Extraction procedure for Chlamydia and Neisseria antigens

Enhanced extraction of solubilized antigens is obtained from bacteria such as Chlamydia and Neisseri by the use of a buffer containing a zwitterionic surface active agent, especially CHAPS or CHAPSO, in the absence of divalent cations. The extraction is conducted at elevated temperature, and provides a sample useful in assays for the presence of the bacteria.

The present invention relates to procedures for extracting antigenic 
material in solubilised form from cellular biological materials such as 
bacteria. The solubilised antigenic material can be used thereafter in 
assay procedures to determine the presence or identity of the cellular 
material. 
The use of surface active agents in extraction media at elevated 
temperature has been proposed. Examples are given in EP 167395 and EP 
183383, both of which relate to extraction procedures especially 
applicable to species of Chlamydia. 
EP 183383 says that it is beneficial to have divalent cations, specifically 
magnesium and zinc, present during the heating stage of an extraction 
procedure. 
However, in complete contrast, we have found that a better extraction of 
antigenic material can be obtained in the absence of divalent cations. 
The invention provides a procedure for extracting solubilised antigenic 
material from cellular biological material, such as bacteria, wherein the 
cellular material is treated with an aqueous solution of a surface active 
agent, the solution being substantially free from divalent cations. 
Preferably the surface active agent is zwitterionic. More preferably, the 
surface active agent is 
3-(3-chlolamidopropyl)dimethylammonio-1-propanesulfonate (conveniently 
known as CHAPS) or 
3-(3-chlolamidopropyl)dimethylammonio-2-hydroxyl-1-propanesulfonate 
(conveniently known as CHAPSO), or mixtures thereof. 
In particular, we have found that an especially effective extraction of 
lipopolysaccharide antigen from Chlamydia species such as Chlamydia 
trachomitis, C. psittaci and C. twar, is achieved if the extraction is 
performed using an aqueous solution of a zwitterionic surface active 
agent, especially CHAPS and/or CHAPSO. 
We have also found that an especially effective extraction of a 
proteinaceous antigen from Neisseria gonorrhoeae is achieved if the 
extraction is performed using an aqueous solution of a zwitterionic 
surface active agent, especially CHAPS and/or CHAPSO. 
Preferably, the extraction is conducted at elevated temperature, for 
example in excess of about 50.degree. C., for a period of time sufficient 
to solubilise the antigenic material. More preferably, the extraction 
temperature is at least about 60.degree. C. In general, the extraction 
temperature need not be greater than about 100.degree. C., and is 
preferably not greater than about 90.degree. C. Ideally, the extraction 
temperature is about 80.degree. C. The stage of the extraction conducted 
at such elevated temperature should generally last for at least about 5 
minutes. 
Preferably, the quantity of surface active agent in the aqueous extraction 
medium is at least about 0.1% by weight. Preferably the quantity of 
surface active agent is not greater than about 2%, and more preferably not 
greater than about 1% by weight. 
The pH of the extraction medium should generally be in the range of about 
7.5 to about 9. 
As stated above, the extraction medium should be substantially free from 
divalent cations. In particular, zinc and magnesium ions should not be 
present in any appreciable quantity. This can be achieved by using 
ion-free water and other components when preparing the extraction medium. 
Alternatively, or in addition, chelating agents such as EDTA, EGTA or DPTA 
can be incorporated in the extraction medium to remove, in effect, any 
divalent cations that may be present. If the performance of the subsequent 
assay applied to the extracted antigen solution may be adversely 
influenced by ionic strength, it is preferable that the chelating agent is 
used in the free acid form, rather than as a water-soluble salt such as 
its sodium salt; this appears to be an important consideration in the case 
of extraction from Neisseria. We believe that the effective absence of 
divalent cations enhances disruption of epithelial cellular material which 
in turn enhances extraction of any bacteria such as Chlamydia which is an 
intra-cellular parasite. 
In a typical extraction procedure according to the invention, a biological 
sample obtained from a patient suspected of carrying a Chlamydia 
infection, for example, is contacted with an extraction medium containing 
the CHAPS and/or CHAPSO. Appropriate samples can take the form, for 
example, of genital, rectal or ocular swabs, or centrifugal pellets from 
liquids such as early morning urine. Extraction, for example at 80.degree. 
C. for 10 minutes, is followed by a brief period, for example 5 minutes, 
during which the extraction medium is allowed to cool. Thereafter the 
extraction medium can be separated from solid matter, for example by 
removal of the swab and filtration of the solution to provide a sample 
liquid containing any extracted antigen ready for use in any suitable 
assay procedure. The subsequent assay can involve any conventional assay 
technique, such as radioimmunoassay or enzyme-linked immunoassay. The 
extracted sample is ideal for use in an immunochromatographic assay 
procedure such as described and claimed in GB 2204398 A, especially using 
a nitrocellulose solid phase and an antibody reagent labelled with a 
direct particulate label such as coloured latex particles. The use of the 
CHAPS and/or CHAPSO enhances the sensitivity of a Chlamydia assay which 
involves anti-lipopolysaccharide antigen antibodies as specific binding 
reagents.

EXAMPLE 1 
An extraction procedure for Chlamydia trachomatis can be performed as 
follows. This provides an extract suitable for use in an immunoassay. 
The extraction procedure utilises: 
a) Disposable, flexible plastics "test tubes" each capable of holding a 
volume (e.g. 5 ml) of liquid and of accommodating the end of a 
conventional sampling swab. 
b) A heating block in which such tubes can be inserted to permit the 
contents of the tube to be heated to a temperature in the range 
50.degree.-100.degree. C. and held at that temperature for at least a 
number of minutes. 
c) Means for filtering the liquid contents of the tube at the end of the 
extraction procedure. Conveniently this can take the form of a filter plug 
incorporated in a perforated stopper with which the tube can be closed and 
through which the liquid contents can be expelled. 
d) An extraction buffer having the following formulation: 
0.1M TRIS pH 8.5 containing 
0.85% Sodium chloride 
0.25% CHAPSO 
1% Bovine serum albumin 
5 m M EDTA 
To conduct the extraction, a pre-determined quantity (for example 600 .mu.l 
) of extraction buffer is placed in a tube. A genital swab from a patient 
suspected of carrying a Chlamydia infection is placed in the tube, and the 
tube and contents are then incubated in the heating block at a temperature 
of approximately. 80.degree. C. for 10 minutes. The tube is removed from 
the heating block and allowed to cool for 5 minutes. The swab is lifted 
out of the extraction buffer and, before the swab is removed completely 
from the tube, the sides of the tube are pressed in gently by hand to 
squeeze liquid from the swab. The swab can then be removed completely from 
the tube and discarded. A perforated stopper containing a filter plug is 
inserted in the top of the tube, and the liquid contents of the tube can 
be expelled therethrough to provide an essentially clear liquid sample 
containing any extracted Chlamydia lipopolysaccharide antigen for use in a 
subsequent assay. If desired, one or more assay reagents, such as labelled 
antibodies, can be added to the contents of the tube before the 
filter/stopper is applied to the tube. 
EXAMPLE 2 
The same extraction procedure as Example 1 is followed. However, the 
extraction buffer has the formulation: 
0.1M TRIS pH 8.5 containing: 
1.25% Sodium Chloride 
0.25% CHAPSO 
1% Bovine Serum Albumin 
5 m M EDTA 
10 m M L. Ascorbic Acid. 
An increased level of sodium chloride helps to prevent non-specific 
binding, which may occur in the subsequent immunoassay. The presence of 
ascorbic acid (preferably L form) has been found to increase the storage 
life of the extraction buffer, where it acts as a stabilizer, when the 
buffer is subjected to adverse conditions (e.g. increased temperature 
during distribution, or long-term refrigerated storage).