CA1195926A - Immunometric and inhibition assays using monoclonal antibodies - Google Patents
Immunometric and inhibition assays using monoclonal antibodiesInfo
- Publication number
- CA1195926A CA1195926A CA000473725A CA473725A CA1195926A CA 1195926 A CA1195926 A CA 1195926A CA 000473725 A CA000473725 A CA 000473725A CA 473725 A CA473725 A CA 473725A CA 1195926 A CA1195926 A CA 1195926A
- Authority
- CA
- Canada
- Prior art keywords
- particles
- antibody
- antigenic substance
- monoclonal antibody
- antigen
- 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.)
- Expired
Links
Abstract
ABSTRACT OF THE DISCLOSURE
An inhibition assay technique using a monoclonal antibody for the determination of the presence or concentration of an antigenic substance in a fluid comprising the steps: (a) contacting a sample of the fluid with a known quantity of added antigenic substance and a monoclonal antibody to the antigenic substance, and (b) measuring the inhibition of formation of a complex between the antibody and added antigenic substance by combination of said monoclonal antibody and the antigenic substance in the fluid to form a second complex.
An inhibition assay technique using a monoclonal antibody for the determination of the presence or concentration of an antigenic substance in a fluid comprising the steps: (a) contacting a sample of the fluid with a known quantity of added antigenic substance and a monoclonal antibody to the antigenic substance, and (b) measuring the inhibition of formation of a complex between the antibody and added antigenic substance by combination of said monoclonal antibody and the antigenic substance in the fluid to form a second complex.
Description
No.72~-1371D
This invention relates to an inhibition assay technique for detecting and/or determining the concentration of an~igenic substances in fluids such as serum.
This application is divided from Canadian Application Serial Number 382,964 filed on July 31, 1~81, which discloses the use of immunometric assay techniques with monoclonal antibodies.
According to Serial No.382,964, there is provided a process for the determination of the presence or concentration of an antigenic substance in a fluid comprising the steps: (a) contacking a sample of the fluid with a known quantity of added antigenic substance and a monoclonal antibody to the antigenic substance, and (b) measuring the inhibition o formation of a complex between the antibody and added antigenic substance by combination o~ said monoclonal antibody and the antigenic substance in the fluid to form a second complex.
The determination of the presence or concentration o~ antigenic substances, for example, those associated with a wide ~ariety of physiological disorders, in serum or other body fluids relies increasingly upon immunoassay techniques. ~lese techniques are based upon formation o~ a complex between the antigenic substance being assayed and an antibody or antibodies ln which one or tllC other member of the complex may be labelled, ~or example, by a radloactive element such as 125I, which permits its detec~ion and/or ~u;m-titatLve analysis a~ter separation of the complexed labelled antigen or alltibo~y from uncomplexed labelled an~igen or antibody.
In the case of a competition immunoassay ~echnique, the antigenic substance in a sample of fluid being tested for its presence competes with a known quantity of labelled antigen for a limited quantity of antibody binding sites. Thus, the amount of labelled antigen bound to the antibody is inversely ,~
~s~
proportional to the amount of antigen in the sample. B~ contrast, immunometric assays employ a labelled antibody. In such an assay, the amount of labelled antibody associated with the complex is proportional to the amount of antigenic substance in the fluid sample.
Immunometric assays have been found to be particularly ~ell suited for the detection of polyvalent antigens, i.e., antigenic substances that are able to complex with two or more antibodies at the same time. Such assays typically employ a quantity of unlabelled antibody bound to a solid support that is insoluble in the fluid being tested and a quantity of soluble antibody bearing a label such as a radioactive isotope that permits detection and/or a cluantitative estimate of the amount of the ternary complex formed between solid phase antibody, antigen~ and labelled antibody.
In immunometric assays known to the prior art, typically "fo~ard"
assays~ in which the antibody bound to the solid phase is first contacted with the sample being tested to ex~ract the antigen from ~he sample by formation of a binary solid phase antibody: antigen complex, are employed.
After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with a solution containing a known quantity of labelled antibocly.
~0 Ater a second incubation period to permit the labelled antibody to com~lex with the antigen bound to the solid support through the unlabelled antibodyl the solid support is washed a second time to remove the ~mreacted labelled antibody. In a simple "yes/no" assay to determine whether the antigen is present in the sample being tested9 the washed solid support is tested to detect the presence of labelled antibody, for example, by measuring emitted radiation i~ the label is a radioactive element. The amount of labelled antibody detected is compared to that for a negative control sample
This invention relates to an inhibition assay technique for detecting and/or determining the concentration of an~igenic substances in fluids such as serum.
This application is divided from Canadian Application Serial Number 382,964 filed on July 31, 1~81, which discloses the use of immunometric assay techniques with monoclonal antibodies.
According to Serial No.382,964, there is provided a process for the determination of the presence or concentration of an antigenic substance in a fluid comprising the steps: (a) contacking a sample of the fluid with a known quantity of added antigenic substance and a monoclonal antibody to the antigenic substance, and (b) measuring the inhibition o formation of a complex between the antibody and added antigenic substance by combination o~ said monoclonal antibody and the antigenic substance in the fluid to form a second complex.
The determination of the presence or concentration o~ antigenic substances, for example, those associated with a wide ~ariety of physiological disorders, in serum or other body fluids relies increasingly upon immunoassay techniques. ~lese techniques are based upon formation o~ a complex between the antigenic substance being assayed and an antibody or antibodies ln which one or tllC other member of the complex may be labelled, ~or example, by a radloactive element such as 125I, which permits its detec~ion and/or ~u;m-titatLve analysis a~ter separation of the complexed labelled antigen or alltibo~y from uncomplexed labelled an~igen or antibody.
In the case of a competition immunoassay ~echnique, the antigenic substance in a sample of fluid being tested for its presence competes with a known quantity of labelled antigen for a limited quantity of antibody binding sites. Thus, the amount of labelled antigen bound to the antibody is inversely ,~
~s~
proportional to the amount of antigen in the sample. B~ contrast, immunometric assays employ a labelled antibody. In such an assay, the amount of labelled antibody associated with the complex is proportional to the amount of antigenic substance in the fluid sample.
Immunometric assays have been found to be particularly ~ell suited for the detection of polyvalent antigens, i.e., antigenic substances that are able to complex with two or more antibodies at the same time. Such assays typically employ a quantity of unlabelled antibody bound to a solid support that is insoluble in the fluid being tested and a quantity of soluble antibody bearing a label such as a radioactive isotope that permits detection and/or a cluantitative estimate of the amount of the ternary complex formed between solid phase antibody, antigen~ and labelled antibody.
In immunometric assays known to the prior art, typically "fo~ard"
assays~ in which the antibody bound to the solid phase is first contacted with the sample being tested to ex~ract the antigen from ~he sample by formation of a binary solid phase antibody: antigen complex, are employed.
After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with a solution containing a known quantity of labelled antibocly.
~0 Ater a second incubation period to permit the labelled antibody to com~lex with the antigen bound to the solid support through the unlabelled antibodyl the solid support is washed a second time to remove the ~mreacted labelled antibody. In a simple "yes/no" assay to determine whether the antigen is present in the sample being tested9 the washed solid support is tested to detect the presence of labelled antibody, for example, by measuring emitted radiation i~ the label is a radioactive element. The amount of labelled antibody detected is compared to that for a negative control sample
2~
known to be free of the antigen. Detection of labelled antibody in amounts substantially above the background levels indicated by the negative control is interpreted to indicate the presence of the suspect antigen. Quantitative determinations can be made by comparing ~he measure of labelled antibody with that obtained for calibrated samples containing known quan~ities of the antigen.
This kind sf assay is frequently referred to as a "two-site" or "sandwich" assay since ~he antigen has two antibodies bonded to its surface at different locations. This and related techniques are described by Wide at pp. 199-206 of "Radioimmunoassay Methods", Edited by Kirkham and Hunter, E. ~ S. Livingstone, Edinburgh, 1970. An assay based on this technique for the detection of the antigen associated with serum hepatitis using an 125I
labelled antibody is described in United States Patent 3,867,517.
Despite their great utility, the prior art immunometric assays have been recognized to be slow procedures, in part ~ecause two washing steps are required and because lengthy incubation periods are required to approach equilibriuln, i.e., the point at which the amount of complex formed does not change with increasing time.
To eliminate at least one o~ the washing steps associated with ~0 this procedure, so-called "simultaneous" and "reverse" assays have been proposed. A simultaneous assay involves a single incubatlon step as the antibody bound to the solid support and the labelled antibody are both added to the sample being tested at ~he same time. After the incu~ation is completed, the solid support is washed to remove the residue of fluid sa~ple and uncomplexed labelled antibody. The presence of labelled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.
~g~
A reverse assay involves the stepwise addition first of a solution of labelled antibody to the fluid sample ~ollowed by the addition of unlabelled antibody bound to a solid support after a suitable incubation period. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being ~ested and the solution of tmreacted labelled antibody. The determination of labelled antibody associated with the solid support is -then determined as in ~he simultaneous and forward assays.
Both the simultaneous and reverse assay techni~ues require a sufficient excess amount of solid phase antibody to bind most or all of the antigen present to avoid a high dose hook efect where artifically negative or low quantitation of antigen is observed at extremely high concentrations of antigen. For this reason, the forward assay has been the approach preferred by the prior art. That is because ward assay has been the approach preferred by the prior art. That is `because large amounts of highly purified, active antibody specific to the antigen of interest for preparing a solid phase with sufficient antigen binding capacity is difficult to obtain from the "polyclonal"
antibodies used in prior art processesO When an immunogenic substance is introduced into a living body, the body's immune system reacts by generating antibodies to every site on the imm~mogen it recognizes. A large im~lunogell;c proteln molecule may l~ave dozells of sites and a foreign cell may have hutldreds. Thus, while each antlbody producing cell produces an~ibody specific for a single antigenic site the immune system has generated a specie of specific antibody producing cells for each immunogenic site recognized.
In addition, the body has produced relatively large quantities of antibodies to antigens other than the one of interest such that most of the antibody in the polyclonal mixture is not specific for the antigen of interest.
Accordingly, the antibodies used in prior immunometric assays are necessarily ~ _ 2~
"polyclonal" in na~ure since the antibodies are derived from antis0ra ralsed in a conventional manner in anîmals and their puri~ication is difficult.
Methods ~or affinity puri~ying such antibodies have generally been time consuming and resulted in low yields and loss o~ high af~inity antibodies.
~ hen employing conventional polyclonal antibody mixtures in the reverse and simultaneous assays, the formation o~ a "sandwich" comprising the antigen complexed by two or more labelled antibodies which complex with the antigen at different sites is possible. These comple~es could remain soluble in the sample being tested, be removed by $ubsequent washing steps, and not "coun~ed" when the solid phase is analyzed ~or solid phase bound labelled antibody. I this happens to a signiicant extent, sensitivity of the assay is recluced and erroneous results may arise. However~ if tha unlabelled bound antibody is added to ~he sample first as in the orward sandwich assay, steric considerations prevent formation o~ a sandwich comprising the antigen complexed to two or more unlabelled antibodies where labelled antibody is excluded from also binding to the antigen. Accordingly, the antigen is free to react ~ith a labelled antibody molecule. Nevertheless, it has been proposed to use a simultaneous assay for human thyroid stimulating hormone ~HTSH) by employing a large excess o~ the unlabelled i~() ant:ibody bound to a solid phase to minimi~e ormation of a soluble complex l)y soluble labelled antibodies. See Jeong et al., "Cornparison o l~adiolmlnunoassay ~RtA) with a Uni~ue, Single-lncubation Two-Site Immuno-radiollletric Assay ~IRMA) as Applied to the Determination o~ Human Thyroid Stimulating Hormone ~HTSH)", Bio-Rad Laboratories, 1979.
A variation o~ a simultaneous assay is described in Uni~ed States 4,174,3S4. In that assay, separate por~ions o~ ~ntiIgG ~lluman) are labelled, respectively, with a fluorescing chromophore (:~luorescein) and a chromophore ~s~
(rhodamine) which absorbs light emitted by the fluorescein. Both antibodies~
in a soluble formJ are contacted with a sample containing human IgG.
Reaction of the Anti-IgG with the IgG may bring the ~wo chromophores close enough together, i.e., within 100 angstroms or less, that the emission of light by the fluorescing chromophore is absorbed ~quenched) by the other.
The percentage of ~xim~lm fluorescence for the sample is determined and used as a measure of the amount of Ig~ in the sample.
It has also been proposed to use a reverse assay for HTSH, hepatitis associated antigen (~l~A~ and carcinoembryonic antigen ~CEA) by employing a quantity of labelled an~ibody sufficient to assure a labelled antibody: antigen complex but insufficien$ to fo-rm a "sandwich" of all the antigen present in a sample. See United States Patent No.4,098~876.
Since all three of the procedures known to the prior art use a polyclonal mixture of antibodies, the potential for cross-reaction with other materials in serum or other fluid than the antigen for which the test is intended is increased. The occurrence o:E cross-reactivity with other antigens also reduces the sensitivity of the test for the suspect antigen and increases the prospect of a "false-positive" assay. Purthermore, the use of polyclonal antibodies in a simul~aneous or reverse assay requires a careful consideration of the amount of labelled antibody used relative ~o the amount of solid phase antibody and/or antigen present. In the case o~ using ~luorcscellce quenciling, sensitivity is reduced because the n~;nimllm spacing between the fluorescing chromophore and the quenching chromophore is not assured when polyclonal antibodies are employed.
In view of these shortcomings, the limitations to ~he immunometric procedures known to the prior art are readily apparent. The conventional forward assays are accomplished with fewer steps but require large quantities of solid phase specific antibody and are not well suited to determination of small concentrations of antigen since formation o~ a sandwich of the an~igen with a multiple number of labelled antibody molecules competes with formation of ~he sandwich comprising bound antibody: antigen:
labelled an~ibody or, in the case of using fluorescence quenchin~, the formation of a sandwich without pairing of a fluorescent chromophore with a quènching chromophore is possible; and all are subject to misinterpretation of false-positives due to the polyclonal nature of the antibody.
Accordingly, the invention of the aforementioned Serial No.3S2,96 concerns an improved process for the immunometric assay for antigenic substances, polyclonal antibodies being used in an imm~mometric assay, for example, as the unlabelled antibody bound to a solid support and the antibody used as the soluble labelled antibody or, in the case of assays relying upon ~luorescence quenching, the an~ibodies carrying a fluorescing or quenching chromophore are replaced by at least one and usually two or more different monoclonal antibodies, i.e., each antibody specific to a single antigenic site and separately produced bv clones derived from unique cell lines, i.e., usually to within about 100 angstroms. The advan~ages of the present ~t) invelltion, parti.cularly in simultaneous and reverse assays, over prior art metllods will become clear after consideration of the accompanylng drawings and the following detailed description of the invention.
In the present invention, monoclonal antibodies are employed in inhibition assays. In such assays, a known quantity of an antigen and mono-clonal antibody is contacted with a sample suspected of containing an antigen corresponding to the known antigen added within the monoclonal antibody. The 5~
extent to which inhibition of the complex between the antibody and antigen occurs because a complex comprising the monoclonal antibody and antigen from the sample is formed is a measure of the presence and/or amount of anti.gen in the sample assayed.
Thus, according to the present in~en~ion, there is provided a process for the de~ermination of the presence or concentration of an antigenic substance in a ~luid comprises the steps of:
~ a) contacting a sample of the fluid with a known quantity of ad~ed antigenic substance and a monoclonal antibody to ~he antigenic lo substance, and (b) measuring the inhibition of formation of a complex between tlle antibody and added antigenic substance by combination o~ said monoclonal antibody and the antigenic substance in the fluid to form a second complex.
In a preEerred embodiment, the antibody and antigen are bound, respectively, to one of the members of a pair of fluorescing and quenching chromophores~ Inhibition of the formation of a complex between the labelled .mtigen and antibody by antigens in th0 sample being assayed leads to a reduction in quenching and an increase in fluorescence. The extent of the inhibition of cluenching is a measure o antigen concentratioll in the sample.
In a.nother preferred embocliment of an inhibition assay, the ~nown antigen and allti~ody the ori.ginal complex are bound to particles, :Eor example, latex ~art:icles~ of a size which permits agglomerates to form. When a sample suspected of containing anti.gen is contacted with the antibody and bound antlgell, inhibition of agglomerate formation occurs because of complexing between the bound antibody and sample antigen which cannot form agglomera~es.
The reduction in agglomeration can be measured using turbicdimetric techniques.
~s~
The present in~ention~ together with that of application serial number 382,964 will now be further described, by way of example only, with reference to the acco~panying drawings, in which:
Figure 1 is a graph illustrating the results obtained using polyclonal antibodies in four types of immlmometric assay for human IgE.
Figure 2 is a similar graph illustrating the difference in results obtained using monoclonal antibodies in the same four types of immunometric assay for human IgE.
As indicated above, the polyclonal antibody used in an immunometric assay for an antigenic substance is replaced by a monoclonal an~ibody.
Similarly, monoclonal antibodies are used in inhibition assays. The present inven~ion is useful for ~he determination of the presence and concentration of a wide variety of antigenic substances including polyvalen~
antigenic substances. Accordingly, as used herein, the term antigen or antigenic substance refers broadly to substances to which antibodies can be produced. Among such substances may be mentioned haptens, hormones such as insulin and human thyroid stimulating hormone ~IITSH), gamma globulins, allergens, viruses, virus subunits, bac~eria, toxins such as those associated wlth tetanus and with animal venoms~ aTId even some drugs. Among the specific antigens which may be assayed by the processes of the present invention may be mentiolled carcinoembryonic antigen (CEA), hepatitis A and B, hepatitis Non A -Non B, IgE and alphafetoprotein.
l'hc monoclonal antibodies useful in the present invention are obtained by the process discussed by Milstein and Kohler and reported in Nature 256 495-497, 1975. The de~ails of this process are well known and will not be repeated here. However, basically i~ involves inj~cting a mouse, _ g ~5~
or other suitable animal, with an immunogen. The mouse is subsequently sacrificed and cells taken from its spleen are fused with myeloma cells.
The result is a hybrid cell, referred to as a "hybridoma", that reproduces in vitro. The population of hybridomas is screened and manipulated so as to isolate individual clones each of which secretes a single antibody species to the antigen. Each individual antibody species obtained in this way is the product of a single B cell from the immune animal generated in response to a specific antigenic site recognized on the immunogenic substance.
When an immunogenic substance is introduced into a living host, the host's immune system responds by producing antibodies to all the recognizable sites on the substance. This "shotgun" approach to producing antibodies to combat the invader results in the production of antibodies of differing affinities and specificities for the immunogenic subs~ance.
Accordingly, after the different hybridoma cell lines are screened to identify those that produce antibody to the desired antigen, the antibodies produced by the individual hybridoma cell lines are preferably screened to identify those having the highest affinity for the immunogenic substance stimulating their original production before selection for use in the present invention. Selection based on this criterion is believed to help provlde the increased sensitivity in the immunometric and inhibition assays of the present invention using monoclonal antibody compared to the polyclonal antibody used in the prior art which, at best, has an af~inity for the antigen which is roughly the average of the affinities of all antibodies produced by the immune system. Preferably, the monoclonal antibody selected will have an affinity compatible with the desired sensitivity and range for the test system under consideration. Preferably the antibody will have an _ 10 -~ ~ ~5~2~
affinity of at least 108 liters/mole and, more pre~erably, an affinity of at least about 109 liters/mole.
Furthermore, those monoclonal antibodies having the highest a-ffinities can be further screened by runnin~ a simulated assay on specimens known to give false positive-results with processes employing conventional polyclonal antibodies to identify those monoclonal antibodies which do not cross-react and give false positive resu]ts.
Because the two-site immunometric assay relies upon formation of an antibody: antigen: antibody sandwich, usually two different monoclonal antibodies which do not interfere with the binding of each other to the antigen are selected to be the bound antibody and the soluble labelled antibody or the antibody pair when fluorescence quenching is used. Since both are necessary to complete the sandwich, reverse and simultaneous assays can be conducted without concern, for exampleg that a complex of labelled antibody: antigen: labelled antibody will form which will preclude formation of a complex between the anti~en and the antibody bound to the solid phase and therein lies a particular advantage of the present invention.
Furthermore, a forward assay can be accomplished wi~hout the intermediate washing step since the two an~ibodies bind to two different sites. We refer to such a process as a "fas~ forward" assay.
However, particularly in the case of a ~orward assay, the same monoclonal antibody can be used for both the labelled antibody and the alltibody bound to the solid support when the antigenic substance possesses identical antibody binding sites sufficiently remote from each other to allow more ~han one antibody molecule to be bound a~ ~he same time. In such a system, the addition first of the bound antibody to the sample precludes ~ ~ ~35~
formation of a sandwich bccause of steric considerations. When the labelled monoclonal antibody is subsequently added, it is also able to complex with the antigen bound to unlabelled antibody on the solid phase.
The unlabelled monoclonal antibody used in the process of the present invention to extract the antigenic substance from the sample being tested may be immobili~ed on any of the common supports used in immunometric assays. ~mong these may be mentioned filter paper, plastic beads or ~est tubes made from polyethylene, polystyrene, polypropylene or other suitable material. Also useful are particulate materials such as agarose, cross-linked dextran, and other polysaccharides. The technlques for bonding antibodies to such materials are well known to those skilled in the art.
For example, antibodies may be bo~ld to polysaccharide polymers using the process described in United States Patent No.3,645,852.
The la~elled monoclonal antibody used in the present invention maybe provided with the same labels used in prior art immunometric assays.
Among these may be mentioned fluorogenic labels for detection by fluorimetry as described in United States Patent No.3,940,~75 and enzymatic markers as described in United States Patent No.3,654,090. It is presently preferred to label the antibody with a radioi50topesuch as 125I using, 2() ~or example, the procedure of Hunter and Greenwood, Nature, 144 (1962), page 94S or that of David et al., ~iochemistry, Vol. 13, pp. 1014-1021, 1974.
In a typical assay, the amount of labelled antibody associated with tho insoluble sandwich complex is determined by examination of the insoluble carrier material by suitable means. However, it is also possible to relate the prasence or absence of antigen in the fluid sample being assayed to the amount of labelled antibody which does nt~t react durin~ the assay and remalns in soluble form.
The advantages of *he present invention in which monoclonal antibodies are used in immunometric assays as compared to polyclonal antibodies are seen by reference to the ollowing example. In ~his example, ~our comparative assays, a simultaneous assay, a reverse assay, a forward assay, and a "fast" forward assay, w~re run using both monoclonal antibody and polyclonal antibody using a standard serum containing 100 IUlml of human LgE
as the positive sample. Normal horse serum containing no IgE was used as a negative control.
The polyclonal antibody to IgE used as the labelled antibody in the example was obtained from Pharmacia Diagnostics of Piscataway, New Jersey.
The polyclonal antibody bound to the solid suppor-t w~s obtained from Tago, Inc.
ot Burlingame, California.
Monoclonal antibody to IgE was obtained using the ~nethod of Milstein and Kohler discussed above. The two antibodies selected each exhibited an affinity for IgE of greater than lO liters/mole and did not interfere with the other's binding to IgE.
T}le assays were run using unlabelled antibody bound to agarose by the process of United States Patent No. 3,6~5,852. Labelling of antibody was by 125I according to the process of David et al. referred to above.
2() Phosphate buffered saline~ pH 7.~, was used to wash all samples.
r.x;.l,~p le .
1) Slmultaneous Assay Method -Uuplicate sanlples were run in wllich 100 ~1 of a suspension of antibody imll~obilized on agarose particles was mixed with lOO ~1 of specimen ~serum) and 100 ~l of soluble l25I-labelled antibody. This mixture was incubated for the specified times shown in Table I (for polyclonal antibody) and Iable [I (monoclonal antibody) set forth below, plus 30 minutes. The extra 30 minutes inc-ubation period was added to equalize this assay method with the other assay methods which required an additional 30 minutes incubation time for a second added reagent. Following the incubation periods the agarose particles were washed by addition of bu~fer and centrifuged~ A:fter removal of the was~hing liquid by aspiration3 the resulting pellet of agarose particles was then counted for bound lZ5I-labelled antibody. The counts obtained for each of the complexes after ~he specified incubation ~ime are set forth in Tables I and II.
2) Reverse ~ssay Method -1() Duplicate samples were run in which lOO ~l of specimen (serum) was m:ixed with lOO ~l of l25l-labelled soluble antibody and incubated ~or the specified times shown in Tables I and II. lOO ~l of a suspension of antibody immobilized on agarose particles is then added and the mixture was allowed to incubate for another 30 minutes. The agarose particles were then washed and counted as in the simultaneous assay method. The counts are repor~ed in 'l'ables I and II.
known to be free of the antigen. Detection of labelled antibody in amounts substantially above the background levels indicated by the negative control is interpreted to indicate the presence of the suspect antigen. Quantitative determinations can be made by comparing ~he measure of labelled antibody with that obtained for calibrated samples containing known quan~ities of the antigen.
This kind sf assay is frequently referred to as a "two-site" or "sandwich" assay since ~he antigen has two antibodies bonded to its surface at different locations. This and related techniques are described by Wide at pp. 199-206 of "Radioimmunoassay Methods", Edited by Kirkham and Hunter, E. ~ S. Livingstone, Edinburgh, 1970. An assay based on this technique for the detection of the antigen associated with serum hepatitis using an 125I
labelled antibody is described in United States Patent 3,867,517.
Despite their great utility, the prior art immunometric assays have been recognized to be slow procedures, in part ~ecause two washing steps are required and because lengthy incubation periods are required to approach equilibriuln, i.e., the point at which the amount of complex formed does not change with increasing time.
To eliminate at least one o~ the washing steps associated with ~0 this procedure, so-called "simultaneous" and "reverse" assays have been proposed. A simultaneous assay involves a single incubatlon step as the antibody bound to the solid support and the labelled antibody are both added to the sample being tested at ~he same time. After the incu~ation is completed, the solid support is washed to remove the residue of fluid sa~ple and uncomplexed labelled antibody. The presence of labelled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.
~g~
A reverse assay involves the stepwise addition first of a solution of labelled antibody to the fluid sample ~ollowed by the addition of unlabelled antibody bound to a solid support after a suitable incubation period. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being ~ested and the solution of tmreacted labelled antibody. The determination of labelled antibody associated with the solid support is -then determined as in ~he simultaneous and forward assays.
Both the simultaneous and reverse assay techni~ues require a sufficient excess amount of solid phase antibody to bind most or all of the antigen present to avoid a high dose hook efect where artifically negative or low quantitation of antigen is observed at extremely high concentrations of antigen. For this reason, the forward assay has been the approach preferred by the prior art. That is because ward assay has been the approach preferred by the prior art. That is `because large amounts of highly purified, active antibody specific to the antigen of interest for preparing a solid phase with sufficient antigen binding capacity is difficult to obtain from the "polyclonal"
antibodies used in prior art processesO When an immunogenic substance is introduced into a living body, the body's immune system reacts by generating antibodies to every site on the imm~mogen it recognizes. A large im~lunogell;c proteln molecule may l~ave dozells of sites and a foreign cell may have hutldreds. Thus, while each antlbody producing cell produces an~ibody specific for a single antigenic site the immune system has generated a specie of specific antibody producing cells for each immunogenic site recognized.
In addition, the body has produced relatively large quantities of antibodies to antigens other than the one of interest such that most of the antibody in the polyclonal mixture is not specific for the antigen of interest.
Accordingly, the antibodies used in prior immunometric assays are necessarily ~ _ 2~
"polyclonal" in na~ure since the antibodies are derived from antis0ra ralsed in a conventional manner in anîmals and their puri~ication is difficult.
Methods ~or affinity puri~ying such antibodies have generally been time consuming and resulted in low yields and loss o~ high af~inity antibodies.
~ hen employing conventional polyclonal antibody mixtures in the reverse and simultaneous assays, the formation o~ a "sandwich" comprising the antigen complexed by two or more labelled antibodies which complex with the antigen at different sites is possible. These comple~es could remain soluble in the sample being tested, be removed by $ubsequent washing steps, and not "coun~ed" when the solid phase is analyzed ~or solid phase bound labelled antibody. I this happens to a signiicant extent, sensitivity of the assay is recluced and erroneous results may arise. However~ if tha unlabelled bound antibody is added to ~he sample first as in the orward sandwich assay, steric considerations prevent formation o~ a sandwich comprising the antigen complexed to two or more unlabelled antibodies where labelled antibody is excluded from also binding to the antigen. Accordingly, the antigen is free to react ~ith a labelled antibody molecule. Nevertheless, it has been proposed to use a simultaneous assay for human thyroid stimulating hormone ~HTSH) by employing a large excess o~ the unlabelled i~() ant:ibody bound to a solid phase to minimi~e ormation of a soluble complex l)y soluble labelled antibodies. See Jeong et al., "Cornparison o l~adiolmlnunoassay ~RtA) with a Uni~ue, Single-lncubation Two-Site Immuno-radiollletric Assay ~IRMA) as Applied to the Determination o~ Human Thyroid Stimulating Hormone ~HTSH)", Bio-Rad Laboratories, 1979.
A variation o~ a simultaneous assay is described in Uni~ed States 4,174,3S4. In that assay, separate por~ions o~ ~ntiIgG ~lluman) are labelled, respectively, with a fluorescing chromophore (:~luorescein) and a chromophore ~s~
(rhodamine) which absorbs light emitted by the fluorescein. Both antibodies~
in a soluble formJ are contacted with a sample containing human IgG.
Reaction of the Anti-IgG with the IgG may bring the ~wo chromophores close enough together, i.e., within 100 angstroms or less, that the emission of light by the fluorescing chromophore is absorbed ~quenched) by the other.
The percentage of ~xim~lm fluorescence for the sample is determined and used as a measure of the amount of Ig~ in the sample.
It has also been proposed to use a reverse assay for HTSH, hepatitis associated antigen (~l~A~ and carcinoembryonic antigen ~CEA) by employing a quantity of labelled an~ibody sufficient to assure a labelled antibody: antigen complex but insufficien$ to fo-rm a "sandwich" of all the antigen present in a sample. See United States Patent No.4,098~876.
Since all three of the procedures known to the prior art use a polyclonal mixture of antibodies, the potential for cross-reaction with other materials in serum or other fluid than the antigen for which the test is intended is increased. The occurrence o:E cross-reactivity with other antigens also reduces the sensitivity of the test for the suspect antigen and increases the prospect of a "false-positive" assay. Purthermore, the use of polyclonal antibodies in a simul~aneous or reverse assay requires a careful consideration of the amount of labelled antibody used relative ~o the amount of solid phase antibody and/or antigen present. In the case o~ using ~luorcscellce quenciling, sensitivity is reduced because the n~;nimllm spacing between the fluorescing chromophore and the quenching chromophore is not assured when polyclonal antibodies are employed.
In view of these shortcomings, the limitations to ~he immunometric procedures known to the prior art are readily apparent. The conventional forward assays are accomplished with fewer steps but require large quantities of solid phase specific antibody and are not well suited to determination of small concentrations of antigen since formation o~ a sandwich of the an~igen with a multiple number of labelled antibody molecules competes with formation of ~he sandwich comprising bound antibody: antigen:
labelled an~ibody or, in the case of using fluorescence quenchin~, the formation of a sandwich without pairing of a fluorescent chromophore with a quènching chromophore is possible; and all are subject to misinterpretation of false-positives due to the polyclonal nature of the antibody.
Accordingly, the invention of the aforementioned Serial No.3S2,96 concerns an improved process for the immunometric assay for antigenic substances, polyclonal antibodies being used in an imm~mometric assay, for example, as the unlabelled antibody bound to a solid support and the antibody used as the soluble labelled antibody or, in the case of assays relying upon ~luorescence quenching, the an~ibodies carrying a fluorescing or quenching chromophore are replaced by at least one and usually two or more different monoclonal antibodies, i.e., each antibody specific to a single antigenic site and separately produced bv clones derived from unique cell lines, i.e., usually to within about 100 angstroms. The advan~ages of the present ~t) invelltion, parti.cularly in simultaneous and reverse assays, over prior art metllods will become clear after consideration of the accompanylng drawings and the following detailed description of the invention.
In the present invention, monoclonal antibodies are employed in inhibition assays. In such assays, a known quantity of an antigen and mono-clonal antibody is contacted with a sample suspected of containing an antigen corresponding to the known antigen added within the monoclonal antibody. The 5~
extent to which inhibition of the complex between the antibody and antigen occurs because a complex comprising the monoclonal antibody and antigen from the sample is formed is a measure of the presence and/or amount of anti.gen in the sample assayed.
Thus, according to the present in~en~ion, there is provided a process for the de~ermination of the presence or concentration of an antigenic substance in a ~luid comprises the steps of:
~ a) contacting a sample of the fluid with a known quantity of ad~ed antigenic substance and a monoclonal antibody to ~he antigenic lo substance, and (b) measuring the inhibition of formation of a complex between tlle antibody and added antigenic substance by combination o~ said monoclonal antibody and the antigenic substance in the fluid to form a second complex.
In a preEerred embodiment, the antibody and antigen are bound, respectively, to one of the members of a pair of fluorescing and quenching chromophores~ Inhibition of the formation of a complex between the labelled .mtigen and antibody by antigens in th0 sample being assayed leads to a reduction in quenching and an increase in fluorescence. The extent of the inhibition of cluenching is a measure o antigen concentratioll in the sample.
In a.nother preferred embocliment of an inhibition assay, the ~nown antigen and allti~ody the ori.ginal complex are bound to particles, :Eor example, latex ~art:icles~ of a size which permits agglomerates to form. When a sample suspected of containing anti.gen is contacted with the antibody and bound antlgell, inhibition of agglomerate formation occurs because of complexing between the bound antibody and sample antigen which cannot form agglomera~es.
The reduction in agglomeration can be measured using turbicdimetric techniques.
~s~
The present in~ention~ together with that of application serial number 382,964 will now be further described, by way of example only, with reference to the acco~panying drawings, in which:
Figure 1 is a graph illustrating the results obtained using polyclonal antibodies in four types of immlmometric assay for human IgE.
Figure 2 is a similar graph illustrating the difference in results obtained using monoclonal antibodies in the same four types of immunometric assay for human IgE.
As indicated above, the polyclonal antibody used in an immunometric assay for an antigenic substance is replaced by a monoclonal an~ibody.
Similarly, monoclonal antibodies are used in inhibition assays. The present inven~ion is useful for ~he determination of the presence and concentration of a wide variety of antigenic substances including polyvalen~
antigenic substances. Accordingly, as used herein, the term antigen or antigenic substance refers broadly to substances to which antibodies can be produced. Among such substances may be mentioned haptens, hormones such as insulin and human thyroid stimulating hormone ~IITSH), gamma globulins, allergens, viruses, virus subunits, bac~eria, toxins such as those associated wlth tetanus and with animal venoms~ aTId even some drugs. Among the specific antigens which may be assayed by the processes of the present invention may be mentiolled carcinoembryonic antigen (CEA), hepatitis A and B, hepatitis Non A -Non B, IgE and alphafetoprotein.
l'hc monoclonal antibodies useful in the present invention are obtained by the process discussed by Milstein and Kohler and reported in Nature 256 495-497, 1975. The de~ails of this process are well known and will not be repeated here. However, basically i~ involves inj~cting a mouse, _ g ~5~
or other suitable animal, with an immunogen. The mouse is subsequently sacrificed and cells taken from its spleen are fused with myeloma cells.
The result is a hybrid cell, referred to as a "hybridoma", that reproduces in vitro. The population of hybridomas is screened and manipulated so as to isolate individual clones each of which secretes a single antibody species to the antigen. Each individual antibody species obtained in this way is the product of a single B cell from the immune animal generated in response to a specific antigenic site recognized on the immunogenic substance.
When an immunogenic substance is introduced into a living host, the host's immune system responds by producing antibodies to all the recognizable sites on the substance. This "shotgun" approach to producing antibodies to combat the invader results in the production of antibodies of differing affinities and specificities for the immunogenic subs~ance.
Accordingly, after the different hybridoma cell lines are screened to identify those that produce antibody to the desired antigen, the antibodies produced by the individual hybridoma cell lines are preferably screened to identify those having the highest affinity for the immunogenic substance stimulating their original production before selection for use in the present invention. Selection based on this criterion is believed to help provlde the increased sensitivity in the immunometric and inhibition assays of the present invention using monoclonal antibody compared to the polyclonal antibody used in the prior art which, at best, has an af~inity for the antigen which is roughly the average of the affinities of all antibodies produced by the immune system. Preferably, the monoclonal antibody selected will have an affinity compatible with the desired sensitivity and range for the test system under consideration. Preferably the antibody will have an _ 10 -~ ~ ~5~2~
affinity of at least 108 liters/mole and, more pre~erably, an affinity of at least about 109 liters/mole.
Furthermore, those monoclonal antibodies having the highest a-ffinities can be further screened by runnin~ a simulated assay on specimens known to give false positive-results with processes employing conventional polyclonal antibodies to identify those monoclonal antibodies which do not cross-react and give false positive resu]ts.
Because the two-site immunometric assay relies upon formation of an antibody: antigen: antibody sandwich, usually two different monoclonal antibodies which do not interfere with the binding of each other to the antigen are selected to be the bound antibody and the soluble labelled antibody or the antibody pair when fluorescence quenching is used. Since both are necessary to complete the sandwich, reverse and simultaneous assays can be conducted without concern, for exampleg that a complex of labelled antibody: antigen: labelled antibody will form which will preclude formation of a complex between the anti~en and the antibody bound to the solid phase and therein lies a particular advantage of the present invention.
Furthermore, a forward assay can be accomplished wi~hout the intermediate washing step since the two an~ibodies bind to two different sites. We refer to such a process as a "fas~ forward" assay.
However, particularly in the case of a ~orward assay, the same monoclonal antibody can be used for both the labelled antibody and the alltibody bound to the solid support when the antigenic substance possesses identical antibody binding sites sufficiently remote from each other to allow more ~han one antibody molecule to be bound a~ ~he same time. In such a system, the addition first of the bound antibody to the sample precludes ~ ~ ~35~
formation of a sandwich bccause of steric considerations. When the labelled monoclonal antibody is subsequently added, it is also able to complex with the antigen bound to unlabelled antibody on the solid phase.
The unlabelled monoclonal antibody used in the process of the present invention to extract the antigenic substance from the sample being tested may be immobili~ed on any of the common supports used in immunometric assays. ~mong these may be mentioned filter paper, plastic beads or ~est tubes made from polyethylene, polystyrene, polypropylene or other suitable material. Also useful are particulate materials such as agarose, cross-linked dextran, and other polysaccharides. The technlques for bonding antibodies to such materials are well known to those skilled in the art.
For example, antibodies may be bo~ld to polysaccharide polymers using the process described in United States Patent No.3,645,852.
The la~elled monoclonal antibody used in the present invention maybe provided with the same labels used in prior art immunometric assays.
Among these may be mentioned fluorogenic labels for detection by fluorimetry as described in United States Patent No.3,940,~75 and enzymatic markers as described in United States Patent No.3,654,090. It is presently preferred to label the antibody with a radioi50topesuch as 125I using, 2() ~or example, the procedure of Hunter and Greenwood, Nature, 144 (1962), page 94S or that of David et al., ~iochemistry, Vol. 13, pp. 1014-1021, 1974.
In a typical assay, the amount of labelled antibody associated with tho insoluble sandwich complex is determined by examination of the insoluble carrier material by suitable means. However, it is also possible to relate the prasence or absence of antigen in the fluid sample being assayed to the amount of labelled antibody which does nt~t react durin~ the assay and remalns in soluble form.
The advantages of *he present invention in which monoclonal antibodies are used in immunometric assays as compared to polyclonal antibodies are seen by reference to the ollowing example. In ~his example, ~our comparative assays, a simultaneous assay, a reverse assay, a forward assay, and a "fast" forward assay, w~re run using both monoclonal antibody and polyclonal antibody using a standard serum containing 100 IUlml of human LgE
as the positive sample. Normal horse serum containing no IgE was used as a negative control.
The polyclonal antibody to IgE used as the labelled antibody in the example was obtained from Pharmacia Diagnostics of Piscataway, New Jersey.
The polyclonal antibody bound to the solid suppor-t w~s obtained from Tago, Inc.
ot Burlingame, California.
Monoclonal antibody to IgE was obtained using the ~nethod of Milstein and Kohler discussed above. The two antibodies selected each exhibited an affinity for IgE of greater than lO liters/mole and did not interfere with the other's binding to IgE.
T}le assays were run using unlabelled antibody bound to agarose by the process of United States Patent No. 3,6~5,852. Labelling of antibody was by 125I according to the process of David et al. referred to above.
2() Phosphate buffered saline~ pH 7.~, was used to wash all samples.
r.x;.l,~p le .
1) Slmultaneous Assay Method -Uuplicate sanlples were run in wllich 100 ~1 of a suspension of antibody imll~obilized on agarose particles was mixed with lOO ~1 of specimen ~serum) and 100 ~l of soluble l25I-labelled antibody. This mixture was incubated for the specified times shown in Table I (for polyclonal antibody) and Iable [I (monoclonal antibody) set forth below, plus 30 minutes. The extra 30 minutes inc-ubation period was added to equalize this assay method with the other assay methods which required an additional 30 minutes incubation time for a second added reagent. Following the incubation periods the agarose particles were washed by addition of bu~fer and centrifuged~ A:fter removal of the was~hing liquid by aspiration3 the resulting pellet of agarose particles was then counted for bound lZ5I-labelled antibody. The counts obtained for each of the complexes after ~he specified incubation ~ime are set forth in Tables I and II.
2) Reverse ~ssay Method -1() Duplicate samples were run in which lOO ~l of specimen (serum) was m:ixed with lOO ~l of l25l-labelled soluble antibody and incubated ~or the specified times shown in Tables I and II. lOO ~l of a suspension of antibody immobilized on agarose particles is then added and the mixture was allowed to incubate for another 30 minutes. The agarose particles were then washed and counted as in the simultaneous assay method. The counts are repor~ed in 'l'ables I and II.
3) Forward Assay Method -Duplicate samples were run in which lOO ~l of specimen (ser-lm) was m1xed with lOO ~l of a suspc-~nsion of antibody immobilized on aga-rose ~0 pllrt;cles an-l:incul)atecl for the spec:ified times shown in Tables I and II.
'I'~lo agarose part:ic1es were then washed once by the addition of 2.5-3.0 ml oE l~uffor wllicll, after mixlllg9 was centrifuged, ancl the liquid removed by aX~il`lltiO11. 100 ~1 O~ l25I-labelled soluble antibody was then aclded and the mixture incuhated an additional 30 minutes. The agarose particles were then washed and coun~ed as in the simultaneous assay method. The counts are reported in Tables I and II.
'I'~lo agarose part:ic1es were then washed once by the addition of 2.5-3.0 ml oE l~uffor wllicll, after mixlllg9 was centrifuged, ancl the liquid removed by aX~il`lltiO11. 100 ~1 O~ l25I-labelled soluble antibody was then aclded and the mixture incuhated an additional 30 minutes. The agarose particles were then washed and coun~ed as in the simultaneous assay method. The counts are reported in Tables I and II.
4) Fast Forward Assay Method -The assay was performed, in duplicate, in a similar manner to the orward assay method except that the wash s~ep between the initial incubation of specimen with antibody immobili~ed on agarose particles and the addition of soluble 125I~labelled antibody was omitted.
The counts/minute for the duplicate controls and the duplicate assays of the samples COntaiTIing IgE using polyclonal antibody and monoclonal antibody are shown in Tables I and II, respectively. These data were used to prepare Figures I and II in the ollowing way. The average of the counts/
minute for a control for a given incubation period was subtracted from the average of the co~ts for the corresponding IgE assay. The difference was calculated as a percentage of the total counts/minute of labelled antibody added to the sample and is plotted on the Y axis as the percentage of total counts/minute o antibody bound to ~he solid phase. The incubation time is plotted on the X axis.
A comparison o the plots shown in Figure 2 displaying the results of assays using monoclonal antibody with those of Figure 1 of assays using polyclonal antibody shows ~ilat in each kind of assay, simultaneous, rcverse, forward,and fast forward, the assay using monoclonal antibody was more sensitive as indicated by the higher percentage of total counts bound to tlle solid phase with 100 IU Ig~/ml specimen. Unexpectedly, in the case of the simultaneous and reverse assays, we have ound that those run with monoclonal antibody reach cquillbrium more rapidly -than does the corresp~nding assay using polyclonal antibody. Therefore~ by using a monoclonal antibody in these procedures, the time or the assay can be reduced signiicantly beyond the time saving achieved by merely eliminating a washing s~ep. In that regard, the reverse assay with monoclonal antibody reached equilibrium in less than one hour. The same assay run with polyclonal assay did not reach equilibrium until after 4 hours. Similarly, in the casa of simul~aneous assays, the assay using monoclonal antibody reached equilibrium within 8 hours whereas the assay with polyclonal antibody did not reach equilibrium within 24 hours. Accordingly~ the presen~ invention provides substantially more rapid and sensitive simultaneous and reverse assays than the prior art processes and eliminates the concern that formation of a soluble "sandwich"
LO complex will compete with formation of ~he desired insoluble complex.
In the forecgoing discussion, the focus has been upon two site or sandwich assays in which one of the antibodies is insolubilized while the other is soluble in the medium analyzed. Other variations are possible. A
preferred variant ernploys antibodies bound to particles, such as particles of latex, in a manner which results in each particle carrying a plurality of antibodies. When a quantity of particles to which a first monoclonal ant:ibody is bound is admixed with, for example, quantity of particles to which a second monoclonal antibody is bound, a milky suspension results. However, i a sample containing polyvalent anLigen for which the antibodies are spoci~:ic is introduced to the suspension, agglomeration or agglutination of tlle particles w:ill occur to form easily detectable particle clumps.
111e v:isual detection of agglomerate formation can be used in a screening test for presence of the antigen. ~is detection can be aided by coloring the particles carrying one monoclonal antibody differently from the particle carrying the other. However, the exten~ of agglomera~ion can also be determined as a measure of the amount of antigen present in the sample~
For example, the change in turbidity can be measured using standard techniques such as nephelometry.
Tt~BLE I
Assay ~esults Using Polycion31 Antibody sim~ltaneous Fast Assay Reverse Assay Forward AssayFo~ard Assay Incuhatioll Control IgE Control IgE Control IgE Control IgE
~`ime ~llrs~ Samp~es SamDles San~les Samples Samples Samples Samples Sa~ples 0.25 - 372,314 2705,2667 302,243 25613,2581 357,326 2092,2077 396,293 2271,22 0~50 348,265 2391,2366 2B4,262 295B,2999 238,233 1905,1B17 ,~
l.OQ 315,277 2793,270B 305,277 3154,3218 355,4Z4 2157,2255 304,284 1789,17 2.00 342,356 2B97,2887 290,274 3377,3212 302,314 1946,2019 288,312 1728,18 4.00 421,385 3~96,3746 28,280 3413,3651 274,255- 2019,23~2 283,292 1720,1 6.00 447,435 4028,4101 296,281 3762,3643 241,267 1750,1452 301,257 12~3,14 24.00 526,577 4554,4628 233,263 3651~3546 320,277 1553,1604 273,256 1450,14 ) 'l'A13L~: }I
Assay nesults Usinq l-lonoclonal Antib~x~y Simultaneous Fast Assay . ~everse AssayFor~ard Assay Pon~ard Assay Incubation Control IgE Control IgE Control IgE Control I~E
r i~re (llrs) Salr4~1es _nples S n~les Samples Salllples Samples S~oplcs Sa~ les 0.25 135,132 5610,5803 3U3,594 13407,8358 210,205 4618,41394 194,1l33 4U5~,49()G
0.50 558,459 7472,7115 240,231 823G,8271 223,228 4987,5273 1913,197 502-1,5152 cO
1.00 2G8,265 6289,6529 325,265 8010,B377 230,187 3453,430U 215,192 4887,49Ul 2.00 282,275 67a7,67~4 255,3QS 7856,7644 226,197 4834,4268 192,210 . 4937,49~
4.00 30B,272 0150,al55 343,305 8017,7788 231,216 5269,4420 218,187 4929,5071 6.00 549,667 8884,949Ç 698,850 7870,7358 . 226,361 2006,3631 qOS,243 3033,3713 ~
25.55 426,420 8669,90~4 497,323 7037,7359 194,201 2465,2586 246,233 3945,29~3 S~S
3~L9~
It is presently preferred to use latex particles to which the antibody is covalently bound using techniques well known to those skilled in the art. However, other particulate supports can be used. Among these may be men~ioned silica, glass, cells, polyacrylamides~ polymethyl methacrylate and agarose. Pre-ferably, the particles vary in size between about 0.2 ~ to about 10 ~. Visual screening, however, requires particles of at least about 1.0 11.
In yet another variant, one of the antibodies is insolubilized on a bead, test tube wall or other macroscopic solid support, and the other is bound to small particles o~ latex or other suitable material. In the presence oE antigen~ a sandwich of the antigen between the macroscopically bound antibody and the particle bound antibody will form. By, -for example, coloring the particles, formation of the sandwich can be determined visually.
A fluorescent, enzymatic, radioactive or other label on the particle bound antibody can be used for quanti~ative determinations just as in the case of using a soluble antibody described above.
In another preferred variant of the two-site assay, at least one of two different monoclonal antibodies is bound to an 0nzyme which catalyzes a reaction involving a substance bound to the other monoclonal antibody to ~n produce either a detectable substance or in some other way interacts w:ith the substance on the second antibody to permit detection of ~he antibody:
antigen: antibody complex. Detection may be, or exampla, by colorimetry, fluorimetry, luminescence, spectrophotometry, or the like. It will be appreciated that, using such techniques, neither antibody needs to be insol-ubilized, greatly simplifying the assay.
In a presently preferred embodiment, the substance on the second Z~
antibody is also an en~yme and the assay employs the pair of enzy~e labelled antibodies to catalyze sequential reactions, one of which produces a product required by the other. In those reactions~ the two antibodies are selected so that when they bind with the antigen, they are sterically positioned so that the produc~ of the first enzymatic reaction is generated in such close proximity to the second enzyme labelled antibody, tha~ the second reaction occurs before significant diffusion of the produc~ of the first reaction into the surrounding medi~n can take place.
This process can be illustra~ed using a pair of monoclonal antibodies, one of which is labelled with hexokinase (HK), the other with glucose-6-phosphate dehydrogenase (~-6-PDH), in thé following series of reactions.
HK
(1~ adenosine triphosphate + glucose >
(ATP) adenosine diphosphate -~ glucose-6-phosphate ~ADP) (2) glucose-6-phosphate + nicotinamide adenine dinucleotide (NAD ) G-6-PD~I
gluconolactone-6-phosphate ~
dillydronicotinamide adenine dinucleotide (NADH) The assay is conducted by adding to the sample containing the suspect antigen the labelled antibodies to the antigen, ATP, glucose and ~s~
the coenzyme NAD . I~ the antigen is present~ a complex as illustrated below is formed:
ab(HK) Ag ab(G-6-PDH) The HK labelled antibody catalyzes the forma~ion of glucose-6-phosphate in proximity to the G-6-PDH Labelled antibody where it is converted to gluconolactone-~-phosphate. The NADH formcd in this reaction by reduction o NAD cain be detected spectrophometrically because of the strong absorption at 340 nm characteristic o a dihydronicotinamide.
The same conversion of glucose to gluconolactone-6-phosphate with formation of NADH also may occur in the medium itself catalyzed by the uncomplexed labelled an~ibodies, but at a much slower rate than when the two antibodies are positioned near each other in the antibody: ~lntigen:
antibody complex. Accordingly, an increase of absorption at 340 nm compared to a control sample confirms the presence of antigen in the sample. The increase in absorption can also be correlatedtO the quantity of antigen in the complex.
Any other pair of suitable sequential enz~natically catalyzed rcacr.ions may be used in a two-site assay with appropriately labe:Lled antibodies to a suspect antigen. Among those may be mentioned the reaction o~ glucose catalyzed with glucose oxidase to form glucono-~-lactone and llydrogen peroxide followed by ~he reaction of the hydrogen peroxide with a-o -phenylenediamine catalyzed by peroxidase to produce a colored moie~y. In this assay, one of the monoclonal ~ntibodies is labelled with glucose oxidase and the other wi~h peroxidase. The intensity of the color produced 2~
coinpared to a control can be correlated to the presence and/or amount of antigen in the sample assayed. It will be appreciated that other subs*ances oxidizable to a colored moiety in the presence of an enzyme can be substituted for o-phenylenediamine.
Yet another suitable pair of sequential reactions using a pair of antibodies to a desîred antigen labelled, respectively, with NAD oxidore-ductase and luciferase is the following:
NAD oxido-reductase (1) NADH ~ riboflavin-5'-phosphate (FMN) ~MNH2 ~ NAD
luciferase ~2) FMNH2 ~ RCHO ~ 2 ~~~~~i PMN* -~ RCOOH + H20 ~CHO is typically a straight chain aldehyde of lO or more carbon atoms.
L0 The generation of FMN*, an excited state of FMN, is followed by the emission of a photon which can be detected photome~rically for correlation with a control sample to indicate the presenca and/or quantity of antigen in a sample being assayed.
In another embodiment using a pair of antibodies labelled with enz~nes, tha product of the first enzymatically catalyzed reaction can be e;.ther an allosteric activator or inhibitor of the subsequent enzyme catalyzed reaction. An allosteric activator, rather than being consumed in the second reaction, interacts with the enzyme to increase its affini~y for a substrate or to increase the rate of conversion of the substrate ~o product after the enzyme-substrate complex is formed. Allosteric inhibitors, on the ~ 5~
other hand, have the opposite effec~ and reduce the anzyme's ~ffinlty for a substrate or reduce the rate of conversion of substrate to product. Allosteric inhibition may be of the compe~itive or non-competitive type.
An example of an assay involving allosteric activation employillg a pair of an~ibodies labelled, respectively, with phosphofructoki~ase and phosphoenolpyruvate uses the following reaction scheme:
phosphofruc~okinase (1) fructose-6-phospha~e t ATP
fructose-1,6-diphosphate ~ ADP
(2) HCO3 + phosphoenolpyruvate (PEP) phosphoenolpyruvate carboxylase 3 oxaloacetate (OAA) malate dehydrogenase (3) OAA * NADH ~malate + NAD
The fructose-1,6-diphosphate formed in reaction (l) allosterically interacts witll the phosphoenolpyruvate carboxylase and activates its catalysis of reactioll (2), the formation of oxaloacetate from PEP. Reac-tion (3) occurs in the surrounding medium, i.e.~ there is no necessi~y to bind the malate dellydrogenase to a third monoclonal antibody. The presence and/or quan~ity of suspect antigen is determined by correlating the reduction in the absorption at 340 nm by NADH which is oxidi7ed in reaction (3) to NAD .
An exampla of an assay involving allosteric inhibition employing a pair of antibodies labelled, respectfully, with aspartate amino transferase - 2~ -2~
(AST) and phosphoenolpyruvate carboxylase can use the ~ollowing reaction scheme: AST
(1) oxaloacetate ~ glutamate ~ aspartate -ketoglutorate phosphoenolpyruvate carboxylase (2) PEP ~ NADH > OAA + NAD
The aspartate formed in reaction ~1) inhibits the second reaction by allosterically interacting with the phosphoenolpyruvate carboxylase.
This reduces the rate at which NADH is oxidized to NAD . Therefore, the decrease in the absorption at 340 nm exhibited by NADH can be correlated to the presence and/or quantity of antigen in the sample being assayed, a smaller decrease than occurs with a control sample indicating that antigen is present.
Those skilled in the art will appreciate that numerous other reaction pairs involving activation or inhibition of the second enzymatically catalyzed reaction can be substituted for the examples set forth above for use in a two-site assay. In another embodiment, only one of the antibody pairs ls labelled with an enzyme, the second being labelled with a substance, for e~alnple, that ~dergoes a reaction catalyzed by the enzyme to produce a second product which can be detected and/or quantified by colorimetric, fluorimetric, luminescence, spectrophotometric or other technique. One such example uses a pair of monoclonal antibodies, one of which is labelled with peroxidase and 2n the other with luminol, and takes advantage of the following reaction:
- ~'1 -~ ~3'~
NH O NH O
2 1I peroxi- 12 NIH + 2H O ~ N + 2H2O ~ hv +
-H
luminol The photon (hv) emitted by the reaction can be d~tected using photornetric techniques and related to the presence and/or quantity of an antigen in a sample being assayed.
In yet another preferred variant of the two-site assay, the two monoclonal antibodles are, respectively, conjugated with a fluorescing chromophore and a quenching chromophore which absorbs light at the wavelength emitted by the fluorescer. The two antibodies are selected so that, when they combine with the antigen for which they are specific, the two chromophores are positioned close enough to permit the light emitted by the fluorescer to be absorbed by the other chromophore. Usually, this will place them within about 100 angstroms of each other and, preferably, within about sa angstroms of each other. The selection o suitable antibodies can be done through a screening procedure in which a mixture of fluorescent and quencher labelled monoclonal antibodies are contacted with a sample containing a known quantity of an~igen. Reduction of fluorescence is indicative that the two chromophores are positioned closely enough together.
Using 1uorescence quenching, it is unnecessary to insolubilize either of the two antibodies. Quantitative measurements can be made simply by measuring the decrease in r~x; fluorescence, i.e., the amount of fluorescence exhibited by a control sample free of any antigen or by comparing the fluorescence of tlle sample with that of control samples containing a known quantity of antigen. However, fluorescent-quenching chromophore pairs can also - ~5 -~L9~2~
be used in combination with the particle agglomeration ~echnique and in the technique whereby one of the antibodies is insolubilized by being bound to a solid support such as a test tube wall or bead, since pairing of the fluorescent-quenching chromophores will occur. A decrease in fluorescence again is indicative of the presence or amount of antigen in the sample.
Suitable fluorescing and quenching chromophores and ~echniques for conjugating ~hem with antibodies are described in United States Patent No.~,17~384~ Presently, it i5 pre~erred to use ~luorescein and rhodamine as the fluorescer and quencher chromophores, respectively.
In the preceding discussion o~ our invention, we have described techniques of fluorescence quenching in which antibody pairs carrying the necessary chromopnores are caused to bond to an antigen, if present in the sample being analyzed, in a steric arrangement which permits the quenching chromophore to absorb light emitted by the fluorescent chromophore.
Quantitative determinations of the amount of antigen present are made by m~asuring the decrease in ~ m fluorescence.
These techniques are well suited to determining the presence of antigen in a sample over a wide ran~e of concentration. However, the small decreases in fluorcscence which are associa~ed with low antigen concentration are hard to detect and measure accurately. By contrast, small increases in fluorescence are relatively easy to detect and measure accurately. Accordingly, in another aspect or our invention, we prefer to exploit the inhibition of quencllillg and measure increases in fluorescellce.
To accomplish this in an assay for a particular antigen, quantities of the antigen and monoclonal antibody to the antigen are indi~idually labelled with one or the other of the pair of fluorescent-quencher chromophores. The ~9s~
chromophore labelled antigen and antibody are then combined to ~orm a complex in which the~luorescent chromophore is positioned so that the light it emits is absorbed by the quenching chromophore. To achieve this the antigen may be labelled with ~he fluorescer and the antibody with the quencher or vice versa.
A sample suspected of containing the antigen being assayed is then contacted with the chromophore labelled antigen and antibody. After a suitable incubation period, fluorescence is measured. If antigen is present in the assayed sample~ i~ inhibi~s, at least in part, the formation of a complex between the chromophore labelled antigen and the antibody by itself forming a complex with the monoclonal antibody. To the extent this occurs, the fluorescer chromophore is no longer positioned so that the light it emits is absorbed by the quenching chromophore. This results in an increase in fluorescence. l'he increase in fluorescence can be measur0d and related to the concentration of antigen in the sample undergoing analysis by comparison with the ~luorescence exhibited by control samples free of antigen or containing known amounts of antigen.
From the foregoing, it will be apparent that ~he chromophore labelled antigen: antibody complex may be a soluble one. However, it is presently preferred to employ chromophore labelled antigen and monoclonal antibody which ~0 are bound to latex or other suitable particles, for example, those listed above, o a size that will ~orm agglomerates when the complex is formed. Particles varying in size from about 0.2 to about 10 ~ are usually suitable for this purpose. When an ~known s~nple containing the suspect antigen is contacted with the agglomerate forming particles of antibody and antigen, inhibition of agglomeration will occur because o~ sample antigen combining with particle-bound antibody. An increase in 1uorescence will result, since quenching can no longer occur, which can be detected and measured to correlate with the - ~7 -s~
amount o$ antigen in the sample by comparison with the fluorescence observed for a sample containing a known quantity of antigen.
It is also within the scope of our invention to employ bound antigen and particle bound monoclonal antibody in an assay that directly measures the inhihition of agglomeration. In this tec~miquel nei~her the antigen nor antibody is labelled. When a sample containing antigen is contacted with the particles, inhibition of agglomeration during tlle incubation period will occur. This ~esults in, at least, a partial reduction in the agglomerate $ormation, The inhibition is detected using nephelometry or other techniques for measuring turbidity. The decrease in turbidity can be correlated to the amount of antigen in the sample.
The counts/minute for the duplicate controls and the duplicate assays of the samples COntaiTIing IgE using polyclonal antibody and monoclonal antibody are shown in Tables I and II, respectively. These data were used to prepare Figures I and II in the ollowing way. The average of the counts/
minute for a control for a given incubation period was subtracted from the average of the co~ts for the corresponding IgE assay. The difference was calculated as a percentage of the total counts/minute of labelled antibody added to the sample and is plotted on the Y axis as the percentage of total counts/minute o antibody bound to ~he solid phase. The incubation time is plotted on the X axis.
A comparison o the plots shown in Figure 2 displaying the results of assays using monoclonal antibody with those of Figure 1 of assays using polyclonal antibody shows ~ilat in each kind of assay, simultaneous, rcverse, forward,and fast forward, the assay using monoclonal antibody was more sensitive as indicated by the higher percentage of total counts bound to tlle solid phase with 100 IU Ig~/ml specimen. Unexpectedly, in the case of the simultaneous and reverse assays, we have ound that those run with monoclonal antibody reach cquillbrium more rapidly -than does the corresp~nding assay using polyclonal antibody. Therefore~ by using a monoclonal antibody in these procedures, the time or the assay can be reduced signiicantly beyond the time saving achieved by merely eliminating a washing s~ep. In that regard, the reverse assay with monoclonal antibody reached equilibrium in less than one hour. The same assay run with polyclonal assay did not reach equilibrium until after 4 hours. Similarly, in the casa of simul~aneous assays, the assay using monoclonal antibody reached equilibrium within 8 hours whereas the assay with polyclonal antibody did not reach equilibrium within 24 hours. Accordingly~ the presen~ invention provides substantially more rapid and sensitive simultaneous and reverse assays than the prior art processes and eliminates the concern that formation of a soluble "sandwich"
LO complex will compete with formation of ~he desired insoluble complex.
In the forecgoing discussion, the focus has been upon two site or sandwich assays in which one of the antibodies is insolubilized while the other is soluble in the medium analyzed. Other variations are possible. A
preferred variant ernploys antibodies bound to particles, such as particles of latex, in a manner which results in each particle carrying a plurality of antibodies. When a quantity of particles to which a first monoclonal ant:ibody is bound is admixed with, for example, quantity of particles to which a second monoclonal antibody is bound, a milky suspension results. However, i a sample containing polyvalent anLigen for which the antibodies are spoci~:ic is introduced to the suspension, agglomeration or agglutination of tlle particles w:ill occur to form easily detectable particle clumps.
111e v:isual detection of agglomerate formation can be used in a screening test for presence of the antigen. ~is detection can be aided by coloring the particles carrying one monoclonal antibody differently from the particle carrying the other. However, the exten~ of agglomera~ion can also be determined as a measure of the amount of antigen present in the sample~
For example, the change in turbidity can be measured using standard techniques such as nephelometry.
Tt~BLE I
Assay ~esults Using Polycion31 Antibody sim~ltaneous Fast Assay Reverse Assay Forward AssayFo~ard Assay Incuhatioll Control IgE Control IgE Control IgE Control IgE
~`ime ~llrs~ Samp~es SamDles San~les Samples Samples Samples Samples Sa~ples 0.25 - 372,314 2705,2667 302,243 25613,2581 357,326 2092,2077 396,293 2271,22 0~50 348,265 2391,2366 2B4,262 295B,2999 238,233 1905,1B17 ,~
l.OQ 315,277 2793,270B 305,277 3154,3218 355,4Z4 2157,2255 304,284 1789,17 2.00 342,356 2B97,2887 290,274 3377,3212 302,314 1946,2019 288,312 1728,18 4.00 421,385 3~96,3746 28,280 3413,3651 274,255- 2019,23~2 283,292 1720,1 6.00 447,435 4028,4101 296,281 3762,3643 241,267 1750,1452 301,257 12~3,14 24.00 526,577 4554,4628 233,263 3651~3546 320,277 1553,1604 273,256 1450,14 ) 'l'A13L~: }I
Assay nesults Usinq l-lonoclonal Antib~x~y Simultaneous Fast Assay . ~everse AssayFor~ard Assay Pon~ard Assay Incubation Control IgE Control IgE Control IgE Control I~E
r i~re (llrs) Salr4~1es _nples S n~les Samples Salllples Samples S~oplcs Sa~ les 0.25 135,132 5610,5803 3U3,594 13407,8358 210,205 4618,41394 194,1l33 4U5~,49()G
0.50 558,459 7472,7115 240,231 823G,8271 223,228 4987,5273 1913,197 502-1,5152 cO
1.00 2G8,265 6289,6529 325,265 8010,B377 230,187 3453,430U 215,192 4887,49Ul 2.00 282,275 67a7,67~4 255,3QS 7856,7644 226,197 4834,4268 192,210 . 4937,49~
4.00 30B,272 0150,al55 343,305 8017,7788 231,216 5269,4420 218,187 4929,5071 6.00 549,667 8884,949Ç 698,850 7870,7358 . 226,361 2006,3631 qOS,243 3033,3713 ~
25.55 426,420 8669,90~4 497,323 7037,7359 194,201 2465,2586 246,233 3945,29~3 S~S
3~L9~
It is presently preferred to use latex particles to which the antibody is covalently bound using techniques well known to those skilled in the art. However, other particulate supports can be used. Among these may be men~ioned silica, glass, cells, polyacrylamides~ polymethyl methacrylate and agarose. Pre-ferably, the particles vary in size between about 0.2 ~ to about 10 ~. Visual screening, however, requires particles of at least about 1.0 11.
In yet another variant, one of the antibodies is insolubilized on a bead, test tube wall or other macroscopic solid support, and the other is bound to small particles o~ latex or other suitable material. In the presence oE antigen~ a sandwich of the antigen between the macroscopically bound antibody and the particle bound antibody will form. By, -for example, coloring the particles, formation of the sandwich can be determined visually.
A fluorescent, enzymatic, radioactive or other label on the particle bound antibody can be used for quanti~ative determinations just as in the case of using a soluble antibody described above.
In another preferred variant of the two-site assay, at least one of two different monoclonal antibodies is bound to an 0nzyme which catalyzes a reaction involving a substance bound to the other monoclonal antibody to ~n produce either a detectable substance or in some other way interacts w:ith the substance on the second antibody to permit detection of ~he antibody:
antigen: antibody complex. Detection may be, or exampla, by colorimetry, fluorimetry, luminescence, spectrophotometry, or the like. It will be appreciated that, using such techniques, neither antibody needs to be insol-ubilized, greatly simplifying the assay.
In a presently preferred embodiment, the substance on the second Z~
antibody is also an en~yme and the assay employs the pair of enzy~e labelled antibodies to catalyze sequential reactions, one of which produces a product required by the other. In those reactions~ the two antibodies are selected so that when they bind with the antigen, they are sterically positioned so that the produc~ of the first enzymatic reaction is generated in such close proximity to the second enzyme labelled antibody, tha~ the second reaction occurs before significant diffusion of the produc~ of the first reaction into the surrounding medi~n can take place.
This process can be illustra~ed using a pair of monoclonal antibodies, one of which is labelled with hexokinase (HK), the other with glucose-6-phosphate dehydrogenase (~-6-PDH), in thé following series of reactions.
HK
(1~ adenosine triphosphate + glucose >
(ATP) adenosine diphosphate -~ glucose-6-phosphate ~ADP) (2) glucose-6-phosphate + nicotinamide adenine dinucleotide (NAD ) G-6-PD~I
gluconolactone-6-phosphate ~
dillydronicotinamide adenine dinucleotide (NADH) The assay is conducted by adding to the sample containing the suspect antigen the labelled antibodies to the antigen, ATP, glucose and ~s~
the coenzyme NAD . I~ the antigen is present~ a complex as illustrated below is formed:
ab(HK) Ag ab(G-6-PDH) The HK labelled antibody catalyzes the forma~ion of glucose-6-phosphate in proximity to the G-6-PDH Labelled antibody where it is converted to gluconolactone-~-phosphate. The NADH formcd in this reaction by reduction o NAD cain be detected spectrophometrically because of the strong absorption at 340 nm characteristic o a dihydronicotinamide.
The same conversion of glucose to gluconolactone-6-phosphate with formation of NADH also may occur in the medium itself catalyzed by the uncomplexed labelled an~ibodies, but at a much slower rate than when the two antibodies are positioned near each other in the antibody: ~lntigen:
antibody complex. Accordingly, an increase of absorption at 340 nm compared to a control sample confirms the presence of antigen in the sample. The increase in absorption can also be correlatedtO the quantity of antigen in the complex.
Any other pair of suitable sequential enz~natically catalyzed rcacr.ions may be used in a two-site assay with appropriately labe:Lled antibodies to a suspect antigen. Among those may be mentioned the reaction o~ glucose catalyzed with glucose oxidase to form glucono-~-lactone and llydrogen peroxide followed by ~he reaction of the hydrogen peroxide with a-o -phenylenediamine catalyzed by peroxidase to produce a colored moie~y. In this assay, one of the monoclonal ~ntibodies is labelled with glucose oxidase and the other wi~h peroxidase. The intensity of the color produced 2~
coinpared to a control can be correlated to the presence and/or amount of antigen in the sample assayed. It will be appreciated that other subs*ances oxidizable to a colored moiety in the presence of an enzyme can be substituted for o-phenylenediamine.
Yet another suitable pair of sequential reactions using a pair of antibodies to a desîred antigen labelled, respectively, with NAD oxidore-ductase and luciferase is the following:
NAD oxido-reductase (1) NADH ~ riboflavin-5'-phosphate (FMN) ~MNH2 ~ NAD
luciferase ~2) FMNH2 ~ RCHO ~ 2 ~~~~~i PMN* -~ RCOOH + H20 ~CHO is typically a straight chain aldehyde of lO or more carbon atoms.
L0 The generation of FMN*, an excited state of FMN, is followed by the emission of a photon which can be detected photome~rically for correlation with a control sample to indicate the presenca and/or quantity of antigen in a sample being assayed.
In another embodiment using a pair of antibodies labelled with enz~nes, tha product of the first enzymatically catalyzed reaction can be e;.ther an allosteric activator or inhibitor of the subsequent enzyme catalyzed reaction. An allosteric activator, rather than being consumed in the second reaction, interacts with the enzyme to increase its affini~y for a substrate or to increase the rate of conversion of the substrate ~o product after the enzyme-substrate complex is formed. Allosteric inhibitors, on the ~ 5~
other hand, have the opposite effec~ and reduce the anzyme's ~ffinlty for a substrate or reduce the rate of conversion of substrate to product. Allosteric inhibition may be of the compe~itive or non-competitive type.
An example of an assay involving allosteric activation employillg a pair of an~ibodies labelled, respectively, with phosphofructoki~ase and phosphoenolpyruvate uses the following reaction scheme:
phosphofruc~okinase (1) fructose-6-phospha~e t ATP
fructose-1,6-diphosphate ~ ADP
(2) HCO3 + phosphoenolpyruvate (PEP) phosphoenolpyruvate carboxylase 3 oxaloacetate (OAA) malate dehydrogenase (3) OAA * NADH ~malate + NAD
The fructose-1,6-diphosphate formed in reaction (l) allosterically interacts witll the phosphoenolpyruvate carboxylase and activates its catalysis of reactioll (2), the formation of oxaloacetate from PEP. Reac-tion (3) occurs in the surrounding medium, i.e.~ there is no necessi~y to bind the malate dellydrogenase to a third monoclonal antibody. The presence and/or quan~ity of suspect antigen is determined by correlating the reduction in the absorption at 340 nm by NADH which is oxidi7ed in reaction (3) to NAD .
An exampla of an assay involving allosteric inhibition employing a pair of antibodies labelled, respectfully, with aspartate amino transferase - 2~ -2~
(AST) and phosphoenolpyruvate carboxylase can use the ~ollowing reaction scheme: AST
(1) oxaloacetate ~ glutamate ~ aspartate -ketoglutorate phosphoenolpyruvate carboxylase (2) PEP ~ NADH > OAA + NAD
The aspartate formed in reaction ~1) inhibits the second reaction by allosterically interacting with the phosphoenolpyruvate carboxylase.
This reduces the rate at which NADH is oxidized to NAD . Therefore, the decrease in the absorption at 340 nm exhibited by NADH can be correlated to the presence and/or quantity of antigen in the sample being assayed, a smaller decrease than occurs with a control sample indicating that antigen is present.
Those skilled in the art will appreciate that numerous other reaction pairs involving activation or inhibition of the second enzymatically catalyzed reaction can be substituted for the examples set forth above for use in a two-site assay. In another embodiment, only one of the antibody pairs ls labelled with an enzyme, the second being labelled with a substance, for e~alnple, that ~dergoes a reaction catalyzed by the enzyme to produce a second product which can be detected and/or quantified by colorimetric, fluorimetric, luminescence, spectrophotometric or other technique. One such example uses a pair of monoclonal antibodies, one of which is labelled with peroxidase and 2n the other with luminol, and takes advantage of the following reaction:
- ~'1 -~ ~3'~
NH O NH O
2 1I peroxi- 12 NIH + 2H O ~ N + 2H2O ~ hv +
-H
luminol The photon (hv) emitted by the reaction can be d~tected using photornetric techniques and related to the presence and/or quantity of an antigen in a sample being assayed.
In yet another preferred variant of the two-site assay, the two monoclonal antibodles are, respectively, conjugated with a fluorescing chromophore and a quenching chromophore which absorbs light at the wavelength emitted by the fluorescer. The two antibodies are selected so that, when they combine with the antigen for which they are specific, the two chromophores are positioned close enough to permit the light emitted by the fluorescer to be absorbed by the other chromophore. Usually, this will place them within about 100 angstroms of each other and, preferably, within about sa angstroms of each other. The selection o suitable antibodies can be done through a screening procedure in which a mixture of fluorescent and quencher labelled monoclonal antibodies are contacted with a sample containing a known quantity of an~igen. Reduction of fluorescence is indicative that the two chromophores are positioned closely enough together.
Using 1uorescence quenching, it is unnecessary to insolubilize either of the two antibodies. Quantitative measurements can be made simply by measuring the decrease in r~x; fluorescence, i.e., the amount of fluorescence exhibited by a control sample free of any antigen or by comparing the fluorescence of tlle sample with that of control samples containing a known quantity of antigen. However, fluorescent-quenching chromophore pairs can also - ~5 -~L9~2~
be used in combination with the particle agglomeration ~echnique and in the technique whereby one of the antibodies is insolubilized by being bound to a solid support such as a test tube wall or bead, since pairing of the fluorescent-quenching chromophores will occur. A decrease in fluorescence again is indicative of the presence or amount of antigen in the sample.
Suitable fluorescing and quenching chromophores and ~echniques for conjugating ~hem with antibodies are described in United States Patent No.~,17~384~ Presently, it i5 pre~erred to use ~luorescein and rhodamine as the fluorescer and quencher chromophores, respectively.
In the preceding discussion o~ our invention, we have described techniques of fluorescence quenching in which antibody pairs carrying the necessary chromopnores are caused to bond to an antigen, if present in the sample being analyzed, in a steric arrangement which permits the quenching chromophore to absorb light emitted by the fluorescent chromophore.
Quantitative determinations of the amount of antigen present are made by m~asuring the decrease in ~ m fluorescence.
These techniques are well suited to determining the presence of antigen in a sample over a wide ran~e of concentration. However, the small decreases in fluorcscence which are associa~ed with low antigen concentration are hard to detect and measure accurately. By contrast, small increases in fluorescence are relatively easy to detect and measure accurately. Accordingly, in another aspect or our invention, we prefer to exploit the inhibition of quencllillg and measure increases in fluorescellce.
To accomplish this in an assay for a particular antigen, quantities of the antigen and monoclonal antibody to the antigen are indi~idually labelled with one or the other of the pair of fluorescent-quencher chromophores. The ~9s~
chromophore labelled antigen and antibody are then combined to ~orm a complex in which the~luorescent chromophore is positioned so that the light it emits is absorbed by the quenching chromophore. To achieve this the antigen may be labelled with ~he fluorescer and the antibody with the quencher or vice versa.
A sample suspected of containing the antigen being assayed is then contacted with the chromophore labelled antigen and antibody. After a suitable incubation period, fluorescence is measured. If antigen is present in the assayed sample~ i~ inhibi~s, at least in part, the formation of a complex between the chromophore labelled antigen and the antibody by itself forming a complex with the monoclonal antibody. To the extent this occurs, the fluorescer chromophore is no longer positioned so that the light it emits is absorbed by the quenching chromophore. This results in an increase in fluorescence. l'he increase in fluorescence can be measur0d and related to the concentration of antigen in the sample undergoing analysis by comparison with the ~luorescence exhibited by control samples free of antigen or containing known amounts of antigen.
From the foregoing, it will be apparent that ~he chromophore labelled antigen: antibody complex may be a soluble one. However, it is presently preferred to employ chromophore labelled antigen and monoclonal antibody which ~0 are bound to latex or other suitable particles, for example, those listed above, o a size that will ~orm agglomerates when the complex is formed. Particles varying in size from about 0.2 to about 10 ~ are usually suitable for this purpose. When an ~known s~nple containing the suspect antigen is contacted with the agglomerate forming particles of antibody and antigen, inhibition of agglomeration will occur because o~ sample antigen combining with particle-bound antibody. An increase in 1uorescence will result, since quenching can no longer occur, which can be detected and measured to correlate with the - ~7 -s~
amount o$ antigen in the sample by comparison with the fluorescence observed for a sample containing a known quantity of antigen.
It is also within the scope of our invention to employ bound antigen and particle bound monoclonal antibody in an assay that directly measures the inhihition of agglomeration. In this tec~miquel nei~her the antigen nor antibody is labelled. When a sample containing antigen is contacted with the particles, inhibition of agglomeration during tlle incubation period will occur. This ~esults in, at least, a partial reduction in the agglomerate $ormation, The inhibition is detected using nephelometry or other techniques for measuring turbidity. The decrease in turbidity can be correlated to the amount of antigen in the sample.
Claims (14)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the determination of the presence or concentration of an antigenic substance in a fluid comprising the steps:
(a) contacting a sample of the fluid with a known quantity of added antigenic substance and a monoclonal antibody to the antigenic substance, and (b) measuring the inhibition of formation of a complex between the antibody and added antigenic substance by combination of said monoclonal antibody and the antigenic substance in the fluid to form a second complex.
(a) contacting a sample of the fluid with a known quantity of added antigenic substance and a monoclonal antibody to the antigenic substance, and (b) measuring the inhibition of formation of a complex between the antibody and added antigenic substance by combination of said monoclonal antibody and the antigenic substance in the fluid to form a second complex.
2. A process according to claim 1 whereby a fluorescent chromophore is bound to the added antigenic substance and a chromophore capable of absorbing light at the wavelength emitted by the fluorescent chromophore is bound to the monoclonal antibody and wherein, after said contacting, the intensity of fluorescence is determined and compared to the fluorescence of a standard sample free of said antigenic substance or containing said antigenic substance in a known amount.
3. A process according to claim 1 whereby a fluorescent chromophore is bound to the monoclonal antibody and a chromophore capable of absorbing light at the wavelength emitted by the fluorescent chromophore is bound to the added antigenic substance and wherein, after said contacting, the intensity of a standard sample free of said antigenic substance or containing said antigenic substance in a known amount.
4. A process according to claim 2 or 3 wherein the fluorescent chromophore is fluorescein and the chromophore capable of absorbing emitted light is rhodamine.
5. A process according to claim 2 or 3 wherein the monoclonal antibody and the added antigenic substance are bound to particles and the first complex comprises an agglomerate of the particles binding the monoclonal antibody and the particles binding the antigenic substance.
6. A process according to claim 2 or 3 wherein the monoclonal antibody and the added antigenic substance are bound to particles and the first complex comprises an agglomerate of the particles binding the monoclonal antibody and the particles binding the antigenic substance, the particles being selected from particles of latex, silica, glass, cells, polyacrylamide, polymethyl methacrylate and agarose.
7. A process according to claim 2 or 3 wherein the monoclonal antibody and the added antigenic substance are bound to particles and the first complex comprises an agglomerate of the particles binding the monoclonal antibody and the particles binding the antigenic substance, the particles being latex particles.
8. A process according to claim 2 or 3 wherein the monoclonal antibody and the added antigenic substance are bound to particles and the first complex comprises an agglomerate of the particles binding the monoclonal antibody and the particles binding the antigenic substance, the particles ranging in size from about 0.2 µ to about 10 µ.
9. A process according to claim 2 or 3 wherein the monoclonal antibody and the added antigenic substance are bound to particles and the first complex comprises an agglomerate of the particles binding the monoclonal antibody and the particles binding the antigenic substance, and ranging in size from about 0.2 µ to about 10 µ.
10. A process according to claim 1 wherein the monoclonal antibody and the added antigenic substance are bound to particles and the first complex comprises an agglomerate of the particles binding the monoclonal antibody and the particles binding the added antigenic substance, and wherein, after said contacting, the turbidity of the sample is determined and compared to the turbidity of a standard sample free of said antigenic substance or containing said antigenic substance in a known amount.
11. A process according to claim 10 wherein the particles are selected from particles of latex, silica, glass, cells, polyacrylamide, polymethyl methacrylate and agarose.
12. A process according to claim 11 wherein the particles are latex particles.
13. A process according to claim 10 wherein the particles range in size from about 0.2 µ to about 10 µ.
14. A process according to claim 11 wherein the particles range in size from about 0.2 µ to about 10 µ.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/175,133 US4376110A (en) | 1980-08-04 | 1980-08-04 | Immunometric assays using monoclonal antibodies |
| US175,133 | 1980-08-04 | ||
| US06/323,498 US4486530A (en) | 1980-08-04 | 1981-06-24 | Immunometric assays using monoclonal antibodies |
| US323,498 | 1981-06-24 | ||
| CA000382964A CA1281640C (en) | 1980-08-04 | 1981-07-31 | Antigen bound by two monoclonal antidodies |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000382964A Division CA1281640C (en) | 1980-08-04 | 1981-07-31 | Antigen bound by two monoclonal antidodies |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1195926A true CA1195926A (en) | 1985-10-29 |
Family
ID=27167103
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000473725A Expired CA1195926A (en) | 1980-08-04 | 1985-02-07 | Immunometric and inhibition assays using monoclonal antibodies |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1195926A (en) |
-
1985
- 1985-02-07 CA CA000473725A patent/CA1195926A/en not_active Expired
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1281640C (en) | Antigen bound by two monoclonal antidodies | |
| US4486530A (en) | Immunometric assays using monoclonal antibodies | |
| US4098876A (en) | Reverse sandwich immunoassay | |
| Llenado et al. | Surfactants | |
| US4062935A (en) | Immunoassay involving the binding of RF to the antigen-antibody complex | |
| JPS6188155A (en) | Fluorescent energy by phycobilin protein | |
| JPH09511582A (en) | Antibody detection method | |
| JPH01282447A (en) | Immunoassay system for internal total reflection scattered | |
| EP0711414B1 (en) | Method for assaying an immunological substance using magnetic latex particles and non-magnetic particles | |
| EP0249357B1 (en) | Methods of immunoassay | |
| JP2003531378A (en) | Multi-subject diagnostic test for thyroid disorders | |
| EP0369362B1 (en) | Substrate composition for alkaline phosphatase and method for assay using same | |
| CA1186622A (en) | Homogeneous immunoassay with labeled monoclonal anti- analyte | |
| JPH0421819B2 (en) | ||
| JPH01262471A (en) | Ckmb assay and monoclonal antibody used therefor | |
| CA1195926A (en) | Immunometric and inhibition assays using monoclonal antibodies | |
| US20060035295A1 (en) | Method for determination of thymidine kinase 1 activity and the use thereof | |
| CA1289874C (en) | Anti-enzyme antibody immunoassay | |
| WO1987002779A1 (en) | Idiotypic-antigenic conjunction binding assay | |
| Maggio et al. | Immunology: current and future technology assessment | |
| Salomons et al. | Immunoassay: A survey of patents, patent applications and other literature 1980-1991 | |
| JP3569074B2 (en) | Immunoassay method | |
| EP0181762B1 (en) | Method for detecting enzymatic activity using particle agglutination | |
| JPS5890169A (en) | Immunological inhibition test using single chron antibody | |
| Fortunato | Principles of Immunochemistry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |