CA2056447A1

CA2056447A1 - Method for the collection of antibodies and the detection of a specific antibody species in a fluid sample and a device for carrying out said method as well as a kit comprising said device

Info

Publication number: CA2056447A1
Application number: CA002056447A
Authority: CA
Inventors: Stefan Gavojdea
Original assignee: TENTA PROPERTIES NV
Current assignee: TENTA PROPERTIES NV
Priority date: 1990-03-28
Filing date: 1991-03-27
Publication date: 1991-09-29
Also published as: AU7684791A; WO1991014944A1; BR9105168A; OA09402A; EP0474858A1

Abstract

2056447 9114944 PCTABScor01 A method for the collection of antibodies and the detection of a specific antibody species in a fluid sample. Said fluid sample is brought into contact with a solid phase matrix comprising a porous membrane and latex particles bound with recombinant protein A
or G, resulting in a specific binding of antibodies from the sample. An antigen that is specific for the antibody that is to be detected is subsequently brought into contact with the matrix and forms a detectable complex. The invention is also directed at a device and a kit for carrying out immunodiagnostic assays.

Description

WO 91/14944 1 2 0 5 6 4 4 7 PCI~/NL91/000~1 A ~e~
~pecif'i~ anti~dv sDe~i~s ~ ~_ out ~ai~ ~ethvd as w~ kit c~

~is invention concerns B ~ethod for t~e .coll~csion of ~ntibodies and the detection o~ ~ ~pec~fic ant~bod~ ~psc~es tn A fluid ~ample ~nd 8 device for carrying out ~aid l&ethod ~s well ~s a kit co~prising said device.
European patent application 0200381 describes a solid ph~se syste~ for use in B ligand-receptor Elssay for the detection Or a ~elected analyte in a fluid sample comprlsing a porous matrix, wherein ~icrospheres are entrapped, wherein said microspheres are bound wi~h a receptor capAble o~ c~pturing a specific target ligand. Upon use Or differen~ receptors either different target ligands cEn be bound or one species Or target ligand c~n be bound to di~ferent plsces for examp}e upon use of different antibodies that bind 'che antigen at different non-interfering epitopes. The s~oli~ phase according to EP-A-0200381 can consist of 8 membrane or filter made of glass fiber, nylon or cer~mic ~: ~aterial , wherein microspheres are ~ntrapped , whereby said microspheres 20 ~ ~re made of a polymeric material such as for example latex thht CE~Il bind to the receptor substsnce. Preferably a ~y~tem is used here consisting of membrsn2-microspheres-i~munological p~ir, whereby the : : pa~ir consists of antibody~antigen or ~ntigen-antibody. The ~ethod thst ; is described in this`EP-A comprises the ap,plication of fluid sample ~; 75 Oh the solid phase system, whereby ~he receptor that is b~und to the icrospheres specifioally binds ~he selected target ligand, as the luid through pssses the solid phase system. Su~seguently a sslution of a receptor eonjuga~e~is added, whereby s~id conjuEate is eapable of specirically binding ~nd labelling~ ~he target ligand in order for the bound target ligand to be ~detected ~n the ~olid~phase.: The term "reeeptor conJuga~e" is directed at a complex co~prising a receptor and a label~that can be !del~ec~ed.,I~ the ~arget ligand is ~n ~ntigen: the ~eceptor conjugate can be a labelled antibody preferably ~ ~onoclon~l ntibody. 9n the other hand If the ~ar~et~ ligand is an antibody ~ ~ 35 labelled an~igen can ~e used~ as ~cepto~ conjuga~ç. Non-bound receptor : ~ conjugate can subsequently be re~oved by rinsing. The pre~ence of bound recep~or conjugste on the ~olid phase system implies the: presence of

2~64~7 W O 91/14944 2 PCT/NL91~000~1 ~''" , .
.'the analyte in the ~emple. The EP-A also describes a device for carrying out such a ~ethod, whereby ~id device compri5es the solid phase sy~tem as described, whereby s~id device is provided with ~eans permit~ing the flow of a fluid s~mple through the ~olid ph~se ~ystem. ::
It is known that protein A generally binds i~munoglobulin and that it binds to Fc, it's constant region. (J.J. L~ngone, Advances in ~m~unology, Vol. 32, page 179-211).
Natural protein A is derived from aPl~yloco~cus aur~us, a pathogen, and is thersfore not free o~ ~$~h~1Q~Q~suQ toxins.
American patent 4,189,466 desrribes the detection Or the presence of rheumatoid f~ctor (RF) in an animal sample by mixing the s8mple with a suspension of microbial cells contsining protein A that have been sensitised with free Fc rrsgments of IgG for which RF i5 specific. If the mixing results in agglutination this indicates a 15^ positive result. ~:~
Further protein A is coupled to for example agarose beads via chemically stable amide bonds and this complex is used in affinity chromatography for sntibodies such as for example in American patent 4,879,211.
DuPont has ~o~e on ~o`the ~arket with a recombin~nt protein A
coupled to Seph~rose, whereby said protein is expressed in ~.coli, a non~pathogen rather than in the pathogen StaDhvlococ~us aur~ from ~ which the natural protein A is o~tained, which implies that ~he ligand ~::
:~ is fre~ of toxins. DuPont uses recombinant protein A in :~ 25 the~ per~lex 35RS ~ffinity column. The molecule contains 5 ho~ologous binding regions which are highly speci~ic for the Fc portion of munoglobulins. Immunoslo~ulin ~ (IgG) is~ the ~jor immunoglobulin which binds to recombinant protein A. Other immunoglobulins will also be sdsor~ed, however t~e class and sub-class ~pecificity varies fro~
species ~o ~pecies. The pro~ein A can therefore be us~d for purifying IgG or ~or selectively removing I~G for analysis of the XG classes or for absorbing i~munocomplexes ~or the purification of an`~igen`s. I
The ~-thod according to the inven ion is directed at ~ ~ethod :~ ~or the collect~on o~ antibodies and the detectio~ of a specific ~:
~ntib~dy species in~a fluid shmple comprising the step~ of : a) con~acting ~aid tluid sample with a solid phase ~a~rix. said ~ ~a~rix compr~sing porous ~embrane, latex particles distributed in said ~ ~ .
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W O 91/14944 2 0 5 6 4 4 7 PCT/NL~l/OVo~1 ~
,~.. ~, .
porous ~ewbrane, recombinant protein A or G being covalently bound to ~aid latex particles resulting in an aspec~fic blndin~ ~f antibodies present in the fluid ~mple to ~orm a complex [ant~bodies-protei~ A/G-latex-membrane], b) optiorlally removing excess fluid ~ample rrom sald 50lid phase ~atrix, ~ ) adding an antigen t9 8aid complex tantibodie~-protein A/G-; latex-membrane], ~aid antigen ~eing ~pecific for the antibody species to be detected to allow for~tion of a complex containing said specific antibody species: [antig~n-antibody-protein A/G-latex~m~mbrane], d) optionally removing excess ~ntigen from ssid porous m~trix, e) using a detection ~ethod specif$c for th~ antigen of step c).
The invention is directed a~ a method whereby ~ll antibodies in a fluid sample are entrapped on a ~olid phsse matrix and 1~ ~ubsequently one or optiona11y more antibodies can specifically be detected.
;The antigens and ~nti~odies that can be used with this ~ethod ,c:an be associated with bac~eria, fungi, parasites or virus. Gne can consider a retrov:irus:such as for example a~HIV-1 virus. This invention ~: 20: :is directed 8~ a new method for the collec~ion of i~unoglobulins from : for;example ~a body fluid for example ~or a diagnostic test. The invention is directéd a~ the collection and dete~tion of ~ntibodies f:rom:body fluids of a mammal (including ~ human being) such as ~west, tears~,:blood~, urine or preferably sa1iva.~t is ther~fore possible to use body~ fluids in which the a~ounts of antibodies ~re 1GW as fluid sample. : : The ~ubstance that can bind i~munoglobulin is a recombina~nt protein G ~r recombinRn~ pr~tein A derived ~rom E.coli or recombinant pro~ein A and :G and thi~ pr~tein is cova1ently~ bound to 1 tex-: 30 parti~les. ~hese latex particles are ~urface modified for ~example by carboxylate, amide and j~r hydroxy to ~na~le them to bind protein (page ; 30, Uniform ~atex Pa~ti~cles L~iR. Bangs). The 1atex~psrticle.~ boun~ with protein G, A or G/A are entrapp~d ~n a porous ~embr~ne and form the 801id phase ~trix. The porous ~e~brane can ~e ~de ~r example ~rom ~ 35 c~llul~se, ceramic ~aterial, gla~s ~iber. nylon or plostic.
: Polystyrene l~tex~icrop~rticles possess an in~rinsic lsrge surface whi~h allows a large contact area ~or ~he :immunogl~bulin :: :

W O g1/14Q44 4 2 0 5 6 4 4 7 PcT/NL9l/ooQ~l ~
~j;J binding substance, r~combinant protein A/G, and also for the sample fluid, resulting in a large amount of immunoglobulins that can be entrapped on the natrix, whereby ~he sub~tra~e for a very sensitive immunodiagnostic test is cr~ated. The high affinity of proteins G and A
for the Fc re~ion of i~munoglobulin permits a fast collection of immunoglobulin G's [IgG) and in connection with the lar~e surface area th~t is offered by latex microparticles this combination of protein A/G-l~tex microparticles provides ~n ideal ~ystem for the collection and detection of immunoglobulins in ~luids, p~rticularly in body fluids such as saliva which contain a snaller smount of antibodies than seru~.
Si~ultaneously the antigen position of the ceptured an~ibodies remains free as protein G and A bind to the Fc region of the antibodies so that these captured antibodies can react wi~h their free specific regions with the corresponding speci~ic antigens. The matrix consisting of membrane-latex-protein A/G plays the p~rt of coll~ctor of immunoglobulins and also serYes as solid phase carrier for the identification of antibodies in an im~unodiagnostic test. It is ~herefore only necessary ~o use one specifically binding antigen for ~ ~he detectLon, in contrast to~ the test described in EP-A-0200381, : 20 whereby at least two specifically blnding recepsors Or the analyte are necessary.
,, Furthermore 8 sample that is analysed according to the ~ethod :
.
of~ ~he invention can be examined for the presence o;f various ~pecific antibodies by use of dirferent solutions w~th specif:ic antigens. This : 25 was also~ possible w1th the method sccording ~to EP-A-0200381 however:
only by fillin~ various areas:of the matrix with microspheres bound to : varlous specific receptors,~ wherebg the detection poss1bilities of ~: different antibodies with such a ~atrix are~ restricted ~o a few: antibodies determ1ned at the preparatio~ ~r the matr:ix. This contrast `~
w~th the ~atrix used in ~he method according ~o the invention in which, in accordance with the antibody that is to be detected, a specific ~:corresponding anti~e~ s adde~after bindinglthe anti~odies ~nd whereby~, ~he matrix is suited ~or the binding of ~very antibody. In ~his ~ethod : every poss~le antibody species that is present in~:the ~a~ple is bound and therefore every antibody can be detected by use o~ a specific : ~ antigen. ,~ :
The specificity Or ~his detection methcd depends on the :;.

20~64~7 W O 91/14944 5 PC~/NL91/~00~1 t J antigen ~hat is selected as means of detection. In the ~ethod according to ~he invention one can 5elect the detection nethod of step e) from l~belling with an enzyme, with a radionuclide with ~luorescent agents, phosphorous s~ents, polymers contai~ing eolouring agents or chemilu~inescent portions, whereby preferably a detectio~ procedure is used that will ~enerate a colour change which can be detected by ~nstruments or prefer~bly by ~ight. When lsbelling with on enzy~e lt is possible So subsequently apply a substrate that brings about a colour change. For example 4-rhloro-l-n~phthol or TM~ (tetr~methy}benzidin) esn be used as substr~tes.
This invention is also directed at the use of ~ non-toxic, non-infectious complex of [recombinant protein A or G-latex-membrane]
for immunological tes~s as well BS a device for carrying out immunological tests according to the method of the inven~ion. This 15- device comprises a carrier containing the complex [protein A/G~latex-~embr~ne]. I~e carrier comprises an absorbent means incorporated in ~n elongated cont~iner with an opening at one end, where~y ~aid end contains said complex and said opening enables the entrsnce Or fluid via ~he co~plex absorbent ~eans. The absor~ent Leans is 2ssociated with the solid phase sys~em complex iD such a manner that the absorbent ~eans draws fluid ssmple, that comes into contact with the sol~d ph~se system, through th~-complex. Said absorbent means can ror example contain capillaries~in a direction perpendicular to the surface over whieh ,the solid phase system is placed so thst the capillaries are in direct communication with the pores Or the solid phase system.
embodiment of the device accordin~ to the invention is schematically illustrated in cros~s section 1n the drawing. In said drawing l represents an elongated container of synthetic ~aterial provided with an opening at one end in which a ~embrane permeablc for f1uid 2 has been plac~d~ Porous carrier 3 containing ~he complex ~protein A or G-latex] i5 present under ~enbr~ne 2 in the container l.
Carrier 3 is in c~ntact with a fluid ~bsorbing ~a~eri~l 4 tha~ can completely o~ partially fill the cont~iner l. Yarious ~aterials c~n be u~ed ss absorbent ~e~ns ~uch as cellulose acetate ~bers, cotton.
psper, ~la tic or polyester. Th~ device can further ~or exsmp1e be msde o~ plastic with the following dimensions, ~ length of approx~ately 4-14 cm, a depth o~ 1-5 mm and a wid~h o~ 0,5-5 cm with ~n opening with a W O 91/149~4 2 0 5 6 ~ 4 7 pT~g~uoo~l .. . .
-' di~meter of ~pproximately l-4 cm ~t one end. The absorbent means can have a length of 3-13 cm, a depth o~ 0,5-4,5 m~ and ~ width of 0,3-4,5 cm. ThB open end of th~ device cRn be cov~red with 8 membr~ne.
A preferred embodiment o~ ~he device is a device ~hereby the open end of the device containing the substance that is capable of binding immunoglobulin can be ~nserted ~nto the mouth Or a test person.
In particular for this ~pplication the i~port~nce of the use of recombinant protein A without toxins that sre present upon use of natural protein is an advantage. This is a new and extremely simple ~sst and handy method for collecting i~munoglobulins ~ and the further use of said~im~unoglobulins in an immunodi~gnostic tes~ on ~ solid phase matrix. It is only nece~sary to keep the device in the mouth for the duration of l to 3 minutes. Saliva that comes into contact with ~he ~atrix passes through the latex particles bound with protein A, whereby the immunoglobu}ins that are pre~ent wiIl bind quickly to protein A.
The size, and shape of the device Gske it simple to use and the : discsmfort of collecting blood or urine for an immunodiagnostic test is ~ prevented. ::~
:~ Furthermore this same device in which ~he immunoglobulins ~re collected serves as carrier to~be used in the identifiG~tion of desired ~: ~peci~ic antibodies associated with a cert~in dis~ase. Once the ..
sntibodies heve been bound to the protein A~latex particles membrhne;~
antigen of choice can be used to detec~ ~he specific antibodies from ; the collected saliva in an enzyme i-munotest. An e~ample of such a specific experiment in which saliva of a patient~ was coI1ected is escribed below. : ~
A patien:t known to be HIV-l-antibody~positive k~pt the device according to: ehe invention under the tongue ~or: 1 minute. Af~er c411eeting th~ saliva:O.5 ml of a buffer containing PBS/0,5X Tween~
20/2Z BSA/0.01% Thi~eros~l with pH 7.8 was pipet~ed on to the membrane ~o rinse the exces~ proteins.;Af~er the buffer~hsd ~een c~pletely ; ~bsorbed 0.2 ~l of a HIV-l-solution containing 1 micr~gram of`
:~iot~nilated protein recombinant envelope and ~ore HIV-~ in PBS/0,5%
. Tween-?0/0.l% gelatine was :add~d,and alss:~ab~orbed. The ~embrane was &ubsequently washed wi~h 0.5 ~l PBS/0.5% Tween-20/2% BSA/0.01%
~: Thimeros~l pH 7,8 and l.5 microgra~ o~ HRP labelled avidin was pipetted ~ on t~ the ~embrane. 1~ is also possible to bind biotin to avidin in : :

W O 91/14944 7 PCT/NL91/OOO~l ;.
`~;J ndvsnce 80 ~hat this step can be onitt~d during the tes~. The Dembrane was rinsed ~gain with 1 ml of PBS/0.5~ Tween-20/2% BSA/D.01% Thimerosal ~th pH 7.8. The buffer was absorbed again. Then 100 ~icroliter of a substrate, 4~chloro-1-naphthol, was pipetted on to ~he ~e~brane and 5 within 1 ~inute a positive blue colour developed, ~hara~terizing an sn~ibody positive reaction. Ihe total time required for the test was less than 5 minu~es. A ~imilar experiment was carried out on B patient known to be HIV~ ntibody ne~ative ~nd no colouring reaction of the device was observed, which is characteristic Or an antibody negative reaction. These examples show the u~ility o~ the device for oollecting antibodies from body fluid with a lower concentration ~f ~ntibodies than ia serum. The antibodies ~hat are ~ollected in this ma~ner are bound to a matrix in a concentration that is suited for a specific immunodiagnostic test. Furthermore the ~pe~lficity of the test has been : 15 demonstrEted upon use of a speci~ic entigen for detection of a certain antibody.
The protein A/polystyrene nicrop~rticles were ooupled covalently to each other in the rollowin~ manner:
: la~ex particles were used that had been c~rboxylate modified , (GML): with a diameter of 0.3-95 ~m and are co~ercially svailable from Serydin, Inc., Indian~poIis. The COOH-groups on the surface enable a ~: fast and simple formation o~ æ~ide b~nds via activa~ion of water soluble:carbodiamide. 1 ml of 10% solid latex particles with a diameter of~ 0.3-95 mm was rinsed with 10 ~; o~ delonised water via :25 centrifugation during 10 minu~es at 10-12,000 rpm. The water was ; c~refully~ removed by suction without sbsorbin~` the latex beads. The b~ads were resuspended in lO::ml deionised H20 and soni~ied for a short ; period. The bçads were mixed by using a vsrtex:~nd centrifuged during 10 ninutes at 1~-12,000 rpm. T=hi~ ~tep was repeated twice. ~ ring this rin~ing procedure the 0.07 uol~r carbQdiamide ~olution was prep~red: 67 ~g of DC (ethyl-3-t3-dimethylamino-pr~pylcarbodiamide-HCl~ (Pearce Rockford Ii~ with 5 ml of deionised H20 with i N NaOH~with pH 10 and 40 ~g of sulf`o-~ (N-hydroxy~ulfosuccina~ide ~ were ~dded ~d this was quickly dissolved. Af'ter the last rinsing step of the beads the w~ter

3~ w~s poured of~ and 3 ~l of the above-~entioned EDC/~ulfo-NHS-solution ~ere added. m e besds were uixed vigorously, onitied ~or a shor~
period ~f time and incubated at room te~perature under gentle agitation W O 91/l4944 ~ 2 0 5 6 417 PcT/NLgl/ooosl ,,~t !, t room temperature during 2 hours. After 2 hour~ o~ incubation the ~eads were rins~d another ~hree times with the dei~nl~ed waker as described ~efore. During this rinsing ~tep the -amino-N-caproic acid 801ution was m~de by dissolving 1 g ~-amino-N-caproic acid in 0.5 ~olar potassium phosphate with pH 7.4. The beads were resuspended in 3 ~1 of ~his ~olution. This wa5 power~ully sh~ken on a vortex and ~ncubated at room tempersture in a ~ixer for 8 hours. After said 8 hours of lncub~tion 0.3 ml of 1 molar ethanolamine was added to stop ~he reaction ~nd the solution was incubated at room temperature ~or 1 hour:
the beads were wzshed snoter three ti~es in 10 ~1 of deionised water and centrifuged during ten ~inutes st 12,000 rpm. During the rinsing steps ~ new carbodi~ide solution was prepared a~s described earlier.
The carbodiami~e activstion was repeated and ~t the end of the 2 hours of incubation the beads were rinsed ag~in in the deionised water as is 15 - described previously . The beads were resuspended in 1 ~l of 0. 5 KP04 with pH 7 . 4 and 1 mg of recombinsnt protein A was ~dded that had dissolved in 2 ml of O . 5 KP04 of pH 7 . 4 and was incuba~ced at room temperature over night. To stop the coupling reac~ion 0.3 ml of 1 nolar ethanolamine was added and wss ~ncubated for 1 hour Bt room ~emperature. The latex beads coupled to protein were ri.nsed again ss described ~ earIier wi~h deionised water. The latex beads were .

~ resuspended in 2 ml of PBS and ~ixed vigorously. The ~ixture was :~ sonified for 10 se~onds and vortexed again. This procedure was repeatedone more time. It is known that latex ~icr4particles:bind ~trongly to 25~ ~icroporous glass fiber. The selection of glass fiber ~embrane should ~: be: ~sde so that the latex particles cen be incorporated in the p~res ~f : the ne~br~ne. In this example a Schleier and Schuell gla~s fiber ~embrane No. 34 was used. The freshly prepared microparticles bound with protein A were dissolv~d 1:20 in PBS. 0.2 ml o~ dilu~ed letex ~: 30 microparticles/protein A was pipetted in the shape of a cros~ on~o thegl~ss ~iber ~embrane and allowed to dry at r~om te~perature. To prevent the non-speci~ic binding ~ o'ther proteins that cQuld distu~b the l~mun~çnzyme test the glass fi~er ~e~br~ne that contained la~ex-protein ~-c~plex was blocked with a blocklng buffer containing PBS/2~ BSA/0.1%
~elatine/~.5X Tw2en-20. In order to complcte the blockage khe ~embrane was plac~d ~n an ~bsorbent cushion/filter paper snd 2 nI of the b1ocking buffer were, added to the ~e=brane in order for the bufrer to W 0 91~14944 9 2 D 5 6 ~ 4 7 PCT~NLgl/ooo~l be absorbed by the membran~ fiO that ~n in depth block~ge of the glass fiber m~trix was acco~plished. The ~atrix was allowed to dry at room te~perature. The block~d ~atrix was stor~d before use in a plastic bag with a desiccator. These glsss f'iber-protein bound lstex particles serve as solid matrix for collecti~g immuno~lobulin ~ an increased concentrstion, which is obtained by 8) the large surface ~rea that is ~ffered by the latex microparticles and b) the known high affinity of protein A for immunoglobulin G.
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Claims

C L A I M S

1. A method for the collection of antibodies and the detection of a specific antibody species in a fluid sample comprising the steps of a) contacting said fluid sample with a solid phase matrix, said matrix comprising - a porous membrane, - latex particles distributed in said porous membrane, - recombinant protein A or G being covalently bound to said latex particles resulting in an aspecific binding of antibodies present in said fluid sample to form a complex [antibodies-protein A/G-latex-membrane].
b) optionally removing excess fluid from said solid phase matrix c) adding an antigen to said complex [antibodies-protein A/G-latex-membrane]
said antigen being specific for the antibody species to be detected to allow formation of a complex containing said specific antibody species:
[antigen-antibody-protein A/G-latex-membrane].
d) optionally removing excess antigen from said porous matrix, e) using a detection method specific for the antigen to step c).

2. The method according to claim 1, wherein said antigens and antibodies are associated with a bacterium, fungus, parasite or virus.

3. The method according to claim 2, wherein said virus is HIV-1.

4. The method according to any of claims 1-3, wherein said latex is surface-modified, e.g. modified by carboxylate, amide and/or hydroxy.

5. The method according to any of claims 1-4, wherein said fluid sample is a body fluid of a mammal including a human being, such as sweat, tears, blood, urine or preferably saliva.

6. The method according to any of claims 1-5, wherein said porous matrix is made of ceramic material, nylon, synthetic material or paper, preferably glass fiber.

7. The method according to any of claim 1-6, wherein the antibody to be detected is associated with the HIV virous, steps b) and d) comprise rinsing with a buffer having a pH-value in the range of 7-8.5, comprise rinsing with a buffer of HIV-1 solution containing biotin labelled recombinant envelope and core HIV-1 and step e) rinsing with a buffer having a pH-value in the range 8-8.5 and addition of a suitable labelled enzyme solution and addition of an non-mutagenic, non-carcinogenic substrate for detection.

8. Method according to claim 7, wherein said enzyme solution com-prises avidin labelled with HRP (horseradish peroxidase).

9. Method according to claim 7 or 8, wherein the substrate of step e is chosen from 4-chloro-1-naphthol or TMB (tetramethylbenzidin ).

10. Method according to any of claims 1-9, wherein the detection method of step e) is selected from labelling with an enzyme, with a radionuclide with fluorescent agent, phosphorous agents, polymers containing dyes or chemiluminescent moieties, preferably a labelling which will generate a colour change which may be detected by instrumental means or preferably visually by the human eye.

11. Use of a non-toxic, non-infectious complex [recombinant protein A or G-latex-membrane] for the collection of antibody from body fluids such as saliva.

12. Use of the complex according to claim 11 for immunological assays.

13. Device comprising a carrier which contains the complex [protein A/G-latex-membrane] suitable for the collection of antibodies from the mouth and/or carrying out immunological tests according to a method of any of the claims 1-10.

14. Device according to claim 13, wherein the carrier comprises an absorbent means in an elongated container with an opening at one end whereby said opening contains the complex according to claim 11 which is connected to the absorbent means in such a way that the absorbent means draws fluid that comes into contact with the complex through the complex, whereby the complex is also covered with a membrane layer which lets fluid through.

15. Device according to claim 14, wherein the absorbent means is chosen from an absorbent material such as cellulose, acetate fibers, porous synthetic material, cotton or wood.

16. Kit for carrying out immunodiagnostic assays comprising at least the following components:
1) at least one device according to the claims 13-15.
2) at least the components required for making buffers necessary for carrying out a method according to any of the claims 1-10.
optionally in the shape of one or more solutions, 3) at least a solution containing antigen for a specific antibody which is to be detected optionally already containing a label.

17. Kit according to claim 16 comprising at least a solution for making the antigen solution mentioned in point 3 of claims 16 detectable.

18. Kit according to claim 16 or 17 for detecting a HIV-1 antibody, characterized in that point 2 of claim 16 contains components for making a PBS/0.5 % Tween-20/2 % BSA/001 % Thimerosale buffer with pH 7.8 and the solution of antigen mentioned under point 3 of claim 16 contains a HIV-1 solution which contains 1 microgram of biotinilated recombinant envelope and core HIV-1 in PBS/0.5 % Tween-20/0.1 %
gelatine, that the detection mentioned in point 4 consists of addition of a solution of avidin-labelled with HRP and the addition of a solution of 4-chloro-1-naphthol substrate.