CA2189748A1 - Method and reagents useful for improving immunoassay specificity - Google Patents

Abstract

An improved method for detecting antibodies is disclosed. The method involves the steps of: a) mixing the specimen with a diluent comprising superoxide dismutase, and b) constacting the diluted specimen with at least one recombinant antigen expressed as a fusion protein with superoxide dismutase.

Description

2 1 8 9 7 ~ 8 PCT/US94105152 METHOD AND REAGENTS USEFtlL FOR IMF'ROVING IMMUNOASSAY
SPECIFICITY
t '5t ' Of Thl~. T
The invention relates generslly to a method for improving the specificity of ,~ D~ and more palticularly, relates to a method for adding denatured and~or purified bacterial enzyrne to a specimen diluent to incresse the specUicity of jtD which utilize ~ antigens expressed as fusion proteins with a bacterial enzyme.
Historicslly, ~ D have b~en lcnown to be prone to false positive reactions. Virsl Iysates u. ' protein l , which compriDe the typical antigen solutions which are utilized on the solid support, also csn contain other proteins. These other proteins can bind to the solid suppo,rt, introducing the possibility of reactivity with antibodieD in the patient's Darnple. A~ , a component in the patient's sample can bind to the solid support and interfere with the assay.
For exarnple, McFarlane et al., h~ (1990) 335:75~57, reported a high prevalence of antibody to hepatitis C virus tHCV) in patients with I ~ chronic active hepatitis tAI-CAH). They suggested that the serum of Al-CAH patients may contain a component that gives false-poDi~e resultD in the assay. McFarlane et al.
surmised that tbe assay might have ~een non-specifically detecting IgG since they saw a correlation between IgG levels and OD values, or that factorD contained in the patient sera were responsible for the results. such as an antibody against some which cross-reacts with the antigen used in the assay, or a component which adheres to the solid phase and binds IgG. In addition, some false positive results are due to cross .~t; . ity between the patient ser~ and the fusion protein uDed to express the antigen utilized in the assay.
Since reactivity such as that described herein can be expecled~ assays can be designed to circumvent some of the problems generally associated with utilizing the ' protein. See, for example, P.C.T. Publication No. WO 92/13275, which to U.S. Serial No. 08/059,868 which is . ' herein by reference.
In that l ' ' it is taught that the addition of superoxide dismutase to the spe~imen diluent is useful in increasing the specificity in assays which utilizeproteins produced as fusion proteins with superoxide dismutase (SOD).

wo ss/30so2 2 1 8 9 7 ~ 8 Pcr/uss4/osls2 ~, , , .' I ; ~

One sldlled in the art would be led, based on this teaching, to add such a bacterial enzyme to the specimen diluent in Drder to increase specificity in assays which utilize proteins.
, ~ , however, applicants _ave discovered that the mere addition of 5 such a bacterial enzyrne, even if expressed in tbe same bacterial host system, may ~ot lead to increased specificiq in all types of fusion proteins. Thus, it would be to provide a method and reagents w_ich could increase the specificiq in , ~ when fusion proteins aTe utilized as the capture and/or indicator reagent in an assay.0 y of th~ J
The present invention provides an improved method for increasing specificiq in ~ ~ which comprises the steps of (a) mixing the specirnen with a diluent comprising a denatured ' bacterial enzyme expressed as a fusion protein 15 with s4id bacterial enzyme, and (b) contacting said diluted specimen with at least one ' antigen expressed as a fusion protein with said denatured bacterial e~zyme. The ' denatured bacterial enzyme comprises a ' protein, and can be either CKS or SOD. Further, the of said ' denatured bacterial enzyme is from abDut Q001 g/L diluent to abDut 1.0 20 g/L diluent. More preferably, the ~ of said ' denatured hacrerial enzyme is from about 0.01 g/L diluent to about 0.1 g/L dilueM. Most prefen~d, the ' bacterial erlzyme is at a . of ab~ut 100 ~Lg/ml.
The method can detect nurnerous analytes, including antibody to hepadtis C virusa~CV) and antihody to human ' ~ , virus (HIV). r can be 25 ~ by a vadety of methoDs, including heat and urea. Further, purified, ' bactedal enzyrne may he utilized.
The present invendon also provides a diluerlt useful for detecting antibDdies in a test sample when performing an assay which uses at least one ' antigen expressed as a fusion protein with a ' bacterial enzyme, which diluent 30 compdses the ' bactedal enzyme which has been denatured. The of said denatured ' bacterial enzyme is abDut 100 ~ug/ml.
Pudfied, ' bactedal enzyme may be utilized WO 95/30902 2 1 8 9 7 ~ 8 : PCT/US94/05152 The present invention also provides a test kit for performing an .
which compli es a container cont4ining a denatured bacterial enzyme selected f~m the group consisting of CKS and SOD.
5 r~ '- ' ~rcrr~r ir~n ~-f t~- Tnv~nti~m The present invention provides an , . to methods for detec~ing antibo~ies in a specimen f~m an irldividual wherein a ' andgen employed in the method is expressed as a fusion protein by a vectL~r and wherein the antigen is encoded with a gene of a bacterial enzyme such as CKS or SOD. The 10 . . _~1 comprises the step of mixing the specimen with a diluent comprising denatured ' bacterial enzyme (such as, SOD or CKS), wherein said enzyme aLo may be purified by methods known in the art as discussed herein, including aDion-exchange resins and cation: ' ~ resins.
The ' of proteins by heat or chemical means is known in assay diagnostics when preparing serum samples for cert4in types of assay procedures.
r of proteins occurs when the tertiary bonds of proteins are brol~en, which results in par~al or complete unfolding of the protein. Usually, however, care is taken not to denature the protein solutions used for assays, si{tce such .' can change the properties of such proteins, adversely affecting the results of the assay.
See, for example, N. W. Tietz, ed., ~ h^mic~y. 2nd Edition, W. B. Saunders Company, r ~ (l976), page 27Q
Although it is known to add the ' bacterial enzyrne utilized in the fusion protein to the assay diluent in açder to i~nprove assay specificity, it heretofore has not been known that the rnere addition of such bacte~ial enzyme to a diluent may not result in the desired effect of increasing assay specificity by reducing the amount of faLe positive reactions. A false positive result is defined herein as one in which a sample is repeataWy reactive in an EI~SA, EIA or PHA but is not confnrned by alternative methods for the presence of analyte antibodies. Alternative testing methods include synthetic peptide ElAs, antibody blocking procedures and ' ' assays.
Applicants bave observed that in certain inst4nces, 4~dition of ' or purified ~ ' bæterial enzymes does not result in increased specificity in aC~says ^I which employ ' proteins. Applicants have . ~ ~ discovered that ~ of tbe, ' bæterial enzyme results im incre4sed specificity in WO 95/30902 ~ 1 8 9 7 4 8 PCT/US94/05152 . ', '.
assays. Also, applicants have discovered that at times it may be ad~ . _ to utiliæ
a purified, denat~d, bacterial enzyme in a specimen diluent to increase the specificity of thc assay.
Il has been discovered that it is especially ,ul~ _ to add denatu~ed, ' baclerial etlzyme to a diluent (heranafter defined) to imp~ve the detection of analyte by increasing assay specificiy. T ,~ ~ can employ antigens which are expressed as a fusion protein by a vector in which the antigen is encoded with the superoxide dismutase (SOD) gene or the CKS (CTP:CMP-3~eoxy--manno- cytidylyl transferase or CMP-KDO synthetase) gene (see, U.S.
Patent No. 5,124,255, issued June 23, 1992, which is , ' herein by reference and U.S. Serial No. 07/903,043, which is , ' herein by reference).
Therefore, those assays which utilize antigen expressed as fusion proteins with bacterial enzymes can exhibit an interfering reaction with and-SOD or anti-CKS
antibo~ies in patient samples. The effect of Lhis anti-CKS or anti-SOD is not removable as a false positive reaction unless the bacterial enzyme which is ernployed is denatured prior to iLs a~didon in Lhe specimen diluenL The bacterial enzyme, preferably l~, engineered, can be added to the diluent in ~
rang;ing from about Q001 g/L to about I g/L, more preferably from about 0.005 g/L to about 05 glL, and most preferably from about 0.01 g/L to about 0.1 g/L. The bacterial er~snne can be addcd as a purified or parlially purified protein from a bacterial extract. However, it has been discovered that the bacte/ial enzyrne must be denatured plior to addition and use irl the diluent.
~n~n~ m can be effected by methods known in the an, including heating at . . '~, 50C to 60C fQr ~, r '~, 20 to 60 minutes, and treatnnent of the bacterial enzyme with chemicals, such as, 8M urea, guanidine, and some orga~uc solvents.
In accordance with the present invention, a "diluent" is defined herein as an aqueous solution of buffer(s) and salt(s) as well understood in the art and illustrated ir~a A preferred buffer is Tris[l.J.Lu~
available under the trade designation Tris from Sigma Chemical Co., St. Louis, MO, Ul I ~ ~ ' buffers; suitaole buffers include, but are not limited to, buffers such as HEPES (N-[2 hJ ~ N'-[2; r acid]), PIPES
-N,N'-bis[2; - r ' acid]), CAPS (3-[~ ~ ] 1-wo 95/30902 2 1 g ~ 7 ~ 8 PCTIUS94/05152 acid)andMOPES(3-[N ' ' ~ '~ acid). Suitable salts includc sodium chlolide (NaCI) and salts such as phosphate salts and sulfate salts.
In additioD, Yarious animal sera, detergents, bloclcing agents and other can be added to improYe specificit,Y. For example, animal serum proteins 5 such as boYine serum, boYine serum albumin (BSA), fetal calf serum and goat serum can be added in - ranging from about QS% v/Y to about 50% Y/Y.
Biological detergents such as ~I,~ aYailable as T~Yeen~9 20, pol.~v~,..Syl~,..., ether, ~ aYailable as Triton~l9 X-100, Nonidel P40 (an ~t~ ., oxide condensate), sodium dodecyl sulfate 10 (SDS) or N ~ ' (N~o~vjl N ' ~Igl~. ) can h added in - ranging ~.om about 0.019b vlY to about 5% Y/Y. Chelators such as acid (EDTA) and ethykne glycol-bis(~
N,N,I~,N'-tetraacetic acid (EGTA) can be added in ranging from about 2 mM to about 20 mM.
In order to neutralize nonspecific reactions due to other proteins contained in the YiTal Iysates or ' proteins that comprise the antigen solutions employed on the solid support, a Iymphocy~ Iysate solution, for example, human T-l.~
soludon or a host cell Iysate solution such as an ~,s~j; Iysate solution, can be added in typically ranging from about 0.01% Y/Y to about 10% Y/Y.
20 I~;D~ D such as sodium azide can also be added.
The specimen dilueM can be used to dilute test specimens in Yarious assay formats. It is: . ' ' that the diluent reagent employedi for the assay can be proYided in the form of a Icit with one or mr)re containers such as Yia1is or bot~les, with each container containing a separate reagent such as a diluent, a ' ' antibody 25 or, ' thereof, or a ' protein employed in the assay.
"Solid phases" ("soliid supports") which can be used in the assay formats are known to those in the a{t and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, " ' st[ips, ' ,..~
such as latex paTticles, and others. The "solid phase" is not critical and can be selected 30 by one skilled in the aTt. Thus, latex particles, . ~ rnagnetic or non-magnetic beads, ' plastic tubes, walls of microtiter wells, glass or siliicon chips and red blo~d cells are all suitable examples. Suitable methods for peptides on soliid phases include ionic, h,.' , ' ' , coYalent interacdons and the lil~e.

WO 95/30902 ~ : PCTIUS94/051~2 ~lg~ 8'' The present invention provides a diluent, test ldt and method for inereasing speeifieiy in ~O which utilize speeific binding memb~rs. A "speeifie binding member," as used herein, is a member of a speeific binding pair. That is, two different molecules where one of the molecules through chemical or physical means 5 specifieally binds to the seeond molecule. Therefore, in a~idition to antigen and anti~ody speeific binding pairs of eommon .~O~ other speeific binding pairs ean inelude biotin and avidin, L~l,uh~ ' and leetins, c ~' ~ nucleotide sequences, effeetor and reeeptor moleeules, eofaetorO and enzymes, enzyrne inhibitors and enzymes, and the like. r. speeific binding pairs can include memb~rO
10 that are analogs of the original specific binding members, for example, an analyte-analog. T ~. specific binding memb~rs include antigens, antigen fragments, antibDdies and antib~dy fragments, both mn-~lnn~l and polyclonal, andearnplexes thereof, including those formed by ' DNA moleeules. The term "hapten", as used herein, refers to a partial antigen or non-protein binding member 1~ which is eapable of binding to an antibDdy, but which is not capable of elieiting antibody formation unless eoupled to a carrierprotein.
"Analyte," as used herein, is the substanee to be detected which may be present in the test sample. The analyte can be any substance for which there exists a naturally o~urring specific binding member (sueh as, an antibody), or for which a speeific20 binding member can be prepared. Thus, an analyte is a substanee that ean bind to one or more speeifie binding members in an assay. "Analyte" also includes any antigenic substanees, haptens, antibDdies, and ' thereof. As a member of a speeific binding pair, the analyte ean be deteeted by means of naturally oeeur~ing speeific binding partners (pairs) such as the use of int insic faetor prDtein as a member of a 25 speeific binding pai~ for the ~' of Vitamin B12, the use of folate-binding protein to detemune fohc aeid, or the use of a leetin as a mernb_rof a speCiflC binding pair for the i'~ of a ~l~h~ The analyte ean include a protein, a peptide, an amino aeid, a hDrmone, a steroid, a vitamin, a drug including those ' for therapeutic purposes as well as those ' ' fi~r illieit purposes, 30 a bacterium, a virus, and metabolites of or antibDdies to any of the above substanees.
The details filr the preparation of such antibDdies and the suitability for use as speeific binding memb~rs are well Imown to those skilled in the arL Viruses which ean be tested include hepatitis-eausing viruses (for example, hepatitis A virus, hepatitis B
virus, hepatitis C virus, hepatids delta, and hepatitis E vtrus), hurnan wo ss/30so2 2 1 ~ ~ 74 8 PCTIUS94/05152 viruses (such as E~V-I, HIV-2), the HTLV-I and HTLV-II viruæs, and the lilce.
"Indicator Reagents" may be used in the vaTious assay falmats discusæd herein. The "indicator reagent "comprises a "signal generating compound" (label)5 which is capable of generating a measurable signal detectabk by external meansconjugated (attached) to a specific birlding member for the analyte. "Specific binding member" as used herein means a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical orphysical meansspecificaDy binds to the second molecule. In addition to being an antib~dy member of 10 a specific binding p2ur for the analyte, the indicator reagent also can be a memoer of any specific binding pair, including either h~t.,.. ' , systems such as biotin or arlti-biotin, avidin or biotin, a ~I,uh~ ' or a lectin, a ~ nucleotide se~uence, an effector or a receptor lecul,e, an enyme cofactor and an enyme, arenyme inhibitor or an enyme, and the lilce. An .~. specific binding 15 member can be an antib~dy, an antigen, or an ~ ' 'y~'2t.O~complex that is capable of binding either to the analyte as in a sandwich assay, to the capture reagent as in a ~ assay, or to the anciDary specific binding member as in an indirect assay.
The vaTious "signal generating: . " (labels); . ' ' include O catalysts such as enzymes, I compounds such as fluorescein and rhodarnioe, - . . ' radioactive elements, and direct visual labds. Examples of enymes include al~aline ~ , horseradish peroxidase, and the like. The selection of a paTticular label is not critical, but it ~ill be capable of producing a signal either by itself or in . , with one orrnore additional substances.
The term " test sample" includes biological samples which can be tested by the metho~s of the present invention descTibed heTein and include human and animal b~dy fluids such as whole blood, serum, plasma, ~ p - ~ fluid, urine, Iymph fluids, and vaTious external secretions of the respiratoTy, intestinal and O ~ t~acts, tears, saliva, millc, white bloed cells, myelomas and the lil~e, biological fluids such as ceD culture r ' ', fixed dssue specimens and fixed ceD specimens. Any substance which can be diluted and tested with ' proteins and assay forrnats described in the present invendon are , ' ' to be within the scope of the present invendon.

WO 95/30902 P~T/US94/05152 ~
21837~8 -8-lt is, , ' ' that whatever assay format is chosen will utilize a diluted test samplc. Numerous assay fortnats are Icnown in the art, and all are considered to be within the scope of the invendon if ' antigens are utilized as capture or in~iicator reagents in them. Thus, one and two-step sandwich 1.
5 i ' assays, competitive assays, ' assays, itnrnunodot assays, arc all considered within the scope of the present invention.
The use of haptens is Icnown in the art. It is . ' ' that haptens also can be used in assays e}nploying fusion proteins in order to enhance l ~ of the assay.
The following e,Yamples are n eant to illustrate, but not to lirnit, the spirit and scope of the invention.
FXA~ .F.~:
FY~ 1. Pr~ n of r ~R~-r~36forHlVAccsu~
15 A. pr~ ~hrm<lf~ APD lOcolutnn(availablefrom Pharmacia) was equiiibrated with lOmM phosphate buffer contauung 8M urea (pH
7.5). CKS-rp 36 antigen in 6M G- " HCI solution was hulted with this column and 10 rnM phosphate buffu containing 8M urea (pH 7.5), resuldng in CE~S-rp 36 antigen in 8M urea solution. It was found that a dialysis method also can be used 20 instead of this solu~ion exchange method, Such processing resulted in a purified and denahlred bacterial enzyme preparation.
Then, DFAE ~.. - ' . resin or a QAE iorl . ' ~ resin was f~ed up in the column, e.g., DEAE-5PW (available from Toso), Resource-Q (available from Pharmacia), was . "' ' with the IO mM phosphate buffer containing 8M urea 25 (pH 7.5). Next, CKS-rp 36 antigen m 8M urea solution was passed OVN the a~ove . ' ~ resin column. CKS-rp 36 antigen was passed by this step at a flow rate of ImV~nin. The resultant CKS-rp 36 antigen was used in a passive ~ ---a,,~' assay (PHA) as an antigen, as follows. The denahuled CKS-~p 36 antigen was coated onto human red blood cells type O and used in the PHA assay kit 30 (available from Dainaibot, Tokyo, Japan). Three thousand (3,000) volunteer serum ~ionors were tested usmg this PHA test kit in which the test serum was diluted at the various noted. The results obtained are - ,-- - ; l in the following Table 1.

W0 95130902 ~ 8 PCT/lrS94/05152 g TABLE I
rlh~r Af Fslc~ ~. gr iAnc ~ ' by E (~nli 1:8 1:16 >1:32 Untreated 1 3 56 DEA~Treated 1 2 0 5 B. Pn~l?s ~tiAn of l'~ iAn Fyrhsr~o~ A PD-10 column (available from Pharmacia) was ~, - ' ' with lOmM phosphate buffer contairling gM urea (pH
7.5). CKS-rp 36 antigen in 6M Guanidine-HCI solution was treated with this column and 10 mM phosphate bufer containing 8M urea (pH 7.5), resulting in CKS-rp 36 antigen in 8M urea solution being obtuned. It was found that a dialysis method also 10 can be used as the solution exchange method.
Then, CM ion - ' _ resin or SP ion-exchange resin was filled up in columns, e.g., CM-3WS (available from Toso), Resource-S (available frAm Pharmacia) and , ' ' with the 10 mM phosphate buffer containing 8M urea (pH
7.5). Next, CKS-rp 36 antigen was passed over the above cation exchange resin 15 column. The column then was extensively washed, and the CKS-rp 36 antigen waseluted by step elusion with salt at a flow rate of I ml/min. The denatured CKS fusion protein then was coated onto human red blood cells type O, and used as an anigen in the PHA assay described r ' ._. Three thousand (3000) volunteer serum donArs were tested using the PHA kit described I ' . _ and wherein the test serum was20diluted at va~ious The results obtained were as ' in Table 2.
of F71c.~ R~srtiAnc (~ ' by E Coli 1:8 1:16 >1:32 Untreated 1 3 56 CM-Treated 4 1 0 25C. p~ tiAn for 9n~1 rstiAn~ ' ~ CKS-rp 36 antigen obtained by both of the above treatments were coated onto human red blood cells type O and tested by the PHA assay described I ~ . _. The dght (8) specimens which tested as false positive by the methods described in part (A) and (B) herein were WO 95/30902 ~ 1 ~ 9 ~ 4 8 ~ PCT/I~S94/051~2 retested using the antigen described herein and wherein the test specimens were diluted at various . The results obtained at~ ' in Table 3 li~LE~
~I-mh~ f FPICr Rr~rtitmc (`~--c.-A by E (~nli Number 18 1:16 >1:32 (e<gl 8) Unt~ated 0 0 0 8 D_AE Trated ~ 1 2 8 CM-Treated 3 4 1 0 FYPm,~l~ 2 Prel~aration nf HIV-R ' ' Antibody ~cc~y Rit H= red blood cells type O first were washeA with ~ , ' sale, pH
7.Q and then incubated with a solution contauling DEAE-CM treated HIV CKS-rp 3610 antigen. The cells were incubated in the antigen solution for two hours at room and washed u phosphate buffered saline. The antigen coated cell solution consisted of 1.0% (v/v) of the antigen coated cells in l~h.~ saline solution. F~.
XL 1 Iysates. fi,~ JM 103 Iysates and ~,~ pTB 21q/XL~1 Iysates, 20 llg/ml ' CKS Iysates and 20 ~Lg/ml purified ' CKS was utilized in the 5 PHA assay described herein when testing 3,000 volunteer serum donors. The results are ' in Table 4 below.
Blocking Agent Nurnber of False Reactions Caused by CKS
Control 16 QliXL,I Iysates 16 _~ilM 103 Iysates 16 ~,5~ pTB 210.XL,1 Iysates 0 P ' CRSIysates 0 Purified r ~ CKS~ o These dau indicat~ thatDEAE andlor CM-treatment of CKS-rp 36 HIV antigen can be 20 used to reduce the numbQ of false positive reactions caused by F, coli when the CKS

WO 95130902 2 ~ 8 ~ 7 4 8 PCT/US94/05152 materials used are denatured, as they were in this exarnple and Example 1, with 8M
urea Ey~m~nl~ 3. pr~n~ n r.fR~
A synthetic sequence of the DNA was prepared using the method described in U.S. Patent No. 5,124,255.
EVq~l.~ 4. ~rim~.n Dil The sample diluent comprisu g 10% (vh) bovine serum and 20% (vh) goat serum in 20 mM Tris phosphate buffer containing 0.15% (v/v) Triton X-100, 1%
(wh) BSA, and 500 ~Lg/ml or less CKS bacterial enzyme (prepared as described in Example 3) was prepared, wherein the ' CKS bacterial enzyme was denatured at 50C for a. . - 1~ 30 minutes before adding to the diluent.
r ~ 5. .~r1fi~ty 1` T~ f~ TlV Acc~y r, were conducted in order to assess false reactions due to CKS and determine if heat treatment of the ' bacterial enzyme would reduce false positive reactions. Former data had shown that the addition of CKS Iysate to the 2nd generation HIV assay (available from Abbott T ' Abbon Parlc, IL) did not increase specificity m the 2nd generation HIV assay by reducing the number of false ive reactions believed to be o~curring. C' , ~ r ' were designed ~ aetermine whether ~ ' of the ' CKS bacterial enzyme would improve specificity. A specifi~ity panel consisting of 10 memb~rs was selected for testing, along with positive and negative controls. The Abb~tt 2nd generation HlV
assay (available ~m Abb~tt T ' Abbott Par~, IL) was used in the Specimens were run m duplicate, with controls being run five times each.
The comparison was made between the diluent available as the standard assay reagent and a diluent which contained CKS ' bacterial enzyme (denatured at 50C
for 30 minutes), as described in Example 4. Assay protocol followed was as ....... ...... 1.. 1 by the ' of the assay, with readings obtained reported as optical density readings (QD.). The results are ' in the following Table 5.
/

WO 95130902 ~ 1 8 g ~ ~ 8 PCI'IUS94/05152 Sample Tested O.D. r ~ ~ ~ OD. Reading/Addition of Reagents Heat-Treated CKS to Negative Control ~ . a r,., O.r'f~;
C. ~: f~.f~
f. ~_ 0.~'~
Cj.: (I' Q(ll -Positive Control .11 . .' .
U.". ~ . 'I
' ' 7~
PandMemberl 0.-'- -~17' . _ r~ .
Pand Member 2 C . ~. Ci.
o. l~ r. o~
PandMember3 C~ u ~ ~-r,.o - ~j r PandMember4 C~ Cl.Ct3_ C~ Cl.f l_ Panel Membs 5 C~.J-t ~ Cl.rl~
~.' ' C~.C~
PanelMembcr6 C.:.~' Cj. JI~
Cl._ C. 3.
Pand Member 7 C~ J
C;.:~S J rJ.C3:
Pand Member 8 0.' ' ~.~ 3' ~ 11 O.Ci, Pand Member 9 .~ 1~ 0.r >,.C~)O c~.r Panel Member 10 0.__ . C .' J.
O.~ r~
These data; that several =bers of the 10-=ber specificity pand showed significant ~ .. (>50% OD. reduction) including members 1, 4, 5, 7, 8 and 9. Members 7 and 9 went from a positiYe test . to a negative test with pand member 9 showing the most significant effect.
F~ lr 6. ~r~ifirity r ' T~ c~i~ for HlV I~V 2 Test Parl~ A ccl~y r . were conducted in order to assess false reac~ons due to CKS and 10 determine if heat treatment of the . ' bactaial enzyme would reduce false positive reactions in the Abbott Test Pack HIV1/HIV2 Assay. Folmer data had shown WO 95/30902 PCTtUS94~05152 21~g748 that the addition of CKS Iysate to the HIV1/HIV2 Test Pacl~ Assay (available from Abbon I ' Abbott Parl~, IL) did not increase specificity in the ~V assay by reducing the number of false positive reactions believed to be occurring " 1 , were designed to determute whether .' of the ' CKS bacterial enzyme would improve specificity. A specificity panel consisting of the same 10 memb~rs described in Example 7 was selected for testing, along with positive and negative controls. The Abbott HIV l/HIV 2 Test Pack assay (available from Abbott T ~ Abbott Par!c, ~) was used in the ~ r ' Specimens including positive and negative controls were run in duplicate, and va~ious , of heat treated CKS ' bacterial enzyme were tested (12 ~nl, 25 llglml, 50 ~Lgfml, 75 ,ug/ml and 100 ~lg/ml). All heat treatment to denature the CKS bacterial enzyme was performed at 50C for 30 minutes. Assay protocol followed was as .~.. --- .. l.. ~ by the ~ of the assay, with readings obtained reported as positive, negative or I . ~Jil~L. _. The data obtained fromthis experinlent were similar to those obtained itt Example 5, with an , . . in specif~city being !' ' for the HIV1/HIV2 Test Pack assay when denatured CKS ' bacte ial enz~me was utilized. It was noted tltat ilt most instances, 12 llg/rnl was a sufficient - for iltcreasing specificity; however, it was noted that for panel member 9, the preferred of denatured CKS
bacterial enzyttte was 100 llg/ml.

_e~

Claims (15)

WHAT IS CLAIMED IS:

1. A method for detecting antibodies in a specimen which comprises the steps of:
a) mixing said specimen with a diluent comprising a recombinant denatured bacterial enzyme; and b) contacting said diluted specimen with at least one recombinant antigen expressed as a fusion protein with said denatured bacterial enzyme.

2. The method of claim 1, wherein said recombinant denatured bacterial enzyme comprises a recombinant protein.

3. The method of claim 1, wherein said recombinant denatured bacterial enzyme is CKS

4. The method of claim 1, wherein said recombinant denatured bacterial enzyme purified by passing it through an exchange column.

5. The method of claim 1, wherein the concentration of said recombinant denatured bacterial enzyme is from about 0.001 g/L diluent to about 1.0 g/L diluent.

6. The method of claim 5, wherein the concentration of said recombinant denatured bacterial enzyme is from about 0.01 g/L diluent to about 0.1 g/L diluent.

7. The method of claim 1, wherein said recombinant bacterial enzyme is at a concentration of about 100 µg/ml.

8. The method of claim 1, wherein said method detects anti-HCV antibodies.

9. The method of claim 1, wherein said method detects anti-HIV antibodies.

10. The method of claim 1, wherein said recombinant bacterial enzyme is denatured by heat.

11. The method of claim 1, wherein said recombinant bacterial enzyme is denatured by urea.

12. A diluent useful for detecting antibodies in a test sample when performing an assay which uses at least one recombinant antigen expressed as a fusion protein with a recombinant bacterial enzyme, which diluent comprises said recombinant bacterial enzyme which has been denatured.

13. The diluent of claim 12, wherein the concentration of said denatured recombinant bacterial enzyme is about 100 µg/ml.

14. A test kit useful for performing an immunoassay which comprises a container containing a denatured recombinant bacterial enzyme.

15. The test kit of claim 14, wherein said denatured recombinant bacterial enzymes is selected from the group consisting of CKS and SOD.