabihty of CplA was unaffected by the presence of Tween in the blocking buffer (compare Table 25 to Table 24), while Tween even seemed to have a beneficial effect on the stabihty of C.p 2A (compare Table 28 to Table 27, sera M92 and H163). However, even in the case of C.p 2A, the addition of sucrose was superior to the addition of Tween with regard to enhanced stability (compare e.g. sera M95 and H163 in Tables 26-28). Table 17

CpVDIII , blocker with sucrose

37°C/4°C serum 4°C 3 day 7 days 10 day 3 day 7 days 10 day

M 92 0.488 0.462 0.448 0.482 0.946 0.917 0.988

H171+H228 0.497 0.534 0.476 0.479 1.074 0.958 0.963

H 228 0.448 0.476 0.414 0.415 1.061 0.923 0.926

H 163 0.720 0.672 0.692 0.692 0.933 0.962 0.961

H 171 0.488 0.511 0.490 0.494 1.047 1.003 1.011

H 247 0.590 0.553 0.537 0.542 0.938 0.910 0.919

H 156 0.371 0.356 0.366 0.368 0.960 0.985 0.991

H 203 0.185 0.186 0.188 0.223 1.005 1.019 1.206

Table 18

CpVDIII , blocker without sucrose with PBS.

37°C/4°C serum 4°C 3 day 7 days 10 day 3 day 7 days 10 day

M 92 0.499 0.409 0.434 0.409 0.819 0.870 0.820

H171+H228 0.477 0.641 0.425 0.436 1.343 0.891 0.914

H 228 0.461 0.421 0.485 0.537 0.913 1.052 1.166

H 163 0.753 0.768 0.581 0.590 1.019 0.771 0.783

H 171 0.480 0.437 0.448 0.455 0.910 0.934 0.948

H 247 0.597 0.555 0.553 0.605 0.929 0.926 1.013

H 156 0.357 0.352 0.335 0.366 0.986 0.938 1.025

H 203 0.191 0.199 0.213 0.288 1.039 1.115 1.505

Table 19

CpVDIII , blocker without sucrose with PBS-T.

37°C/4°C serum 4°C 3 day 7 days 10 day 3 day 7 days 10 day

M 92 0.545 0.413 0.416 0.406 0.758 0.763 0.744

H171+H228 0.495 0.415 0.405 0.422 0.837 0.817 0.852

H 228 0.462 0.427 0.454 0.493 0.925 0.983 1.067

H 163 0.749 0.607 0.579 0.591 0.810 0.774 0.790

H 171 0.636 0.416 0.420 0.414 0.654 0.661 0.651

H 247 0.585 0.558 0.514 0.538 0.954 0.879 0.920

H 156 0.328 0.313 0.322 0.326 0.953 0.980 0.994

H 203 0.200 0.215 0.200 0.228 1.075 1.000 1.138 Table 20

CP MIX , blocker with sucrose

37°C/4°C serum 4°C 3 day 7 days 10 day 3 day 7 days 10 dav

M 92 2.528 2.452 2.486 2.498 0.970 0.983 0.988

H171 +H228 0.445 0.521 0.568 0.517 1.172 1.277 1.163

H 228 2.126 2.136 2.075 2.190 1.005 0.976 1.030

H 163 1.180 1.172 1.192 1.210 0.994 1.010 1.026

H 171 0.712 0.746 0.721 0.745 1.048 1.013 1.046

H 247 0.721 0.710 0.734 0.735 0.984 1.017 1.019

H 156 0.249 0.272 0.271 0.235 1.093 1.089 0.944

H 203 0.137 0.157 0.138 0.143 1.147 1.007 1.044

Table 21

CP MIX , blocker without sucrose with PBS.

37°C/4°C serum 4°C 3 day 7 days 10 day 3 day 7 days 10 day

M 92 2.65 2.33 1.72 1.96 0.88 0.65 0.74

H171 +H228 0.55 0.41 0.38 0.52 0.75 0.69 0.94

H 228 2.09 1.84 1.76 1.70 0.88 0.84 0.82

H 163 1.17 0.93 0.87 0.88 0.79 0.74 0.75

H 171 0.63 0.49 0.43 0.45 0.78 0.68 0.71

H 247 0.71 0.57 0.51 0.52 0.81 0.72 0.73

H 156 0.23 0.22 0.24 0.25 0.96 1.01 1.05

H 203 0.16 0.16 0.17 0.19 1.00 1.02 1.19

Table 22

CP MIX , blocker without sucrose with PBS-T.

37°C/4°C serum 4°C 3 day 7 days 10 day 3 day 7 days 10 dav

M 92 2.601 1.734 1.448 1.262 0.667 0.557 0.485

H171+H228 0.668 0.431 0.386 0.424 0.644 0.577 0.635

H 228 2.071 1.754 1.475 1.446 0.847 0.712 0.698

H 163 1.094 0.831 0.734 0.703 0.760 0.671 0.643

H 171 0.711 0.558 0.439 0.578 0.785 0.617 0.813

H 247 0.684 0.459 0.466 0.417 0.671 0.682 0.610

H 156 0.257 0.276 0.267 0.243 1.074 1.041 0.945

H 203 0.148 0.166 0.165 0.172 1.122 1.119 1.163 Table 23

Cp1A, blocker with sucrose

37°C/4°C serum 4°C 3 dav 7 days 10 day 3 dav 7 days 10 day

M 92 1.830 1.749 1.732 1.729 0.955 0.946 0.945

H171+H226 0.875 0.885 0.803 0.837 1.011 0.918 0.957 -

H 228 2.272 2.278 2.233 2.243 1.003 0.983 0.987

H 163 1.300 1.315 1.295 1.320 1.012 0.996 1.015

H 171 0.471 0.484 0.575 0.472 1.029 1.222 1.003

H 247 0.658 0.669 0.681 0.635 1.017 1.035 0.965

H 156 0.591 0.552 0.573 0.548 0.935 0.970 0.928

H 203 0.242 0.299 0.269 0.276 1.236 1.112 1.140

Table 24

Cp1A, blocker without sucrose with PBS.

37°C/4°C serum 4°C 3 dav 7 days 10 day 3 day 7 days 10 day

M 92 1.593 1.143 0.865 0.798 0.717 0.543 0.501

H171+H226 0.719 0.603 0.529 0.554 0.839 0.736 0.770

H 228 2.119 1.771 1.407 1.421 0.836 0.664 0.671

H 163 1.146 0.980 0.831 0.753 0.855 0.725 0.657

H 171 0.429 0.475 0.474 0.399 1.106 1.105 0.929

H 247 0.623 0.544 0.566 0.547 0.872 0.908 0.877

H 156 0.465 0.450 0.460 0.445 0.968 0.990 0.957

H 203 0.253 0.245 0.214 0.234 0.966 0.844 0.923

Table 25

Cp1A, bloi :ker without sucrose with PBS-T.

37°C/4°C serum 4°C 3 dav 7 days 10 day 3 day 7 davs 10 day

M 92 1.574 0.878 0.813 0.737 0.558 0.517 0.468

H171+H226 0.803 0.628 0.776 0.693 0.783 0.967 0.863

H 228 2.129 1.681 1.554 1.466 0.790 0.730 0.689

H 163 1.145 0.892 0.811 0.748 0.779 0.708 0.653

H 171 0.395 0.476 0.423 0.467 1.204 1.070 1.181

H 247 0.594 0.520 0.478 0.579 0.875 0.805 0.975

H 156 0.524 0.430 0.467 0.471 0.820 0.890 0.898

H 203 0.231 0.336 0.291 0.268 1.458 1.262 1.163 Table 26

Cp2A, blocker with sucrose

37°C/4°C serum 4°C 3 day 7 days 10 day 3 day 7 days 10 dav

M 92 2.703 2.539 2.307 2.151 0.939 0.853 0.7956

H171+H226 0.815 0.786 0.842 0.714 0.964 1.033 0.8761

H 228 0.686 0.644 0.627 0.638 0.939 0.914 0.9300

H 163 0.550 0.672 0.582 0.677 1.221 1.058 1.2309

H 171 1.062 1.108 0.899 0.888 1.043 0.847 0.8357

H 247 0.608 0.535 0.507 0.522 0.880 0.833 0.8586

H 156 0.487 0.480 0.448 0.497 0.986 0.920 1.0205

H 203 0.247 0.415 0.264 0.241 1.678 1.067 0.9757

Table 27

Cp2A, blocker without sucrose with PBS.

37°C/4°C serum 4°C 3 dav 7 days 10 dav 3 day 7 davs 10 day

M 92 2.518 1.927 1.548 1.477 0.765 0.615 0.587

H171 +H226 0.714 0.576 0.598 0.528 0.807 0.837 0.740

H 228 0.585 0.504 0.487 0.561 0.862 0.833 0.959

H 163 0.503 0.461 0.409 0.371 0.916 0.813 0.738

H 171 0.875 0.785 0.722 0.636 0.897 0.825 0.727

H 247 0.498 0.487 0.490 0.490 0.978 0.984 0.985

H 156 0.360 0.378 0.371 0.367 1.050 1.031 1.021

H 203 0.176 0.182 0.186 0.192 1.034 1.054 1.091

Table 28

Cp2A, blocker without sucrose with PBS-T.

37°C/4°C serum 4°C 3 day 7 days 10 dav 3 dav 7 days 10 day

M 92 2.606 1.852 1.703 1.692 0.711 0.654 0.649

H 71+H226 0.729 0.551 0.521 0.520 0.756 0.714 0.714

H 228 0.634 0.525 0.764 0.514 0.828 1.205 0.810

H 163 0.470 0.502 0.355 0.503 1.067 0.754 1.070

H 171 0.943 0.792 0.659 0.646 0.840 0.699 0.685

H 247 0.503 0.414 0.402 0.437 0.824 0.800 0.870

H 156 0.350 0.318 0.290 0.316 0.910 0.830 0.903

H 203 0.173 0.354 0.185 0.172 2.049 1.070 0.994

In summary, the above experiments show that the C. trachomatis and C. pneumoniae peptides used herein display several epitopes, that these epitopes are independently affected by storage without sucrose and by the addition of Tween into the blocking solution. They further show that storage without sucrose may lead to the exposure or formation of epitopes that bind unspecifically, giving rise to high background levels and therefore raising the likelihood to obtain a false-positive output in the assay. In very rare cases, the addition of Tween-20 may help to stabilize a specific epitope, as in the case of reactivity of Cp2A to H163 (compare Tables 28 and 27). The omission of Tween-20 and the addition of sucrose to the blocking buffer, which is in contrast to the current understanding in the art of ELISA, overcome all these problems. This is demonstrated using a variety of peptides that are derived from different areas (VD1, VDII and VDIV) of the MOMP of two different Chlamydia strains. The peptides therefore have no common structure or sequence homology, so that the results obtained here are valid for peptides in general.

Example 4 The influence of detergents on antibody binding

The composition of the serum diluent must assure specific and stable binding of the antibody to the coated antigen, while at the same time minimizing background resulting from non-specific binding. It is therefore desirable to provide the components of the diluent which achieve a maximum signal-to-noise ratio. Another requirement of the diluent is an enhanced stabihty so it can be used as component of a kit. The influence of detergent on antibody binding and on the stabihty of the serum and conjugate diluents was determined as follows: three sera were tested for their IgG binding activity on plates coated with the C.t. mix peptides (as described for Table 4) with serum diluent (0.03% gelatin, 10% NRS, 1% blue food color in PBS) with or without 0.05% Tween-20, and conjugate diluent (0.03% gelatin, 10% NRS, 0.5% yellow) with or without 0.05% Tween-20. The results show that the addition of Tween-20 to the conjugate diluent was unnecessary (Table 32, compare row 1 with row 2). In contrast, Tween-20 in the serum diluent was essential for binding of Antibodies of serum V27 (which reacts with the Ct4D peptide) and serum V22 (which reacts with the Ct4B peptide, see Table 32, compare row 1 to row 3). Therefore, addition of Tween-20 to the serum diluent was necessary in order to cover the binding reactivities of all peptides used in Table 4.

However, stabihty studies showed that serum diluent containing Tween 20 was ineffective after storage for only two days at 4°C (data not shown). Binding reactivity could be restored if the Tween-20 was added to the SD freshly prior to use, even if the SD had been stored for extended periods, e.g. two weeks at 37°C (Table 32, compare row 1 to row 5).

CD, conjugate diluent; SD, serum diluent. For fresh diluents, 0.05 % Tween-20 was added as indicated when preparing the diluent; for diluent stored for two weeks at the indicated temperatures, Tween was added after storage, just prior to use to both SD and CD.

In order to provide stable, ready-to-use mixtures of SD as component of a kit, several alternatives to Tween-20 were tested. As shown in Table 33, the addition of either Triton-X 100 or NP-40 was able to replace Tween-20 in freshly prepared SD. Moreover, the activity of Triton-X 100 or NP-40 was not lost after incubation of SD for 5 days at 37°C (Table 33, 37°C). Since the use of Triton-X 100 gave shghtly better reactivities after 37°C incubation, it was chosen as optimal detergent for the formulation of stable SD. A further test with longer periods of storage proved Triton-X 100 to be stable for ten days at 37°C (Table 34).

Table 33 Influence of different detergents on Antibody binding and SD stability

Table 34 Stability of Triton-X 100 containing SD

Example 3 Use of the enhanced stability of immobilized peptides and serum diluent in diagnostic kits.

The diagnostic kits described herein are essentially improved Enzyme- Linked Immunosorbent Assay (ELISA) kit designed for the diagnosis of C. trachomatis infections. Two basic kits are exemplified, both being manipulatable to vary various of the constituents thereof to meet the users' needs. One kit is intended for the determination of specific C. trachomatis IgG antibodies in human sera, this being designated herein as the "IgG kit". The second kit is intended for the determination of specific C. trachomatis IgA antibodies in human sera, this being designated herein as the "IgA kit". Generally, the above kits of the invention will comprise at least some of the following constituents:

(i) A C. trachomatis antigen-coated microtiter plate with a plate cover, usually of the standard multiwell type having 96 wells per plate arranged in the form of 12 columns and 8 rows, i.e., 8 wells per column for a total of 96 wells. With such plates, there will be provided 12 removable 8-well strips coated with the C. trachomatis antigen. The C. trachomatis antigen will preferably be a mixture of new peptides as set forth in the aforementioned copending patent application, incorporated herein by reference, the most preferred mixtures being those designated in said apphcation as "MIX 1" (Ct2A, Ct4A, Ct4B and Ct4C) and "MIX 2" (Ct4A, Ct4B, Ct4C, Ct4D). Hence, for each well of the microtiter plate, there will be a strip coated with the C. trachomatis peptide mixture in the manner described in the invention.

(ii) A concentrated wash buffer, usually being a concentrated PBS- Tween buffer of the standard type well known in the art. (iii) An improved serum diluent as described in example 1, usually in the form of a ready-to-use buffer solution.

(iv) A conjugate diluent, usually in the form of a ready-to-use buffer solution.

(v) A negative control which is usually a C. trachomatis IgG or IgA negative human serum in a ready-to-use form.

(vi) A positive control which is usually a C. trachomatis IgG or IgA positive human serum in a ready-to-use form.

(vii) A concentrated HRP-conjugate, usually in the form of horseradish peroxidase (HRP) conjugated to anti-human IgG or anti- human IgA (gamma chain specific).

(viii) A concentrated TMB-substrate, usually in the form of 3,3\5,5'-te-ramethyl-benzidine (TMB) in dimethylsulfoxide (DMSO) as chromagen and urea hydrogen peroxide as substrate for peroxidase (HRP).

(ix) A stop solution, usually containing 1 M H2S0 in a ready-to- use form.

(x) Detailed instructions for use, inclusive of warnings and precautions.

Of all of the above constituents, those unique to the present invention, and hence essential to the kits of the invention, are: (i) the peptide-coated microtiter plate, and (hi) the serum diluent. All the other constituents, (ii, iv-x) may be the improved or modified ones noted below in accordance with the invention, or standard, commercially available equivalents well known in the art of ELISA.

Using the above kit components and new coating procedure, the basic assay procedure is as follows:

A) Assay Procedure

1. Incubation of the sera samples and controls:

1.1 Dilute each patient serum 1/10 to 1/21 with the serum diluent (as in example 1, usually supplied with the kit; see also below).

1.2 Pipette 50 μl from positive control, negative control and from the diluted patient serum (from step 1.1) into separate wells of the test strip (as noted above, each strip is an antigen-coated 8-well strip, with a possibility of 12 such strips per microtiter plate when using 96-well plates).

1.3 Cover the strips (i.e., cover the whole plate with the plate cover) and incubate for 1 hour at 37°C in a humidified environment.

1.4 Discard the liquid contents of the wells.

1.5 Washing step: Fill each well with wash buffer and discard the hquid; repeat this step six times.

1.6 Dry the strips and ELISA plate, gently tapping them over clean absorbent paper.

2. Incubation with conjugate:

2.1 Dilute the concentrated (usually 300x concentrated) HRP conjugate anti-human IgG 1/300 with conjugate diluent.

2.2 Pipette 50 μl of diluted conjugate into each well. 2.3 Cover the strips and incubate for one hour at 37°C in a humidified environment.

2.4 Discard the liquid content and wash as described in step 1.5.

2.5 Dry the strips and ELISA plate by gently tapping them over clean absorbent paper.

3. Incubation with TMB substrate:

3.1 Dilute the concentrated (usually lOx concentrated) TMB-Substrate 1/10 in DDW. Alternatively, ready-to-use (RTU)-TMB substrate may be used, and the dilution step omitted.

3.2 Pipette lOOμl of diluted TMB-Substrate into each well, cover the strips and incubate at room temperature for 10 minutes only. Alternatively, the RTU-TMB substrate may be used, and incubation extended to 15 minutes.

3.3 Stop the reaction by adding 100 μl of 1 M H₂S0 (chromogen stop solution) to each well.

3.4 Determine the absorbence at 450 nm and record the results.

B- Improved serological diagnostic kits:

The following is an outline of the development of the improved kits:

1) Making the kits more "user friendly": a. Reducing the number of washing steps. b. Adding different colors to the serum diluent and the conjugate diluent. c. Stabilizing the kit for longer shelf life, e.g. by using the improvements as set forth in example 1.

2) Clinical evaluations of the improved kits. In order to achieve goal la) above, namely, to reduce the number of washing steps from six to three, different wash buffers were tested and compared to the original one usuaUy used in ELISA, which is a PBS-based buffer. These wash buffers contained an increasing amount of non-ionic detergents, or ionic detergents.

Results: The wash buffer that enabled only three washing steps was the one that contained non-ionic detergent, this being the above-noted PBS- Tween buffer (see constituent no. (ii) in the above hst). Further, it was found that this PBS-Tween buffer could be readily prepared in a preferred concentration of 20x concentrated, which 20x concentrated wash buffer was stable for one month at 37° C and for 11 months at 4°C

To achieve the goal set forth in lb) above, the foUowing was carried out: The following colors were added either to the serum diluent or to the conjugate diluent and tested: violet powder, evans blue, mocca brown powders and from the food colors: blue brilliant, yellow sunset and their combination (green color).

Results: The violet, evans blue and mocca brown colors had some interference with the test, by either increasing the background signal and/or decreasing the actual test signal. The only colors that worked well in the test were the blue brilliant, yellow sunset and their combination (green color).

It should also be noted that the blue brilliant and yellow sunset colors and their combination (green color) were stable for one month at 37°C and for one year at 4°C Based on these results, in the preferred kits of the invention, the serum diluent is provided with blue color and the conjugate diluent is provided with green color, thereby providing for optimal distinction between the two diluents on a color basis, while at the same time, these colors do not interfere with the assay. To achieve the goal lc) with respect to the shelf-life of the peptide-coated plates, peptide mix (0.3 μg/well) was dissolved in 0.05 M phosphate buffer, pipetted into the wells and left to adsorb to the plastic for 1 hr at 37°C The unbound peptide solution was then removed and the wells filled with improved blocking solution as described in example 1. After blocking for 1 h at 37°C, the blocking solution was removed and the plates dried overnight at room temperature.

To achieve the goal lc) with respect to the shelf life of the serum diluent, a serum diluent composition as described in Example 1 was used (PBS containing 0.03% gelatin, 10% normal rabbit serum, 0.05% Triton-X 100).

Regarding the goal lc) with respect to the shelf life of TMB substrate, it was found that RTU-TMB substrate was stable for one month at 37 °C and for 12 months at 4°C This is also the case for the other reagents used in the improved kit, as mentioned above. Further ongoing stabihty tests show that all of the above reagents used in the improved kit are stable for a period of 18 month when stored at 4°C

Regarding point 2 above, the following examples are illustrating the evaluation of the improved kits.

Example 6 Comparison of the sensitivity and specificity of IgG and IgA kits as compared to MIF MRL

To evaluate the sensitivity and the specificity of the IgG and IgA kits, sera from uninfected individuals (negative sera), or those already determined to have been positively infected with only C. trachomatis (positive sera) were tested according to the above procedure detailed in Example 5. The sensitivity and specificity were calculated as compared to the results obtained by MIF MRL (a commercially available, standard microimmunofluorescence (MIF) assay kit used in accordance with the manufacturer's instructions and employing the relevant C. trachomatis antigens for detecting the IgG and IgA antibodies in the sera).

Results: The IgG and IgA kits are able to detect both IgG and IgA levels in sera from C. trachomatis-infected individuals as determined by MIF MRL. The sensitivity and specificity of the peptide assay are high and were 94% and 90%, respectively, for IgG and 95% and 90%, for IgA. These results are summarized in Figure 1, in which the light bars in the bar graphs represent the results obtained with the MIF-commercial reference kit and the dark bars represent the results obtained with the C. trachomatis peptide assay, i.e., with the IgG kit (left-hand graph) and with the IgA kit (right-hand graph). N represents the number of sera tested.

Example 7 Comparison of the sensitivity and specificity of IgG and IgA kits as compared to Culture

Human sera from individuals infected with C. trachomatis as determined by culture were tested for C. trachomatis IgG and IgA antibodies with the IgG and IgA kits. Sera which were IgG negative were also tested by another serological test, MIF (as noted above). The sensitivity of the IgG and IgA kits were compared to culture.

Results: The results are summarized in Figure 2, wherein the left-hand, brick-patterned bar represents the number of sera tested positive by culture assay, the cross-hatched bars represent the number of sera tested positive by the C. trachomatis peptide assay, and the right-hand bar represents the number of sera tested negative by the C. trachomatis peptide assay for IgG antibodies. The open part of the right-hand bar represents the part of the sera that was tested negative by MIF assay, while the cross-hatched part of this bar represents the remaining sera tested negative by C trachomatis peptide assay for IgG antibodies. N represents the number of individual sera.

From this experiment it is apparent that the sensitivity of C. trachomatis assay (cross-hatched bars in Figure 2), as compared to culture (brick- patterned bar in Figure 2) was 78% for IgG and 78% for IgA. Five percent of the sera showed only IgA reactivity. Therefore, the overall sensitivity of the kits was calculated to be 83%. 70% of the sera that were negative for IgG (22%o of the total sera) were also negative by MIF (open part of the right-hand bar, bar, Figure 2).

Example 8 The Specificity of the IgG kit as Compared to Different MIF Tests

The specificity of the IgG kit was determined, as compared to different MIF tests (MIFl, MIF2 and SeroFIA tests). AU the MIF-tested sera were C. trachomatis negative (C.t-) and a portion of the sera were also C. pneumoniae positive sera (Ct/C.p+). MIFl and MIF2 are standard MIF tests as noted above, while SeroFIA is a new microimmunofluorescence test for the differential detection of C. trachomatis, C. pneumoniae and C. psittaci.

Results: The results are summarized in Figure 3, from which it is apparent that the IgG kit is highly specific, and showed at least 90% specificity (horizontaUy striped, hght bars in Figure 3), as compared to the various MIF assays (black bar, MIFl assay, obhquely double-striped bar, SeroFIA assay, of Savyon, cross-striped bar, MIF 2 assay). The IgG kit did not cross-react with C. pneumoniae positive sera.

All the above and other description and examples have been provided for the purpose of illustration and are not intended to h it the invention in any way. Many modifications can be effected in the various reagents, kits and pathogens tested, all without exceeding the scope of the invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Savyon Diagnostics Ltd.

(B) STREET: Kiryat Minrav 3 Habosem Str.

(C) CITY: Ashdod

(E) COUNTRY: Israel

(F) POSTAL CODE (ZIP) : 77101

(G) TELEPHONE: +972 8 856 2920 (H) TELEFAX: +972 8 856 3258

(ii) TITLE OF INVENTION: Stabilization of Polypeptides for Use in Iπurtunoassay Procedures

(iii) NUMBER OF SEQUENCES: 10

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: lie Phe Asp Thr Thr Leu Asn Pro Thr lie Ala Gly Ala Gly Asp Val 1 5 10 15

Lys

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Val Asp lie Thr Thr Leu Asn Pro Thr lie Ala Gly Cys Gly Ser Val 1 5 10 15

Ala Lys (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Cys Val Phe Asp Val Thr Thr Leu Asn Pro Thr lie Ala Gly Ala Gly 1 5 10 15

Asp Val Lys

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Leu Ala Glu Ala lie Leu Asp Val Thr Thr Leu Asn Pro Thr lie Thr 1 5 10 15

Gly Lys Ala Val Val Ser Lys 20

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Cys Asp Asn Glu Asn Gin Ser Thr Val Lys Thr Asn Ser Val Pro Asn 1 5 10 15

Met Ser Leu Asp Gin Ser Lys 20 (2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Leu Asp Val Thr Thr Asn Ala Thr lie Ala Gly Lys Gly Thr Val Val 1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Cys Phe Ser Met Gly Ala Lys Pro Thr Gly Ser Ala Ala Ala Asn Tyr 1 5 10 15

Thr Thr Ala Val Asp Arg Pro Asn Pro Ala Tyr Asn Lys 20 25

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Ala Phe Pro Leu Pro Thr Asp Ala Gly Val Ala Thr Ala Thr Gly Thr 1 5 10 15

Lys Ser (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Val Lys Gly Thr Thr Val Asn Ala Asn Glu Leu Pro Asn Val Ser Leu 1 5 10 15

Ser Asn Gly Lys 20

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Leu Asn Leu Thr Ala Trp Asn Pro Ser Leu Leu Gly Asn Ala Thr Ala 1 5 10 15

Leu Ser Thr Thr Asp Ser Phe Lys 20

Claims

Claims:

1. A method for improving the stabihty of reagents for detection of antibodies in a sample, comprising:

(a) providing a first buffer for the stabihzation of polypeptides or proteins attached to a sohd support, said buffer containing about 1-20% sucrose,

(b) providing a second buffer for the dflution of the antibody- containing sample and for reacting said immobihzed polypeptides with the antibodies present in said sample, said second buffer comprising about 0.05% (v/v) detergent.

2. A method according to claim 1, wherein the detergent is a detergent other than Tween-20.

3. A method according to claim 1 wherein the first buffer is a blocking solution in an immunoassay, said blocking solution containing about 0.05 to 4 % (w/v) gelatin, about 1 to 30 % (v/v) normal rabbit serum, 1 to 20% (w/v) sucrose in PBS; and wherein the second buffer is a serum duuent containing about 0.05 to 4%% (w/v) gelatin, about 1 to 30% (v/v) normal rabbit serum, and about 0.01 to 2 % (v/v) Triton-X 100 in PBS.

4. A stabilizing solution for the stabihzation of a polypeptide or protein attached to a sohd support, comprising about 1-20% sucrose in a buffer.

5. A buffer solution for the dilution of an antibody-containing sample to be reacted with an immobilized polypeptide, comprising about 0.05% (w/v) of a detergent other than Tween-20.

6. A kit comprising an immunoassay for the detection of antibodies to said immobihzed polypeptide, said kit comprising a solution according to claim 3 or 4.

7. A kit according to claim 5, said kit being an ELISA kit.

8. A kit according to claim 6, for the diagnosis of Chlamydia trachomatis infections.

9. A kit according to claim 6, for the diagnosis of Chlamydia pneumoniae infections.

10. A method for detecting an infection in a mammal, said method comprising:

(a) obtaining a serum or other body fluid sample from the person or animal, and diluting said sample using a dflution buffer comprising about 0.05% (w/v) of a detergent according to claims l or 2,

(b) contacting said sample with said immobihzed polypeptides, which have been with a stabihzation solution containing about 1-20% sucrose in a buffer; and

(c) determining the extent of reaction between antibodies present in said sample and said immobihzed polypeptides using conventional immunoassay technology.

11. A method for improving the stabihty of reagents for the detection of antibodies in samples, essentiaUy as described and with particular reference to the examples.