CA2046606A1 - Method to immobilize cardiolipin, phosphatidyl choline and cholesterol to solid phase and immunoassay - Google Patents

Method to immobilize cardiolipin, phosphatidyl choline and cholesterol to solid phase and immunoassay

Info

Publication number
CA2046606A1
CA2046606A1 CA 2046606 CA2046606A CA2046606A1 CA 2046606 A1 CA2046606 A1 CA 2046606A1 CA 2046606 CA2046606 CA 2046606 CA 2046606 A CA2046606 A CA 2046606A CA 2046606 A1 CA2046606 A1 CA 2046606A1
Authority
CA
Canada
Prior art keywords
solid phase
antigen
reagent
cardiolipin
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA 2046606
Other languages
French (fr)
Inventor
Dinesh O. Shah
Rajen C. Gandhi
John A. Todd
Jack Phillips
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baxter International Inc
Baxter Healthcare Corp
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of CA2046606A1 publication Critical patent/CA2046606A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Steroid Compounds (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

This invention relates to a method to immobilize cardiolipin, phosphatidyl choline and/or cholesterol individually or in combination on a solid phase and a sensitive and rapid assay for reaginic antibodies, such as syphilis, in human serum of plasma, using said immobilized cardiolipin, phosphatidyl choline and/or cholesterol.

Description

WO 91/10138 ;~09~6~6 Pcr/US90/07368 METHOD TO IMMOBILIZE CARDIOLIPIN, PHASE AND IMMUNOASSI~Y
_ _ BA~BQ~LQF TH~ INVENTION
1. Field of the Invent70n The present lnventlon relates to a method to immobll~ze cardlollpln (CARD), phosphatldyl chollne (PC), and cholesterol ~CHOL) lndlvldually or 1n co~blnat~on on a solld phase and the use of thls solld phase to assay for reagln1c ant~odles ln human serum or plasma, such as the ant~body present ln the serum of patlents wlth syphlli 5 .
: 2. Dlscusslon of Prlor Art 15The presence of reagln, an 'lantlbody-llke" substance ln human ; plasma or serum ls lndlcatlYe of syphllls d1seas~. Reagln ~or reaglnlc antlbodles) 1s measured uslng a varlety of tests. All of the above assays use the cardlollpln ant1g~n to detect reagln. Thls antlgen 1s a mlxture of phosphatldyl c~ol1ne or leclthln:
cardlollp~n:cholesterol in the ~elght ratlos of 2:0.3:9.
.~. . .The most cammon 1nvolve ~ard ~loeculatlon tests, such as the RPR
(Rapid Plasma Reag1n) card test. The RPR card t~st uses a carbon partlcle card1011pln antigen suspenslon that flocculates ~hen exposed to plasma con~a~n~ng Reag1n. The flocculat~on ~s noted ~lth the naked eye as a clumplng of black c~rbon p~rtlcl~s agalnst the ~hlte background of the card. Other tests, such as ~he VDRL
(Venereal Dlsease Ressarch Lab) or RS~ (Reagln Screen Test~ are based on ~he same flocculatlon prlnciple and use the cardlol~pln antlg~n. For example, one patent, U.S. Patent 4,73~,932, dlscloses 30 an ag9lutlnat1Gn test for syphtl~s asssclat~d an~lbod~es. The test uses an antlgen rsagent that comprls~s a buffered aqu@ous suspenslon of card~ol1pln antlgen lonlcally couple to la~ex partlcles vla a polyp2ptlde br~dge. The floc~ulatlon t~sts sre llm~ted 1n tha~ they are labor lntenslve ~hen scr~ening large nu~bers of spec~mens and ' WO 91~10138 PCr/U~i~0/07368 .~, . ,~
2~ 6 ~ 0~ - 2 -results are based on sub~ective lnterpretatlon.
Numerous lnvestlgators have trled to lmprove reagin tests by lncorporatlng the cardlol~pin antlgen lnto ELISA-type procedures.
These enzyme llnked lmmunosorbent assay (ELISA) methods are documented ln the sclentlflc llterature and lnvolve the adsorptlon of CARD PC and CHOL onto commerc1ally available polystyrene mlcrotltre plates. Further a R~agln ELISA ls commerc1ally avallable from ADI Dlagnostlcs. These ELISA assays are better than card tests for screening large numbers of spectmens (e.g. less labor interlslve) and do provlde for a numerlcal or seml-quantltatlve result; however they are not as speclfic or sens1tlYe as the RPR or VDRL tests ~l.e. they yleld more fals~ posltlve results). For example ADI Dlagnostics clalm a speciflclty of 95.4% and sensltlvlty of 82.2% compared to the VDRL test.
These ELISA types of assays however do not perform satls~actorlly 1n the presence of detergents such as Nonldet P-40 or Tween 20. Th~ use of detergents tn EIA or ELISA reduces reagent nonspeciflc blndlng and hence greatly enhances the sensltlvlty and speclf~clty of the assay. Slnce the detergent cannot be used ln these previously documented assay ~CARD PC and CHOL are presumably removed from the solld phase) they are not very sensltlve or speclflc. One advantaqe of the present lnYentlon ls that these detergents can be used 1n the assays wlthout comprom1slng assay performance.
2S SUMMARY OF T~E INVENTIQN
In accordance ~lth the present 1nventlon a method ls dlsclosed for lmmoblllzlng cardlolipln phosphatldyl chollne and cholestero1 tndlv~dually or in comb1natlon on a solld phase elther by passlve adsorptlon andlor covalent coupllng and the use of thls solld phase ln an assay for r@aglnlc antlbodies 1n human serum or plasma.
Add~ttona11y the current lnvent~on descrlbed hereln ~s an ~ssay that measur~s reagln seml-quantltatlvely ls designed for automatlon and ls as sensitlve and speclftc as the RPR card test.

' ' ' , ' . ' ., "' ' ' , ~ : . ` ' ;. ' ' ` " .

wo s1Jl~138 Pcr/US90/07368 20a~ 06 DET~IlEp DE~CRIPTI~N ~F THF INVENTIC)N AND BEST MODE
Thls tnventton relates to methods of tmmoblllzatlon of card1011ptn phosphat1dyl ehol1ne and cholesterol lndlv1dually or 1n combtnatton on solld phases elther by passlve adsorptton or covalent coupllng or a cc~blnat10n o~ both. The solld phase may be paramagnet1c partlcles nonparamagnet1c partlcles or any other solid phase. The 1mmoblltzat10n can be done by partlcular types of pass1Ye adsorpt10n or by covalent coupl!ng chem~stry. The sol1d phase compr1slng lmmobtlized eardtollptn phosphat~dyl chol1ne and/or cholesterol can be used ln an ~munoassay to detect the presence of antl-cardtollp1n antl-phosphatidyl chollne and/or ant1-cholesterol ant1bodtes (1.e. serolog1eal tests ~or syph11ts).
Card~oltptn (CARD) phosphat1dyl chol1ne (PC) andtor cholesterol (CHOL) ~hen coupled to a sol1d phase by the means descrlbed here1n have the follow1ng charaeterlsttcs:
a. Reta1n thetr ant1gentc character.
b. Are res1stant to the presence of detergents eammonly used 1n l~munoassays ~e.g. Nontdet P-40 or T~een 20) and remaln 1mmoblllzed at least 1n part to the soltd phase dur~ng the ~ourse of an assay.
c. Can be used 1n a serologlcal test for syphllts (l.e. detect the presence of antlbodies speclf1c for CARD PC and/or CHOL
~Reagin) ~h1ch are ~nd1cat~ve o~ a syphtlittc J1sease state) or other s2rolo~tcal tests to measure the presence of ant1-CARD PC
and/or CHOL anttbodles.
d. Are stable and retatn the1r anttgentc character after being - sub~ected to te~peratures of -20 ~ 3C 5 ~ 3~C 25 ~ 3C and 37 Another adv~ntage of th1s 1nventlon 1s that the serologleal test - for syph111s descr1bed above can pr~vlde a sensttlvtty and spectftclty stm11ar to those currently marketed to detect reag1n ~l.e. RPR or VDRL tests).
Yet another advantage of th1s 1nventlon 1s that the serologtcal test for syphilts descr1bed above can be used in an automated or manual system.

~; . . ,-:, , , . .. .,: .. . .

.. . . . ..

WO 9~/10138 PCI/US90/07368 2~ 0ti 4_ Stlll another advantage of the lnventlon ls the us~ o~ antigen ~mmobll~zed partlcles ln agglutlnatlon assays. Thls type of assay ls s~mllar tn style to the RPR test however the agglutlnatlon or clumplng of ant1gen lmmob111zed partlcles in the presence of a posltlve (reaglnlc reactlon) sample ts observed. ~he part1cles do not agglutlna~e or clump in the presence of a negatlve sample.
For pass1ve adsorptlon chemlstry we used dlfferent functlonallzed Pandex~ paramagnetlc part~cles (approxlmately ~ m - 100 but preferably 4.0 ~m 1n dlameter) U.S. Patent Appllcat~sns numbers 71113 294 7l337 511 7/337 513 7/337 244 and 7 337 234 herelnafter Wang and Shah appllcatlon (lncorporated by reference)~.
The functlonal group~ on these partlcles are amino dlmethylamlno and tr~ethylammonlum or other functlonal groups that lnteract strongly ~lth the negatlYe charge of the phosphate m~lety of CARD
and/or PC. Adsorpt10n chem1stry ls achleved us~ng elther physlcal adsorpt10n or concentratlon of CARD and PC on the surface of the paramagnet1c partlcles.
Sultabllity of partlcles prepared ln thls manner ~5 measured as good 1mmunoassay performance 1n the presence of low ~onc~ntratlons _. 20 of detergents. Sultablllty of the partlcles 1s dependent upon the funct10nal group used. Partlcles contalnlng trlethylammonlum are m~re sultable than dl~ethylamlno or amlRo Punctlonal groups.
Partlcles contalnlng carboxylated groups or hydrophob1c polystyrene partlcles are less sultable compared to trlethylamlno d~methylam1no and am1no functlonal partlcles. Thus passtve adsorpt~on does allow for the preparatlon of par$1cular partlcles that are seml-reslstant to the presence of low concentratlons of detergents. Covalent coupllng chemlstry however provldes for the lmmoblllzat~on of CARD PC and CHOL ln a form that 1s more res1stant to low concentrat10ns of detergent.
The fol10wtng covalent coupl1ng methods v1a polar head group and/or vla fatty acld mvletles can be used for CARD and/or PC:
a. SeO2 ox1datlon.
b- PCC (pyr~dlnlum chloroFhromate~ ox~dat10n.

.

.. . ~ , . , " .. . .. .. .

wo ~1/10138 P~/U~90/07368 c. M-chloroperbenzo1c acld ox~atlon.
d. 1 4-Butanedtol dlglycldyl ether ~oxlrane) coupllng.
e. Blotln coupllng ~n the presence of EDC tl-ethyl-3 (3-d~methylamino propyl) carbodllmlde].
f. Succln~c anhydrtde coupllng The ~odlfled CARD and/or PC ls coupled to elther amlno functlonallzed or carboxyl functlcnallzed or avld~n coated part~cles ln the presence of coupllng reagent 1f requ~red.
A11 the eoupling methods as mentloned above provlde for resultant part~cles that can be us~d 1n the assays to detect antl-CARD anti-PC or antl-CHOL antlbod~es ~serolog~eal ~ests for syphllls). However a cc~binatlon of SeO2 and PCC or SeO2 and EDC
chemistry for CARD and PC performs best. To lncrease the sens~tl-vlty and specif1clty of the t~st lncorporated ls cholestercl along ~lth CARD and PC. These mod~f1cations have been observed to greatly lmprove the test per~ormance. The r~tto (wel~ht) of ~ARD PC and CHOL u~ed for covalent coupl1ng 1s ln the range of 0.01-1 0.01-3 and 0.1~10 respectlvely but the best assay ls obtalned uslng the ratlo 0.1 0.3 and 8.
The technlques of ~mmoblllz~ng CARD PC and/or CHOL caD be applled to a var1ety of solld phases. These solid phases 1nclude but are not l~mlted to par~magnet~c partleles nonparamagnetic -- part~cles or mlcrotltre plates.
Ex~mples 1. Pass1ve adsorpt10n of CARD and PC on Solld Phase.
- 625 ul of trl~ethylal~momiu~ funetlcnaltzed paramagnetic part1cles (4~ w/v approx1~ately 4 ~m Wang and Shah appllcatlon) ~ere separated on a magnetic s~parator and clear supernatant ~s - removed by asp~ratton. To the r~sldual p~rtlcles was added 2 ml of ~ 30 CARD (Roach Labs Loganvllle GA; 5 mg 2.5 mg/ml 1n ethanol) 238 - ~1 of PC tRoach- Labs i 15 mg 63 mglml 1n ethanol) 5 ml of nonidetwP40 ~NP-40~. Ar~on ~5 purged to thts partlcles suspenslon to dryness. lhe pass~vely adsorbed part9tles were washed wlth delonized water and separated by etther ~agnetic separat~on or . ~ . ., , .. ,. ., . ,. . . , . , . - , . .

WO 91/10~38 PCI/U!~i9(~/0736$

~0~6~j~t~
centrlfugatlon untll the supernatant was clear. Flnally, partlcles were resuspended ln 5 ml of O.OlM acetate buffer, pH 6.5, contalnlng O.lX sodium az7de to glve 0.5% w/v concentratlon of partlcles.
2. Covalent Coupllng of CARD and FC Antlgens on Solld Phase.
6 ml of CARD (15 mg) and 682 ul of PC (45 mg) were evaporated to dryness. To the res1due were added 4 ml of methylene chlorlde and 135 mg of SeO2. The reactlon mlxture was tumbled for 4-6 hours at room temperature. In the meantlme, 30 ml of amlno ~unctlonallzed cross-llnked paramagnetlc part~cles (4.0% ~/v, approxlmately 4 ~m;
Hang and Shah application) were washed once w~th absolute ethanol for 5 mlnutes, followed by three times ln anhydrous d~methylfor-mamlde. To the resldual partlcles was added 1.2 gm of cholesteryl chloroformate ln 30 ml dlmethylformamlde follo~ed by the addltlon of 3 ml of trlethylamlne. After belng tumbled for 4-6 hours, the ~ 15 partlcles were separated on a magnetlc separator ind unreacted - cholesteryl chloroformate was removed by asplratlng the supernatant.
CARD and PC solutlon was flltered through an 0.1 u Teflon~
~11ter membrane, then drled by evaporatlon. The residue was redlssol~ed ln 30 ml of anhydrous dlmethylformamlde. Thls CARD and 20 ~ C- 501utlon ~as then added to the cholesteryl chloroformate treated res1dual partlcles, followed by the add1tlon of 1.05 gm of EDC and 3 ml of tr7ethylamlne. Thls reactlon mlxture was tumbled for approx1mately 16 hours at room temperature, then 530 mg o~ EDC and 18.0 ml of Q.05 M MES ~4-morphol~neethanesulfonlc acld) buffer, pH
6.0 was added. After belng tumbled for 6 hours, 250 mg of sodlum borohydrld~ ~as added and further tumbled for 1 hour. The coated partlcl~s were washed wtth delontzed water and s~parated by e~ther - magnetlc separatlon or centrifugatlon tlll the supernatant was clear. ~lnally, partlcles ~ere resuspended ln 200 ml of ~.01 M
acetate buffer, pH 6.5 contaln~ng O.lX w/v sodlum azldc to glve approxl~ately 0.5X w/v concentration of partlcles.
3. Magnetlc Part~cle Assay for Syphllls A. A human serum or plasma speclmen ~as dlluted 1:10 ln Solutlon A ~3.15 9 Trls.HCl, 1.21 9 Trls.base, and 0.2 9 sodlum .
.

W~ 91/10138 PCr~1S90/~7368 ;~46~0~

az~de; br1ng volume to lOOO ml ~lth Ml111-Q ~ater; pH 7.8). An aliquot of the d11uted specimen ~as then added to a ~ell ln a mlcrotitr~ plate ~5 ~l~ con~alnlng 20 ~l Solu~on A and 5 ~l Solut10n B ~4.346 9 sod1um phosphate dlbaslc, 0.524 9 sodluM
phosphate monobaslc, 5.~ ml Nonlclet P-40, 29.22 ~ sodlum chlorlde, and l.O g sodtum azlde; bring volume to l900 ml; pH 7.4). The ~oncentrat10n of Non1det-P40 (NP-40) and sodlum chlorlde were ad~usted to mlnlmlze nonspec1~1c blnd~ng o~ human IgM and IgG to the plate. Up to 16 dlluted spec1~ens ~16 ~ells) can be added to one p1ate, as the assay 1s run as part of a panel of 51x assays. The assay can be run 1ndependent of the panel as well ln whlch 96 di1uted speclm~ns can be added to one p~ate.
B. 20 ~l of speclmen dllutlon buf~er (750 ml calf serum, 43.83 g sodlum chlor~de, l.O g sodlum az1de, 9.58 9 TRIS base, ani 43.4 g . 15 adenoslne-5-monophosphate (AMP), herelnafter l'SDB") was then added.
The ca1~ sera and sod1um chlor1de concentratlons were deslgn~d to mlnlmlze nonspec1f1~ blnd1ng of human IgG and IgM to the - paramagnetlc partlcles added ln the next step. AMP m1n1mlzes false react10ns to the ant19ens coupled to the paramagnet1c partlcles and ls an essentlal component oF ~hls lnventlon. The phosphat~ of AMP
presumably competes for I~G and IgM blnd1ng to slmllar ep1topes on the partlcles. ~aramagnetlc part1cles dlluted ln phosphate buffered sallne ~PBS) ~ere then added. Paramagn~tlc part kles ~ere ~oated w~th CARD, PC, and CHOL tn a we1ght ratlo of 001;0.3:8. CARD, PC, and CHOL partlcles ~ere overc~ated for two hours ~lth heat ; lnactlvated calf sera (56C for 45 mln.), uslng rotatlon, ~ashed, ~ magnet1cally separated, supernate removed, and brought to volume : wlth Solutlon C (40346 9 sodlum phosphat~ dlbas1c, 0.524 9 sodlum . phosphate ~onobasic, 8.76 9 sod~um chlor1de, and 1 9 sodlum azlde;
br1ng ~olume to 1000 ml w1th Mllll Q water; pU 7.4).
- The chemlstrles of CARD, PC, and CHOL attachment to the part1cles has been opt1m1zed for eovalent coupl1ng. The presence of NP-40 ln the reactlon ~ell ~ith the part1cl~s do~s not totally re~ove CARD, PC, and CHOL fr~ the part1cles. In contrast, the : .. -, :: . . .
. . . . .. .... . .. . . . .... .

W O 91/lU13~ P~T/US90/07368 20~6606 - 8-presence of NP~40 ln the ELISA-type assays slgn~f~cantly dimlnishes posltive slgnal reactivlty presumably by removlng PC CARD and cholesterol from the wall of the ~LISA mlcrotltre plate. In the eurrent assay lnventlon the NP-40 besldes mlnlmlz~ng IgG and IgM
blndlng to the mlcrotltre plate also ~inlmlzes nonspeclflc binding to the partlcles thus lncreaslng the 5petlflclty of the assay (e.g. mlnlm~zlng false posltive reactlons). The antlgen coated paramagnetic partlcles ~partlcles) allow for maximum Reagln blndlng. Thls ls because of the large surface area added (4 X 105 part~cles/~ell; 4.0 u dlameter of partlcle) and the klnetics of reactlon. The klnetlcs are enhanced because the part~cles slowly settle to the bottom of the mlcrotltre plate well durlng the 30 mlnute lncubatlon. Maxlmum exposure to Reagln and resultant blndlng takes place durlng the 1ncubatlon.
In addltlon lt was observed that AMP mlnlmlzed false react~ons to the antlgens coupled to the paramagnetlc partlcle~ and was an essentlal component of thls lnventlon when paramagnetlc partlcles are used. The phosphate of AMP presumably competes for IgG and IgM
blndlng to s1mllar epltopes on the part1cles. The range sf AMP ls from 10 - 200 mmolar and pre~erably 50 100 mmolar. Phosphollp~ds and tn partlcular negatlvely charged phosphollplds such as phospha-tldyl serlne ~ork as ~ell to mlnlmlze false reactlons. Also tt should be noted that Thymldlne-s-~onophosph~te ~TMP) mlmlcs AMP
effect.
Along ~lth the partlcles coated ~lth CARD PC and CHOI a dlfferent type of part~cle ls added. Whereas the CARD PC and CHOL
coated partlcles blnd Reagln (lf present) the other type of partlcle ls deslgned to be nonblndlng. Instead lts role ls to provlde a marker for correct number of partltle dellYery per well and for ~ 30 partlcle loss ln subsequent steps. These paramagnetlc particles ; conslst ~f a Nlle red fluorescent core carboxylated surface and a coatlng of calf serum. The nu~ber of Nlle red partlcles added ylelds a predetermlned number of fluorescent counts per ~ell. (See Wang and Shah U.S. Patent Appllcatlons: Process to Make Wo 91/10138 PCI`/US90/07368 z~G60~i Magnetically Responsive Fluorescent Polymer Particles, U.S. Serial - , No. 07/452,0999 filed December 14, 1989; Magnetically Responsive Fluorescent Polymer Particles and Application Thereof, U.S. Serial No. 07/451,483, filed ~ecembPr 14, 1989; Method to Use Fluorescent Magnetic PolymPr Particles as Markers in an Immunoassay~ U.S. Serial No. 07/451,274, filed December 14~ 1989; Method to Use Magnetically Responsive Fluorescent Polymer Particles in a Molecular Diagnostic Assay, U.S. Serial No. 07/451,494, filed December 14, 1989.
C. Upon completlon of the lncubatlon, the partlcles 1n the wells ~ere ~ashed ~1th Solutlon D ~2.06 9 sodlum phosphate d~bas1c, 0.3l3 g sodlum phosphate monobaslc, 0.5 ml Tween~20, B.76 9 sodlum chlorlde, and l.O ~ sodtum azlde; brlnQ volume to 70~0 ~l w~th mlll1-Q water; pH 7.4) (PBS conta1n~ng ~e~n-20~. It was observed ; that the T~een-20 removed nonspec1flcally ~ound IgG and I~M. Agaln,dve t~ the coupllng chemlstry, CARD, PC, and CHOL rema~ned bound to the particles ln the presence of Tween-20. In contrast, lt has been obser~ed that Tween-20 ~sed ~n the ELISA procedures dlmln1shes posltlve spec1~en sl~nal generatlon, presumably by remov1ng antlgen from the surface of the mlcrotltre plate ~ells. Thus, th~ current 1nventlon allows for ~re thorough washgng and fewer falsê poslt~ve reactlons caused by nonspeclflc bindlng ~f IgG or IgM. Durlng the ~ash steps, the par~magne~1c part~cles ~ere held 1n the ~l~rotltre ~ell vl~ ~agnetlc ~leld applled to the bo K ~m of the plate.
Part1cles ~er ~ashed ln th1s manner S:x t1me5.
D. Partl~les were resuspended 1n 30 ~l of Solutlon C ~4.346 g sod1um phosphate dlbas1c, 0.524 9 sod1um phosphate monobas1c, 8.76 sod1um chlorlde, and l 9 sodtum azlde; brlng volume to lO00 ml ~1~h ml l l 1-Q ~t~r; pH 7.4) . 20 1ll of goat ant1-human IgG (H ~ L) ron~ugated ~lth B galactos1dase (Icon~ugate) was then added to the ~ells (d11uted~ 1n a solut10n of 300 mL ne~born ealf sera, sod1um chloride and phosphate buffer ~1.e. con~ug~te dllutl~n buffer ~240 ~l phosphate buf~er O.lM, pH ~.4, 60 ml glycerol, 1.2 9 sodlum az1de, 300 ml ne~born calf serum, 0.487 ~ magnes1um ehloride, 35.06 .,' . ' ., , ., .. : , : .

PCI`/VS90/07368 ., ~

2~L6606 - lo-.

of sod~um chlorlde, brlng volume to 1000 ml wlth mllll-Q H20, pH
7.2)). Any human IsG or IgM (lncludlng reaQ~n) that was bound to the partlcles will be recognlzed by and assoc1ated wlth conJugate.
The con~ugate solutlon was deslgned to glve maxlmum llquld stab~llty and reactlvlty. In partlcular, newborn calf sera ls preferred over calf sera. ~fter lncubatlon wlth con~ugate for 15 mlnutes, the partlcles ln the wells ~ere washed to remove essentlally all of the unbound con~ugate. Agaln, the Tween-20 ln the wash solutlon enhances the wash~ng process and removes nonspeclflca11y bound con~ugate.
E. Flnally, a substrate solutlon of 4-methyl-umbelliferyl-~-galactos1de (MUG) was added to the wells (0.178 9 4-methylum-belllferyl-b-D-galactopyronoslde, 3.58 9 tr1ctne, 5.1 ml dlmethyl sulfox1de, 30 ml methyl alcohol, 0.20 y sodlum a21de, 0.5 ml Tween-20, brlng volume to 1000 ml w1th ~ Q water, pH 8.5). The presence of B-galactosldase (e.s. con~ugate) ln the wells trlggered the cleavage of MUG to generate a fluoreseent product. Thls reagent and con~ugate ls used as a sensltlYe detectlon system. Fl~orescence (wavelength 400/450) ls measured at two tlmed lntervals (l.e. 2 and 14 mlnutes) post MV5 addltl~n. The dtfference bet~een the two values ~as a klnetlc measurement of flu~rescent product generatlon and 1s a d~rect measurement of con~ugate and human IgG/IgM (reagln) bound to the partlcles.
Posltlve spec1mens ~lve klnetlc values that were equal to or greater than the cutoff valuec The cutoff va1~e ~as approxlma~ely 4-5 tlmes the mean of negatlve test values.
To valldate the assay, each ~ell ~as evaluated for the pressnce sf Nlle red partlcle fluorescence (~avelength 525J580) at a prede-termlned level, 1nd1catlng no loss of partlcles and correct addltlon of part1cles to the ~ell. See Wang-Shah U.S. Patent Appllcatlon Ser~al No. 113,294, 337l511, 337,513, 337,244, and 337,234 relitlng t~ ~luorescent magnetlc partlcles and thelr use ln assays.
The ELISA procedure prevlously descrlbed ~y others, base thelr results on statlc Yalues (e.g. adsorbance value from one ;

.. : ~ .... ~. ;, . .. .

wo 91J10138 PC~/US90/07368 46~

determ~natlon). A statlc value conslsts of at least ln part opt~cal/system noise. Thls type of system nolse ls removed ~lth ~ klnetlc values. Thus the klnet~c value is mvre accurate.
The senslt~v1ty and speclflclty of thls rapld Reagln assay ls - 5 98.gX (94/95) and 99.8% (973/975) respectlvely compared ~o the Macrovue~ RPR test.
G. The Magnetlc Partlcle Assay Performed ln an Automated or Manual Mode.
The manual mode uses the above descrlbed steps of the assay except as follows: Specimens were dlluted ~1:100) dlrectly ~nto a comb~natlon of the Solutlons A B and SDB ln the rat10s o~ 25.5:20 respectlvely. Con~ugate was added dlrectly to the partlcles and ~as predlluted ~lth Solutlon C prlor to addlt~on.
4. VDRL ELISA Procedure (Reference: N.S. Pedersen et al J. Cl~n.
Mlcrsb1010gy 25 19) 1711-1716 (1987)) VDRL ant~gen (Cardlollpln O.~lSX phosphatldyl chol~ne O.lX
cholesterol 0.45X ~n ethanol) ~50 ~1) was drled to clear m~crotitre plate wells. The dr1ed YDRL antlgen was ~ashed wlth phosphate buffered sallne (PBS) three t1mes and then blocked w~th lOX bovlne serum albumln (BSA) ln PBS for one hour ~t rc~m temperature. Thls ; mixture was then ~ashed wlth PBS three t1mes. Sample was added for one hour at room temperature (dlluted 1:50 1n PBS ~ 19X BSA). Th1s mlxture was then washed ~lth PBS three t~mes. Conjugate ~as added ~beta-galactosldase eon~ugated to goat antl-human IgG (HIL chalns) dlluted ln PBS ~ lOX BSA) for 30 mlnutes at room temperature. Thls mlxture was washed wlth PBS flve tlmes. 50 ~1 of substrate ~as added (25 mg O~nltrophenyl beta-galactose m~xed ~nto 300 ~1 dlmethylformamide then added to 10 ml 0.1 M potasslum phosphate buffer pH 7.4 contalnlng 1 mM magneslum chlorlde) and ~ncubated for 40 mtnutes at 37C. The plate was read ~or absorbance at a wavelength of 405 uslng a B10tek mlcrst~tre plate reader.
The perfor~ance of the magnet~c part~cle assay for syph~l1s was compared to that of the VDRL LISA ustn~ the procedures as descrlbed above . Identl cal speclmens from the same donor ~ere tested wl th .
' w V 91/~013~
P~T/US90/0736s ~09~6606 12 -both assays. Cutoff values ~ere t:alculated ustng the same formula for both assays. From the results presented ln Tables lA and lB
the sensltlvlty and spectflcity of each assay compared to Macrovue~
RPR card te~t was determtned. 7he sensttlvtty and spectflclty of the magnetlc parttcle assay ~as lOOX ~or both wh~le sensltlvtty and spectftc~ty for the VDRL ELISA was 85.7X and 80X respectlvely.
5. The Speclftclty of the Magnetlc Partlcle Assay for Syphllls Relatlve to the Macrovue~ RPR Card Test ~as Determlned.
EDTA plasma spectmens (900) and serum speclmens (1025) were obtalned from random donors. Klnetlc fluorescent values were obtalned for each speclmen as well as posltlve and negatlve contro1s as described ln Example 3. An assay cutoff was calculated as:
PoS~tive ~oqtrol ~ Negattve Control ~ cutoff Z
For each speclmen the ktnet~c fluorescent value was dlvtded by the cutoff value to obtatn an lndex value. These lndex values were plotted ln a d7strtbut1On format to obtain the graphlc d~strlbuttons for the serum and EDTA plasma speclmens see Ftgures 1 and 2. Out of the total spectmens there ~ere three that ~ave lndex values greater than 1.0 ~e.g.: pos7ttve relattve to cutoff) yet were RPR
test nonreactlve. The rema7nlng 1922 were RPR nonreactlve as well.
Thus the ~agnettc part1cle assay speclf~c1ty ~as calculated to be ~1922/1925 X 100) - 99.84X. Plasma and serum spec~mens ylelded very stmllar performances.
; 25 6. The SenstttYtty of the Ma~netlc Partlcle Assay for Syph111s Relat1ve to Macrovue~ RPR Card Te~t ~as Determlned Us1ng the Assay Procedure Descrlb~d ln Example 3.
RPR reactlve spectmens (195) that ~ere conflrmed ~o possess antlbodles to treponema pal1tdum as ~ell (e.g.: conf~rmed pos~ttve ~0 speclmens) ~ere evaluated (see Table 2). F~ve spec~mens that were RPR reactlve were found negatlve wtth the magnettc partlrle assay.
The rema~nlng 190 spec1mens were reacttYe uslng e7ther test. Thus the senstttv1ty of the magn~ttc partlcle assay was calculated to be: l90/l9S X 100 ~ 97.4X.

- . ~ . . . . . . . . .

WO 91/10138 60~
20~ Pcrtus9o/o736x The 1nvent~on ~5 not llmlted to appllcatlons ~n serology of syphllls but may be appl~ed to other test systems ~hlch measure antl-CARD antl-PC or antl-CHOL antlbodies.
The use of paramagnet~c partlcles wlth a fluorescent core ls not 5 requ~r~d for optlmum assay performance. ~hey serve only as a marker for correct number of partlcle dellvery per ~ell and for partlcle loss measurement. Thus the assay ean be performed wlthout these part~cles as well.

~ 15 ., .
. ,.

~,, .
. .

.

W o 91/1013~ PCT/U~9OJ07368 ;~3466~6 - Table lA
Index Values of ELISA Test Relative to Cutoff RPR NEGATIVES RPR POSITIVES CALIBRATORS
Spec~men Spec1men I.D. Tnd~x* I.D. Ind~x~ _ . .Inde~
SP15 0.376 SP2 1.740 POS, CALIB. 1.55 (0.084 OD) SP16 0.413 3134 1:15 0.753 NEG. CALIB. 0.42 (0.023 OD) SP17 0.191 OLJ 4.438 SP18 1.611 OLK 9.549BLANK0.09 (0.005 OD
SPl9 0.734 SP6 1.919 SP20 0.746 SP39 1.722 SP21 2.407 OLL 8.092 SP22 0.654 SP23 0.481 SP2q 0.524 G3-3 0.567 SP43 . 0.555 SP4~ 0.2~3 SP14 0.5 SPZ6 0.462 SP27 0.919 - ` SP28 1.648 RPR ~ 2PR -SP29 0.~39 SP30 2.141 ELISA ~ 6 4 SP31 0.845 FLIsA - 1 16 Index ~s deflned as O.D. of Spec1men/Cutoff O.D.
Cutoff O.D. ls defined as (O.D. POS CALIB ~ O.D. NEG CALIB~/2 CUTOFF ~ (0.084 1 0.023)/2 ~ 0.054 O.D.
SENSITIVITY ~ 6i7 X 100 ~ 85.7 i SPECIFICITY . 16/20 X 100 ~ 80 X
MEAN ~EGATIVE VALUE ~ 0.84 , .

wo 91/1013~ PCrtU!~;90/0736X
6~)6 Table lB
Index Values of Magnetic Partlcle Assay Relatlve to Cutoff RPR NEGATIVES RPR POSITIVES CALIBRATORS
S Spec~men ~peclmen I.P. lQd~* I.D. Ind~x~ _ _Index~
SP15 0.2 SP2 2.31 POS. CALIB. 1.561 SP16 0.12 3134 1:15 1.57 NEG. CALIB. 0.438 SP17 0.13 OLJ 7.7 - 10 SP18 0.27 OLK 7.87 BLANK 0.104 - SPl9 0.42 SP~ 1.73 SP20 0.13 SP39 2.33 SP21 0.22 OLL 18.27 SP22 ~.14 SP~3 O.lS
: SP24 0.19 G3-3 0.44 SP43 0.26 SP44 0.~6 SP14 0.89 SP~6 0.31 : SP27 0.13 SP28 0.09 B~EL~ R~R -SP29 0.3 SP30 0.15 MAGNETIC PARTICLE ~ 7 0 SP31 0.19 MAGNETIC PARTICLE - O 20 d Index ls defined as fluorescence of Speclmen/Cutoff fluorescence Cutoff fluorescence ~s ~efined as (f1uorescence POS CALIB
~luoresce~ce NEG CALIB)/2 ~ 3368 fluorescence un~ts .
5EN5ITIVITY . 7/7 X 100 ~ 100 ~
~PECIFICIrY . 20l20 X 100 ~ 100 X
MEAN NEGATIVE VALUE ~ 0.22 .

wo 91/10138 Pcr,~US90/0736~

2~ 66~)6 Tab 1 e 2 Pandex~ Assay Performance w1th RPR React1ve Spec1mens Conf~rmed Poslt~ve for Syphll1s (F'oslt~ve T.pallidum Antlbodles) DEGR~E OF RPR REACTIVITY. PANDEX
Pos~t~ve Negatlve '/- lO 3 1~ 19 2~ 34 3~ 121 0 4i ~ 0 - '

Claims (29)

We Claim:
1. A stable, detergent resistant, bioreactive solid phase comprising the antigen reagents cardiolipin, phosphatidyl choline and cholesterol individually or in combination adsorbed or covalently coupled to a solid phase.
2. The composition of claim 1 wherein the weight ratio of cardiolipin, phosphatidyl choline, and cholesterol is in the range of 0.01-1, 0.01-3, and 0.1-10 respectively.
3. The composition of claim 2 wherein the weight ratio of cardiolipin, phosphatidyl choline, and cholesterol is 0.1;0.3:8Ø
4. The composition of claim 1 wherein the solid phase is neutral or modified to have positively or negatively charged or reactive surface moieties.
5. The composition of claim 1 wherein the solid phase is particles of size range of about 0.1 to. 100 µm.
6. The composition of claim 1 wherein the solid phase is particles of size range 2 to 8 µm.
7. The composition of claim 1 wherein the solid phase is particles functionalized with functional groups for passive adsorption of cardiolipin and phosphatidyl choline.
8. The composition of claim 1 wherein the solid phase is paramagnetic.
9. The composition of claim 1 wherein the solid phase is cross-linked amino functionalized paramagnetic particles for passive and/or covalent coupling of cardiolipin, phosphatidyl choline, and cholesterol in the presence of organic solvents.
10. The composition of claim 1 wherein said antigen reagents are modified with a reagent prior to covalent coupling to said solid phase.
11. The composition of claim 10 wherein at least one of the antigen reagents is oxidized to obtain a functional group for covalent coupling to said solid phase.
12. The composition of claim 1 wherein cardiolipin is chemically modified prior coupling to obtain a function group for covalent coupling to said solid phase.
13. The composition of claim 12 wherein said chemical modification occurs by mixing cardiolipin with 1,4- butanediol diglycidyl ether (oxirane), with biotin in the presence of EDC or with succinic anhydride.
14. An antigen reagent for use in a reaginic test for syphilis comprising of cardiolipin phosphatidyl choline, and cholesterol individually or in combination adsorbed or covalently coupled to a latex particle.
15. The composition of claim 14 wherein the weight ratio of cardiolipin, phosphatidyl choline, and cholesterol is in the range of 0.01-1, 0.01-3, and 0.1-10 respectively.
16. The composition of claim 14 wherein the weight ratio of cardiolipin, phosphatidyl choline, and cholesterol is 0.1;0.3:8Ø
17. The reagent of claim 14 wherein the solid phase is neutral or modified to have positively or negatively charged or reactive surface moieties.
18. The reagent of claim 14 wherein the solid phase is paramagnetic particles of size range of 0.1 to 100 µm.
19. The reagent of claim 14 wherein the solid phase is paramagnetic particles of size range 2 to 8 µm.
20. The reagent of claim 14 wherein the solid phase is paramagnetic particles functionalized with functional groups for passive adsorption of cardiolipin and phosphatidyl choline.
21. The reagent of claim 14 wherein the solid phase is cross-linked amino functionalized paramagnetic particles for passive and/or covalent coupling of cardiolipin, phosphatidyl choline, and cholesterol in the presence of organic solvents.
22. A reaginic test for syphilis associated antibodies comprising:
a) incubating a diluted specimen sample suspected of containing said antibodies with the antigen reagent of claim 14 under conditions that permit binding between said antibodies and antigen in said antigen reagent;

b) determining whether the antigen reagent has agglutinated, agglutination indicating the presence of said antibodies in said sample.
23. The method of claim 22 wherein the specimen is either serum or plasma.
24. The method of claim 22 where said sample is diluted with a buffer containing calf sera and a phosphate containing compound.
25. The method of claim 22 wherein said phosphate containing compound is either AMP, TMP or phosphatidyl serine.
26. A serologic test for syphilis associated antibodies comprising:
a) incubating a diluted specimen sample suspected of containing said antibodies with the antigen reagent of claim 14 under conditions that permit binding between said antibodies and antigen in said antigen reagent;
b) determining whether said antigen reagent has agglutinated, agglutination of said antibodies in said sample.
27. An assay for syphilis-associated antibodies comprising:
a) incubating sample suspected of containing said antibodies with the antigen reagent of claim 9 under conditions that permit binding between said antibodies and antigen in said antigen reagent;
b) washing to separate material unbound to said latex particles;
c) incubating said sample antigen reagent with a labeled immunoreactive agent; and d) washing to, detecting, or determining the unknown by detecting or determining the labeled immunoreactive agent.
28. The assay of claim 14 wherein the labled is radioactive, enzymatic, chemilumenescent, or fluorescent.
29. An assay for a reaginic antibody comprising:
a) incubating sample suspected of containing said antibodies with the antigen reagent of claim 9 under conditions that permit binding between said antibodies and antigen in said antigen reagent;
b) washing to separate material unbound to said latex particles;
c) incubating said sample-antigen reagent with a labeled immunoreactive agent.
CA 2046606 1989-12-27 1990-12-12 Method to immobilize cardiolipin, phosphatidyl choline and cholesterol to solid phase and immunoassay Abandoned CA2046606A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US45746689A 1989-12-27 1989-12-27
US457,466 1989-12-27

Publications (1)

Publication Number Publication Date
CA2046606A1 true CA2046606A1 (en) 1991-06-28

Family

ID=23816853

Family Applications (1)

Application Number Title Priority Date Filing Date
CA 2046606 Abandoned CA2046606A1 (en) 1989-12-27 1990-12-12 Method to immobilize cardiolipin, phosphatidyl choline and cholesterol to solid phase and immunoassay

Country Status (5)

Country Link
EP (1) EP0460172A1 (en)
JP (1) JPH04503865A (en)
AU (1) AU7030991A (en)
CA (1) CA2046606A1 (en)
WO (1) WO1991010138A1 (en)

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5780319A (en) * 1996-04-19 1998-07-14 Pasteur Sanofi Diagnostics Immunoassays to detect antiphospholipid antibodies
US5776487A (en) * 1996-04-19 1998-07-07 Pasteur Sanofi Diagnostics Liposome reagents for immunoassays
US6177282B1 (en) 1997-08-12 2001-01-23 Mcintyre John A. Antigens embedded in thermoplastic
GB0104057D0 (en) * 2001-02-20 2001-04-04 Babraham Inst Antiphospholipid antibody syndrome
DE10250368A1 (en) * 2002-10-29 2004-05-19 Viramed Biotech Ag Means and methods for diagnosing treponema infection
CA2603814C (en) 2005-04-18 2013-08-27 Bio-Rad Laboratories, Inc. Solid phase immobilization of phospholipids and cofactor proteins via covalent attachment
CN101243321A (en) 2005-06-21 2008-08-13 美国政府健康及人类服务部，疾病控制和预防中心 Methods, immunoassays and devices for detecting anti-lipid antibodies
US8148057B2 (en) 2005-06-21 2012-04-03 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention Methods, immunoassays and devices for detection of anti-lipoidal antibodies
US8778619B2 (en) 2005-11-18 2014-07-15 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Oxidized cardiolipin and uses to detect cardiolipin antibodies
EP1949108B1 (en) 2005-11-18 2013-09-25 THE GOVERNMENT OF THE UNITED STATES OF AMERICA as represented by THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES Modified cardiolipin and uses therefor
JP4629563B2 (en) * 2005-12-07 2011-02-09 積水メディカル株式会社 Antiphospholipid antibody measurement reagent
EP1956373B1 (en) * 2005-12-07 2017-01-25 Sekisui Medical Co., Ltd. Method for assaying anti-treponema pallidum antibody
FR2994482B1 (en) * 2012-08-13 2014-08-22 Repropharm COMPOSITION, METHOD AND KIT FOR DETECTION OF PREVULATORY PIC OF LH
CN107533060B (en) 2015-03-10 2019-11-05 生物辐射实验室股份有限公司 Combine treponema and the test of non-treponema syphilis
RU2633087C2 (en) * 2016-03-23 2017-10-11 Сейфаддин Гашим Оглы Марданлы Stabilized cardiolypine antigen for the reaction of microprecipitation with syphilisis diagnosis and the method of its reception
WO2018000381A2 (en) * 2016-06-30 2018-01-04 深圳市亚辉龙生物科技股份有限公司 Modified cardiolipin-coated magnetic nanobeads and preparation method therefor
WO2018000339A1 (en) * 2016-06-30 2018-01-04 深圳市亚辉龙生物科技股份有限公司 Modified cardiolipin-coated magnetic nanobead and preparation method therefor

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3564089A (en) * 1966-09-29 1971-02-16 Sandra Jean Kiddy Diagnostic reagent for syphilis
US4738932A (en) * 1985-12-03 1988-04-19 Advanced Polymer Systems, Inc. Reaginic test for syphilis
JP2736467B2 (en) * 1987-10-26 1998-04-02 デイド、インターナショナル、インコーポレイテッド Manufacturing method of magnetically responsive polymer particles and its application

Also Published As

Publication number Publication date
WO1991010138A1 (en) 1991-07-11
EP0460172A4 (en) 1991-10-01
JPH04503865A (en) 1992-07-09
AU7030991A (en) 1991-07-24
EP0460172A1 (en) 1991-12-11

Similar Documents

Publication Publication Date Title
CA2046606A1 (en) Method to immobilize cardiolipin, phosphatidyl choline and cholesterol to solid phase and immunoassay
EP0267317A1 (en) Method for the detection of proteins and viruses
CA1308349C (en) Agglutination immunoassay and kit for determination of a multivalent immune species using a buffered salt wash solution
CA1336395C (en) Indicator reagents, diagnostic assays and test kits employing organic polymer latex particles
EP1049808B1 (en) Force discrimination assay
CA1264662A (en) Immunoliposome assay - methods and products
US4738932A (en) Reaginic test for syphilis
US5145784A (en) Double capture assay method employing a capillary flow device
JP3330604B2 (en) Solid-phase immunoassay
US5375606A (en) Bio-analytical separation method
US4060597A (en) Serological reagent and preparation thereof
JPH0296567A (en) Detection of analyte using acrydinium ester and liposome
EP0280559A2 (en) Agglutination immunoassay and kit for determination of a multivalent immune species using a buffered salt wash solution
JP6607735B2 (en) Method and reagent kit for measuring cholesterol uptake ability of lipoprotein
US20160327555A1 (en) Method for avoiding influence of endogenous lipoprotein and reagent
JP2003528289A (en) A method for detecting binding reactions using measurements of the relaxation of the birefringence of magnetic particles
ES2575902T3 (en) Chemiluminescence Enhancer
US6258607B1 (en) Indirect agglutination immunoassay and apparatus therefor
JPH0467916B2 (en)
KR20010025027A (en) Immunoassay reagents and immunoassay method
WO1990002334A1 (en) Reagents and methods for determination of analytes
WO2019117585A2 (en) Composition for diagnosing tuberculosis and method for diagnosing tuberculosis on basis of change in optical characteristics
US20050282237A1 (en) Immunological analysis carrier and an immunological analysis method using the same
EP2653867A1 (en) Biotracer and its use in an immunoassay
CA1039648A (en) Visual method for the detection of syphilis and other treponemal diseases

Legal Events

Date Code Title Description
FZDE Dead