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1 This applica~ion is a divisional applica-tion oE
Canadian patent application serial number 262,333 fi.led on September 29, 1976 and assi.gned to Cordis Corporation.
BACKGROUND OF THE INVEllTION
This inventi.on rela~es, in general, to a me-thod for detecting the presence of an-tigens associa-ted with hepati-tis.
In particular, it rela-tes to an irQmunoassay for antigens associa-ted with hepatitis involving the use of an enzyme-tagged antibody which reacts with an antigen to detect the presence of hepatitis.
Hepatitis, which means "an inflammation o~ the livex", is due to an infection or obstruction of the bile channels.
There are thought to be two varieties of viral hepatitis, one hav- :
ing a longer incubation period than the other~ In the past, when a patient contracted hepatitis and had a known parenteral ; exposure, the hepatitis was termed "serum hepatitis". If the ` patient did not have a known parenteral exposure and he con-tracted hepatitis orally it was called "infectious". However~
it has been documented that in addition to having overlapping , ~ ~o incubation periods, "infectious hepatitis" can be contracted ~ ~ .
parenterally, and, the so-called "serum hepatitis" can be contracted orally. Thus, although there would appear to be two ~orms of hepatitis caused by at least two distinct agen-ts, the terms~"serum~hepatitis" and~"infectious hepatitis" should not he used to distinguish them. Accordingly, it has been suggested that the terms "hepatitis A" be used to desisnate the form most ~ closely resembling "infectious hepatitis", and, the term 'ihepatitis i ~ B" be used to designate the orm most closely resembling "serum hepatitis~
~: 3~ The examples appearing in this specification are directed to the detection of the antigen or antigens a.ssoc.iated with hepatitis type B. Patients who contract the form most ~closely resembling serum hepatitis, no matter how contracted~
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1 ~ ten have these antigens in their blood. A-t this point, it should be noted that there is no reliable assay for determining the presence of an antigen associated with hepatitis A or a hypothesized hepatitis C. Thus, the examples in this specifi-cation are directed to the detection of the presence of those antigens associa-ted with hepatitis s. However, there is no reason why the process of the present invention could not be used to detect the presence of antigens associated with other types of hepatitis once their antigens have been identified.
Contraction of "serum hepatitis" or hepatitis B
creates a serious clinical problem that cannot be ignored.
Because of the severity of this problem, a variety of test methods for the detection of hepatitis have been developed These included Micro~Ouchterlony, immunodiffusion, complement fixa-tion, immunoelectro-osmophoresis, haemagglutination and haemagglutionation inhibition, electron microscopy, and solid ! phase radioimmunoassay. See British Medical Bulletin, 1972, ; Vol. 28, No. 2 (Viral Hepatltis) pages 138 - 141 for a brief description of each.
~o Immunoelectro-osmophoresis or counterelectrophoresis .
(CEP) provides a rapid, simple method for the de-tection of the hepatitis antigen and its antibody. However, this technlque is somewhat less sensitive then, for example, complement fixation. Its principle advantage is that tests can be completed within two hours~ However, because of its low sensltivity level, CEP is no longer approved by the Food and Drug Administration.
The application of radio-immunoassay ~RIA) for routine diagnos~ic purposes is believed to be somewhat limited, : :
not only because of the relatively complex, specialized/ and 30 -expensive equipment necessary ~or conducttng the -test, but also
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1 ecause of the stric-t precautions required in handl:ing radio-active isotopes. Isotope tagging presents a serious potential health hazard, requires monitoring, and ~tomlc En~rgy Co~nission licensing ~for user and manufacturer), and presents was-te disposal problems. Nevertheless, this technique is now rather well established for il~nunoassay.
Immunological methods depend, of course, upon a primary characteristic of all antibodies and an-tigens~ i.e., their ability to react with a specific complementary antigen or ; 10 antibody. Thus, if an antibody is added to a serum con-taining its antigen, the antibody and antigen will complex and may precipitate from the solution. In most of the above-mentioned test methods, the presence of antigens in human sera is detec-ted by making use of this simple fact.
Labeled antibodies have been used previously for identifying various antigens. If an antibody known to be specific for a particular antigen is isolated from the globulin portion of serum or plasma of a host animal which has been stimulated to produce that antibody, it can be labeled or ZO tagged by known means. By conjugating the antibody with a labeling agent, e~g., a physically detectable substance such as a radio isotope, as above-mentioned, or fluorescent chemicals, the presence of the anitbody can be detected. Thus~ when used diagnostically, if the counterpart antigen is present in some prepared test sample, the labeled antibody will attach itself :: : :
to that antigen, and the presence of t~e antigen can be confirmed through detection of the labeled antibody in the sample.
A labeled antibody, for diagnostic purposes, should be made sufflciently specific so that it will react only with those ~30 antigens~whose detection is desired and without cross-reaction ~ -
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th other closely related antiyens which may have quite dis-similar or insi~niEicallt conse~uences. Thus, i-t is apparent that bo-th the source and -the manner of prepara-tion of the anti-body is quite critical ln any immunoassay.
One manner of detectin~ hepatitis associa-ted antigen, as earlier mentioned, involves solid phase radio-i~nunoassay.
Such a precedure is disclosed in United States Pa-tent No. 3,867,517.
~s disclosed therein, in the performance of the assay, a tube well, or insert for use therewith, of molded polystyrene 10 is first coated with antibody. This is accomplished by incubating the member to be coated with an antibody solution.
~fterwards, the unknown sample is incubated with the coated well or insert to react the antibody with antigen present in the sample. The well is then washed and incubated with an antibody labeled with the radioactive isotope I-125. It is then again washed to remove any unbonded labeled antibody~ Thus, in the event any an-tigen is present in the test sample, a sandwich is formed from the polystyrene well (or insert)~ the antibody, the antigen, and the I-125 tagged antibody. The radiation emitted from the I-125 tagged antibody is then counted and compared against a control.
It has also been disclosed that a disc of poly-tetrafluoroethylene onto which is grafted a substituted poly-styrene, e.g., isothiocyanostyrene, might be useful in performing radioimmunoassay. This polystyrene is an insoluble material having specifically desi~ned surfaces of protein-reactive groups which ma~ be used to covalently bond proteins to provide ~: ~ . . , a reagent useful in bioassay procedures.
Recently, an important alternative to labeling anti-bodies with radio-isotopes or fluorescent chemicals has been ~':
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1 veloped. This involves :Labeling or tagqillcJ an antibody wi-th an enæyme. Such a procedure is descri.bed irl U~S. Paten-ts 3,654,090 and 3,791,~32. In Clinica Chemica Acta, 48 (1973) 15-18, an enz-yme-~linked immunoassay for alphaEetoproteln hy either the competi-tive or the sandwich procedure is disclosed.
Compared to radio-immunoassay, enzym~ labeling ofers severcll important advantages. For example, every enzyme tagged molecule in the final mix-ture participates in the readout. On the other hand, only a very small proportion of iso-top:ic atoms lO in the final mixture undergo decay duxing rea~out to participate in the assay. In enzyme tagging, every tag repeatedly parti-cipates in readout, by attacking many substrate molecules to form a detectable end product, e.g., up to 100,000 times per minute, hence greatly enhancing sensitivity. An isotope tagged atom decays only once during readout, after which it is lost from participation. An enzyme tagged reagent has a long shelf lifè; whereas, the isotope tagged reagent is constantly decaying, and presents serious shelf life problems. While there is minimal health hazards associated with using an enzyme ~O tagged rea~ent, serious potential health haza~ds are encountered ; wi-th isotope tagging. Lastly, an immunoassay involving enzyme tagging can use simple, relatively inexpensive equipment for readout. The success o isotope immunoassay~ in contrast, is dependent on the efficiency of detecting decay, and hence on the ~ quallty of very expensive detection equipmen-t.
`~ Although the finding of an antigen associatecl with hepatitis in one's blood may not be the equivalent ~f obtaining , a clinical history of hepatitis, investi~ations have revealed a high incidence o hepatitis infection when a patient has received~blood which tests positively for antigens associa-ted with - 5 ~
' ' 76~ ( 1 ~epati-tis. Since decisions on whether -to use particular bloocl units available from a blood center must often be made in a realtively short time period, a sensitive,rapid, easy to per~orm screening test Eor hepati-tis, withou-t need for expensive equipment, is of extreme importance. ~lthc,ugh the ~arious -tests used in the pas-t for the detection of the an-tigens associated with hepatitis have been satisfactory to a degree, they are all attendant with one or more disadvantages.
Each year hepatitis causes thousands of deaths and hospitaliza-tions. It has long been thought that the key to bringing the disease under control would be a technique for screening blood which could be made available world-wide and could be conducted simply and routinely. While, as indicated above, there are now a number oE techniques available, because they are relatively insensitive, require a lengthy and detailed procedurè, require the use of sophisticated equipment not 1 readily available in most hemotology labs, or require the use of reagents which are highly unstable and thus cannot be main-tained at hand, they have not satisfied the demands of the ideal test.
SUMMARY OF THE INVENTION
The invention broadly involves a direct immunoassay for antigens associated with hepatitis involving the "sandwich"
principle. II1 the performance of the assay r antigen to be de-tected is sandwiched between antibody layers which react with it. One antibody layer is labeled with an enzyme. The other layer is covalently bonded to an insoluble member. The en~yme is exposed to a chemical substrate which will undergo a chemicaI change to produce a reaction product in the presence of the enzyme catalyst. The presence of hepatitis is determined -:
' 1 ~y d~terminin~ -the presence of the reaction product.
In accordance with another aspect of -the invention, a hepa-titis detec-tion kit or set is provided. 'rhe three main reagen-ts of the test se-t are the insoluble polymeric solid having antibodies reac-tive with antigens associated with hepatitis bonded the:reto, enzyme tagged hepatitis ant.ibody reagent, and an enzyme substrate capable oE being chemically changed under the ca-talytic influence of the enzyme to ~orm a detec-table end product. In the preferred embodimen-t, the enzyme of the con-jugate is an alkaline phosphatase and the enzyme substrate is p-nitrophenylphosphate. The -test set may also contain control sera including sera nega-tive, weakly positive, and strongly positive for hepatitis antigens, a solution of a horse globulin additive for the tes-t sample for minimizing the frequency of non-specific reactions between the test sample and the insoluble ; polymeric solid, a buffer designed to maintain the pH of the 1 enzyme substrate solution in the optimum range for reaction and a plurality of vials of a size calculated to promo-te contact between the insoluble solid and the small quantities of 2;0 reagents used in the incubations. When alkaline phosphatase and p-nitrophenylphosphate are used as the enzyme-enzyme sub~trate system, the buffer is an aqueous solution 0.02 8M in Na2CO3 and 0.001 M in Mg+ .
In anothex aspect of the invention, an immunologically actlve purified hepatitis antibody conjuga-ted, i.e., chemically linkedl with a functional enzyme such as alkaline phosphatase is provided for use in the immunoassay.
In still another aspect of the invention, a disc-like insolub]e ~ember or matrix is provided for use in the immunoassay.
The matrix has a plurality of groups reactive with proteins gxafted uniformly to its surface layer. Puri~ied hepatitis - ' ~
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1 alltibody is covalen-tly boncled ~o thc reactive CJXOUpS -to provide an exterior layer of hepatitls antibody.
In the immunoassay, -the insoluble member is placed in a flat-bottomed vial and jus-t covered with a liquid test sa~p]e.
For the test to be reproducible, it is important that the antigens in the test samp]e be exposed to the entire surface of the antibody coated discs. It has been discovered that use of a flat or planar surfaced disc resulted in loss of sensitivity.
Accordingly, the two opposed surfaces of the disc of the invention are distorted to an irregular configuration to reduce the disc surface-vial bottom contact. In one preferred embodi-ment, the opposea surfaces are rendered waffle-like by passing the disc through a press prior to attachment of the antibody.
Accordingly, it is a primary object of the invention to provide a hepatitis detection kit and a proc~dure for its use such that the presence of antigens associated with hepatitis ~may be rapidly, simply, accurately, and rou-tinely determined -in body Eluids.
Another o~ject of the invention is to provide a unitized test set which is adapted for the performance of a sensitive, reproducible immunoassay of antigens associated with hepatitis on a routine basis by relatively unskilled persons.
Another ob3ect of the invention is to provide such a test set which is designed to minimize procedural errors in the performance of the immunoassay and which contains all the :~
necessary reagents, reaction containers, etc. in a form designed to optimlze the accuracy and sensitivity oE the test.
~;~ Anot~her object of the invention is to provide a test~set~containlng lahoratory equipment designed to standardize ;the various steps performed during the assay.
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1 Still another object of the invention is to provide a -test set containing standard control samples, negat:ive~, weak:Ly positive, and strongly positive for hepatitis associated antigen, with which the test samples may be compared.
Another object of the invention is to provide a hepati-tis antibody-enzyme conjugate for use with -the immunoassay wherein both -the antibody and the en~yme retain their respective desired chemical characteristics.
A further objec-t is to provide such a conjugate which can be stored for extended periods without losing its immunological or catalytic characteristics and which is highly reactive with antigens associated with hepatitis.
Still another object of the invention is to provide hepatikis antibody conjugated with an enzyme which is capable of ~ catalyzing a reaction to produce an end product at a high ; reaction rate, the concentration oE the end product being precisely detectable using a photometric detector.
Another object of the invention is to provide a conjugate as disclosed above which is highly purified, and thus 2~ contributes heavily to promoting sensitive and accurate immuno-assays of antigens associated with hepatitis.
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Another object of the invention is to provide an insoluble matrix useful as described in the above-mentioned test which has a high ooncentration of purified, hepatitls associated antibody distributed uniformly over its entire surface.
A further object i5 to attach hepatitis antibody to a solid such that it will not be dislodged by mechanical or chemical forces to which it may be subjected during use. In this regard, it is imperative to effect the attachmen-t such that ~30 the antlbody remalns functionalt i.e., capa~le of participating in its immunologicaI reac-tion and immunochemically unaltered ~ ' ' 7~
by its attachment.
A further objec-t of -the invention i.s to provide an improved shape for an insoluble antibody coated solid which is easy to handle duriny immunoassa~ and which :is designed to expose a constant and a greater porti.on of its coated sur:Eace to the solutions used during.incubations.
To this end, in one of its aspects, the invention provides a test set useful for detecting the presence of antigens associated with hepatitis in a test sample comprisi:ng, in combination:
(a) an insoluble polymeric solid having antibody reactive wlth antigens associated with hapatitis bonded thereto for immobilizing hepatitis antigens from the sample, the surfaces on the solid being distorted to comprise a field of high and low points to provide a solid which, when placed in a flat-bottomed vial will be substantially in contact with any solution in the vial while minimizing surface-to-surface contact;
(b) an alkaline phosphatase - antibody conjugate reagent :-: ~2.0 for labelling ~epatitis antigen immobilized on the solid;
(c) an enzyme substrate capable of being chemically :.
.: changed under the catalytic influence of the alkaline phos~
phatasa; and (d) a flat-bottomed vial sized to receive the solid.
; In another of its aspects~ the invention provides : ~ a test set useful for detecting the presence of antigens assoclated with hepati.tis in a test sample comprising, in . ~ combination:
(a) an insoluble polymeric solid having antibody reactive 3~ wlth antigens associated with hepatitis bonded thereto ~or immobilizing hepatitis antigens from the sample~ the surfaces ';::: ~
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on the polymeric solid being distorted to comprise a field of high and low points to provide a solid which, when placed in a flat-bottomed vial, wi.ll be substantially in contact with any solution in the vial while mi.ni.mizing surface-to-sur:Eace contact;
~ b) an alkaline phosphatase-hepati.tis antibody conjugate reagent for labelling hepatitis antigens immobilized on the solid;
(c) a quantity of p-nitrophenylphosphate for being exposed to the alkaline phosphatase to detect the presence thereoE on the solid;
td) quantities of control sera negative, weakly positive, and strongly posi.tive ~or antigens associated with hepa-titis for comparison with the test sample;
(e) an enzyme substrate buffer solution having a pH on the order of 9.8 for optimizing the catalytic effect of the i~ alkaline phosphatase on the p-nitrophenylphosphate;
(f) a horse globulin solution for minimizing nonspecific immobilization of enzyme on the soluble solid; and a flat~
~ bottomed vial sized to receive the polymeric solid;
(g) a flat-bottomed vial sized to receive the solid.
These and other objects and features of the invention will be apparent to those skilled in -the art from the following description of a pre~erred embodiment :~ ~ BRI:EF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a flow chart summarizing the steps of the process of the present inventionî
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Fig. 2 is a perspective view of the preferred insol~le member used in the test set and the process o~ the present 3~ invention;
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Fiys~ 3~ 4 and 5 depj.ct three incubation stages o~
the immunoassay of the presen-t invention; and Fig. 6 is a schematic representation showiny -the "sanclwich" struc-ture developed duriny positive :immunoas.say oE
a test sample tested in accordance wi-th the presen-t invention.
DESCRIPTION OF THE PREFERRED EMBODIMENT
At the ou-tset the invention is described in its broades-t overall aspects with a more detailed description Eollow-ing. The method of detecting hepatitis according to the presentinvention i5 carried out in four di.stinc-t stages as is illustra-ted in Fig. 1.
The first stage involves reacting the immobilized antibody on the insoluble member 10 (see Fig. 2) with an antigen present in the test serum as is shown in Fig. 3. This reection .
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lmmobilizes antigerl so -tha-t on reaction in the next stage with an enzyme tagged antibody, as is shown in Fiy. 4, the tagged antibody is also immobilized. As is sho~ln in Eig. 5, the enzyme is exposed to a suitable subs-trate which is reacted upon by the enzyme to produce changes in colour. The colour change acts as an indication of the presence of antigens associated with hepatitis. The last stage involves reading the degree of colour and comparing the value obtained with a standard of control.
The hepatitis detection test set of the instant invention which contains all materials needed in the test out-lined above is designed for lOO tests~. Obviously, larger or smaller sets may be manufactured by proportional]y increasing or decreasing the quantities of reagents, etc. disclosed herein-after. The kit is distributed, in, e.g., a box, containing reagents and controls to be stored at 2 8C until used. In addition, vials sufficient for conducting lOO tests are provided.
One hunclred to one hundred and five polymeric discs, having waffle-like surfaces, as shown in Fig. 2, and having a :
layer of antibody reactive with hepatitis antigens honded to --their exterior and lyophilized are supplied. The exact nature of these insoluble solid discs and their method of manufacture are disclosed hereinafter.
The second reagent in the set comprises a sample of :
~ ~ hepatitis associated antibody-alkaline phosphatase con~ugate.
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The~exact nature of this reagent, the method for its preparation and alternative useful reagents are disclosed hereinafter.
This reagent is supplied as a solution ready for use. ~-A third reagent supplied in the test set is 400 mg of p-nitrophenylphosphate enzyme substrate. This compound is stable in its powdered form, but, when dissolved in buffer to ::
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1 orm a 1 mg/ml so:Lution, becomes relat:ively unstable. Con-sequently, the solution must be prepared just prior to use.
l'he o-ther materials usecl during the procecl~lre inclucle a quantity oE horse globulill, sa~ple cliluent, various buffer solutions, and v~rious wash solutions. The exact nature and method of preparation of these reagents and materials are indicated below.
A. Preparation of Antibody In order to practice the present invention, it is lt) necessary to obtain antibody that will react with an antigen or antigens associa-ted with hepatitis. It should be noted that such an antibody exists, and thus, the present invention is no-t intended to be limited to the use of any particular antibody.
A reactive antibody may be prepared by purifying bleeding from a host animal which has been injected with a known sample of antigen. An antibody which is reactive with an antigen associated with hepatitis may, in general, be prepared by the process disclosed in British Patent No. 1,387,625.
The preparation of hepatitis antibody depends first of all on obkaining blood which is known to be positive for antigens associated with hepatitis. Consequently, hlood units obtained from various sources must first be evaluated to determine their suitability for preparing the immunospecific purified antibody of the invention.
A blood bag segment, containing blood which is believed ; to be positive for hepatitis antigens, is held at 2~8C in an upright position to allow the blood cells to settle to the lower half. Tbe plasma îs separated from the cells and a ti~er is run on the undiluted plasma and a sample diluted 1:16 in normal saline against a standard antibody by the well-known technique :: ~
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1 ~f counterlec-trophoresis (CEP). I~hen both the unclilu-ted plasma sample and the 1:16 diluted sampl~ are positive, -the blood unit is considered acceptable for use in the preparation oE the purified an-tibody of the invention as clescribed hereinafter.
Por-tion of antigens isolated from blood units are use~
for ei-ther stimulating an-tibody production in a host animal or purifying the antibody produced by that animal. The antigens must be subjec-ted to a preliminary isolation process pr:ior to either of the above uses.
1~ The plasma is transferred to a sterile vacuum container and clotted by adding a 5M solution of a CaC12 on the basis of 0.75 ml CaC12 per 200 ml of plasma. This solu-tion is then in-cubated at 37C in a water bath for one hour or until a clot forms. After a firm clot forms, the plasma is frozen at -20C
and allowed to thaw at 2-8C to allow clot retraction. The serum is separated from the clot and filtered if necessary, then is ready for preparation of a hepatitis antigen pellet for immunization or for use in immunoabsorhent columns.
(1) Preparation of Antiyen Pellet for Immunization ; 20 Hepatitis positive sera which have been ~ubjected to the above process are centrifuged at 10,000 rpm for 3Q minutes at 4C. The supernatant of this centrifugation is distributed into ultra centrifuge tubes and centrifuged in, for example, -~
a Beckman L2-65B ultra-centrifuge, at 40,000 to 50,000 rpm for ; 4 - 20 hours at 4C. The supernatant in each tube is removed and discarded~ the pellet, which contains antigens, is given ~ a pxeli~inary xinse with normal (.15M) saline.
; ~ A small volume of normal saline is -then added to each centrifuge tube and the contents are subjected to son:ication to break up the pel]et. The suspensions in the sonicator tubes .. . : . . .. , ~ - - - .
Lil.764L ~' 1 re then pooled and redistributed equally into clean tubes which are filled with normal saline. This solution is agail1 centrifuged in the Beckman L2-65B centrifuge at 40,000 to 50,000 rpm Eor 20 hours a-t 4C, as mentioned above.
The procedure in the preceeding paragraph may be repeated 5 or more times.
The pellet material, after removing the supernatan-t from each centrifuge tube, is pooled in the miliimal volume of normal saline. A sample is assayed against a standard hepatitis antibody. If the pellet titers at l:25 or higher by CEP, it can be used for immunization. The pooled antigen pellet may be divided into 3 ml aliquots and frozen at -20C for future use.
(2) Produc-tion and Pre]iminary Purification of Hepatitis_Antibody A sample of the antigen pellet, prepared as described above, is added to an equal volume of FREUND'S complete adjuvant j the morning of the immunization and an emulsion is prepared in .;`
accordance with procedures well known to those skilled in the ~, ' , art. The antigen is then injected into a host animall e.g., a horse, in accordance with techniques known per se, -to produce hepatitis antibody. The immunized horses are bled or subjected , to plasmaphoresis according to conventional techniaues. Alter-natively, or in addition, preparations without adjuvant can he used with other routes of immunization.
~; These ble~dings must be treated to isolate the hepatitis antibody in anticipation of the final immunoabsorption purification step. ~Broadly, this preliminary purification is :
~; accomplished in three steps. First, plasma from the host animal is reoalcified. Second, the serum is mixed with a sufficient amount of normal human plasma ~NHP) to precipitate antibodies other than those associated with hepatltis by inducing insoluble - 14 ~
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1 ntigen-antibody complex Eormation. The absorbed antiserum is assayed for hepatitis antibody using CEP. Third, the an-tibody reac-tive with antigens associ~ted with hepatitis is precipi-tated wi-th ammonium sulfate. Th:is ma-terial can be Ero~en untll used.
(3) ~k~aratioll of Ch r_ al :[mmunoab_orbe_t Column The prepara-tion of the purified antibody which is used to produce the reagents of this inventlon is accomplished by subjecting the hepati-tis antibody produced as disclosed above to an il~unospecific extraction process. For a general discussion of this procedure, reference should be made to British Paten-t No. l,387,625 to ~radish et al., March 19, 1375, entitled "Immunospeclfic Separation of Antigens and Antibodies" .
In general, this puriEication process as utilized in the present invention, takes advantage of the ability of anti-bodies reactive with hepatitis antigens to complex with these antigens to the exclusion of other extraneous antibodies and proteins which are inevitably present in the an-tibody sample extracted from the bleedings.
A column is prepared by packing prewashed, sorbent carbon into a glass or plastic tube by using conventional techniques. A pool of antiyen is prepared from at least six individual serum specimens to obtain a diverse mixture of hepatltis antigens. The pool is then adjusted to a protein concentra-tion of between l and 2 mg protein per ml solvent, ::
based~on W absorption.
To attach the antigen onto the carbon, this diluted solution is introduced at a flow rate within the range of 300 to 1,000 ml per hour. Seventy five mg protein should be added per gram of charcoal in the column. The effluent from the column is `~ 30 ; - 15 - -:
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lill764 1 ollected in 500 ~1 aliquots, each of which are checked for protein content. The column is considerecl saturated wi-th the antigen when the effluent has a pro-tein content approximately equal -to that of the starting material. The bed is washed by flowing phosphate huffered saline (PBS) through the column un-til the effluent shows no appreciable detectable absorption at 280 nm.
To elute any loosely attached protein, the charcoal bed is flushed with freshly prepared 5M sodium iodide solution containing 200 mg per liter of sodium thiosulfate. Afterwards, the sodium iodide solution is flushed from the column by running a sufficient volume of PBS therethrough. After a final washing of the bed with PBS containing 1 mg per ml sodium azide --(pxeservative), the column can be stored at 2-8C until ready for use.
(4) Preparation of Antibody for Use in Enzyme Conjugate and ~l Insoluble Solld The antibody, purifled as described above, is freed of ammonium sulfate and diluted with PBS on the basis of 1 part ~ antibody to 2 parts buffer. The column is set up and situated ;:
so that fractions can he collected. The antibody solution is added continuously to the column with a flow rate of about 200 ml/hr. Effluent is collected and tested for protein and hepatitis antibody content to determine when the column is saturated~with antibody. After saturation is achieved, the column bed is washed with PB5 to remove loosely absorbed protein.
At this point, antigens immobilized on the charcoal column have formed a bond with the antibodies reactive with them.
Other extraneous proteins and antibodies, nonspecific to the 30~absorbed antlgens having passed through the column and have ; ~ b en separated from th~ antibody.
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To brea]i this an-tibody-antigen bonci, and to elute -the purified antibody, a 5M solution oE NaC prepared immedia-tely prior to use is in-troducecl into the column. The volume of NaI
solution used should b* sufEicient to remove all an-tibody bound to the column. With -the Elow rate of the column se-t at leas-t 200 ml per hour, the elua-te is collected in fractions of appro-priate volumes. The to-tal amount collec-ted should be at least equal to the volume of sodium iodide solution added.
As each fraction of the puri-Eied antibody is collec-ted, it is subjected to a dual filtration; -Eirst, through a 0.~5 ~
membrane, and second, through a 0.22 ~ membraneO The filtrate is diluted 1:3 using distilled water at 2-8C, e.g., 200 ml of filtrate is added to 400 ml of distilled ~7ater. These diluted antibody fractions are then added to, for example, an ~MICON*
concentrator equipped with an XM-50*membrane, and the fractions are concentrated.
As a last purification step, the concentrated, purified, antibody is dialyzed. Following dialysis, the antibody is removed and centrifuged. The supernatant is dialyzed for at least 24 hours against 0.01M sodium phosphate solution, as com-pared to against PBS in the flrst dialysis. After completion of this final dialysis, protein concen-tration of the antibody is measured.
The antibody is then assayed for activity against the standard anti~en according to the CEP technique to determine antibody content and, if found acceptable, is lyophilized and -stored until used.
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; B. Pre aration of ~n-tibody~Enzyme Conju~ate Çalf intestinal alkaline phosphatase is mixed with a solution of the reconstituted antibody in a ratio of 3:1~ enzyme ~ I`rade Marks :
I!L764 1 ~o antibody, -to a final concentrat:ion cJreater than 10 mg of -to-tal protein per m]. o.~ solution in PBS (pEI 7.4). The solution i.s dialyzed thoroughly to remove N.~I~ ions.
The dialyzed antibody enzyme mixture is then centri-fuged to remove any insoluble material. The proteln content of the supernatant is adjusted to 10 mg/ml by adding the PBS-Mg~+ solution. To thls solution, 8% glutaraldehyde is added on the basis of 1 ml gluteraldehyde solution per 10 ml antibody-enzyme solution. After stirring slowly for 3.5 to 20 minutes, during which time the antibody and enzyme are chemically .
: linked by the glutaraldehyde, the solution of conjugate is dialyzed against PBS containing O.OOlM Mg to rernove the glutaraldehyde.
The dialyzed material is then centrifuged and the supernatant is diluked by addition of an aqueous solution of 0.05M in tris(hydroxymethyl) aminomethane (pH 8~ buffer, 1%
normal human albumin ~crystalline), .02~ NaN3, and .OOlM in ~ MgC12.
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In addition to alkaline phosphatase (EC 3.1.3.1), ~ ::
other enzymes are useable in the procedure of the present in_ vention. Indeed, there is an almost limitless list of enzymes which can be covalently bonded to the antibody. Of the various enzymes, the following table indicates enzymes of particular interest.
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1. alcohol dehydrogenase EC 1~1.1.1 2. glycerol dehydrogenase ~ EC 1.1.1.6 3. glyoxylate reductase ~ EC 1.1.1.26 4. L-lacta~e dehydrogenase ~ EC 1.1.2.3
5. malate dehydrogenase ~ EC 1..101.37
6. glucose 6-phosphate dehydrogenase ~ EC 101.1.4 :~ :
: 3~:~ 7. ma~nitol l-phosphate dehydrogenase EC 1.1~1.17 ;~ : 8. glucose oxidase EC 1.1.3~4 . 9. galactose oxidase ~ EC 1.1~3.9 10. L~amino acid oxidase ~ EC 1~4.3.2 : : :
~ ~ 18 -- ~
11. D~amino acid o~idase - EC 1.~.3.3 12. polypIIetlol ox:idase - E:C ]..14.18.1 13. ascorbate oxidase - E`C 1.10.3.3 1~. c~-talase - EC 1.11.1.6 15. pero~idase - EC 1.11.1.7 16. cholinesteras~- EC 3.1.1.7 17. phospholipase C - EC 3.1.4.3 18. a-amylase - EC 3.2.1.1 19. lysozyme - EC 3.2.1.17 20. ~-galactosidase - EC 3.2.1~23 : 21. amyloglucosldase - EC 3.2.1.3 .. 22. ~-glucuronidase - EC 3.~.1.31 , l~ 23. carboxypeptidase A - EC 3.4.12.2 24. urease - EC 3.5.1.5 ''`
25. inorganic pyrophosphatase - EC 3.6.1.1 26. aldolase - EC 4.1.2.13 - 27. carbonic anhydrase - EC 4~2.1.1 28. histidase EC 4.3.1.3 The enzyme that is used for tayginy the antibody is selected with severai considerations in mind. These consi-derations include the stability of the enzyme, the ease of , assay of the enzyme, the ability of the enzyme to withstana thP
condikions of the covalent bonding of the antibody, the availabili-20 ty of the enzyme, and the cost of the enzyme. `:
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.C. Preparation of Antibody Coate.d Insoluble,Member 'rhe next step in the prep~ration o-f the reagents'is '~.
; to covalently bond a portion of the purified antiboay to an : insoluble m mbPr. To effect this bonding, the insoluble member used must be~proviaed with reactive groups or sites capable of : .reacting with the specific antibody used in the bioassay.
::: -U.S~ Patent No. 3,700,609 entitled "Graft Copolvmers", : to G.W. Tregear et al. discloses an insoluble continuous poly-:
; meric: ~ubstance comprising a polymeric backb'one onto which , ~: 30 side~chains ~ 1 9 ~: ' ' ."
1 -~f another polymer or copolymer are grafted. By suitable choice of the grafted polymer, i-t is possible to chemically link biolog-ical substances to the insoluble ~-trix. ~ product which is disclosed in the above patent is commercially available in a disc Eorm under the tradename P~o~PO~ DI/l from Imperial Chemical :[ndustries oE ~ustralia and New Zealand ~ICIANZ).
The PROT~POL ~I/l comprises a polytetrafluoroethylene backbone having iso-thiocyanopolyst~rene groups grafted unifcrmly over its surface and is designed for use in radioimmunoassay.
~O The discs, as presently available, are approximately .01 inches thick and 0.5 inch in diameter.
In accordance with one important embodiment of the matrix of the present invention, as shown in Fig. 2, each disc 10 is provided with a waffle like pair of surfaces 11 comprising a first series of linear ridges 12 and a second series of linear ridges 14 which form grids. Ridges 12 and 14 are preferably perpendicular to each other and henca define a plurality of square depressions 15. The sides of each ridge 12 and 14 taper upwardly from adjacent pairs of depressions 16 to form a line defining the top of the ridge. It should be noted that , in order to facilitate the description of the invention, the ; ridges 12 and 14 are greatly exaggerated in -the drawing.
The desired configuration of the disc is achieved by ; passing the disc through rollers having projections on the surface of the rollers designed to impart the desired confiyuration on the disc. As is obvious, the rollers are designed to provide a ~sufficient amount of pressure to disfigure the polymeric material in the disc without actually puncturing the disc. This is important because the disc has a reactive layer on its surface.
~ ~ :
~ Thus, penetra-tion oE the disc would expose interior por-tions , ~:
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l76~
1 o wl~ich no antibody can be bonc~e~. Exposure of the poly-tetraEluoroethylene layer woulcl actucllly result in a disc which would have a lower bonding capacity.
The main consideration is to prov:ide a disc matrix with surfaces which, when placed in a flat bot-tomed vial, will be substantially in complete contact with *he test sample, i.e., there should be a minlmum of surfaca-to-surface contact between the matrix and the bottom of the vial. In another important embodiment of the disc of the invention, the disc's surfaces ~0 are configured to have a field of high and low points.
Thè antibody, produced as disclosed above in -the lyophilized form, is xeconstituted by adding 100 ml of O.lM
NaHC03 ~pH 9.6) for each 5.0 mg of an-tibod~. In general, the procedure for attachment involves contacting the wa~fled dises with the dilute solution at ~-8C for 8 to 16 hours, with agitation. Afterwards, the antibody solution is disearded and the discs are washed twice with suceessive volumes of O.lM
NaHC03, pH 9.6, phosphate buffered saline, and cold (2-8C) 0.3~ bovine serum albumin in phosphate buffered saline with 0.5~ T~EEN*2~. After an adclitional washing with erystalline ~, bovlne;serum~albumin, and freezing over dry iee, lyophilization is carried out and the discs are stored at 2-8C until ready for use~
Aithough the description in this specifieation relates to~the preparation of dises having hepatitis antibody bonded there-tor it should~be apparent to those skilled in the ar-t that the dise of the present invention is useful to immobilize an almost llmitless number of proteins. For example, the inereased ::
eontact between the test sample and the disc enables the disc to 3~ be used in tests whieh involve the bonding of the followin~
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1 proteins thereto: antibod:ies -to drugs such as cli~oxin, opia-tes, and steroicls; antibodies to natural produc-ts, for exarnpl.e insulin and other hormonesi and speclfic enzymes to me-t~bolities :Eound in blood ancl ot.her body fluids.
.Example The fo:Llowi.ng procedure ~as used to prepare 8,000 discs, each of wh.ich were first treated with the press to produce the desired configura-tion as described. A batch of 8,000 discs r~quires 40 mg of hepa-ti-t;.s antibody, i.e., 5 mg per l,000 discs.
The protein content of the reconst:ituted hepatitis antibody is adjusted to .05 mg/ml in a final volume of 800 ml in O.lM
NaHCO3 (pH 9.6). The entire 800 ml of buffered antibody is -then added to a l,000 ml screw-c~p bottle provided wi~h a leak proof liner containing the 8,000 discs, and the bottle is ~ rotated for 16 hours, e.g., overnight, a-t 2-8C to slowly -. tumble the discs through each rotation cycle. Afterwards, the liquid is poured from the bottle and discarded and the discs are transferred to a wide-mouth 2 liter flask.
~: The discs are washed twice with successive l liter ~ ~ ~ volumes of cold (2 8C) O.lM NaHCO3, pH 9A6/ following which the buffer is removed. The discs are then washed again, this time using two successive l liter volumes of cold buffer (0.01~
sodium phosphate, O.lSM NaC1~ pH 7.4). After removing resi~ual buffer, the discs are washed for a third -time, using two ~: ; successive one liter volumes of cold bovine serum albumin solution : ~0 3%~.
The discs are finally washed with two successive l liter volumes of a solution of cold crystalline bovine serum albumin ~pH 8) at a concentration of 2 mg/ml. This step i5 3~
: performed to provide a protein environment for t~e protein on ~ 22 - ~;
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1 he disc. The cliscs, after removing the residual wash, are then transferred to dishes or -trays (9 x 9"), each oE which is lined with a sheet of fil-ter paper and each of which con-tains 200 ml of -the crystalline bovine serum alhumin solution. When tl1e transfer is complete, a sheet of fil-te:r paper is used to cover them. Buffer is thoroughly removed. The discs are then quick frozen for 30 minutes on dry iceO
The contents of the tray are then lyophilized and the dry discs are removed and stored in stoppered containers.
(l) Preparation of Control Serum In order to obtain meaningful data from the immuno- -assay, it is necessary to provide negative and positive control sera for proper comparison with a given tes-t sample. The preparation of -these controls is disclosed in detail below.
The negative control is made from human plasma which has been tested and found negative for hepatitis associated antigen, by, for example, the radioimmunoassay technique.
To each unit that is clearly negative for hepatitis antigen, 5M CaCl2 is added to induce clotting, on the basis of 0.75 ml :,~ X~
of CaCl2 solution per 200 ml o:E plasmaO This plasma is then lncubated~at 37C in a water ba-th until a clot forms. The clotted plasma units are then frozen at --20C and stored for at least 12 hours. The plasma units are then allowed to thaw at 2~8C and the serum is collected. If the serum appears excessively turbid, it may be desirable to clarify it by centrifugation, e.g~, at 9,000 rpm for 30 minutes at 2-8C~
Twenty grams of sllica, e.g., AEROSIL-380, is added per liter of serum and mixed for 2 hours at room temperature to remove lipoproteins and stabilize the serum. The mixture is ~ then centrifuged and the precipltate is discarded. :[f desired, ;~ * Trade Mark
7~L
1 _he silica can be removed by filtration through appropriate fil.-ter media.
The supernatan-t is then further processecl by filtration through, for example, MILLIPORE or HORM membranes and pads of successively decreasing porosity, -the last being ~ 0.~5 micron membrane. Before fil-tration through -the 0.~5 mlcron membrane, sodium azide (NaN3) is added to the liquid in sufEicient amount to provide a concentration of 0.1% by weight. As is well known in the art, sodium azide acts as a preservative. The fina:L
~ filtration, through a 0.45 micron or smaller porosity filter, should be done in a laminar flow environment using sterile equipment and -techniques.
The st~rile solution may be then stored at 2-8C un-ti.l ready for subdivision into reagent sized containers for use in the immunoassay. In the preferred, 100 test set, 7..5 m] of negative control serum are provided.
The pQsitive control serum is produced from recalcified : . plasma from blood units which test positively for antigens -.
associated with hepatltis. From each positive unit, a l~
:`
sample is taken, and these are pooled together to form a trial pool. The trial pool is first heat treated for 10 hours at 60C to inactivate any hepatitis causative agent in the sample.
When the pool has cooled to room temperature~ a porti.on is removed and titrated against a standard antibody usi.ng the CEP -~
technique to check that the antigen activity has been retained.
To the trial~pool is then added a sufficient amount of silica to provide a concentration of 20 g per liter of serum. The serum is then stirred using a stirring bar at room temperature fo:r two hours after which it is centrifuged at 9,000 rpm fox 30 mlnutes at 2-8C. The precipltate is cliscarded.
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1 The superl-atant is -then tltrated using CEP against a standard re:Eerence antibody. If the titer has remalnecl at satisfacto.ry levels, -the -total volume of all serum units may be - pooled toge-ther and sub~ec-ted -to the same p:rocess as jus-t described for the trial pool.
The -trial pool and main pool are then combined and diluted with a sufEicient amount of nega-tive control serum -to obtain optimurn results with the positive control serum in the immunoassay of the invention. Preferably, the reading of the positive control serum in the test of the invention, i.n absor~ence units x 10n0, should be greater than 2000. This diluted positive control serum is then filtered through suitable media as before described, using a successive range of decreasing po.rosity. Before the final filtration through a 0.45 micron or smaller membrane, 0.1% by weight soclium azide is added. As with the negative pool, the final filtration should be done under aseptic conditions in a laminar flow environment.
The weakly positive control serum may be made by diluting the strong pos.itive control with negative serum. The . . .
reading of the weakly positive serum as determined by the test of the invent1on should be between 600 and 1000. In the 100 test set of the preferred embodiment of the invention, 2.5 ml of . strong~positive control and 2.5 of weak positive control are supplied.
~: D. iscellaneous Rea~ents and E~uipment ' Two hundred or more glass, disposable, flat bottomed : vials are supplied which have a diameter sli~htly greater than .
.5 inch, i.e , sized to match the 0.50 inch diameter of the ~ discs. One hundred of these vials are used for the initial .~ 30 incubations and washings of the test, the other 100 are employed :: ~
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1 or the final incubation with the enzyme substrate. ~ccorclingly, the -tes-t set enables 100 assays -to be conclucted simultaneous]y.
As set forth in more de-ta:il below, beEore one insoluble solid disc is added to each of the 100 vials, a 0.05 ml portion of horse globulin test sample diluent is ad~ed to each vial. This step is taken as a precaution -to eliminate non-specific reactions in -the firs-t incubation oE the test samples wi-th the inso]uble discs. Although the antlbody coated on the disc is purified and hi~hly reactive with hepatltis antigens~ there is occasiona]ly present in human serum or plasma a substance capable of reac-ting with horse globulin per se that can thus form a bridge between the disc and the enzyme labeled conjugate, thus resulting in a false positive reaction. The horse globulin added in the first step binds this substance so it is not free to react with the disc. As disclosed above, the antibody coated on the disc is produced by immunizing a horse with hepatitis associated antigen collected from hurnan blood. In the 100 -test set of the invention, a 5.5 ml solution comprising 330 mg of horse globulin dissolved in phosphate buffered saline (PBS) is supplied.
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F. Preparation of Substrate ~ ' The preferred substrate for the enzyme reaction in the test is p-nitrophenylphosphate which is dissolved to a con-centration of 1 milligram per ml in sodium carbonate buffer, ~` the concentration of which is 0.028 molar sodium carbonate and 0.001 molar magnesium (pH 9.8).
At this point, it should be noted that other substrates with suitable pH buffering agents, as appear in Table I below, may be used.
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~ 26 -' .7~4 1 TAsl.E I
glycerol phospha-te (serum), 7.4 ~-glycerol phospha-te barbital, 8.6 phenyl phosphate carbonate-hicarbonate, 9-10 ~-naph-thyl phosphate barbital, 9.1 p-ni-trophenyl phosphate 2A2MlP, 10.25 phenolph-thalein phospha-te 2A2MlP, 9.90 p-ni-trophenyl phosphate 2A2MlP, 10.17 thymolphthalein phosphate carbonate-bicarbonate, 10.0 p-nitrophenyl phosphate diethanolamine, 9.8 4-methylumbelliEeryl phosphate carbona-te-bicarbonate, 9.2 The substrates indicated in the Table are all organic phospha-te esters. It should be apparent that other organic phosphate esters could be used as substrates for the preferred enzyme, alkaline phosphatase. Furthermore, it should be noted that ~lcse skilled in the art will have little diEficulty selecting a suitable substrate if an enzyme other than alkaline phosphatase is used in the antibody-enzyme conjugate.
G Procedure To perform the assay according to the invention, 10~
vials are se-t out in racks and each is identified to correspond to a test sample. To each vial is added O.OS ml of the horse globulin solution, then O.S ml of sample is added to 95 of the vials. At the same time, three 0.5 ml samples of negative control ;
; serum are placed in each of three vials, a 0.5 ml sample of strong posi-tive control serum is placed in one vial, and a 0.5 ml sample of weakly positive control serum is placed in .
another vial. To each vial con-taining the horse globulin and sample, including the control vials, is then added 1 antibody coated disc~ The vials, with contents r are incllbated for 0.5 3~
hours at 43C in, e.g., a shaking water bath. During this , ~ :
' incubation, hepati-tis antiyens present in the test sample or COIl-trols will combine with the antibody on the disc.
Prior to the addition of -the enzyme-tagged antibody reagent to the vials containing the insoluble memhers, the super--natant from the firs-t incubation must be removed and the insoluble members mus-t be washed to remove any unbonded antiyen.
The wash solut:ion is preferably a 0.85% solu-tion of sodium chloride, pH 6.5 - 7.5. After two 2.5 ml washes using this solution, 0.3 ml of the antibody-enzyme conjugate is added to each vial, and the vials are again incubated at 43C for 1 hour with shaking, during which time the enzyme tagged antibody will react with the hepatitis antigens that were fixed to the antibody coated disc during the first incubation.
After addition of -the enzyme tagged antibody reagent and a second incubation, the supernatants are aspirated off and the discs in each vial are washed three times with 2.5 ml aliquots of wash solution. This remove$ unreacted enzyme-antibody conjugate. Each insoluble member is then transferred to a clean vial and 2.5 ml of p-nitrophenylphosphate enzyme substrate-buffer solution is addecl to each vial (1 mg pNPP per ml). Since the optimum operational pH of the alkaline phosphatase-p nitrophenyl--phosphate system is 9.8, the enzyme substrate is dissolved in carbonate-Mg ~ buffer (pH 9.8 -~ .1). This buffer as used comprises an aqueous solution 0.028M in Na2C03 and 0. OOlr~ in ~Mg+~. Forty ml of concentrate may be supplied with the set which, when diluted to 400 ml with distilled water, may be added directly to the 400 mg of pNPP. After addition of the buffexed sukstratet the vials are su~jected to a third incubation for one hour at 43C with sha~ing. Different substrates must be 3~ employed if a different enzyme antibody conjugate is used.
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-1 T~o drops (0.1 ml) of 3L~ NaO~I solution are then added ,o each vial -to terminate -the reactiorl. Each test set is suppLied ~7ith 15 ml o~ 3M sodium hydroxide Eor this purpose.
The enzyme substrate solution disclosed above changes from a colorless liquid -to one having a yellow colour in the event enzyme is present on the disc, i.e., in -those vials containing samples posi-t:ive for antigens associated with hepatitis.
The superna-tant from the negative control are pooled together in a suitable vial and their absorption is read at 405 nm in a spectrophotometer against a distilled water blank. When the negative controls read less -than 600 ~absorbance units x 1000), they are considered as proper standards against which to com-pare the test resultsn Using the pooled negative control samples as a blank, tes-t sample and positive control values are read, and the results are recorded as absorbance units x 1000. With some spectrophotometers, it is possible to insert a neyative control in the instrument, ad~ust the reading to zero, and ~ read the value of the test samples directly. An unknown test ; sample whose optical density times 1,000 is greater than 100, using the pooled nega~ive con-trols as a blank, is considered positive for antigens associated with hepatitis. Thls value has been selected to l1mit nonrepeatable positives which, if present, generally result from errors in laboratory technique.
The readings of the test sa~ples may also be compared with the weak positive and strong positive control samples.
Thus, not only the presence, but an indication of the concen-tration of hepatitis antigens in the sample may be obtained.
Examples , Five controls should be assayed wi-th each group of 3~
unknowns--three negative controls, one strong positive control, , ~ ' ..
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., 1 ~nd one weak posltive control. I'hese shouLd he sub-jec-ted to the same process and incuba-tion -times as the test sa~ples.
Caution: Use a clean pipet or disposale t:ip for each transfer to avoid cross-contamination.
1. Prese-t water bath at 43C.
2. Num~er two sets of vials to correspond to test sample iden-tification and controls, and place vials in vial holders.
The firs-t set of these vials will be used for the incubation of test specimens and controls with the antibody coated disc and with the antibody-enæyme tagged reagent. The second - set of vials will be used in Step 12 for the substrate reaction.
3O Pipet 0.05 ml (one drop) of horse globulin reagent into the bottom of the first set of vials. Nonspecific false positives resulting from antibodies present in certain human sera which react with horse globulin, advantageously are essentially eliminated ~y using horse globuline in the test sample diluent.
4. Into this first set of vials pipet 0.5 ml of each test sample into the bottom of the vial having the corresponding sample identification; pipet 0.5 ml of the posi-tive and negative controls into the bottom of their respective vials~
5. ~Transfer an antibody-coated disc to each vial of the first ~ set. Keep the surface of the discs clean. They should be ;~ ~ transferred with clean forceps or a suction-tipped cannula.
~ They shouId not be handled with the fingers.
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6~ Incubate the ~ials at 43C in a water bath with shaking attachment set for mild agitation for 30 minutes.
30 ~ 7. After incubating the samp~e with the antibody disc, com-pletel~ aspirate all supernatants from each vial. Wash the :
~ 30 ;
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1 discs by adding 2.5 ml of iSO'tOlliC saline solution to all -the vials. Repeat 't~'liS procedure so that each disc is washed t~o times. To aid in removing all fluid, tilt the vial holder while asplratin~. Shake the vial holder after each addition of wash solution. Th~ liquid waste collected in the con-tainer at-tached -to the aspirator should be autoclaved before disposal (minimum of one hour at 121~).
8. After final wash and aspiration, add 0.3 ml of the enzyme-labeled antibody solution to each vial.
9. Incubate the vials for one hour at 43C in the water bath with the shaking attachment set for mild agitation.
10. Prepare p-nitrophenyl phosphate substrate by rinsing the contents of one vial of p-NPP (100 mg) into 100 ml of diluted substrate buffer. (The latter is prepared by adding 10 ml of concentrated sodium bicarbonate buffer to 90 ml of distilled water.) Rotate gently to mix; solution should i occur immediately.
Note: This substrate solution must be prepared on the day it is to be used. When not being used it should be refrigerated. Any solution remaining after 24 hours . .
should be discarded.
11. Aspirate the supernatant and wash three times as in 5tep 7.
12. Transfer the discs to the second set of identically numbered clean vials prepared in 5tep 2.
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13. To each vial containing a disc, add 2.5 ml of the p-nitro-phenyl phosphate substxate solution prepared in Step 10.
14. Incubate vials~ for one hour at 43C in a water bath with the shaking at-tachment se-t for mild agitation.
15. Add two drops of 3M sodium hydroxide solution (approximately : .
0.1 ml total) to all the vials to terminate the reactions.
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1 Shake v.ials ln holder to mi~ reagents well.. Tile absorption readings mus-t be made wi-th;n four hours after term:inating the reactions.
16. Pool the three negative controls and read the absorption at ~05 nm in a photometer agains-t a blank of d:istllled water. Record -the resul-t as absorbance units X 1000. If the negative con-trol reads greater than 600, the assay is unsatisfactory and must be repeated. When a flow-through photometer is used and strong positive samples are encountered, the curvette should be rinsed with distilled water before rèading the absorbance of the next sample.
When nondisposable curvettes are used r the curvette must be rinsed with distilled water ~ollowing any positive reading.
17. With the poolèd negative control, adjust the instrument to 0 absorbance. Determine the absorbance of each reactlon mixture, recording -the results as absorbance units X 1000. With some photometers, the instrument cannot be adjusted to 0 with the pooled negative control samples.
: 2~ In this case, the reading of the negative controls must be subtracted from the reading or each sample.
Evaluation o Resul-ts Unknown test samples whose absorbance units X 1000 values are greater than 100 are considered to be reactive, using the pooled negative controls as a blank. It may be desired to repeat the test on samples considered reactive. Be~ore classi- :
~ying a reactive serum as positive for antigens associated with : hepati-tis, confirmation must be obtained by -testing with the ~ . Cordis Confirmatory Test Set (Cat. No. 783-950). ~his assay : 3:0 must be performed on all reactive samples. A reactive serum, ~ ~ - 32 ; ' !
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'76~ ( 1 onE.irmed by neutrali.za-tion wit~ horse antiserum, mus-t be considered poslt:ive Eor hepatitis B antiyen. As is noted above, con-Eirmation tes-ting is requlred in order to evaluate the results. Confirmation tes-ting is re~uired by the Food and Drug ~dminis-tration as well as by the laws of many states.
13.rie:Ely, confirmation testiny in accordance with the present inven-tion is accomplished as :Eollows:
The positive test sample is -tes-ted in duplicate. After the first step in which the sample is exposed to the disc in 1~ each vial, following washing, one disc is exposed to antibodies ~ `
: specific for the hepatitis antigens; and, the other disc is exposed to normal horse serum. After one half hour of : incubation, the subsequent procedures are identical to those carried out in the routine screening assay. If a sample is positive for hepatitis, the specimen which was exposed to the horse antibody will have a very low value; whereas, the disc - : exposed to the normal horse ser~ will have a high value equivalent to that found in the routine screening procedure.
Samples which have low concentrations of the hepatitis ~ antigen, tend to have low final read out values. Those which' have a high concentration of hepatitis antigens will have maximum values. Within a narrow range of concentration differenoes, the resulting read out optical densities will tend to give quantitative information concerning the concentration of : hepatitis antlcJens present in the sample.
Limitations to the Procedure :
:
:: 1. Nonrepeatable reactives: If repeat testing on a reactive sample shows the value is less than the lOO cutoff value, : the test is presumed to be a nonrepeatable reactive and :~: is considered negative for an-tigens associated with hepatitis.
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~' 7 Ei~ ( 1 The original resul-t may be due to errors ln technique, such an inade~uate washincl.
2. Nonspecific positives: The nonspecific false positives resulting from antibodies present in certain human sera tha-t react with horse globulin are essentially eliminated by using horse globulln in the tes-t sample diluent.
3. Plasma from blood collec-ted in EDT~ should not be usecl.
It should be noted that the incubation periods for v2rious steps and the temperature at which incubation is performed can be accomplished over a wide range of times and temperatures. Thus, this invention is not intended to be limited in any way to the time and temperature of incubatiQn.
For example, the time of the incubation of the insoluble member with the sample may range between 10 minutes and 24 hours, with the temperature of incubation ranglng between 2C and 50 C. On the other hand, the time of incubation of the insoluble member with the enzyme tagged binding partner ranges between 30 minutes to 24 hours, with the temperature of ~ incubation of the insoluble member with the enzyme tagged binding .~ ~O partner ranging between 2C and 50C. .
It should also be noted that the process of the present invention can be used to determine the presence of :~
antigens associated with hepatitis in any body fluid where ~ ~the antigens are present~ Thus the process can be used to : :: determine the presence of these antigens in serum, plasma, ~ .
components of plasma, components of serum, urine, saliva, and cere~rospinal fluid.
Results A representati.ve showlng of the results of tests : .
performed in accordance with the present invention appear below~ :~
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1 he results sho~n i:n Tabl.e A are -the resu].ts of -tests in accordance ~ith the p:resent inven-tion for samples which test positive for hepatitis by the CEP method. The values given in Table B a.re the values for -test samples which tes-t positively for hepa-titis by the radioimmunoassay method yet negative for hepatitis by thc CEP me-thod and the values given in Table C test negative for bo-th the RIA and the OE P method~
The numerical values given in Tables A, B and C for the various test samples are the values ob-tained by testing with -the proced~re of the present invention.
TABLE A TABLE B TABLE C
-CEP~ RIA+ CEP- Neg -Test (O.D. x 1000) Test(O.D. x 1000) Test (O.D. x 1000) Sample OD405 ~ leOD405 SampleoD405 ! 82 2822 75 1354 91 23 : 121 2831 77 265 92 42 . 201 2768 81 953 93 0 ~ 202 2780 87 138~ 9~ 0 .~: 20205 2749 123 1688 97. 38 206 2758 12~ 273g 98 0 208 2795 125 17~1 99 0 .-:
~ 210 2764 243 2466 101 0 :~ 213 2776 261 2636 102 o 21~ 2804 103 0 217 2795 104 o , 218 2800 203 o 219 27~6 207 o 2~26 26g5 212 o 228 2782 215 o 232 2760 216 o 234 2774 22~ o 235 27~ 225 o ~ :
~ 35 ~
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1 T~BLE ~ (con-td~) T~I.,E C (contd.) __ _ ___ ___ _ CEP-t Test (O.D. x 1000) Test(O.D. x 1000) 0~405 _ Sample OD405 _ 238 27~0 227 0 251 2761 233 ~5 252 2778 2~0 0 253 2783 2~1 0 ~O 255 27~3 256 2,784 .
The inventlon may be embodied in other specific forms ' without departing from the spirit or essential characteristics thereo. The present embodiment is the,refore to be considered ,in all respects as illustrative and not restrictive, the scope :
of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come "' within the meaning and range of equivalency of the claims are ~ therefore intended to be embraced therein.
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