Patent Application: US-73799509-A

Abstract:
the invention relates to assay methods for use in detecting specific materials such as core oligosaccharides derived from microorganisms , particularly pathogenic microorganisms , in a test sample . the invention further relates to compositions and methods for the rapid growth of such microorganisms enabling detection of same significantly earlier than is currently possible . in particular embodiments the invention is directed towards the rapid growth and / or detection of salmonella , shigella or listeria .

Description:
the assays of the present invention are preferably utilised to identify the presence or absence of core oligosaccharides of bacterial lpss in a given sample . the assays of the present invention are capable of identifying samples containing , or contaminated , with bacteria such as salmonella , shigella or listeria which have species - specific epitopes in the core oligosaccharide region of the lps . the inventions may be better appreciated by reference to the following description and examples which are intended to be illustrative of the methods of the invention . fig1 a illustrates the steps of a direct binding assay utilising a labelled primary antibody . fig1 b illustrates the direct binding assay utilising unlabelled primary antibody and a secondary labelled antibody . a direct binding ( direct or indirect antibody - linked ) chemiluminescence - based immunosorbent assay for the detection of salmonella spp on animal carcasses and in foodstuffs may be carried out as described below . 25 g of a food sample were added to 225 ml culture medium according to the first aspect of the invention . alternatively a surface swab may be taken from a 10 × 10 cm area on a carcass and cultured in 2 - 5 ml culture medium according to the first aspect of the invention . specifically the culture medium comprised 1 % peptone , 8 g / l sodium tetrathionate and 0 . 15 mg / l brilliant green . the sample was cultured for 5 hours at 37 ° c . after 5 hours of culture , a 2 ml aliquot of the sample was removed and sds was added to a final concentration of 0 . 5 % ( w / v ). the sample was heated to 100 ° c . for 5 minutes and allowed to cool . one hundred microlitres of each test sample was added directly to a well of a solid white 96 well high binding microtitre plate ( greiner bio one ) and incubated at 37 ° c . for 30 minutes . during incubation , the lipid a portion of the lps binds to the surface of the plate via non - covalent hydrophobic interactions ( fig1 a ( 1 ), fig1 b ( 1 )). following incubation the plate was emptied and the wells washed three times with a wash buffer comprising 0 . 01m sodium phosphate buffer , ph 7 . 4 , containing 0 . 147m nacl and 0 . 05 ( v / v ) tween 20 . one hundred microlitres of anti - salmonella antibody conjugate , at a concentration of 500 ng / ml in 0 . 01m phosphate buffer , ph7 . 4 , containing 0 . 147m nacl , was added to each well . the final concentration of antibody per well was 50 ng . the plate - bound sample ( salmonella lps / core oligosaccharide ) and antibody were incubated in the coated wells for 30 minutes as 37 ° c . following incubation , plates were washed three times in wash buffer and pat dried prior to detection ( fig1 a ( 2 ), fig1 b ( 2 )). when the anti - salmonella is directly labelled with acridinium ester , plates were placed into a luminometer . 30 μl of trigger solution a and 60 μl of trigger solution b was added to each well of the microtitre plate to initiate light output from conjugated acridinium ester ( fig1 a ( 3 )). the luminometer settings were as follows : measurement time interval 1 — 0 . 0 seconds delay injection m ( for solution b )— 0 . 0 seconds measurement time interval 2 — 1 . 0 seconds trigger solution a comprised : 63 μl 70 % ( w / w ) hno3 and 165 μl 30 % ( v / v ) h2o2 in a total volume of 10 ml distilled water . trigger solution b comprises : 0 . 1 g naoh and 75 mg ctac in 10 ml of distilled water . addition of goat anti - mouse igg2b acridinium conjugate ( if anti - salmonella monoclonal antibody is unconjugated ). when the anti - salmonella antibody is not labelled , a second binding member , a goat anti - mouse igg2b conjugate is used . post column igg2b was diluted 1 : 100 in a diluent comprising 3 % ( w / v ) non - fat milk powder and 0 . 05 % ( v / v ) tween 20 and 100 ul of this solution was added to each well of the plate ( fig1 b ( 3 )). following incubation at 37 ° c . for 60 minutes the plate was washed four times in wash buffer , dried and read as above ( fig1 b ( 4 )). a competitive ( direct or indirect ) chemiluminescence - linked immunosorbent assay for the detection of salmonella spp in foodstuffs may be carried out as described below . salmonella enteritidis lps - coated microtitre plates were prepared as follows . s . enteritidis was cultured in a standard broth culture medium ( 2 % ( w / v ) buffered peptone water — oxoid ) not according to the first aspect of the invention for 18 hours . the number of colony forming units was quantified and approximately 10 8 cfu / ml were placed in a covered but unsealed polypropylene boiling tube containing naedta and sds to achieve final concentrations of 10 mm and 0 . 5 % ( w / v ) respectively . the culture was boiled at a temperature of 100 ° c . for 2 minutes thereby killing the bacteria ( and neutralising any biohazard associated ) whilst also exposing the bacterial lps core oligosaccharide or monomer epitope ( see for example fig1 b ). the boiled stock was further diluted to a concentration of 10 6 cfu / ml by addition of a diluent comprising 2 % buffered peptone water ( bpw ). one hundred microlitres of diluted boiled stock was added to each well of a solid white 96 well high binding microtitre plate ( greiner bio one ) and incubated at 37 ° c . for 60 minutes . during incubation , the lipid a portion of the lps binds to the surface of the plate via non - covalent hydrophobic interactions . following incubation the plate was emptied and the wells washed three times with a wash buffer comprising 0 . 01m sodium phosphate buffer , ph 7 . 4 , containing 0 . 147m nacl and 0 . 05 ( v / v ) tween 20 . washed coated plates were either used immediately or freeze - dried for storage ( fig2 a ( 1 ), fig2 b ( 1 )). 25 g of a test sample of minced meat spiked with 10 cfu of salmonella was added to 200 ml of culture medium according to the first aspect of the invention . specifically the culture medium comprised 1 % peptone , 8 g / l sodium tetrathionate and 15 mg / l brilliant green . the sample was cultured for 5 hours at 37 ° c . after 5 hours of culture , a 5 ml aliquot of the sample was removed and tween 20 was added to a final concentration of 2 % ( v / v ). the sample was heated to 100 ° c . for 2 minutes and allowed to cool . 80 ul aliquots of the boiled sample were added to each well of the coated microtitre plate . twenty microlitres of anti - salmonella antibody conjugate at a concentration of 125 ng / ml in 0 . 01m phosphate buffer , ph7 . 4 , containing 0 . 147m nacl was added to each well ( fig2 a ( 2 ), fig2 b ( 2 )). the final concentration of antibody per well was 25 ng / ml . competing sample lps / core oligosaccharide and antibody were incubated in the coated wells for 60 minutes as 37 ° c . following incubation , plates were washed three times in wash buffer and pat dried prior to detection ( fig2 a ( 3 ), fig2 b ( 3 )). when the anti - salmonella is directly labelled with acridinium ester , plates were placed into a luminometer . 30 μl of trigger solution a and 60 ul of trigger solution b was added to each well of the microtitre plate to initiate light output from conjugated acridinium ester ( fig2 a ( 4 )). the luminometer settings were as follows : delay injection p ( for solution a )— 1 . 6 seconds measurement time interval 1 — 0 . 0 seconds delay injection m ( for solution b )— 0 . 0 seconds measurement time interval 2 — 1 . 0 seconds trigger solution a comprised : 63 ul of 70 % ( w / w ) nitric acid ( hno 3 ) and 165 ul of 30 % ( v / v ) h 2 o 2 in a total volume of 10 ml of distilled water . trigger solution b comprises : 0 . 1 g naoh and 75 mg of ctac in 10 ml of distilled water . addition of goat anti - mouse igg2b acridinium conjugate ( if anti - salmonella monoclonal antibody is unconjugated ) when the anti - salmonella is not labelled , a second binding member , a goat anti - mouse igg2b conjugate is used . post column igg2b was diluted 1 : 100 in a diluent comprising 3 % ( w / v ) non - fat milk powder and 0 . 05 % ( v / v ) tween 20 and 100 ul of this solution was added to each well of the plate ( fig2 b ( 4 )). following incubation at 37 ° c . for 60 minutes the plate was washed four times in wash buffer , dried and read as above ( fig2 b ( 5 )). fig3 demonstrates the effect of sodium tetrathionate at concentrations of between 0 and 16 g / l on the growth of salmonella aberdeen , shigella flexneri , staphylococcus aureus and e . coli . 0 . 1 ml inoculum ( 10 3 cells / ml ) was added to a 100 ml conical flask containing tryptic soy broth with 0 to 16 g / l of sodium tetrathionate . the flask was incubated at 37 ° c . for 18 hours . after this time , the a 620 was measured . each value represents the mean ± sd of three separate experiments . * shows p & lt ; 0 . 05 . at levels of between 2 to 16 g / l growth of shigella , staphylococcus and e . coli are inhibited in contrast to growth of salmonella which is un - affected or promoted . not only does the tetrathionate inhibit the growth of e . coli at levels of & gt ; 4 g / litre but at a concentration of 8 g / litre it has a clear enhancing effect on the growth of salmonella . n . b . a 620 measures turbidity and hence , the higher the value the higher the bacterial growth . at levels above 16 g / l , growth of salmonella is inhibited . fig4 demonstrates the growth response of bacteria to brilliant green . 0 . 1 ml inoculum ( 10 3 cells / ml ) was added to a 100 ml conical flask containing tryptic soy broth with 0 . 05 g to 5 g / l of brilliant green . the flask was incubated at 37 ° c . for 18 hours . after this time , the a 620 was measured . each value represents the mean ± sd of three separate experiments . * shows p & lt ; 0 . 05 , ** p & lt ; 0 . 01 . a 620 is employed as a measure of bacterial numbers by a turbidometric method . * high absorbance values due to absorbance of brilliant green ; at these concentrations the spectrometer could not be blanked against the brilliant green solution . at levels of brilliant green 0 . 3 mg / l or higher the growth of both salmonella and e . coli are limited but at 0 . 15 mg / l it has an inhibitory effect on the e - coli but not the salmonella . fig5 demonstrates the growth response of bacteria to ammonium ferric citrate . 0 . 1 ml inoculum ( 10 3 cells / ml ) was added to a 100 ml conical flask containing tryptic soy broth with 0 . 25 to 1 . 5 g / l of ammonium ferric citrate . the flask was incubated at 37 ° c . for 18 hours . after this time , the a 620 was measured . each value represents the mean ± sd of three separate experiments . * shows p & lt ; 0 . 05 . at levels of ammonium ferric citrate of 0 . 25 g / l or higher the growth of both staphylococcus and e . coli are limited . fig6 demonstrates the growth response of bacteria to sodium citrate . 0 . 1 ml inoculum ( 10 3 cells / ml ) was added to a 100 ml conical flask containing tryptic soy broth with 5 to 25 g / l of sodium citrate . the flask was incubated at 37 ° c . for 18 hours . after this time , the a 620 was measured . each value represents the mean ± sd of three separate experiments . * shows p & lt ; 0 . 05 . at levels of sodium citrate of 5 g / l or higher the growth of both staphylococcus and e . coli are limited . at levels of 15 g / l the growth response of shigella is significantly increased over those of staphylococcus and e . coli . generation study of different bacteria in peptone , tryptic soy broth and modified tryptic soy broth three strains of shigella and other bacteria including salmonella aberdeen , e . coli and staphylococcus aureus were grown in conventional broth cultures to investigate the generation time . 0 . 1 ml inoculum ( 10 3 cells / ml ) was added to a 100 ml conical flask containing either peptone ( fig7 ), trypric soy broth ( tsb ) ( fig8 ), modified tryptic soy broth ( mtsb ) ( fig9 ) or gram - negative broth ( fig1 ). each flask was incubated at 37 ° c . for 18 hours . after this time , the number of viable cells was determined by drop plate technique on nutrient agar . the values in parenthesis are generation times . each value represents the mean ± sd of three separate experiments . * shows p & lt ; 0 . 05 . the doubling time was studied in peptone , tryptic soy and modified tryptic soy broth . the generation time of shigella flexneri , salmonella aberdeen , e . coli and staphylococcus aureus was 36 , 57 , 41 and 44 min respectively when they were grown in gram - negative broth . the growth rate of all bacteria increased in tsb . as a result , tsb was used as the basic growth media in conjunction with other traditional selective agents , alone or in combination to selectively allow better growth of shigella . the doubling time of shigella flexneri , salmonella aberdeen , e . coli and staphylococcus aureus was 48 , 46 , 28 and 33 min in tsb . shigella flexneri and salmonella aberdeen grew significantly better ( p & lt ; 0 . 01 ) in the mtsb , whereas , it took longer for e . coli and staphylococcus aureus to multiply in this base broth . the growth rate of e . coli could be delayed up to 68 min when grown in modified tsb . generation time of shigella flexneri could be shortened to 46 minutes in modified tsb . listeria growth medium was prepared comprising a combination of lithium chloride and nalidixic acid . 0 . 1 ml inoculum ( 10 3 cells / ml ) was added to a 100 ml conical flask containing according to the following recipe : tsbye — 3 . 3 % tryptic soy broth with 0 . 5 % yeast extract , 2 g / l licl , 2 mg / l nalidixic acid and 250 mg / l ammonium ferric citrate . the flask was incubated at 37 ° c . for 20 hours . after this time , the a 620 was measured ( fig1 ). each value represents the mean ± sd of three separate experiments . l . monocytogenes and l . innocua were both able to grow efficiently in the media . the growth of e . coli . lactobacillus acidophilus and erysipelothrix rhusiopathiae were all significantly inhibited . preparation of culture media for the selective growth of salmonella , shigella or listeria utilising the above data , selective culture media were prepared according to the following recipes : preparation of antibodies and antibody fragments for conjugation with acridium ester for use in immunoassays an improved preparation of antibodies can be produced by preparation of pure igg from ascites by protein a chromatography , followed by the optional step of cleaving the igg to give a fab fragment , and conjugation of the fragment or whole antibody to an ester and subsequent purification . alternatively , other isotypes or isoforms of antibody can be used unpurified . 0 . 1m citrate - acid buffers , ph 6 , and ph 4 . 5 : dissolve 29 g dry sodium citrate in 800 ml of distilled water . add 1 m citric acid solution ( 210 g / l ) until a ph of 6 and 4 . 5 , respectively , is obtained . make up to 1 litre . ph 3 buffer , 0 . 1m acetic acid containing 0 . 15 m nacl , to 800 ml of distilled water , add 100 ml 1 m acetic acid and 100 ml 1 . 5 m nacl . 1 . 5 m glycine buffer , ph 8 . 9 , containing 3m nacl : dissolve 112 g glycine and 174 g nacl in 700 ml of distilled water . adjust ph to 8 . 9 with 5m sodium hydroxide solution and make up to 1 litre with distilled water . allow 1 . 5 g of protein a - sepharose ( cl - 4b , pharmacia ) ( protein g may also be used ) to swell in 0 . 1 m phosphate buffer , ph 8 , for 30 minutes ( 1 . 5 g of beads give 5 ml of gel ); fill a 20 × 2 cm column and rinse with starting buffer . after dialysis against starting buffer , load on to the column 1 ml delipided ascites previously precipitated by ammonium sulphate at 40 % ( v / v ) saturation . delipiding of ascites , if used , is carried out by centrifugation at 100 , 000 g for 45 minutes . any pellet formed or floating ‘ lipid ’ is discarded . wash the column with 0 . 1 m phosphate buffer , ph 8 , until the a 280 is & lt ; 0 . 050 . add citrate buffer , ph6 , and wash until the a 280 is & lt ; 0 . 050 . elute the other immunoglobulins in the same manner by successively employing the ph4 . 5 and ph 3 buffers . neutralise with phosphate buffer , ph 8 , containing 0 . 02 % sodium azide , and store the protein a - sepharose in this buffer . after elution , neutralise the antibody with several drops of 1m phosphate buffer , ph 8 , and dialyse against pbs . since enzymatic digestion never goes to completion , the action of papain on igg gives rise to 10 % of undigested igg in addition to the fab and fc fragments , which have the same molecular weight and are hence difficult to separate . protein a is used to simplify their purification . in the first step , the antibody is treated with papain , and then the mixture is passed over protein a in order to isolate the igg and the fab . the fab fragments are then separated from undigested igg by filtration on sephadex or protein a - sepharose . 0 . 1m edta : dissolved 3 . 6 g edta in 100 ml of 0 . 2m naoh . since edta dissolves significantly only at approximately ph 8 , it may be necessary to add a few drops of im naoh in order to ph the solution to obtain complete solubility of the edta . the immunoglobulin fraction was prepared from the antiserum by precipitation with 40 % ( v / v ) saturated ammonium sulphate solution . the immunoglobulins were dialysed against 0 . 1m phosphate buffer , ph 7 . 4 . an approximate determination of the protein concentration was made ( a 1 mg / ml solution of igg gives an a 280 of 1 . 4 ). the concentration of the igg was adjusted to 30 mg / ml and the final volume ( v ) required to give a protein concentration of 20 mg / ml was calculated . for affinity purified antibodies , a final concentration of 2 . 5 mg / ml was used . a volume of v / 20 of 0 . 04m edta ( final concentration : 0 . 002 m ) was added . a volume of v / 20 of 0 . 2m l - cysteine solution ( final concentration : 0 . 01m ) was then added . a 1 mg / ml papain solution to give 1 mg of papain per 100 mg globulins was added . the volume was adjusted to v ml with the 0 . 1m phosphate buffer , ph 7 . 4 . the reaction was allowed to proceed for 2 hours at 37 ° c . a volume of v / 10 of a 0 . 4m iodoacetamide solution ( final concentration : 0 . 04m ) was added . this was left for 30 minutes , and then the preparation was dialysed against pbs overnight at + 4 ° c . igg antibodies binding to the glcnac - glc - gal epitope ( fig1 ) were isolated and the fab fragments were isolated by protein a chromatography . the mixture was fractionated on a column of sephadex g - 100 ( 2 . 5 × 80 cm ) and equilibrated with pbs . the first peak corresponded to igg , and the second peak corresponded to the fab fragments . the fab peak was concentrated to 5 mg / ml . i ) the acridindium ester e . g . ( 4 -( 2 - succinimidyloxycarbonylethyl ) phenyl - 10 - dimethylacridinium - 9 - carboxylate fluorosulphate is weighed in a clean , dry borosilicate vial . dry dimethyl formamide is added ( volume depending on acridinium ester quantity available ) and the solution aliquoted into vials at 5 mg per vial normally . ii ) antibody is dissolved in 0 . 2m sodium phosphate buffer , ph 8 . 0 at a concentration of 0 . 5 mg igg / ml . iii ) add 5 mg acridinium ester solution to 200 ml antibody solution and mix well . iv ) incubate for 15 minutes at room temperature and then stop reaction by the addition of 100 ml 10 % ( w / v ) lysine monohydrochloride followed by a further 5 minutes at room temperature in the dark . ( i ) a gel filtration column may be used to purify the conjugate . a column of 1 . 6 × 100 cm of sephadex g200 ( pharmacia ) is equilibrated with 0 . 1m phosphate buffer , ph 7 . 4 , containing 0 . 147 m nacl and 0 . 5 % ( w / v ) bovine serum albumin ( sigma ). up to 1 . 5 ml of conjugate is placed on the column and separated for 18 hours at a flow rate of 9 ml / h . the effluent is monitored at a 280 and the peak corresponding to 45 - 55 k daltons collected for a fab fragment or 140 - 170k daltons for a whole antibody — this is the conjugate , which should be diluted to a working strength before use . ( ii ) the preferred alternative procedure employs the use of an fplc . the conjugate is purified on a pharmacia superdex 200 hr 10 / 30 column . 50 ml 0 . 007 g / ml solution of bovine serum albumin ( bsa ) is added to the conjugate ( to bring the bsa concentration of the conjugate up to that of the elution buffer ). before applying the sample , the column is equilibrated with two column volumes ( 50 ml ) of elution buffer . the conjugate solution is then centrifuged at 10 , 000 g for 10 minutes to remove any particulate matter and applied to the fplc column . the antibody is eluted from the column in the elution , and storage buffer at a flow rate of 0 . 5 ml / min . after the first 5 ml has passed through the column 0 . 5 ml fractions are collected . the presence of antibody is detected though the use of an ultraviolet ( uv ) monitor and the fractions spanning the antibody peak are collected and analyzed for luminescent activity ( normally fractions 16 - 21 ). the antibody fractions are diluted 1 : 500 in saline and 5 μl samples of each fraction spotted into the wells of an assay plate . the fractions are then tested for luminescent activity by reaction with activating reagents 1 and 2 .- 15 μl of activating reagent 1 is first added to the sample well , followed by 30 μl of activating reagent 2 . this is normally achieved by automatic injectors in the luminometer , which is then activated to read the light emission from the well in question . the results are recorded using a repeat for each sample . samples containing high levels of luminescent activity can then be confirmed in a microbial assay , in this example , a salmonella assay . in order to generate a satisfactory luminescent signal , the antibody may be conjugated to the enzyme alkaline phosphatase and the substrate amppd employed in the immunoassay ( amppd - 3 -( 2 ′- spiroadamantane )- 4 - methoxy - 4 -( 3 ″- phosphoryloxy ) phenyl - 1 , 2 - dioxetane ; 3 -( 4 - methoxyspiro ( 1 , 2 - dioxetane - 3 , 2 ′- tricyclo ( 3 . 3 . 1 . 1 ( 3 , 7 )) decan )- 4 - yl ) phenyl phosphate ). the diluent for this substrate is 0 . 9 g of ctab ( cetyltrimethyammonium bromide ), 1 . 9 ml amp ( 2 - amino - 2 - methyl - 1 - propanol ), 14 . 5 mg magnesium chloride . 6h 2 o , 1 mm , ph 9 . 6 , in 100 ml distilled water . 0 . 2m tris ( 24 . 228 g / litre ) 0 . 2m nacl ( 11 . 688 g / litre )+ 0 . 05 % ( v / v ) tween ( 0 . 5 ml / litre ). dissolve 24 . 228 g of tris and 11 . 688 g of nacl in 900 ml of dh20 . add 0 . 5 mls of tween 20 . adjust the ph to 7 . 4 using hcl , make up to 1 litre and store at room temperature . 2m tris ( 24 . 228 g / iooml ) 2m nacl ( 11 . 688 g / iooml )+ 0 . 5 % tween ( 0 . 5 mls / iooml ). dissolve 24 . 228 g tris and 11 . 688 g of nacl in 80 mls distilled h 2 0 . add 0 . 5 ml of tween 20 and adjust the ph to 7 . 4 with conc . hcl . make up to 100 ml with distilled h 2 0 and store at room temp . to reconstitute the wash buffer , add 100 ml of concentrate to 900 ml of dh 2 0 and store at room temperature . 0 . 1m sodium phosphate buffer ph 6 . 3 with 0 . 15m nacl 0 . 1 % ( w / v ) bovine serum albumin ( bsa ) 0 . 05 % nan 3 . make up a solution of 0 . 1m nah 2 po 4 with 0 . 15m nacl containing 0 . 1 % w / v bsa ( a ) and 0 . 1m na 2 hpo 4 with 0 . 15m nacl containing 0 . 1 % w / v bsa ( b ). add 100 ml of a to 50 ml b . add 0 . 05 % nan 3 , filter through a 0 . 22 mm filter and store at 4 ° c . 0 . 01m nah 2 po 4 ( 1 . 2 g / litre ) 0 . 15m nacl ( 8 . 75 g / litre ) with 0 . 1 % w / v nan 3 and 0 . 25 % w / v bsa . dissolve 1 . 2 g of nah 2 po 4 and 8 . 75 g of nacl in 900 ml dh 2 0 . add 1 . 0 g nan 3 and 2 . 5 g bsa . allow to dissolve completely and adjust the ph to 7 . 4 with 1 . 0m naoh . make to 1000 ml with distilled h 2 0 . filter through a 0 . 22 μm filter and store at 4 ° c . 20 % ( w / v ) sds solution : dissolve 5 g of sds in 25 ml of dh 2 0 . store at room temperature . 8 g sodium tetrathionate and 0 . 15 mg brilliant green added to 1 litre of sterile peptone broth . mix gently until evenly distributed . 6 . 3 ml of 70 % nitric acid ; 16 . 5 ml of 30 % hydrogen peroxide ; 977 ml of distilled water . the wells of an assay plate are coated with standard concentrations of bacteria for 1 hour at 37 ° c . these standard concentrations are : 10 6 , 10 5 , 5 × 10 4 , 2 . 5 × 10 4 , 10 4 and 5 × 10 3 and blank wells containing 10 6 e . coli . the fractions to be tested are diluted 1 : 100 in assay buffer and 50 ml is added to each well and incubated at 37 ° c . for 20 minutes . the wells are then read on the luminometer , as above . those fractions demonstrating good binding in the assay are pooled and the optimal dilution for the pooled conjugate determined — normally 1 : 100 to 1 : 1000 . novel black and white plate ( wallac ) read on a tube luminometer ( berthold lb 9509 ) using the detergent , sodium dodecyl sulphate ( sds ) white plate ( wallac ), read on lucy i plate luminometer ( 1 : 100 conjugate dilution ) effects of various levels of sds on the direct binding salmonella assay ( 1 : 100 conjugate dilution ) using black and white plates effects of various levels of tween 20 on the competitive binding salmonella assay ( 1 : 100 conjugate dilution ) using black and white plates competing 0 . 5 % 0 . 5 % s . enteriditis tween tween 1 % tween , 1 % tween 2 % tween 2 % tween ( cfu / ml ) 2 min boil 20 min boil 2 min boil 20 min boil 2 min boil 20 min boil 10 6 4274 ± 712 3531 ± 154 4497 ± 181 3216 ± 130 6747 ± 223 2074 ± 42 10 5 37803 ± 2376 30413 ± 2985 48390 ± 2811 25614 ± 1030 53067 ± 701 6619 ± 573 10 4 200745 ± 12074 190752 ± 6265 222178 ± 3436 171024 ± 2620 226168 ± 24620 63938 ± 4766 10 3 277066 ± 20343 305744 ± 27327 305343 ± 15168 270782 ± 15522 266820 ± 24059 281305 ± 20480 0 370245 ± 16595 370245 ± 16595 370245 ± 16595 370245 ± 16595 370245 ± 16595 370245 ± 16595 ( mean ± standard deviation , relative light units ) effects of anti salmonella mab incubation times with 1 : 100 anti 2b conjugate monoclonal antibody ( 1 : 100 dilution ) was incubated with either salmonella or listeria to determine optimum incubation times : 30 mins 40 mins 60 mins 10 6 s . aberdeen 598080 609383 593741 10 5 s . aberdeen 716821 644854 629340 10 4 s . aberdeen 276239 328483 371163 10 3 s . aberdeen 20117 19454 29194 10 2 s . aberdeen 5439 7204 17242 10 6 l . innocua 6376 8909 12151 ( read on lucy i plate luminometer , results shown in relative light units ) effects of anti salmonella mab incubation times with 1 : 200 anti 2b conjugate monoclonal antibody ( 1 : 200 dilution ) was incubated with either salmonella or listeria to determine optimum incubation times : ( read on lucy i plate luminometer , results shown in relative light units ) * all + ve ( bacteria positive ) food cultures were contaminated with 10 c . f . u . of s . aberdeen and cultured for 18 hours ( 25 g in 225 ml ) of peptone broth + 8 g / 1 sodium tetrathionate and 0 . 15 mg / l of brilliant green . the bacteria negative cultures (− ve ) were contaminated and also cultured for 18 hours . 5 ml samples of all the food cultures ( with or without sds ) were heated for 20 minutes in a boiling water bath prior to the assay . sds provides the most reliable and reproducible results for dissolution of food sample - based salmonella lps into monomers . however only the tween can be used for this purpose in the competitive assay due to protein - detergent interactions with the other detergents . effects of varying anti - salmonella antibody levels on detection of salmonella in the competitive assay . buzby , j . c . and roberts , t . 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( 2000 ) stressed salmonella are exposed to reactive oxygen species from two independent sources during recovery in conventional culture media . int . j . food microbiol . 60 : 269 - 285 .