JP2598893C - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the invention Chemical characterization and determination of tumor specificity and clinical response of carcinoembryonic antigen (CEA)
A major problem in use is the use of polyclonal antiserum reagents. because
CEA is partially due to cumbersome and mostly inaccurate and incomplete antibody adsorption techniques
Has been shown to contain numerous antigenic sites that can be defined and characterized
It is. Thus, anti-blood prepared against what is termed "purified CEA"
With the use of Qing, at least 12 cross-reactive antigens have been described. However
However, such “purified CEA” still alters these cross-reactive antigenic determinants
Contained amount. For example, anti-blood group A antiserum is CEA and CEA
Copeptide fragments can be linked. CEA molecules are also found in fetal sulfogly, often found in the gastric juice of gastric cancer patients.
It may have determinants that are cross-reactive with coproteins. Gastrointestinal tract cancer, normal colon
In the normal lung, normal spleen, and certain white blood cells, such as granulocytes, including cancerous
Abundant non-specific cross-reactive antigens are the most abundant CEA cross-reactive determinants
It is. In addition to these known CEA cross-reactivity determinants, the
Assays, such as in vitro immunoassays, in vivo radioimmunodetection and therapy, and
And others that can interfere with the specificity of in vitro immunohistochemical detection. Therefore, such cross-reactivity
Material characterization and isolation is of interest. One useful derivation is precise specificity, like
Closely related and found only in one antigen of the cross-reactive group of antigens
Specificity for an epitope, or an epitope common to two or more of such antigens
Production of antiserum having specificity for the These antisera are used in numerous clinical and
Useful in laboratory equipment. An epitope is an antigen that affects its specificity.
Is defined as a single determinant of the antigen or immunogen (Dictionary of Scientific and Tec
hnical Terms, McGraw-Hill, New Yourk, 1976). In the past, attempts to produce a monospecific anti-CEA antiserum have been
Or exhaustive adsorption by other substances, but these methods are cumbersome.
Yes, difficult to control, inaccurate, and still in polyclonal antiserum
The problem of having a cross-reactive immunoglobulin that is present in the Attempts have recently been made to overcome the above problems by using monoclonal antibodies
. However, in some attempts to produce monoclonal antibodies against CEA,
Even based on the categorization of the epitopes of CEA to which they are reactive,
The different species of the CEA monoclonal antibody could not be elucidated. in this way,
The range of true CEA specificity is the range of epitopes found in the CEA antigen family.
Fine characterization and / or epitopes found on antigens cross-reactive with CEA
I still have to wait for the elucidation. CEA family antigen is common with CEA
Glycoprotein or protein having several physicochemical and immunological properties
Defined as a group of protein substances. It is known to use both antigens and specific antibodies in immunoassays
. However, such assays are often misdefined for epitope specificity
The use of sexually active antigens and antibodies was limited. Therefore, they form part of the present invention.
Some applications have not been possible before. Similar limitations in the fields of immunohistochemistry, in vivo imaging and tumor treatment
Overcome by Object of the present invention It is an object of the present invention to precisely define CEA and / or antigens cross-reactive with CEA.
It is to provide a substantially monospecific antibody to a defined epitope. Another object of the present invention is to have precisely defined epitopes and immunoreactivity.
To provide a purified antigen and / or antigen fragment. Yet another object of the present invention is to provide specific epitopes on antigens in the CEA family.
The purpose is to provide a way to detect its presence. Yet another object of the present invention is to provide a method on the same antigen or on two different cross-reactive antigens.
Method for determining the relative proportions of at least two different epitopes present in
Is to provide. Yet another object of the present invention is a method of diagnosing and / or differentially diagnosing a pathogenic condition,
And / or precise identification, characterization and identification of closely related antibodies and / or antigens
And a method for titration. Yet another object of the present invention is to provide methods for detecting, identifying, locating, characterizing and staging cancer, and for treating tumors. Upon further consideration of the specification and claims, one of ordinary skill in the art will recognize further details of the present invention.
The above objectives and benefits will become apparent. Summary of the invention These and other objects which will become more readily apparent when reading the following description
Can be achieved by the present invention. In terms of composition, the present invention
At least one CEA cross-reactivity determinant, such as a substance for substantially pure meconium antigen
It relates to a qualitative monospecific antibody. In a method aspect, the present invention provides a method for treating antigens on CEA or antigens cross-reactive with CEA.
A method for detecting the presence of a particular epitope present on at least one antigen.
The method comprises contacting the antigen with an antibody specific for the epitope.
It becomes. The invention further relates to at least two antigens present on the same or different antigens.
There is provided a method of measuring the relative proportion of different epitopes, said method comprising:
Analyzing the analyte comprising at least two epitopes
Contacting at least one capture antibody capable of binding a Specis having
Combining the resulting epitope with one of the two epitopes.
Contacting with a probe comprising a probe antibody capable of
Measuring the concentration of the probe. The present invention also provides an immunoassay method,
Immunohistochemical methods, in vivo detection, localization and therapeutic methods, and precision antibodies,
Methods for isolating and purifying antigens and antigen fragments are provided. Detailed discussion Since the discovery of CEA, the definition of this antigen has been some physicochemical, immunochemical, and biochemical.
It is based on molecules that meet biochemical criteria. As stated earlier, CEA
Is restricted to fetal and cancerous gastrointestinal tissues and is soluble in perchloric acid,
A 200,000 molecular weight β-glycoprotein having a nooic acid and carbohydrate composition
there were. The identification of this substance was based on the fact that the antiserum was based on immunodominant groups on the glycoprotein
Exclusively for its ability to recognize the same as detected by anti-CEA antiserum
Was dependent. Later, CEA was found to have widespread physicochemical and immunological properties.
It was shown to show uniformity. The feature of CEA is “Immunodiagnosis of Cancer
, Part I ", Herberman et al., Eds., Marcel Bakker, Inc., New York and B.
asel 1979 and “Carcinoembryonic Proteins: Recent Progress”, Norgaad-P
edersen and Axelsen, Eds, Blackwell Scientific Publications, London, 197
8 A list of these references and the references cited therein.
All are incorporated herein by reference. A major problem penetrating numerous studies of CEA, including its clinical applications, is that
The specificity of the various methods used to detect the antigen. Up against the CEA
Conventional antisera raised antibodies that react with a group of substances closely related to CEA.
Contains characteristically. Of the latter antigens, the first to be described is normal human tissue, especially
A 60,000 molecular size group present at higher levels in the lung and spleen than CEA
It is a non-specific cross-reactive antigen (NCA) which is a lycoprotein. Their immunity
Several other substances that seem to be NCA based on chemical similarity have been described and
These include normal glycoprotein, CEA-related protein, colon CEA-2, colon calci
Includes Norma antigen-III, and tumor-associated antigens. NCA and CEA represent the determinants that distinguish them individually.
Because they appear, they can be distinguished immunologically. CEA-like antitumor in meconium and adult stool
A second group of originals was also identified. N characterized by Burtin and collaborators
CA-2 is slightly smaller in molecular size than CEA and either NCA or CEA
Determinants that do not have any. In addition to the common determinant between the three antigens, the second
NCA-2 is an NCA because the group is common only between NCA-2 and CEA.
Seems to be more closely related to CEA. Normal stool antigen in adult stool (
NFA) has recently been separated into three molecular species, all of which are
A-2, different from NCA and CEA. Between meconium CEA-like antigen and CEA
Elucidation of the chemical, antigenic, and developmental interrelationships of these was required. Of these antigens
Some clinical utilities are unknown and they are either cleavage products of CEA or CE
It is unclear whether it represents an A precursor molecule or a truly different gene product
However, their identification by polyclonal antibodies was equal among related antigens.
This resulted in the determination of at least three epitopes on CEA that were not shared. example
See, for example, Burtin, P, Roubertie, P., Chavanel, G., Sabine, MC, and Hirsch-
Marie, H .; “CEA and related antigens: A study of NCA-2”; WH. Fishman
And S. Sell (eds.)., “Onco-Developmental Gane Expression”, pp. 609
-611, New York, Academic Press, Inc., 1976; Kuroki, M., Koga, Y., and
Matsuoka Y. “Puri-fication and characterization of carbinoembryonic an
tigen-related antigens in normal adult feces ”, Cancer Res., 41 : 713-72
0, 1981; Primus FJ, Collins, RW, III, and Blue, A .; “Antigenic relationship of carcinoembryonic, nonspecific cross-react
ing, and meconium antigens ", Proc. Amer. Ass. Cancer Res., twenty two : 298, 19
See 81. The immunohistochemical localization of CEA has been performed using polyclonal antibodies.
Widely studied in epithelial cancer. It is normal and non-cancerous pathological tissue, good
Tumors, and to varying degrees in atypical epithelium of various organs,
Detection of this antigen can be used to distinguish normal or benign cells from cancerous cells.
Absent. Most studies have determined that measuring antigen levels in blood is
Most studies have found that CEA staining and pathological stages of primary tumors
No correlation with prognosis has been found. Several studies have described the development of monoclonal antibodies against CEA,
Their use for immunohistochemical detection of this antigen in scar tissue sections has been adequately explored.
Was not investigated. Similarly, US Patent No. 4,348,376 to Goldenberg and US Patent
In vivo imaging of cancer employing the method disclosed in application No. 414,729 according to the invention
Limited monoclonal applications for CEE family antigens are now pursued
And some benefits for the detection and localization of some cancer types
I do. Similarly, such antibodies to epitopes on CEA family antigens
No. 4,348,376 and No. 4 to Goldenberg for antibody fragments.
Single antibody or antibody combination as described in U.S. Pat.
Including antibodies labeled with isotopes, drugs, toxins, or similar toxic agents
Preferred reagents for cancer immunotherapy and for use in neutron capture therapy. Recent studies to improve the immunospecificity of CEA detection and in vitro immunoassays
Efforts to develop monoclonal antibodies against CEA and compare with conventional assays
Is focused on However, cross-reactivity with CEA-related antigens
Monoclonal antibodies as well as their conventional counterparts, and
A model for CEA family antigens whose epitopes are not properly defined
The use of noclonal antibodies is expected to severely limit this application. Monoclonal antibodies raised against colon tumor CEA show three of the antigenic determinants
Generic classes based on their reactivity with CEA-like antigens, NCA and MA
Differentiation has now been shown (FIG. 1). The minimum percentage of clones is
Epitopes that all antigens have in common and belong to the class I category
Produce antibodies that recognize. Class II monoclonal antibodies between CEA and MA
React with common sites, but those of class III type bind only CEA,
Obviously, it recognizes determinants unique to this molecule. We are NCA cross-reactive products
The affinity of the clonal antibody NP-1 for NCA depends on its affinity for CEA.
It was found to be significantly lower in comparison. So a monoclonal antibody against CEA
The NCA cross-reacting properties of the antibody are such that the appropriate amount of antibody or NCA is
May not become apparent unless used in a competitive inhibition assay. NP-1
Comparable amounts are effective to bind both CEA and MA, and MA
This is similar to CEA in blocking the reaction of NP-1 with CEA,
It doesn't seem like that. We have neutrophils in their CEA
But showed positive staining with NP-1 as expected from the known synthesis of NCA. This
Thus, the immunocytological staining of neutrophils was determined by the monoclonal antibody against CEA.
An alternative method of assessing CA cross-reactivity is provided. The repertoire of CEA epitopes limited by monoclonal antibodies is now
I can understand. Mutual inhibition experiments were performed using NP-1 and NP-1 antibodies of class I and II, respectively.
NP-2 can block each other, but all block the class II antibody NP-3.
It could not be stopped or could not be blocked by NP-3. NP-1
The mutual blocking properties between NP-2 and NP-2 depend on the respective epitaxy recognized by them.
The tops suggest partially overlapping or very close to each other. However,
The lack of mutual inhibition between NP-2 and NP-3 indicates that they are within the Class II category
To recognize two other epitopes. Thus, we
Our monoclonal antibody has at least four antigenicities on colon tumor CEA
It has been found that the sites can be distinguished. One is CEA, MA and N
Held together by CA (α1 in FIG. 1), the other two by CEA and MA
Both are retained (β1 and β2), and the fourth is specific for CEA.
(Γ1), and the other CEA determinants are the three classes shown in FIG.
Expected to be identified within all of the NP-4 monoclonal antibodies and other class III antibodies are typically CEA
Binds less than 50% of CEA molecules recognized by goat antibodies specific for
You. The basis for the partial reactivity of Class III antibodies is the knowledge of the partial number of CEA molecules.
Related to another. One key goal for the development of immunospecific monoclonal antibodies to CEA
Is to accelerate the development of sophisticated applications of CEA detection. If CEA various
If the immune forms are produced in different individuals or at different disease stages,
The ultimate benefit of the antibody is its ability to target monoclonal antibody conjugates with limited specificity.
Will depend on creation. CEA to determine monoclonal antibody epitope specificity
Incorporation of related antigens is an antibody with diagnostic and prognostic relevance for CEA detection
Must be easier to demarcate. This is NCA-2, NFA-2, or MA
Interactions between ER and CEA and the Role of the former Antigen as a Tumor Marker
Will be further strengthened. Finally, the model for CEA
The use of other methods, such as immunohistochemistry, to study noclonal antibodies may be known elsewhere.
Molecular determinants with no prognostic potential can be identified. This disclosure of the present invention is provided by Cancer Research, 43 (1983
Includes full disclosure of three papers, taken as reference.
Put in. Identification of NCA-unrelated, CEA cross-reactive antigen in meconium NCA neutralizing anti-CEA antiserum was tested on individual meconium samples by double immunodiffusion.
When testing, approximately the same number of samples are usually one or two sedimentation elements that are very close to each other.
Give the band. For most specimens with two bands, both have CE
Generates a reaction that partially matches A, but 10% of unextracted or PCA-extracted samples
Below, the second band gives a response consistent with CEA. These two CEs
Precipitin band cross-reactive with A does not fuse with CEA also present in meconium
. These gel diffusion patterns demonstrate two common decisions on CEA in the NCA neutralizing antiserum that are not related to epitopes shared by CEA and NCA.
It indicates the presence of two additional antibody specificities that recognize the group. Radioimmunoassay for NCA-unrelated CEA cross-reactive antibody in CEA antiserum
(RIA) to determine if it contributed to the measurement of CEA in meconium
CA neutralizing antiserum is a meconium sample that does not give a concordant reaction with CEA by the double diffusion method
Is further absorbed by the This meconium absorbed antiserum has a sedimentation activity against CEA.
Maximum binding of the labeled and retained CEA compared to that of the non-absorbing antiserum
About 10%. However, the antibodies originally present in the NCA neutralizing antisera
More than 80% of the activity is eliminated by absorption by meconium. Roche kit CE
A, NCA-neutralizing antiserum or specific anti-CEA antiserum and CEA-labeled
CEA concentration in meconium by RIA, used as racer and inhibitor
Were compared. All three antisera have the same sensitivity for inhibition by CEA.
Adjusted every time. Analysis of 12 meconium samples was performed by Roche and NCA neutralizing antiserum.
The CEA content measured by the method described above was higher than the value obtained with the specific anti-CEA antiserum.
3 to 10 times greater (p <0.001;Student's t-test)
(Table 1). Purification of MA from meconium At various steps during the purification of MA, antigenic activity is determined by NC as a meconium species.
A Roche's kit antibody is considered to measure unrelated CEA cross-reactive substances.
Specific anti-CEA anti-blood depending on the RIA used and to track CEA activity
Monitored by Qing. These antigen activities at various stages during the isolation procedure,
Specific NCA concentrations are shown in Table 2. Meconium in 40% ethanol at pH 8.0
The extraction gave about 75% of the original MA activity in the ethanol supernatant. Large precipitates
Produced by tanol fractionation, but supernatant remains viscous and highly colored
there were. Chromatography of ethanol extract on DEAE cellulose is a dye
Most of the MA was removed, but a subfractionation of MA was generated through the entire chromatogram.
I did After chromatography of 0.1 M NaCl fraction on Sephacryl S-300, MA activity
Appeared overlapping with the elution of the radiolabeled CEA marker at a slightly smaller size. 2
The MA appearing between the elution volumes of 25 to 270 ml was pooled and the two
It was subjected to an affinity chromatography step. The first compounded with MA
In order to remove the small amount of residual CEA, an antibody containing a specific anti-CEA antibody
Includes passage over the epidemic sorbent. This operation removed 95% of the CEA, but MA
Was lost and was not found in the adsorbed fraction (Table 2). Second
At least one of the NCA-unrelated CEA cross-reactive substances in meconium
Contains murine monoclonal CEA antibody, NP-3, cross-reactive with two groups
Immunosorbents were used to select and further purify the MA. Applied
More than 80% of the MA activity obtained was retained by this immunosorbent. 50g meconium
The overall yield of MA activity starting from was 4% or 0.1 M NaCl DEAE fractionation
20% of that present in it. (a) Unextracted meconium samples were first suspended 1: 4 (w / v) in PBS, then
Prior to the above, it was diluted in 0.01M borate buffer pH 8.5. (b) Values are specific levels of NCA measured in NCA RIA (c) μg / g (a) For NCA, RIA and CEA using NCA specificity assay
Goat-specific anti-CEA antiserum, or Roche kit antibody for MA
Measured with the CEA RIA used. Immunological analysis of purified MA Based on the neutralizing activity in the RIA, the finally purified MA contained less than 1.0% CEA or NCA (Table 2). NCA neutralizing anti-CEA
Double immunodiffusion of MA to antiserum is a single form of partial coincidence with CEA
A sedimentary element band was provided. The two cells appearing between unextracted meconium and the latter antiserum
Both fused to a single line formed by purified MA. Purified
The appearance of only one sedimentary element band due to MA was due to the second NCA-unrelated CE.
Suggests that it does not contain A-related antigens. Purified MA is notable for double diffusion.
Did not react with the heterologous anti-CEA antiserum, but this antiserum was capable of precipitating CEA.
Power was possessed. MA is also alpha-sensitive for serum proteins in immunoelectrophoresis.
Single precipitin band against NCA neutralizing CEA antiserum migrating as globulin
Gave. The antibody binding properties of the radiolabeled MA were evaluated in an RIA. As shown in Table 3
Both Roche kit CEA antibody and monoclonal CEA antibody NP-3
One binds a similar amount of MA, comparable to the binding of labeled CEA by these antibodies.
Was. More than 80% of MA was immunoreactive when tested shortly after labeling. Goat
Although only specific anti-CEA antiserum was marginally immunoreactive with labeled MA,
The goat anti-NCA antibody was non-reactive. The former antiserum and Roche kit antibodies
Were similar in their reactivity with labeled CEA. Excess specific anti-CEA
With Roche kit antibodies is more than with Roche kit antibodies themselves
The same major number of labeled CEAs was not increased by both antisera without increasing the maximum binding of CEA.
Is recognized. Inhibition assays and double diffusion were tested for CE in MA.
A did not show a significant amount of A, so that labeled MA was added to goat-specific anti-CEA antiserum.
The low level of binding is most likely due to the presence of residual cross-reactive antibodies in this antiserum, not to the reactivity with labeled CEA contaminants.
It was great. (a) After incubating at 45 ° C. for 4 hours, the antibody was measured in excess. Solid-phase goat anti-mouse I
gG or donkey anti-goat IgG was used to separate bound antigen from free antigen. MA molecular size Chromatography of labeled MA on Sephacryl S-300 equilibrated with phosphate buffered saline
The chromatography shows a single pair eluted only in a slightly larger elution volume than CEA.
Nominal peak was given. This relationship in the MA and CEA elution patterns is 6.
After passing over Sephacryl S-300, which is equilibrated and eluted with 0 M guanidine HCl,
Was held. Overlap of size distribution between MA and CEA was also observed after SDS-PAGE electrophoresis of reduced samples on calibrated gels. Logarithm for standard mobility
The estimation of the molecular size for MA by interpolation from the molecular size plot is
A value of 185,000 was given for 200,000 for EA. Coomassie Brilliant Bulb
Staining of reduced MA in SDS-PAGE with Leu or cyclic acid Schiff reagents
A single diffuse band migrating to a position similar to EA was shown. Binding of MA to Conconavalin-A Binding of radiolabeled MA to concanavalin-A sepharose was determined by CEA and NC.
A compared with that of A. More than 9% of CEA and NCA bound to this lectin
However, less than 20% of the MA was reactive. Pronase digestion of CEA and MA Digestion of CEA by Pronase E shows its reactivity with goat-specific anti-CEA antisera
NP-3 monoclonal antibody or Roche kit goat
This resulted in a 50% reduction in its avidity for the antibody. Loss of this antibody binding activity or
No decrease was observed after a similar treatment in the absence of the enzyme. 6.0M Guani
Chromatography of enzyme digestion solution on Sephacryl S-300 equilibrated with gin HCl
Showed that most of the CEA was degraded into smaller fragments. In contrast,
Enzymatic digestion of MA reduces NP-3 and its binding to Roche kit antibodies by 20%
And resulted in only a slight decrease in its molecular size. Meconium antigen MA and
We for one of the named NCA-unrelated CEA-related molecular species in meconium
Isolation operation avoids the application of an immunoseparation step in the early stages. This is a
Means for providing an extract having advantageous properties for affinity chromatography;
This is because the solubilization of perchloric acid PA alone or in combination with alcohol failed. Different alcohol concentrations and pH ranges tested
In the box, 40% alcohol at pH 8.5 gives the best MA solubilization and maximum
A precipitate of inappropriate protein was obtained. Subsequent chromatography on DEAE cellulose
Feet has desirable characteristics that leave the bulk of the dye, but inappropriate protein and N
Large amounts of CA were not adsorbed. These benefits are due to the M observed on the ion exchanger.
The sub-fraction of A activity was weighted. In the final stage, MA
Specific adsorption to immunoadsorbent containing cross-reactive monoclonal antibody NP-3
Was. The use of monoclonal antibodies may have differences with respect to other determinants.
However, a molecular spectrum that is uniform in the expression of the epitope recognized by the antibody
Having the benefit of choosing cis. MA is NCA-2 and the NFA family of CEA-like antigens in adult stool,
In particular, it is distinguished from NFA-2. NCA-2 and NFA-2 are molecular size,
And CEA in carbohydrate and amino acid composition. Immunologically,
NCA-2 shares two epitopes with CEA, while three epitopes on NFA-2
Cross-reactive sites were indicated. We have a monoclonal antibody against CEA.
MA, which represents one of the two NCA-unrelated CEA cross-reactive substances in meconium, is C
It has been found that it shares at least three epitopes with EA. The second in meconium
The CEA-associated antigen has at least one cross-reactant present on both CEA and MA.
Lacking site. MA can be distinguished from other CEA-related antigens in meconium described earlier. MA is CE
A has a similar molecular size to A, but by the conventional antiserum we have used
Antigenically differing in the lack of at least one determinant distinguished on CEA, and this difference is
Noclonal antibodies are also found. In addition, MA is either CEA or NCA
It has a lower affinity for Concanavalin A than it does. For this lectin
The low affinity of MA for the separation of MA from CEA and NCA
Provides another possible method that does not require an adsorbent. MA and other NCA non
Related CEA-related antibodies are expected to coalesce and appear at approximately 6-fold higher concentrations than meconium CEA
Our observations indicate that these antigens are earlier markers of differentiation than CEA
Maybe. The supply of purified MA was determined by monoclonal antibodies against CEA.
Epitope specificity and of these epitopes between CEA, MA and NCA
We have made it easier to demarcate the distribution. Staining of normal colonic mucosa Staining of morphologically normal colonic mucosa has all four monoclonal antibodies.
Could be visualized. This is fixed in ethanol / acetic acid (EA), not in formalin
Was best observed in the prepared specimens. Positive staining of normal and tumor epithelium is
Did not appear in control strips, including those reacted with riclonal antiserum and ascites
. Specimens fixed in EA rather than formalin often appear in test and control specimens.
Variable non-specific staining of connective tissue was demonstrated. Staining of normal mucosa with goat antiserum and monoclonal antibodies NP1,2 and 4
The color is the cytoplasm and mucous membrane of the columnar cells where the positive staining mainly covers the upper level of the crypts
Localized on the surface. Secretory substances in the glands or gastrointestinal tract were also stained, but
Vesicle mucus was antigen negative. The intensity of the staining reaction with all of these antibodies and
Presence was quite variable between the crypts, but goat anti-CEA and NCA antiserum and NP-1
Most prominent with monoclonal antibodies. No NP-4 monoclonal antibody
It gave a weaker staining reaction which was strangely less pronounced than any of the other antibodies. Cross-reacts with NCA based on labeled antigen binding in addition to normal colon epithelial cells
Class I monoclonal antibody NP-1 is also known to
Neutrophils in the weave were stained. This staining reaction was readily apparent in specimens fixed in EA.
It was clear, but not in formalin. Neutrophil localization is immunope
Was visualized by the reoxytase procedure,
No need to block the activity of the enzyme
Easy identification by immunoglucose oxidase method for easy recognition of primary stains
Was done. In addition to NP-1, only goat anti-NCA antiserum among the other antibodies studied was preferred.
Neutrophils were stained. Colon tumor staining Goat anti-CEA and NCA antibodies, and NP-1, 2, and 3 monoclonals
Antibodies were found in all 22 cases of moderately differentiated colon adenocarcinoma in the origin of their colon
Stained regardless of position. About 70% (15/22) of these colon adenocarcinomas are class II
A positive staining reaction was given with the I monoclonal antibody NP-4. Fixed in formalin
Predominant staining patterns obtained in isolated specimens indicate the apical surface of tumor cells and intraglandular deposits.
It was a sign. Except for the NP-4 antibody, the same area of the tumor specimen is more similar to other antibodies
Stained to the specified intensity, but no absolute correspondence was established in cell localization
Was. Among the monoclonal antibodies, NP-1 is consistent in its strength and range.
And in some cases, more than that obtained with goat anti-CEA or NCA antiserum.
It was great. Intensity of NP-4 staining was obtained with other antibodies in 6 cases.
A little lower, but it dropped significantly in the remaining nine specimens. Tumor gland
Most were stained by NP-4 in 12 cases, but in the other three specimens
30-50% of the tumor tissue reacted with this antibody. Smell all of these examples
Thus, over 80% of the tumor tissue reacted with other antibodies. 2 isolated from NP-4 negative
Examples gave a weak focal response to less than 10% of the tumor glands. For 6 specimens of primary tumor, sufficient material is available for additional fixation in EA
Was. The intensity of staining with all of the antibodies was observed after formalin fixation in each case.
EA fixed specimens were higher than those obtained. Cytoplasmic staining of tumor cells is EA fixed label
It was very clear in the book, but after formalin fixation it was absent or
It has become weak. Enhanced cytoplasmic staining in EA-fixed tissue compared to that of goat antiserum
Was not so large with the NP-4 antibody, but was obtained with the former antibody
The overall staining reaction was significantly greater in EA-fixed tissues when compared to formalin-fixed specimens.
Met. Only one case of degenerative cancer was studied, and this was
Completely negative for all of the bodies. NP-4 staining of metastases Of the 22 moderately differentiated adenocarcinomas examined, Duke's stages A, B, C and
And 1, 5/7, 3/4 and 6/10 of primary tumors from patients with
Reacted with P-4. With a small number of patients, a positive correlation between NP-4 responsiveness and clinical staging
Although preliminary correlations cannot be reached, these preliminary results establish an absolute relationship
Suggest that it will not be. Between NP-4 reactivity and location of tumor in colon or rectum
The relationship between them was not clear. From the above 8 patients, C and D stages of Duke respectively
Regional lymph nodes and / or as formalin fixed specimens from 2 and 6 cases on the floor
Liver metastases were obtained for study. These specimens contain goat anti-CEA and NCA antibodies
And stained with NP-3 and NP-4 monoclonal antibodies (Table 4). N
The P-4 antibody was one of those visualized in a single piece that stained positively with this antibody.
Even in patients with more than 80% of secondary tumor tissue, only a few
Only a positive staining reaction was given. Most of these metastases consist of goat antibody and NP
-3 monoclonal antibody. Regional lymph nodes and liver metastases of two patients C-2 and D-3 were stained for their
Further differences in phenotype were noted (Table 4). Two nodules from patient C-2 are anti-
Did not react with all of the body, but the third nodule gave a negative reaction only with the NP-4 antibody
I got it. One nodule from patient D-3 was not stained by any of the antibodies, plus
Another nodule and liver metastasis were with goat anti-CEA antiserum, NP-3 and NP-
No staining with 4 monoclonal antibodies. In contrast, the remaining 4 nodules and liver
Metastases were NCA positive. These specimens from patient D-3 were subjected to NCA cross-reactivity.
When tested against the monoclonal antibody NP-1, the staining pattern was
The same as that obtained with the NCA antibody. (a) = Percentage of primary gland tumors staining positively with NP-4 (b) = Letters indicate Duke stage. The numbers identify individual patients. (c) = ratio of the number of positives out of all tested The ultimate aggressive behavior of morphologically similar tumors depends on the number of
The potential that can be confirmed by the detection of antigen markers is some cancer types.
Emerged from the immunohistochemical study of Ip. Other markers include molecules of certain antigens
Or the identification of subtle determinant changes can be attributed to epiclones originally recognized by polyclonal antibodies.
It would have been overlooked due to the diversity of the topps. Identification of these molecular changes
Because they really lead to concomitant changes in the biological representation of cancer cells
Anything would be meaningful. The precise immunospecificity of monoclonal antibodies is
It provides a tool by which the original modification can be explored, and
We have polyclonal antibodies and four monoclonals directed against CEA.
The staining characteristics of colonic adenocarcinoma were compared using the antibody of the present invention. Immunohistochemistry using conventional antisera showed the presence of CEA in normal colon
As expected from previous experiments, this tissue contains three cross-reactive monoclonals.
Stained with antibody. This lacks cross-reactivity with NCA and MA
Appears to recognize a small number of CEA molecules based on labeled antigen binding
The same is true for NP-4. Staining with these antibodies is not reactive with individual antibodies.
To be localized within the same cell as visualized in separate tissue sections
Looked like. The monoclonal antibody did not stain the mucous of colonic goblet cells.
Of the Molucnal antibodies, only NP-1 is predicted by their known NCA synthesis.
As can be seen, neutrophils that reacted similarly with goat anti-NCA antibody were stained. Thus, immunohistochemical manipulation
Works to determine the specificity of monoclonal antibodies to CEA as determined by other methods.
Their NCA exchanges when other methods are not readily available.
Useful for identifying differential reactivity. Except for the NP-4 antibody, other monoclonal antibodies and conventional goat antiserum are primary
All of the moderately differentiated colon tumors and the regional lymph examined in this study
Most of the node and liver metastases were stained. From NP-4, about 30% of primary tumors
It is non-reactive, and most of the metastases arising from NP-4 positive primary tumors
A different staining pattern appeared in that it was not stained in the body. We have shown that the staining intensity obtained with the NP-4 monoclonal antibody was not formalin.
It was found that the primary tumor specimens fixed in EA were significantly improved. So
Instead, there is almost no substantial NP-4 staining of metastases in patients with positive primary tumors
The location of recognized determinants or antigens from metastatic tumors
Suggests sexual deletion or cloning of antigen-negative tumor cells. We have not yet compared the determinant representation in the organization with that in the circulation,
The presence of A determinant heterogeneity is determined by the concentration of this antigen in the blood as measured by monoclonal antibodies.
It suggests that the correlation between disease and disease activity arises in a potential problem. Here
Based on the immunohistochemical studies we provided, we
Relative contribution to ring antigens and monoclonal antibodies used in immunoassays
Depending on specificity, predicts poor correlation with various assay results and disease activity
I think Fundamental questions remain unanswered regarding antibody specificity and the presence of reactive antigens in tissues and blood
The answer, but immunohistochemistry that can be conveniently applied to conventional histopathological specimens, is
Monoclonal anti-reactive with CEA variants or determinants of after and diagnostic value
Will play a major role in identifying the body. This is a radioimmunoassay for colon tumors
Appropriate selection of monoclonal antibodies for antibody and antibody-based therapy
. Classes of CEA hybridoma clones The antigen binding profile indicates that the CEA hybridoma clones have their CEA, M
Differentiation into three distinct classes based on comparative immunoreactivity with A and NCA (Table 5)
Clarified what can be done. Most of the clones from the first fusion were CEA and
Produced antibodies that bind MA (class II) and clones from the second fusion were class
II and those showing only CEA reactivity (Class III)
Clothed. Reactivity with all three antigens (Class I) is due to the first and second fusions.
These clones were found in 16% and 2%, respectively. The results described in Table 1 are rare.
Obtained by assaying 50 μl aliquots of the culture supernatant. class
Although the percent binding of labeled CEA by the I and II clones was similar, the class
III clones gave consistently low values. Class I and II for MA
The reactivity of the clones resembled the binding of their CEA, but the
The culture supernatant bound 9.9% (f2.4 SE) of labeled NCA on average. This low
NCA binding has a higher NC as compared to the amount of antibody required for CEA and MA binding.
This is attributed to the need for high concentrations of antibody to obtain A-binding. NCA's best results
All required longer incubation times compared to CEA and MA. None of the CEA-reactive cultures was obtained from humans from both secretor and non-secretor individuals.
Did not bind to erythrocytes. Antigen binding in RIA of selected clones One from class I (NP-1), two from class II (NP-2 and NP
-3) and four hybrid macros of class III (NP-4)
Were selected and recloned for further characterization. NP-1
, 2 and 3 antibodies were purified from media containing gamma-free serum and NP-4 was
Used as ascites. NP-1, 2 and 3 clones are IgG1 (κ) subclasses.
NP-4 clones produced the IgG 1 (λ) isotype. This
These four monoclonal antibodies did not precipitate CEA in double immunodiffusion
. NP-1, 2, and 3 demonstrate the reactivity of goat anti-CEA antibodies with CEA and MA.
Similarly, 60-70% of these antibodies bound. NP-4 does not bind MP
And only up to about 30% of the radiolabeled CEA bound. Normal rat ascites or
Serum did not react with CEA and the highest CEA binding level observed for NP-4.
Bell is a property of other class III hybridomas. For binding CEA and MA
In addition, the class I monoclonal antibody NP-1 is as well labeled as the goat anti-NCA antibody.
Reacted with NCA. However, 2600 ng of NP-1 was
Binds the same level of CEA and MA but was required to bind 30%
For this, 1.1 and 2.5 ng of this antibody were required. A little more than CEA
Large amounts of NP-2 were required to bind MA, but the opposite was true for NP-3
Met. Class III hybridoma clone NP-4 binds up to 30% of labeled CEA to the control and 70% of binding to goat anti-CEA antibody, resulting in NP-4
To recognize the population of CEA molecules also detected by goat antibodies
Two experiments were planned. In the first experiment, NP-4 and goat antiserum were
At the highest antigen binding level and then free antigen by Z-gel.
Was tested for additional binding of CEA by separating bound antigens from E. coli. this
Test results show that CEA binding by combined antigen mixture is obtained with goat antiserum alone
Indicated that it is not bigger than the ones. If NP-4 reacted with goat antiserum
If a different number of labeled molecules were recognized, the mixture of the two antibodies would be
It should have given a percentage of antigen binding approximately equal to the sum of the combined percentages. Second
The experiments consisted of sequentially incubating the labeled antigen with the goat antibody and then with NP-4, followed by
Separating bound antigen from free antigen with a solid-phase goat anti-mouse antibody
The ability of the anti-Gi antibody to block the reaction between NP-4 and the labeled antigen was evaluated. Based on addition experiment
As expected, goat anti-CEA antiserum binds NP-4 to labeled antigen.
Completely blocked. Goat antibodies to normal goat serum or inappropriate NCA are not effective
Did not. (a) 50 μl of hybridoma cultures were individually tested for binding to radiolabeled antigen.
Say. (b) clones belonging to a specific class in the total number of clones exhibiting anti-CEA activity
number. The numbers in parentheses indicate the percentage of clones belonging to each class. (c) Mean ± SE Competitive blocking and antigen affinity Unlabeled CEA is labeled with CEA labeled with 4 hybridoma monoclonal antibodies.
Was tested in competitive RIA for its ability to block the reaction. In this operation,
The amount of monoclonal or goat antibody is 50% of labeled antigen in the absence of unlabeled antigen
Adjusted to give binding. Under these conditions, the four monoclonal antibodies have significantly different antigen inhibition.
A stopping curve was generated. NP-1 antibody is a Roche kit goat anti-
Although not similar to the body, NP-2, 3 and 4 monoclonal antibodies are more likely to block.
And required a large amount of antigen. Using M 200,000 as the molecular size of CEA,
Data obtained by the method of Mueller to determine the average affinity constant
(Table 6). K value obtained by this treatment and required to inhibit binding by 50%
The CEA ng / ml is similar to the antibody affinity between NP-1 and goat anti-CEA antibody.
Figure 7 illustrates similarity and reduced antibody affinity of NP-2,3 and 4. Significantly higher levels of binding labeled NCA than labeled CEA or MA
Since Bell NP-1 monoclonal antibody is required, it is necessary to counteract NP-1 with labeled CEA.
The ability of CEA, MA and NCA to block response was compared. The result is MA and
NCA can compete with this reaction, but compared to CEA,
MA and NCA required about 2 and 8 fold, respectively, to reduce by 50%. MA
And NCA inhibition were determined by RIA with goat-specific anti-CEA antibodies.
The formulation contained less than 1.0% CEA, so it was not due to CEA contamination
Was. Only at the highest concentration of NCA tested does CEA contamination
Start contributing to the induced arrest. 200 for CEA, MA and NCA respectively
Using molecular weights of 000, 185,000 and 60,000, NP-1
Slightly lower for MA and 10-fold lower for NCA compared to affinity
won. (a) Molar concentration of unlabeled CEA required to block 50% of antibody binding of labeled CEA
Degree or ng / ml Blocking monoclonal antibody binding Two monoclonal antibodies, NP-2 and NP-3, were tested for their binding to CEA binding.
First selected for study based on the effect of cubic buffer ionic strength
. Compared to 0.01 M ammonium acetate to obtain 30% binding of labeled CEA
At 0.1 M, a considerable amount of goat, NP-1 and NP-2;
It was discovered that a factor of 6 was needed. In contrast, NP- in these two buffers
Antigen binding by 3 and NP-4 was very similar. The possibility that NP-2 and NP-3 recognize distinct epitopes within the class III category is mutually exclusive
Analyzed by shut-down experiments. In these studies, CEA-sensitized solid-phase poly
Clonal goat anti-CEA using a radiolabeled monoclonal antibody probe
Used for Dwitch system. Unlabeled monoclonal antibody prevents binding of radiolabeled antibody
The ability to stop was evaluated in a cross-over inhibition experiment. Belongs to different class categories
However, NP-1 and NP-2 were very efficient at blocking each other.
NP-3 failed to block NP-1 and NP-2 binding at the highest concentration tested
The latter monoclonal antibody failed to block the binding of NP-3 to CEA. The invention relates to at least two different epitopes present on the same or different antigens.
A general immunoassay method by which the relative proportion of loops can be determined
Including the law. The method comprises: (a) containing at least two different epitopes on the same or different molecules
The analyte that binds to the space carrying these two epitopes
Contacting one of the two epitopes with the other.
(C) measuring the concentration of the bound probe. The capture antibody is bound, can be bound, or forms a solid phase
Antibodies that can precipitate and / or can be separated from the analyte in any manner.
Or a mixture of antibodies. For example, the capture antibody contains the two different epitopes
Polyclonal antibody capable of binding two different antigens, two different
Polyclonal, or monoclonal or substantially monospecific antibody specific for an epitope, different from both the two epitopes to be measured
Can bind an epitope on the same antigen as two such different epitopes
Antibodies, or any other equivalent, including antibody fragments with appropriate specificity
Antibody or antibody mixture. The capture antibody is a solid support, such as various substances
Solid particles such as polystyrene, polyethylene, glass, etc.
Or, for example, by reaction with another antibody specific for the capture antibody, and
Have the ability to combine to form a phase that can be separated later by precipitation or otherwise.
Can be. Instead, the capture antibody may be magnetized or other known process, for example.
To a material that can be separated. Once a Specis carrying two epitopes to be determined is bound to the capture antibody
The generated binding space can bind one of two different epitopes
The other, but not the other, is contacted with a probe antibody. Preferably, a probe antibody
May be a substantially monospecific and / or monoclonal antibody. Probe antibody
Binding permits discrimination between capture antibody binding epitopes. This decision is a routine immuno
It can be performed using various techniques applied from the assay procedure. Appropriate
Such techniques include labeling probe antibodies, eg, radioisotopes, fluorescent markers
Binding enzyme, enzyme, etc., and contains the capture antibody, the two epitopes to be determined.
Separating the complex containing the probe and the probe antibody from the rest of the analyte, and
Measuring the concentration of the label. Instead, competition prevention and / or other
Indirect measurements can be performed. A few or, preferably, a single
Precisely defined antigens and / or antigen fragments, including pitope-bearing antigen fragments, are suitable for such manipulation.
Using another probe specis to detect antibodies by immunoassay
It will be appreciated that it can be used as a capture space. Immunoassay based on antigen detection in in vitro liquid specimens bound to solid support
Broadly divided into competitive and non-competitive manipulations where either antibodies or antigens can be used as well
It is. The specific type of assay chosen is available for purified antibodies and antigens
, The ease with which these reagents can be labeled with markers used to quantify the reaction,
Desired sensitivity level, intended purpose of the assay, and automation of the assay
It depends on the adaptability to the solution. Solid-phase non-competitive immunoassays using enzyme-labeled antibodies make them simple and real
It is well suited for multiple antigen and / or epitope determination due to its ease of application. This
This type of operation involves two main steps. Antigen solution onto inert carrier
Exposure to immobilized antibody. During this antigen capture step, the immobilized antibody reacts with the antigen.
And then the entire complex is superfluous by washing the inert carrier with buffer.
Separated from substance. Next, excess enzyme-labeled free antibody is transferred to the immobilized antibody-carrier-antigen complex.
Is added to During this antibody probe step, free antibody is transferred to the carrier by the capture antibody.
Interacts with the secondary bound antigen. Next, the carrier is washed, and the enzyme substrate solution and
The amount of enzyme-labeled antibody that has been cuvetted and detected
And / or is directly proportional to the concentration of the epitope. The monoclonal antibodies identified in the present invention have a limited epitope specificity
And the enhanced binding constant. As an example, a monoclonal that recognizes a common CEA-MA-NCA epitope
Antibody is 5.3 × 10 ¹¹ M ^-1 , And this value is
1.6 × 10 of antibody ¹² M ^-1 Is only slightly smaller than the constant Immunoassay
Are composed of two types. The first is polystyrene by hydrophobic bonding
A broad antibody that recognizes all of the CEA family as a capture antibody attached to a ball
It has a vector monoclonal antibody. Agarose, cellulose, acrylamid
Other inert carriers such as iron, polycarbonate and iron powder can also be used
You. The ball is then bound to a biological sample such as serum or free carrier probe
Amount of antigen CEA used to correlate with the concentration of antigen in the stet specimen
, MA or NCA. After incubation and washing, binding
Individual balls with captured antigens have specific or common epitope recognition capabilities
Exposed to enzyme-labeled monoclonal and polyclonal antibody probes with
You. Marker enzymes suitable for antibody labeling are peroxidase, glucose oxidase
Or alkaline phosphatase or β-galactosidase. That generation
Instead, use fluorescent or radioactive compounds instead of enzymes for antibody labeling
be able to. After incubation and washing, the enzyme activity
The color developed and measured by incubation with the appropriate enzyme substrate solution
The level of the product is determined by spectroscopy. By this method, the specific level of each antigen
The bell is determined and compared individually between patients with similar or different illnesses
Or compared as a ratio of specific antigens as a whole. Specific antibody levels,
Their aggregation ratios, and partial or total consensus epitope expression, may vary between patients with cancerous versus non-cancerous disease and at different stages of disease activity or degeneration.
Is expected to change. The second type of solid phase assay is configured in the same manner as the first type, but with the primary
The exception is that monospecific monoclonal and polyclonal antibodies bind separately to the ball.
And used for antigen capture. Different from that of capture antibody
Monoclonal having specificity for a second specificity epitope on a suitable antibody
A nal antibody, or a common epitope antibody, is used as the antibody probe. These immunoassay techniques involve specific antigens and / or antibodies in the analyte.
To detect the purity and / or exchange of the heterogeneous antigen and / or antibody mixture.
Drugs or markers, such as enzymes, labels or toxins, can be used to assess differential reactivity.
Antigen and / or antibody purity, including antigen or antibody labeled with
Mixtures of closely related antigens for determining titers or for example CEA families
Ratio of antigens present in the cells, isoenzymes in enzyme extracts,
Ratio of K / L light chain of antibody produced, relative to meconium antigen in blood or amniotic fluid
It can be used to measure the degree of target increase. The presence of a closely related antigen and / or its epitope characteristics and / or
The ability to make qualitative and / or quantitative measurements of relative abundance can be attributed to various diagnostics
It may be useful in situations. For example, the ratio of CEA to NCA is
Identification and diagnosis of gastrointestinal cancers for gastrointestinal diseases such as ulcerative colitis, especially colon cancer
Can be used to help. Other examples include the diagnosis of cystic fibrosis and / or
Or use of the above procedure to detect the presence of meconium antigen in blood as a prognostic indicator. Elevated MA levels in the capsular fluid and / or
Indicates an elevated relative proportion of MA above CEA in the sclerosal fluid, due to stool / maternal problems, especially difficulties
It is a flight indicator. Α-subunit of human chorionic gonand trobin
Detection of relative ratios of β and β-subunits is dependent on the particular type of tumor, e.g.
It can help diagnose tumors. The above method should be adapted to the determination of the ratio of subpopulations of circulating lymphocytes
Can be. This is useful for the diagnosis of infectious diseases such as influenza and hepatitis, and
It is useful for typing cancer cancer. Presence and / or relative ratio of isoenzymes
The use of this refinement of immunoassays to determine percent abnormalities
Various lesions such as creatine kinase-M at high levels into the bloodstream
Myocardial infarction characterized by the release of B and certain hepatic dehydrogenases
Helps detect and manage various lesions characterized by abnormal levels of isoenzymes
it can. Immunoassay techniques and / or immunization for detection of specific tumor-related determinants
The use of cytochemical techniques is useful for diagnosis. For example, the presence of certain determinants is
If the putative cancer is at an ectopic site, this would be a reversal.
It may be a cooperative indication of the presence of a transferred tumor. Demonstrates the existence of determinant features of CEA
And is ectopic, ie in a different organ than the one that originated and possibly a malignant tumor
Detection of CEA-producing tumor metastasis found in cells is strong confirmatory evidence of tumor metastasis
is there. Tumor progression from early stage of cancer to more advanced state is an expression of several determinants
Or can be determined by their loss. This is preferably through the use of monoclonal antibodies against specific epitopes,
Detection by radioimmunoassay and / or immunocytochemical methods
Can be. The presence or absence of certain determinants is thus indicative of tumor progression or characteristic.
It correlates with routine stages and also serves as a prognostic indicator. Another example of the use of this method is the indicator of tumor burden in myeloid leukemia.
One is the detection of NCA in leukemia patients. The present invention provides for organ-specific epitopes on CEA family antigens
Detection using an antibody specific to the probe. As before,
These antibodies are obtained by immunocytochemistry, in vitro immunoassays and in vivo imaging procedures.
For the determination of neoplastic tissue sources, and also for the further selection of such tumors
Can be used for targeted immunotherapy. All here incorporated by reference
As described in U.S. Patent Nos. 4,331,647, 4,348,376 and 4,361,544.
Tumor localization, detection, and treatment methods, as previously indicated, are the methods and methods of the present invention.
And compositions. For example, a specific epitope on CEA
Monoclonal antibodies of specificity are available from the United States for tumor localization and / or treatment.
It can be used according to the method of No. 4,348,376. CEA family antigen
F (ab) from two different antibodies specific for ^Two The mixture of fragments is rice
Bivalent Hybrid Antibody Flag for Use According to the Method of National Patent No. 4,331,647
Can be used in the manufacture of cement. Specificity to the precise epitope of CEA
Antibodies and colon-specific antigen-p (CSAp), together with appropriate radiolabels,
It can be used cooperatively to produce antibody formulations with increased binding for types, especially for colorectal cancer. The new monoclonal antibody of the present invention is used for immunohistochemical techniques or immunoassay.
Use for the detection of myeloid leukemia and certain lymphomas using the procedure
Is also useful. Used for CEA detection in tissue samples by immunohistochemical techniques
Some CEA-specific antibodies that have been identified are
Would be comparable regardless of the polyclonal antibody, but in other cases
The use of monoclonal antibodies according to the present invention has significant benefits, especially
Will have the benefit of giving constant results. Other licenses of the antigen according to the invention
Epidemiological histochemical applications introduced for localization of multiple markers in the same tissue specimen
Use of these antigens with different labels, such as radiolabels, fluorescent labels, etc.
No. Other uses include determination of K / L light chain ratio in lymphoma phenotyping or leukemia
As in the detection of NCA in the tissue of a diseased patient, the detection of the marker ratio in the tissue
You. The antibodies of the present invention can be prepared by conventional techniques such as affinity chromatography, precipitation,
Can be used for purification of antigenic determinants. They are also new
Unraveling undefined determinants in families of canonical and related antigens
Can be used as an aid to Conversely, containing a single epitope
Purified antigens, including antigen fragments, are antibodies, e.g., monoclonal antibodies, heterogeneous
Detect antibody preparations and other preparations such as blood, tissue, other body fluids, etc.
There are uses to make, titrate and characterize. Purified antigen requires fusion
For the production of highly monospecific antibodies without the use of monoclonal antibodies.
Antibodies with qualitatively the same monospecificity can of course be used for their production without the use of hybridoma technology.
Useful. Digestion of highly purified antigen to produce antigenic fragments
Such techniques can be used when the fragment is limited to a single immunochemically active epitope.
To further amplify its usefulness. The antigen of the present invention is, for example, Staphylococcus protea
Tamper like trypsin, chymotrypsin, thermolysin and others
It can be fragmented by exposure to cracking enzymes. Fragment
Conjugated monoclonal antibodies with common or specific epitope specificity
It can be isolated by using a contained immunoabsorbent. Preferably
, Such fragments will contain only a single epitope. Without further elaboration, those skilled in the art will, using the preceding description, understand the invention
It is believed that the entire range can be used. Therefore the following preferred
Specific examples are merely illustrative and should not be considered as limiting the rest of the description in any way.
It is. In the following examples, all temperatures are uncorrected Celsius and all parts
And percentages are by weight unless otherwise indicated. Example 1 Purification of CEA and NCA from tumor tissue The CEA products used in this study, except those for RIA, were tested by Newman et al, (Cancer
Res. 34: 2125, 1974), modified by Krupey et al, (Immunochem. 9: 6).
17, 1972) isolated from liver metastases of colon adenocarcinoma
. Generally, the concentrated PCA extract is 0.1 M NH _Four Ac, DE equilibrated to pH 4.0
Applied to a 1: 1 mixture of AE and CM cellulose (Whatman, Clifton, NJ). Adsorbed
The material was purified on a discontinuous gradient of 0.05M, 0.1M and 0.2M NaCl in the same buffer.
Eluted. Approximately 30% of the original CEA immunoreactivity in the PCA extract was due to Roche CE
Monitor with Assay Kit (Nutly, NV) and apply buffer (A), 0.05M
NaCl (B), which appeared in each of the 0.1M NaCl (C) fractions. The C fraction
Is 0.05M PO _Four Sepharose 6B in pH 5.0 plus 0.15 M NaCl buffer
And sequential chromatography on Sephadex G-200 (Pharmacia)
I did. Final separation step on Concanavalin-A Sepharose (Pharmacia)
Was carried out according to the method of Pritchard and Todd, and
The crude was eluted at room temperature with 20% (w / v) α-methyl-D-glucoside. CEA
Is dialyzed against distilled water, lyophilized, _Two Dry to constant weight on
Was done. Purified CEA has specific activity (unit dry weight) as determined by Roche assay
Neutralizing activity). It is normal human in immunoelectrophoresis
Migrate as α-globulin compared to serum and
A band consistent with the reference CEA was given. Stained for protein or carbohydrate
A single band was observed in the resulting 7.5% polyacrylamide gel. The A fraction from the DEAE-CM cellulose ion exchanger contains NCA and
By chromatography on a 5 × 90 cm Sephadex G-200 column.
It was separated from CEA which was also present in the fraction. The presence of NCA leads to double immunodiffusion
The peak of antigen activity was monitored by the elution volume of 1000-1200 ml.
Appeared inside. This fraction was subjected to Concanavalin-A Sepharose chromatography as described for CEA, and the adsorbed antigen was an anti-NCA immunosorbent.
It was further purified by the above pass. NCA is 0.2 M glycine-HCl, pH 2.
0, eluted from the immunosorbent, neutralized with NaOH, dialyzed against PBS
And concentrated on an Amicon PM-10 membrane (Amicon, Lexington, Mass.). Refined
NCA migrates as β-globulin in immunoelectrophoresis and is double-immunized.
A concordant response with the reference NCA was given in spread. NCA was mentioned earlier
Sea urchin (Primus et al., J. Immunol. 118: 55, 1977) Normal spleen by immunoprecipitation
Was partially purified. Example 2 Purification of MA from meconium 50g meconium with 0.1M PO _Four , Suspended in 300 ml of pH 8.0 and mixed overnight at 4 °
did. After centrifugation at 10P000 xg for 30 minutes, the pH of the supernatant was adjusted to 8.5, and
Then cold absolute ethanol was added to a final concentration of 40%. Ethanol obtained after centrifugation
Aqueous supernatant was added to 0.02M PO _Four , Dialyzed against pH 7.8 and equilibrated with the same buffer
It was mixed with 5.0 g of E52 (Whatman). Unbound material eluted with applied buffer
Afterwards, the adsorbed material was removed with a discontinuous NaCl gradient in the same buffer. 0.1M Nacl min
The antigen activity that appears in the picture is 0.05M PO _Four , PH 5.0 plus 0.15 NaCl
Dialysis, concentrated on a YM-10 membrane (Amicon) and 2.6 × 90 cm Sephacryl S-
Applied to a 300 (Pharmacia) column. Included in 225-270 ml elution volume
Pooled antigen activity and 0.1M PO _Four Dialysis against pH 7.0, and
It was passed over an immunosorbent containing goat-specific anti-CEA antibodies. The latter
The unadsorbed fraction from the dressing was then applied to a second immunosorbent containing meconium monoclonal CEA antibody NP-3 as described above. Cross reaction from goats
Based on inhibition studies with neutral CEA antibodies, NP-3 was expressed in meconium in CEA and NC.
A was shown to recognize determinants shared between unrelated CEA related substances. NP-
Antigen bound to 3 was eluted as above for NCA purification. The adsorbed fraction is medium
, Dialyzed against PBS, and concentrated on a YM-10 membrane. Example 3 Animal immunization and production of antisera Goat anti-CEA antiserum was prepared using an equal volume of methylated bovine serum albumin (Sigma, St. Louis).
is, Mo. ) And an equal volume of complete Freund's adjuvant (Difco, Detro
it, Mich.) prepared by injection of 100-200 μg of purified CEA emulsified in
did. Incomplete adjuvant was used after the first injection. Obtained after 21 injections
The goat anti-CEA antiserum was allowed to ^a And Le ^b Individual
Were repeatedly absorbed by A, B and O red blood cells. The antiserum is then normal colon and NC
A. Chromatography on immunoadsorbent sequentially, fractions that are not adsorbed
Used in the case of The integrity of the adsorption is secondary to normal tissue extracts and NCA.
Analyzed in heavy immunodiffusion. A portion of the NCA neutralizing anti-CEA antiserum is
Per meconium by adding 250 mg (dry weight) of alcohol extracted meconium per
Was absorbed. After separation of the immunoprecipitates, the integrity of the absorption is due to double immunodiffusion to meconium.
More proven. This NCA and meconium-adsorbed antiserum will be
Identified as Qing. Goat anti-NCA antiserum cross-reacts with CEA by passage over CEA immunosorbent
Antibodies were removed. CEA neutralizing antisera did not react with CEA in double immunodiffusion or RIA. Example 4 Antigen and goat antiserum CEA and NCA used for mouse immunization are used in colonic adenocarcinoma liver.
Isolated from metastases, and MA was isolated from meconium by Primus et al., Cancer Res. 43, Feb
, 1983 (hereinafter “Primus '83”).
Lamin T method ¹²⁵ About 30 Ci / g at I (Amersham, Arlington Heights, IL)
Radioiodinated to specific activity. Roche CEA Assay Kit (Nutley, NJ)
Their radiolabeled CEA was the same as that obtained with the CEA used for immunization.
Used routinely when found to give similar results. Rochekit goat anti-CEA antibody was used for RIA. Goat anti-NCA antiserum
(No. 80) courtesy of Edward Newman of Roche Research Center, Nutley, NJ
Supplied and cross-reacts with CEA by passage over the CEA immunosorbent before use
Antibodies were removed. Binding properties of labeled CEA, MA and NCA to goat antiserum are described in Primus '83
Have been. Rochekit antibody and CEA absorbed by NCA and meconium
The opposite sex goat anti-CEA antiserum reacted similarly to labeled CEA. Labeled M
A was bound by the Rochekit antibody, but a goat-specific anti-CEA antiserum
Was not combined. NCA is Rochekit antibody or specific anti-CEA
Does not react with antiserum and does not bind goat-specific anti-NCA antibodies to CEA and MA
won. Example 5 Mouse immunization 3-4 month old BALB / C female mice (Harlan-Sprague-Dawley, Indianapo
lis, IN. ) To 20 μg ip of CEA in incomplete Freund's adjuvant. injection
Three doses were administered. The second and third injections are 2 and 8 weeks, respectively, from the first injection
Released. Six months after the first immunization, two mice showing serum antibodies to CEA
Was selected as a spleen cell donor and described in Stahli et al. Res. Monogr. Immunol
. 3: Final CEA injection of 50 mg each in saline according to the immunization protocol of 201, 1981
I received a shooting series. Four and one days prior to fusion, CEA was ip. Injection and fusion
3 and 2 days prior to And an equal volume for iv injection. Example 6 Cell fusion and cloning Two CEA immunized mice according to the method of McKearn cited in Primus '83.
Two separate fusions were performed with the spleen cells obtained from the spleen. 5 × 10 for each fusion
⁷ Ficoll-Hypaque spleen cells and 5 × 10 ⁶ , P3-x63-Ag8.653 bone marrow
Cells (Salk Institute San Diego, CA.) were mixed in a 60 mm culture dish and 250
Centrifuged at xg for 5 minutes. Remove excess media and modify Dulbecco at 37 ° C
50% (v / v) polyethylene glycol in Eagle's medium (GIBCO, Grand Island, NY.)
Recall (1500, fisher) was replaced with 1.0 ml. 30 to polyethylene glycol
After a second exposure, the cells are washed twice and then 20% gamma-free serum (KC Boilogi
cals, Inc., Lenexa, KS), L-glutamine 2 mM, sodium pyruvate 1 mM, L-
Supplement with 0.55 mM arginine, 0.27 mM L-asparagine and 14 μM folic acid
And incubated overnight in 5 ml of Dulbecco's modified Eagle's medium. Hypoxa cells
0.1 mM, 0.4 μM aminopterin, and 16 μM thymidine
The latter was dispersed in 30 ml or more of the medium. Transfer cell dispersion to 96-well microwell
100 μl per well in a microplate (Castar, Cambridge, Mass.)
Arranged. An additional 100 μl of medium without aminopterin was added to the wells after 7 days.
Was. After 2 to 4 weeks in culture, cells from wells containing growing cell clones
The media was assayed for CEA antibodies by RIA. CEA antibody positive well
To 10 per well ⁷ Of irradiated (1400R) Lewis rat thymocytes
And spread them into 24-well plates (Costar). Antibody positive when reunion is reached
All cultures that have been frozen are frozen, while 4 clones are
Recloned twice. 10 hybridomas per well ⁶ Irradiation Lewis luck
Recloned into 96-well plates containing Thymocytes. Re-selected
The cloned hybridoma cell line was treated with 10% gamma-free serum and L-gluta
Dulbecco-modified Eagle's medium supplemented with 2 mM of min and 1 mM of sodium pyruvate
Was expanded into a culture flask containing. Example 7 Monoclonal antibody purification Monoclonal antibodies were purified from media containing 10% gamma-free serum.
. Generally, immunoglobulins are 50% (NH _Four ) _Two SO _Four Precipitated and distilled
Re-dissolve in water and use polyethylene glycol (7000-9000) for a final concentration of 13% (w / v)
). Polyethylene glycol precipitation is 0.02M PO _Four , PH 5.
6 and applied to CM cellulose (Whatman, Clifton, NJ) equilibrated with the same buffer
. 0.02M PO adsorbed immunoglobulin _Four , PH 7.8 plus 0.2M NaCl
Elution, 0.01M PO _Four , Dialyzed against pH 8.0 and equilibrated with the latter buffer
DEAE cellulose (Whatman) column. Remove adsorbed antibody
025M PO _Four , PH 8.0, equilibrated against PBS, ultrafiltration (Amico
n, Danvers, MA. ). Purified monoclonal antibody is chlorami
It was radioiodinated to a specific activity of 5 to 10 μCi / μg by the 1-T method. Mouse I
In addition to gG, purified antibodies bind radioiodinated products to solid-phase goat anti-horse IgG.
Containing 10 to 40% horse immunoglobulin
I was The percentage of monoclonal antibody in the product is described in Primus et al., J. Am. Immu
nol. CEA of radioiodinated products, as described at 118: 55,1977.
Determined by binding to the immunosorbent. Example 8 RIA (radioimmunoassay) Two different types of RIAs are used in the separation of bound and free labeled antigen.
Was used. Anti-CEA and anti-MA antibody activity from hybridoma cultures
The assay of the supernatant medium was performed according to the method described by Hansen et al, Human Pathol., 5: 139, 1974.
-The gel method was used. Binding of selected monoclonal antibodies to CEA and MA
All studies and all assays for NCA reactivity were
(Newman et al, Proc. Am. Assoc. Cancer Res. 21: 218, 1980).
Was. The basic assay consists of 1.0% normal rabbit serum and approximately 0.5 ng of labeled antigen (30 ng).
2.0 ml of 0.01 M NH3 containing 50 μCi / μg) and a 0.05 ml aliquot of the antibody product. _Four Ac, pH 6.25
Was. Incubation for 1 hour at 45 ° C was performed with CEA and hybridoma cultures.
For 4 hours at the same temperature
Used for detection of A antibody. Labeled antibody binding curves and competitive inhibition measurements
Was induced after incubation at 45 ° for 4 hours and room temperature for 24 hours. Inn
After incubation, Z-gel or solid phase anti-immunoglobulin GAM or DAG
To 1 ml and the tubes mixed. Immediately remove the test tube containing the Z-gel
Centrifuge and add an additional 15 minutes at room temperature for tubes containing GAM or DAG.
Incubation was performed. Test tubes should be 0.1 M NH before counting. _Four Ac (Z
-Gel assay) or PBS (double antibody assay) 2.0 ml.
Non-specific binding was 10% for Z-gel and double antibody assays, respectively.
And 1%. Monoclonal antibody, goat antiserum, unlabeled antigen, and labeled antibody
The stock was diluted in PBS containing 1.0% human serum albumin. Example 9 Antibody affinity measurement Antibody affinity is described by Mueller, J. et al. Immunol. Methods 34; Competitive RIA method of 345, 1980
Therefore, it was measured. The product of the calculated binding constant and antibody concentration is described in Jacobsen et al. J. I
mmunol. Methods. 50: 77, 1982, less than 10. Example 10 Inhibition assay Mutual inhibition experiments used a solid phase competitive sandwich operation. Polyclonal goat anti-CEA serum is made of vinylidene fluoride powder (Kynar, grade 30 / F; P
ennwalt Corp., King of Prussia, PA. ) And 0.01M NH _Four Ac in 45
Incubation for 1 hour sensitized with unlabeled CEA. CE used
The amount of A bound 40% of the maximum amount of radiolabeled monoclonal antibody that could be bound.
0.5 ml of the CEA-sensitized Kynar was added to a 0.05 ml aliquot of the monoclonal antibody dilution and 4
Incubate at 5 ° for 1 hour, centrifuge, and pellet to 1% normal rabbit serum
0.01M NH containing _Four Resuspended in 1.0 ml of Ac. Release contained in 0.05ml
Add radioactive iodinated monoclonal antibody to test tube and incubate at 45 ° for 1 hour
And centrifuged, washed once and the pellets counted. Labeled antibody products
Nonspecific binding to unsensitized Kynar was less than 5%. Example 11 Erythrocyte binding Red blood cells from individuals secreting A, B and O secretors and non-secretors 2% or
Mix as a 10% suspension and a 50 μl aliquot of hybridoma tissue medium
And incubated for 1 hour at room temperature. Wash the blood cells, resuspend in PBS, and
Radioiodinated affinity purified G with heavy and light chain specificity
Incubated with AM for 1 hour at room temperature. After incubation, wash red blood cells
, Counted. Example 12 Tissue processing and fixation Tissue was obtained from 23 people with colorectal cancer. Of these adenocarcinomas
Three were the cecum, six were the ascending colon, and 14 were the rectum sigmoid. One was poorly differentiated, while the rest were well differentiated.
I was Normal morphology of the mucosa adjacent to the tumor and / or at the resection margin
Available from 21 cases. Duke's classification was used for clinical staging (
Duke, GE, J.S. Pathol, Bacteriol. 35: 322, 1932.) Surgical specimens are routinely fixed in 10% buffered formalin pH 7.2, and 6
In Primus et al., J. Am. Natl. Cancer Insti., 67:10, 1981
It was also fixed in EA as previously described. Tissue is embedded in paraffin,
The sections were sequentially cut to a thickness of 5 μm. Sections are mounted on gelatinized slides
Deparaffinized with xylene, decreasing ethanol concentration, and finally with PBS
Rehydrated. Example 13 Immunohistochemical manipulation BRAB immunoperoxidase method for staining with goat and monoclonal antibodies
Used for most of the color reactions (Guesdon et al., J. Histochem. Cytochem.
. 27: 1131, 1979). Basic operation with goat antibody is temporary goat antibody, biotinylated
Rabbit anti-goat IgG (50 μg / ml), free avidin (100 μg / ml), and
The sequential application of biotinylated horseradish peroxidase (50 μg / ml)
I was Biotinylation reagents and avidin are available from Vector Laboratories (Burlingame
, CA). Avidin in 0.05M Tris, pH 8.6 plus 0.15M NaCl
All reagents were diluted in PBS with the exception that they were diluted in. Rat monochrome
For the detection of antigen by nal antibody, the basic operation is the first of monoclonal antibody.
After application modified by the introduction of an affinity purified goat anti-mouse IgG (30 μg / ml) step
. All immunizations and biotin-avidin reactions were performed at 37 ° C in a warm atmosphere.
Performed for 20 minutes and 30 minutes at room temperature. After each step, wash twice in PBS for 5 minutes
Was cleaned. All pieces in each slide receive test antibody, adjacent sections exposed to control
Was done. Goat anti-CEA and anti-NCA antisera were diluted 1/600 and 1/100, respectively.
Used at grade. These antisera controls were similarly diluted antigen neutralizing CEA antisera
And / or consisted of normal goat serum. 5 μg / ml for monoclonal antibody
And normal mouse IgG (Pel-Freeze, Rogers, AR) at 50 μg / ml
Antigen-neutralized purified monoclonal antibody and ascites fluid for control purposes.
Was. Prior to the application of the primary antibody, hydrated sections were 3% H in methanol. _Two O _Two Incubation in
Bait, washed in PBS and diluted with normal rabbit serum for 10 minutes at 37 ° C
For a while. After the biotinylation enzyme step, the histochemical reaction was performed at 0.0.
0.01% diaminobenzidine and 0.003 in 5M Tris-HCl, pH 7.6
% H _Two O _Two For 15 minutes at room temperature. Immunoperoxidase stained slide
Were counterstained brightly with Harris hematoxylin. Unlabeled antibodies using glucose oxidase anti-glucose oxidase conjugate
Body method (Clark et al, J. Histochem. Cytochem. 30:27, 1982).
Used for NCA detection. The basic operation consists of donkey anti-goat IgG and goat anti-goat.
Each of the glucose oxidase-glucose oxidase complex was biotinylated.
Used instead of heron anti-goat IgG and biotinylated horseradish peroxidase.
With the exception of BRAB technology. Avidin and H _Two O _Two The step is also omitted and the plate is replaced with undiluted normal horse serum instead of normal rabbit serum
Cuvitation was used. The enzyme disclosure reaction consisted of: 6.7m
g / ml β-D-glucose (calbiochem-Behringer, LaJolla, CA), 0.67 mg
/ Ml NBT (Reseach Organics Incs., Cleveland, OH) and 0.005M
Squirrel, 0.0167 mg / ml phenazine methosulfate in pH 8.3 (Sigma, S
t. Louis, MO). Preheat glucose and NBT at 37 ° C. for 1 hour,
At the time points phenazine methosulfate and tissue pieces were added. Further at 37 ° C
After a 45 minute incubation, the slides are washed and nuclear fast red
For counterstaining. The above examples illustrate the reactants and / or described generally or specifically for the present invention.
Or by replacing the working conditions with those used in the above examples,
Can be repeated with a degree. From the above description, those skilled in the art can easily ascertain the essential features of the invention,
And without departing from its spirit and scope,
Various changes and modifications can be made to suit.

Claims (1)

Claims [1] An immunoassay method for quantification of carcinoembryonic antigen (CEA) in a serum or plasma sample, comprising: (1) a method comprising the steps of: Anti-C derived from a hybridoma that specifically binds to an epitope shared by a cross-reactive antigen
Contacting a capture antibody bound to a solid support, which is an EA monoclonal antibody, for a time sufficient to allow antibody binding to CEA in the sample; (2) contacting the capture antibody bound to the capture antibody with the capture antibody; The resulting solid support having above,
The detectably labeled anti-CEA probe antibody is contacted with the probe antibody for a time sufficient for the probe antibody to bind to the CEA bound to the capture antibody, and the extent of binding of the probe antibody to the CEA bound to the capture antibody And wherein the probe antibody specifically binds to a certain epitobe on CEA, but does not specifically bind to either meconium antigen MA or a non-specific cross-reactive antigen. The above immunoassay method, which is an induced anti-CEA monoclonal antibody. [Claim 2] The method according to claim 1, wherein the probe antibody is labeled with a radioisotope, a fluorescent marker or an enzyme. [Claim 3] An immunoassay kit for quantifying carcinoembryonic antigen (CEA) in a serum or plasma sample, the kit comprising: (1) CEA, meconium antigen MA and non-human meconium antigen; A capture antibody bound to a solid support, which is an anti-CEA monoclonal antibody derived from a hybridoma, that specifically binds to an epitope shared by a specific cross-reactive antigen; An anti-CEA monoclonal derived from a hybridoma that is a different antibody and specifically binds to an epitope on CEA but does not specifically bind to meconium antigen MA or non-specific anti-differentiated antigen Said kit comprising an antibody, a detectably labeled anti-CEA probe antibody. [4] The kit according to [3], wherein the probe antibody is labeled with a radioisotope, a fluorescent marker or an enzyme. Claim 5: An immunoassay method for quantifying carcinoembryonic antigen (CEA) in a serum or plasma sample, comprising: (1) sharing with a sample CEA, meconium antigen MA and a non-specific cross-reactive antigen; Anti-C derived from hybridomas that specifically binds to the
Contacting a capture antibody bound to a solid support, which is an EA monoclonal antibody, for a time sufficient to allow antibody binding to CEA in the sample; (2) contacting the capture antibody bound to the capture antibody with the capture antibody; The resulting solid support having above,
The detectably labeled anti-CEA probe antibody is contacted with the probe antibody for a time sufficient for the probe antibody to bind to the CEA bound to the capture antibody, and the extent of binding of the probe antibody to the CEA bound to the capture antibody And wherein said probe antibody is derived from a hybridoma, which specifically binds to an epitope shared by CEA and meconium antigen MA, but does not specifically bind to a non-specific cross-reactive antigen. Anti-CEA monoclonal antibody,
And binding of the probe antibody to an epitope shared by CEA and meconium antigen MA is derived from a hybridoma that specifically binds to an epitope shared by CEA, meconium antigen MA and a non-specific cross-reactive antigen. The immunoassay method, wherein the immunoassay method is not cross-blocked by the anti-CEA monoclonal antibody. [Claim 6] The method according to claim 5, wherein the probe antibody is labeled with a radioisotope, a fluorescent marker or an enzyme. [Claim 7] An immunoassay kit for quantifying carcinoembryonic antigen (CEA) in a serum or plasma sample, wherein the kit is used in an amount sufficient to carry out the immunoassay. A capture antibody bound to a solid support, which is an anti-CEA monoclonal antibody derived from a hybridoma, that specifically binds to an epitope shared by a specific cross-reactive antigen; An anti-CEA monoclonal antibody derived from a hybridoma that is a different antibody and specifically binds to an epitope shared by CEA and meconium antigen MA but does not specifically bind to a non-specific cross-reactive antigen And the CEA and meconium antigen M of the probe antibody
Binding to an epitope shared by A binds specifically to an epitope shared by CEA, meconium antigen MA and a non-specific cross-reactive antigen.
A kit comprising: a detectably labeled anti-CEA probe antibody that is not cross-blocked by an anti-CEA monoclonal antibody derived from a hybridoma. [8] The kit according to [7], wherein the probe antibody is labeled with a radioisotope, a fluorescent marker or an enzyme.