A SPECIFIC, RAPID TEST DIFFERENTIATING GRAM POSITIVE AND GRAM NEGATIVE BACTERIA

Abstract

The present invention discloses a specific, rapid test differentiating microorganisms by trait, such as distinguishing gram positive and gram negative bacteria, in bodily fluids at point of care. This is achieved through a method of producing a polyclonal antibody targeted towards the particular trait of the microorganism to be tested for, as well as the polyclonal antibody produced by such a method. Also disclosed is a lateral flow point of care test device comprising such a polyclonal antibody, as well as a method of use of such a device.

Claims

1. A method of producing a polyclonal antibody targeted towards a particular trait of a microorganism, comprising the steps of: a) obtaining antigens specific to said trait from multiple different types of microorganisms expressing said trait; b) mixing said antigens to produce a mixed antigen; c) coupling said mixed antigen to a carrier protein to produce an immunogen; d) injecting said immunogen into an animal; e) obtaining polyclonal antibodies targeted towards said trait from said animal.

2. The method of claim 1, wherein the trait is gram positivity and the antigen is teichoic acid.

3. The method of claim 1, wherein the trait is gram negativity and the antigen is lipopolysaccharide.

4. The method of claim 1, wherein the trait is cell morphology, class, order, family, genus, or species.

5. The method of claim 1, wherein the carrier protein is keyhole limpet hemocyanin.

6. The method of claim 1, wherein the animal is a mammal.

7. The method of claim 1, wherein the animal is a sheep, pig, or ape.

8. The method of claim 1, wherein the polyclonal antibody is purified through a process of salt precipitation and affinity purification.

9. A polyclonal antibody produced by the method of claim 1.

10. A lateral flow point of care test device comprising the polyclonal antibody according to claim 9.

11. The device of claim 10, further comprising monoclonal antibodies.

12. The device of claim 10, wherein the polyclonal antibodies are produced by the method of claim 2 and the device permits the characterisation of gram positive microorganisms.

13. The device of claim 10, wherein the polyclonal antibodies are produced by the method of claim 3 and the device permits the characterisation of gram negative microorganisms.

14. The device of claim 10, wherein some of the polyclonal antibodies are produced by the method of claim 2 and some are produced by the method of claim 3 and the device permits the characterisation and differentiation of gram positive and gram negative microorganisms.

15. The device of claim 11, further comprising: a sample application pad containing a buffer; a conjugate release pad containing the polyclonal antibodies; a nitrocellulose membrane; and an absorbent pad.

16. The device of claim 15, wherein the application pad is CF6 glass fibre/cellulose paper pre-treated with borate buffer.

17. The device of claim 16, wherein the CF6 glass fibre/cellulose paper is pre-treated with borate buffer of pH 7.5, the borate buffer comprising: 1% (w/v) BSA; 0.5% (v/v) Tween 20; and 0.05% (w/v) sodium azide; and being dried at 37 C. for 2 hours prior to assembly.

18. The device of 15, wherein the conjugate release pad has a maximum pore size of 11 m.

19. The device of claim 15, wherein the conjugate release pad has a water absorption of 40 m and a particle retention of 2.3 m.

20. The device of claim 15, wherein the conjugate release pad is a FUSION 5 pad immersed in conjugate suspension and dried at 37 C. for 2 hours before assembly.

21. The device of claim 15, wherein the polyclonal antibodies in the conjugate release pad of the lateral flow device are conjugated to colloidal gold nanoparticles of 20 or 40 nm size.

22. The device of claim 15, wherein the nitrocellulose membrane is untreated Immunopore RP paper.

23. The device of claim 15, wherein the absorbent pad is untreated PALL 165 Paper.

24. The device of claim 15, wherein one end of the nitrocellulose membrane is attached under the absorbent pad and the other end is attached under the conjugate release pad, which is at least partially attached under the sample application pad.

25. The device of claim 15, further comprising a test line comprising a second set of antibodies.

26. A method of use of the device of claim 25, comprising the steps of: administering a sample of microorganisms to the sample application pad; the sample being drawn from the sample application pad through the conjugate release pad and through the nitrocellulose membrane towards the absorbent pad and past the test line; such that the microorganisms in the sample bind to the polyclonal antibodies in the conjugate release pad as they are drawn through if they express the trait the antibodies target, and wherein if this occurs the antibodies from the release pad, now conjugated to the microorganisms in the sample, bind to a second set of antibodies in a test line on the nitrocellulose membrane; the test line becoming visible when bound to by the conjugated antibodies and microorganisms, indicating that the microorganisms express the trait the antibodies target.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The illustrations included are described below:

[0017] FIG. 1: A schematic of a lateral flow strip construct developed during the preliminary studies in accordance with the invention.

[0018] FIGS. 2a and 2b: Examples of the various potential formats of the final Lateral flow strip device in accordance with the invention.

DETAILED DESCRIPTION

[0019] FIG. 1 shows a preferred embodiment of the lateral flow point of care test device of the invention. A sample of microorganisms is administered to a sample application pad 2 containing a buffer which is at least partially overlapping the conjugate release pad 8 infused with a suitable antibody. The sample is drawn from the sample application pad 2 through the conjugate release pad 8 and through a nitrocellulose membrane 10, which is at least partially attached under the conjugate release pad 8. At the other end of the nitrocellulose membrane 10 to the sample application pad 2 and conjugate release pad 8 is an absorbent pad 12 which is responsible for drawing the sample from one end of the device to the other.

[0020] As the microorganisms in the sample are drawn through the conjugate release pad 8, any gram positive or negative bacteria, depending on which the particular device is testing for, bind to the antibodies infused therein. Travelling further, the first antibodies bind to a second set of antibodies located in a test line 4 on the nitrocellulose membrane 10, which accordingly becomes visible, indicating that the sample contains gram positive or gram negative bacteria, depending on which antibodies the test line contains. The device further comprises a control line 6, which indicates whether or not the test has been successful, and a backing 14, upon which the other elements of the device are situated.

[0021] Prototyping work was performed with an aim to produce a preferable device for use in a strip test able to distinguish between gram positive and gram negative bacteria using a lateral flow gold coupled antibody procedure. The objective was to obtain visible coloured lines minutes after direct application of a pathological sample.

[0022] Escherichia coli and Pseudomonas aeruginosa were used as examples of gram negative organisms while Staphylococcus aureus, Bacillus subtilis and Streptococcus agalactiae were used as examples of gram positive organisms. These were sourced from The National Collection of Type Cultures (NCTC), UK and were cultured in LB broth and Nutrient agar (Sigma-Aldrich, UK) for a few generations following which stocks were prepared and stored at 4 C. and 70 C.

[0023] The antibodies selected were chosen for their suitability for use in strip assay procedures and their availability (Table 1). They were either monoclonal or polyclonal antibodies raised against unique components of the surface of either gram positive or gram negative bacteria. The characteristics of the chosen antibodies were initially assessed using ELISA (enzyme linked immunoassay) and Dot blot techniques.

TABLE-US-00001 TABLE 1 Antibody Product Description Abbreviation Company code Anti-gram positive bacteria Mm AntiG+ Abcam Ab20344 antibody [BDI380]. Mouse (ab20344) monoclonal Anti-LTA. 1.8 mg Anti-gram positive bacteria MmAntiG+ Abcam Ab155857 antibody [3801]. Mouse (ab155857) monoclonal. 0.1 mg. Anti-LTA Mouse anti gram positive MmAntiG+ Acris BDI813 bacteria BDI813 - 1 mg. Anti- (Acris-BDI813) Antibodies LTA Mouse anti-gram negative Mm AntiG Abcam Ab31204 bacteria monoclonal antibody, (ab31204) reacts with endotoxin-2 mg/ml. Anti-gram negative endotoxin MmAntiG Abcam Ab20954 antibody [306] -mouse (ab20954) monoclonal-ascites fluid Lipid A LPS Endotoxin GpAntiG Thermo PA1- Antibody (Pierce) - Goat (Pierce) Fisher 73178 Polyclonal -4-5 mg/ml Lipopolysaccharide Gram GpAntiG Acris BP2235 negative antibody - Polyclonal- Antibodies Goat-BP2235-4-5 mg/ml

[0024] The optimum conditions for the interaction of the above antibodies with the selected bacteria were established using ELISA. In order to achieve this, a wide range of concentrations of bacteria were used which replicated the concentrations of bacteria found in pathological samples. Once the optimum concentrations were identified, the antibodies were then tested using Dot Blot technology in order to optimize the antibody reaction conditions on nitrocellulose paper.

[0025] A range of commercially available papers were tested in the prototype strip to achieve the optimum results. The lateral flow strip consisted of 4 componentsa nitrocellulose membrane, a sample application pad, a conjugate pad and an absorption pad (FIG. 1).

TABLE-US-00002 TABLE 2 Sample Paper Potential Strip Component Whatman Rapid 27 Sample application Whatman Rapid 24 Pad Whatman STD 17 Conjugate Pad Whatman STD 14 Whatman Rapid 27 Whatman FUSION 5 Immunopore RP Nitrocellulose Membrane Whatman CF6 Absorbent Pad

[0026] In addition, the following paper present from previous studies was also investigated.

TABLE-US-00003 TABLE 3 Sample Paper Potential Strip Component Pall Cellulose Absorbent Pad 165 Absorbent Pad

[0027] Below are the properties of the various papers investigated for the different components of the prototype strip:

[0028] Sample Application Pad:

[0029] Rapid 24 and 27 were initially investigated for use as a sample application pad. They are made up of treated bound glass fiber material which has good rewetting properties and improved conjugate release without interfering with assay sensitivity.

TABLE-US-00004 TABLE 4 Thickness Wicking Rate Water Absorption Paper (m @ 53 kPA) (s/4 cm) (mg/cm.sup.2) Rapid 24 340 38 55 Rapid 27 365 39 40

[0030] Conjugate Pad:

[0031] Standard 14 and 17 are untreated grades of bound glass fiber material and are suitable for conjugate pad optimization, particularly for sensitive assays. They have a faster flow than cotton and lower sample retention, thus making them a likely choice for the conjugate release pad.

[0032] Rapid 27 has been described above and was selected to be investigated as a conjugate pad due to its ability to release more conjugate than untreated pads.

[0033] The FUSION 5 is a unique type of paper developed by Whatman which is a single material that performs all the functions of a lateral flow strip. It's designed to be used in a wide range of tests simplifying manufacturing and reducing costs.

TABLE-US-00005 TABLE 5 Thickness Absorption Max. % Release of (m @ Capacity Pore size Gold Conjugate Paper 53 kPA) (mg/cm.sup.2) (m) (after 90 secs) Standard 14 355 55 23 75 Standard 17 370 35 23 75 Rapid 27 365 40 22 86 FUSION 370 40 11 >94 5

[0034] Nitrocellulose Membrane:

[0035] The Immunopore membrane is the preferable choice for the most sensitive assays, particularly assays for infectious disease, therefore, was the obvious choice for use with this strip prototype. It offers the best reproducibility, stability and accuracy and is available in three wicking rates. Only the Immunopore RP, the general purpose membrane, was investigated in this study.

TABLE-US-00006 TABLE 6 Capillary Rise Caliper Paper (s/4 cm) (m) Immunopore RP 85-115 200

[0036] Absorbent Pad:

[0037] CF6 is made up of a mixture of cotton and glass fiber and was selected as a possible material for the absorbent pad because of its unique property to minimize any conjugate backflow. In many assays, the sample and conjugate tend to run back up the membrane after the reaching the absorbent pad which can result in false positives.

[0038] Pall 165 membrane was also assessed because of its thickness and high absorption properties. It has been used as the absorbent pad in other tests developed by the inventors and it also successfully reduces backflow.

[0039] The Pall 165 proved to be a better absorbent and is therefore preferable for use as the absorbent pad in the final strip prototype. The CF6 was preferred as the sample application pad as its thickness and water absorption was greater than Rapid 24 and 27, allowing the sample volume to be applied with more ease. Out of the papers investigated, the FUSION 5 proved to be the most superior paper for the conjugate pad, due to its high percentage release of gold conjugate, allowing the maximum amount of gold coupled antibody-bacteria complex to move to the test and control lines. The nitrocellulose Immunopore RP membrane was satisfactory for its purpose, however, there is potential to investigate the other two wicking rates.

[0040] The papers finally selected for the various segments of the preferred prototype construct are listed in the Table 7 below:

TABLE-US-00007 TABLE 7 Strip Prototype Component Specification Sample application pad CFS glass fibre/cellulose paper obtained (1.5 1 cm) from Whatman International Ltd., Uk. The paper was pretreated with Borate buffer, pH 7.5 containing 1% (w/v) BSA, 0.5% (v/v) Tween 20 (RTM) and 0.05% (w/v) sodium azide, dried at 37 C. for 2 hours before assembly. Conjugate pad FUSION 5 pad (Whatman International (1 1 cm) Ltd., UK) immersed in conjugate suspension and dried at 37 C. for 2 hours before assembly. Nitrocellulose membrane Immunopore RP paper (Whatman (2.5 1 cm) International Ltd., UK), untreated. Absorbent pad PALL 165 Paper (obtained from Pall Europe (1.7 1 cm) Ltd., Portsmouth, UK), untreated.

[0041] Optimising the Size of Gold Particles

[0042] The binding of the primary commercial antibody (e.g., mouse anti-gram positive monoclonal antibody against lipo-tecichoic acid, MmAb Gr+ve, 50 l/ml, Abcam plc) with 40 nm colloidal gold particles (BBI solutions) in 2 mM borate buffer pH 9.0 was unsuccessful as none of the conjugates contained enough antibody adsorbed onto the gold nanoparticles to prevent floculation when 10% sodium chloride was added. Similar experiments with 20 nm gold nanoparticles (BBI Solutions, Cardiff) when Mouse anti-gram positive monoclonal antibody (MmAb Gr+ve, Abcam) concentrations up to 60 g/ml were added again resulted in flocculation. Further experiments were carried out in this case using gold nanoparticles (40 nm) with goat anti-lipid A LPS endotoxin Gram ve polyclonal antibody (GpAb Lipid A, 4-5 mg/ml, Pierce Thermo) following removal of sodium azide (Abcam.com/technical). Aliquots (100 l) of different dilutions (10-200 l/ml) of the GpAbaLipidA antibody were incubated with 1 ml of 40 nm 0.15 nM gold nanoparticles (AuNp.sub.40nm) and added to 100 l aliquots and left for 2 min with gentle mixing, no flocculation chloride was found with 10% sodium when antibody concentrations 20, 50 and 200 g/ml were used. Gold particles of either 20 nm or 40 nm in diameter were used in the final construct.

[0043] All the antibodies used were conjugated with gold nanoparticles either directly using 2 mM borate buffer pH 9.0 or using a commercial kit (Innova Biosciences Ltd). Antibodies were applied to the conjugation pad of the strip. The test and control lines were situated on the nitrocellulose membrane. The bacterial or pathological fluid sample(s) were loaded on the sample application pad. Movement along the strip was due to capillary action and driven by the absorption pad. The detection of the bacteria was achieved using either monoclonal or polyclonal antibodies located at the test lines. The validity of each test was established using polyclonal antibodies located on the control line. The sensitivity of the strip was assessed using clinical ranges of gram positive and gram negative bacteria.

[0044] Table 8 below summarizes the performance of the seven commercial antibodies investigated.

TABLE-US-00008 TABLE 8 Gold Antibody ELISA Dot Blot Conjugation Lateral Flow Mm AntiG+ (ab20344) MmAntiG+ + + + +/ (abl55857) MmAntiG+ + +/ + (Acris-BDI813) Mm AntiG (ab31204) MmAntiG + +/ + +/ (ab20954) GpAntiG + + + + (Pierce) GpAntiG + + + (Acris)

[0045] The major challenge posed was finding a suitable, good quality commercial antibody. Some of the antibodies were reacting with both gram positive and gram negative organisms and as a result lowered the sensitivity of the test.

[0046] Polyclonal Antibodies Selecting for a Specific Trait

[0047] The method of obtaining polyclonal antibodies of the present invention overcomes the shortcomings of commercially obtained prior art antibodies, thus enabling the production of a more sensitive test.

[0048] Essential to the procedure is the availability of robust antibodies which are meticulously selected to recognise their target antigens in the lateral flow format. Although the physical components of the test strip, construction techniques and buffers play the major role in optimizing the test, the heart of these processes are the functional properties of antibodies, which need to be carefully designed at the stage of selecting antigens for immunisation and highly purified. Antibodies which perform well in e.g. ELISA may not always be suitable in lateral flow test format immunoassay. The applicants propose a novel approach to synthesising the generation of anti gram negative and anti-gram positive antisera antibodies. Teichoic acid is specific to gram positive organisms and is available from Sigma Aldrich or an equivalent supplier. Teichoic acid from 4 or more different types of gram positive bacteria can be mixed and used as gram positive biomarker antigens. The mixed antigen can then be coupled to a suitable carrier immunogenogenic protein such as keyhole limpet hemocyanin (KLH) to form an immunogen. Similarly, Lipopolysaccharide (LPS) is specific to gram negative organisms and can be obtained from Sigma Aldrich or equivalent supplier. LPs preparations derived from four or more different gram negative bacteria can be mixed and can be used as gram negative biomarker antigens and following coupling to a suitable carrier protein form the immunogen.

[0049] Commercial Teichoic acid and LPS can be contaminated with traces of the other bacterial biomarker (e.g. traces of LPS in teichoic acid preparations and vice versa), which is the principal cause of cross reactivity of the resultant antisera. In order to enhance the specificity of the generated antisera, it is intended to purify the commercial teichoic acid and LPS by e.g. SDS-PAGE electrophoresis prior to subsequent steps. The chemical coupling (conjugation) of the gram positive and gram negative biomarker antigens to carrier proteins will be carried out using methods that avoid masking the critical unique moieties of the bacterial antigens and obviate extreme alteration of the bacterial biomarker molecules. The immunogens can be injected into sheep or other appropriate species in order to generate polyclonal antibodies. The serum from sheep or other appropriate species should be assessed every month, up to 6 months. The affinity of the antibodies towards the antigenic sites on both gram positive and gram negative bacteria can be assessed in the serum obtained at 3 months and 6 months post injection. When high affinity has been demonstrated in the serum the antibody can be further purified using salt precipitation and affinity purification enabling the antibody concentration, activity and titre to be determined.

[0050] Polyclonal antibodies produced according to the preceding method can be incorporated into a lateral flow immunoassay paper strip construct. The efficacy of the antibodies can be assessed using bacteria grown in broth, saline and urine or other body fluid samples spiked with bacteria. The construct and all its constituents are maximised to achieve best results.

[0051] A number of different strip constructs are possible, as shown in FIGS. 1, 2A and 2B. For example, FIG. 1 shows one type which is designed simply for either gram positive bacteria or for gram negative bacteria, but only one of these at a time. FIG. 2 shows another type which can detect the presence of both gram-positive and gram negative bacteria simultaneously. Instead of having one test line 4 as the device in FIG. 1 does, this embodiment has two test lines 4a and 4b. As the sample travels through the conjugate release pad, gram positive bacteria bind to the antibodies which target teichoic acid, whilst gram negative bacteria bind to the antibodies which target lipopolysaccharides. One of the test lines 4a and 4b will contain secondary antibodies which target one of the primary antibodies, whilst the other test line will contain secondary antibodies which target the other primary antibody. The test lines 4a and 4b will therefore become respectively visible when gram positive or gram negative bacteria travel through them.

[0052] The system of FIG. 2 can have alternative designs depending on the control line(s) 6, as shown in FIGS. 2A and 2B. FIG. 2A has two control lines 6a and 6b, one corresponding to each of the two test lines 4a and 4b, whilst FIG. 3B has only one control line 6 acting as a control for both test lines 4a and 4b.

[0053] The lateral flow immunoassay device can be used at the point of care (POC) to establish bacterial groups present in the urine of selected cohorts of patients. The specificity and sensitivity of the test can be compared with the results obtained in the laboratory using standardised culture methods. Different formats of the strip tests, and different selections of strip components, can be used depending on the type of patients and body fluid used. The results obtained at point of care will allow the clinician to recommend the correct antibiotic to be used. The final aim will be to have a working single strip prototype, which is able to distinguish between infections caused by gram positive or gram negative bacteria of sufficient clarity to allow the administration of the correct antibiotics at point of care.

[0054] Other Potential Uses of the Test

[0055] The initial characterisation of infection into either gram positive or gram negative bacteria is crucial in enabling clinicians to administer the correct type of antibiotic and to start treatment. It can be envisioned that other traits could be tested for by the test instead of gram positive or gram negative status, simply by the same method of collecting antigens distinctive to said trait, producing therefrom a mixed antigen, and utilising the mixed antigen to produce a polyclonal antibody and creating a strip test from said antibody. Such traits could include cell morphology, class, order, family, genus, or species. Testing for such traits may be particularly useful in characterising miscellaneous organisms which do not stain well with gram stain tests; examples of these include Mycoplasma, Mycobacteria and Helicobacter.

[0056] The technology described could be modified to allow secondary more specific identification of the bacteria present in either group, by the introduction of species-specific antibodies into secondary strip tests. These would be a valuable addition to the point of care tests available in, for example, hospital infections. For example, the strip construct could be modified using antibodies to detect the common Gram positive bacteria; Staphylococci aureus (MRSA), Clostridium difficile, Streptococci or Enterococci. Alternatively, when Gram negative infections have been identified modified strip tests for the detection of E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter or Proteus could provide more specific information.

[0057] When such secondary strip tests are available, this can then enable a tiered testing process, in which a first test takes place using a strip according to the present invention to determine the presence of gram-negative or gram-positive bacteria (or, conceivably, neither) in a sample. After this initial test, secondary tests for specific species (for example, with secondary strip tests) can take place, taking into account the result of the initial test.

[0058] For instance, table 9 outlines types of fluid that may be extracted in a testing procedure from the human body or from other sample sources, and families of gram positive or gram negative bacteria which may be especially likely to be found there; being able to positively identify (or rule out) the species of bacteria at this stage could be extremely helpful in ensuring that inappropriate antibiotics are not overprescribed and effective, species-specific treatment is provided.

[0059] Conversely, if the initial test suggested neither the presence of gram-positive or gram-negative bacteria, this could prompt explorations of other possibilities such as viral infection, sparing the use of antibiotics altogether. Such tiered tests would also be particularly advantageous in e.g. developing countries and areas of conflict where no laboratory facilities are available.

TABLE-US-00009 TABLE 9 Common infections in accessible body fluids or other sample sources Type of Gram Gram fluid positive negative 1 Peritoneal dialysis Staphylococcus fluid epidermis 2 Urine Enterococci (14%) E. coli (64%), Coliforms (12%) including Klebsiella pneumoniae, Proteus (5%) 3 Wound fluid, Staphylococcus Pseudomonas aeruginosa exudates (ulcers) aureus (chronic wound infections) 4 Blood via venous or arterial Acinetobacter baumannii (80%) catheters in Intensive Care patients 5 Swabs - Streptoccal Throat infections pharyngitis 6 Blood, Bronchoscopy Klebsiella pneumoniae (lung) 7 Methicillin- Staphylococcus resistant Staphylococcus aureus aureus (MRSA) 8 Cystic fibrosis Bukholderia cepacia 10 Catheter fluid infections E. coli and Klebsiella e.g. bladder and intravenous catheters 11 Lacrimal fluid (tears) Staphylococcus aureus, streptococcal pneumoniae 12 Milk -Mastitis (cows) Coliforms a (severe) (E. coli and Klebsiella) Milk -Mastitis (cows) Staphylococcal b (less severe) 15 Food (animal general) E. coli Poultry Salmonella strains 16 Vegetables Clostridium perfringens Contamination (hands) Staph. aureus 17. Environmental water E. coli, Enterococci