MXPA98009346A - A citofluorometric method for determination in serum of antibodies against antigens tumora - Google Patents
A citofluorometric method for determination in serum of antibodies against antigens tumoraInfo
- Publication number
- MXPA98009346A MXPA98009346A MXPA/A/1998/009346A MX9809346A MXPA98009346A MX PA98009346 A MXPA98009346 A MX PA98009346A MX 9809346 A MX9809346 A MX 9809346A MX PA98009346 A MXPA98009346 A MX PA98009346A
- Authority
- MX
- Mexico
- Prior art keywords
- cells
- serum
- antibodies
- tumor
- inactivated
- Prior art date
Links
- 108090001123 antibodies Proteins 0.000 title claims abstract description 29
- 102000004965 antibodies Human genes 0.000 title claims abstract description 29
- 239000000427 antigen Substances 0.000 title claims abstract description 18
- 102000038129 antigens Human genes 0.000 title claims abstract description 18
- 108091007172 antigens Proteins 0.000 title claims abstract description 18
- 210000002966 Serum Anatomy 0.000 title claims description 18
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 12
- 210000004881 tumor cells Anatomy 0.000 claims abstract description 12
- 210000004027 cells Anatomy 0.000 claims description 28
- WSFSSNUMVMOOMR-UHFFFAOYSA-N formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 10
- 238000004458 analytical method Methods 0.000 claims description 5
- 201000011510 cancer Diseases 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- VLTRZXGMWDSKGL-UHFFFAOYSA-N Perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- 229940072221 IMMUNOGLOBULINS Drugs 0.000 claims description 3
- 102000018358 Immunoglobulins Human genes 0.000 claims description 3
- 108060003951 Immunoglobulins Proteins 0.000 claims description 3
- 210000004185 Liver Anatomy 0.000 claims description 3
- 241000124008 Mammalia Species 0.000 claims description 3
- 150000001299 aldehydes Chemical class 0.000 claims description 3
- 201000009030 carcinoma Diseases 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 206010025323 Lymphomas Diseases 0.000 claims description 2
- 206010025650 Malignant melanoma Diseases 0.000 claims description 2
- GYHFUZHODSMOHU-UHFFFAOYSA-N Nonanal Chemical compound CCCCCCCCC=O GYHFUZHODSMOHU-UHFFFAOYSA-N 0.000 claims description 2
- 206010025310 Other lymphomas Diseases 0.000 claims description 2
- 206010039491 Sarcoma Diseases 0.000 claims description 2
- 239000011230 binding agent Substances 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 2
- 201000001441 melanoma Diseases 0.000 claims description 2
- 241000750027 Nestor notabilis Species 0.000 claims 1
- 230000001173 tumoral Effects 0.000 claims 1
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 102000004851 Immunoglobulin G Human genes 0.000 description 3
- 108090001095 Immunoglobulin G Proteins 0.000 description 3
- 230000000259 anti-tumor Effects 0.000 description 3
- 230000000295 complement Effects 0.000 description 3
- 230000001472 cytotoxic Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 206010017758 Gastric cancer Diseases 0.000 description 2
- 108010093001 anti-IgG Proteins 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 238000000053 physical method Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 201000000498 stomach carcinoma Diseases 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 229940027941 Immunoglobulin G Drugs 0.000 description 1
- 229940040511 Liver Extract Drugs 0.000 description 1
- 210000004072 Lung Anatomy 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 101710033558 PSMB3 Proteins 0.000 description 1
- 108010040256 UK114 antigen Proteins 0.000 description 1
- ZBKFYXZXZJPWNQ-UHFFFAOYSA-N [N-]=C=S Chemical compound [N-]=C=S ZBKFYXZXZJPWNQ-UHFFFAOYSA-N 0.000 description 1
- 230000002391 anti-complement Effects 0.000 description 1
- 108010008730 anticomplement Proteins 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 201000003963 colon carcinoma Diseases 0.000 description 1
- 230000001461 cytolytic Effects 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000003287 optical Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001681 protective Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
Abstract
The present invention relates to a cytofluorometric method for detecting the presence of antibodies against tumor antigens, using whole inactive tumor cells expressing tumor antigens
Description
A CITOFLUOROMETRIC METHOD FOR THE DETERMINATION IN SERUM OF THE ANTIBODIES AGAINST THE TUMOR ANTIGENS
Field of the Invention
The present invention relates to a cytofluorometric method for the detection in serum of antibodies directed against tumor antigens.
Background of the Invention
The importance of the detection in the serum of antibodies directed against tumor antigens for diagnostic and prognostic purposes has been appreciated for some time. On the other hand, recently a new class of tumor antigens, recognized by the antibodies arisen from the immunization of mammals with extractable proteins with perchloric acid of the mammalian liver (WO 92/10197), has been described. In particular, one of these antigens, a protein having a molecular weight of about
14 dDa, referred to hereinafter as UK 114, has been isolated and sequenced and is endowed with antitumor activity and capacity to induce, when
REF .: 28822 administers to animals of different species from which they are extracted, antibodies that recognize the tumor antigens expressed on the membrane of the tumor cells. It has also been shown the existence of natural antibodies which bind to UK protein 114 as well as to tumor cells. A high concentration of natural antibodies could possibly be considered a protective factor against the attack of neoplasms capable of influencing the evolution of malignancies. The concentration of antibodies in the serum of patients affected by malignant tumors or induced by the exogenous administration of UK 114, although not directly related to cytotoxic activity, can in any way provide useful indications for the verification of the therapy and for diagnostic purposes. Therefore, the importance of a method that allows the determination of antitumor antibodies, special antibodies of anti-UK 114 either natural or induced, in the serum of cancer patients with the contemporary possibility of determining the different classes of serum immunoglobulins (IgG, IgA, IgM, IgE, IgD) as well as to distinguish between IgGi, IgG2, IgG4, ie the subtypes of immunoglobulin G which are specifically responsible for the cytotoxic effects.
Detailed description of the invention
Now we have found a particularly sensitive and efficient method that allows the determination of antitumor antibodies and their typing. The method is based on the cytofluorometric method and is characterized by the use of whole dead cells, generally of the tumor type, or of microspheres that have the antigen bound to the surface. Dead whole cells are understood to be non-viable cells inactivated by chemical or physical methods which do not cause permeabilization of the membrane or denaturation of the antigen expressed on the membrane itself. The use of dead cells allows the preparation of sets or sets ready for use, which do not involve the problems and risks related to the need to manage viable tumor cells. The method of the invention also allows the desired differentiation between the different classes and types of serum immunoglobulins (IgGi, IgG2, igG4) • The cells that can be used according to the invention are preferably stabilized tumor cell lines either of origin of a carcinoma, sarcoma, lymphoma or melanoma, expressing tumor antigens on its surface, especially the UK 114 antigen: Examples of stabilized tumor cells include the HT 29 cell line of the colon carcinoma, KATO III of the gastric carcinoma, NICH of carcinoma of the lung, CTL-16 of breast carcinoma and the like. Chemical methods for the inactivation of cells according to the invention comprise treatment with aqueous solutions of aldehydes such as formaldehyde or glutaraldehyde or with aldehyde or non-aldehyde binding agents. The use of formaldehyde is preferred, in concentrations from 0.1 to 10%, preferably from 0.2 to 4%, for times ranging from 30 'to 24 hours, preferably from 30' to 60 '. Alternatively, the cells can be inactivated by physical methods such as temperature changes, ß or γ radiations, state changes.
The method of the invention comprises the incubation of the serum sample with the dead tumor cells followed by the addition of anti-IgG species, antibodies conjugated with fluorescent labels. The reading is then carried out by the optical path (microscope) or, preferably, using a cytofluorometer or a FACS device (cell sorter activated with fluoro). This allows the determination of the number of positive cells and, therefore, of the concentration of the antibody. Alternatively, in the case of anti-UK 114 antibodies, the first incubation can be carried out in the presence of the complement, using fractions of anti-fluoroscent complement of the antibodies in the detection phase. It was observed in fact that the components of the complement (particularly the Cg) plays an essential role in the determination of the cytolytic effect of the antibodies. The method described above thus makes possible the study of the cytotoxic effect. It is also possible, to avoid interferences with other antibodies that recognize antigens other than UK 114, preadsorb the serum with UK 114 as a control, to evaluate by difference only the specific interaction of the antibodies that react with the antibodies related to the UK 114. The invention also provides a kit or kit ready for use, to carry out the methods described above, comprising a suspension of the whole killed tumor cells, or microspheres containing the antigen of UK 114, anti-IgG. anti-complement fluorescent antibodies or antibodies in addition to buffer solutions, diluents and reagents suitable for cytofluorometric analysis. Positive and negative test controls will also be included. The following example further illustrates the invention.
EXAMPLE
KLATO III gastric carcinoma cells growing in a culture such as the cells in the suspensions are fixed in 2% formaldehyde in buffered phosphate buffered saline pH 7.2 (PBS). The fixation was carried out at room temperature for 15 minutes. The cells were then washed in a 0.1M solution containing PBS and then kept at 4 ° C in PBS until use.
For the indirect immunofluoroscence reaction, 1 x 106 cells were incubated for 30 minutes at room temperature with the human serum to be tested, diluted 1:10 in PBS, then washed three times in PBS, then incubated with Ig anti-human goat Ig, is labeled with fluoroscein isothiocyanate. After washing in PBS 3 times, the cells were analyzed with a FACScan (R) (Becton-Dic inson) apparatus having an excitation laser beam of 15 mW at 488 nm. Figure 1 shows the FACScan (R) analysis of a serum from a normal subject. After washing, the cells were incubated with rabbit anti-human IgG, labeled with fluoroscein. The bar Ml shows the interval in which the fluorescent positive cells are located. 1.68% of the cells are within this range. The result of the test must be considered negative. Figure 2 shows the analysis of FACScanÍR) with the serum of a patient treated with the liver extract containing the UK 114, called UK 101. In the subject's serum, the antibodies that recognize the antigen (UK 114) present on the KATO III cell surface, are present. The antibodies bind to the cell surface of KATO III and they are themselves bound by fluorescent anti-human Ig antibodies. Most of the cells (87.34%) are positive in the fluorescence test (falling or falling within the range of the bar)
Ml). In Figure 3 the curves of the negative test
(Figure 1) and positive test (Figure 2) are compared. The two curves are clearly different.
It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.
Having described the invention as above, the content of the following is claimed as property
Claims (12)
1. A method for determining the presence and concentration of antibodies against tumor antigens carried by the surface of the tumor cell, characterized in that it comprises the cytofluorometry of the whole dead cells expressing the antigens previously incubated with the serum to be analyzed .
2. A method according to claim 1, characterized in that the cells are inactivated by chemical treatments.
3. A method according to claim 2, characterized in that the cells are inactivated by treatment with formaldehyde, glutaraldehyde, aldehyde or non-aldehyde binding agents.
4. A method according to claim 3, characterized in that the cells are inactivated by the formaldehyde treatment.
5. A method according to claim 1, characterized in that the cells are inactivated by the treatment by means of physical treatments.
6. A method according to claim 5, characterized in that the cells are subjected to temperature changes, to ß or γ radiations, changes of state.
7. A method according to any one of the previous claims, characterized in that it is used for the detection of the antibodies that recognize the protein of 14 kDa extractable from the liver of the mammal by the treatment with perchloric acid.
8. A method according to any of the previous claims, characterized in that it is used for the selective analysis of the antibodies belonging to the different classes of serum immunoglobulins (IgGi, IgG2, IgG4).
9. A method according to any of claims 1-8, characterized in that the cells are of tumoral origin.
10. A method according to claim 9, characterized in that the tumor cells are selected from the stabilized tumor cell lines of origin of the carcinoma, lymphoma, sarcoma or melanoma, which express the tumor antigens on their surface.
11. A method according to claim 7, characterized in that a control serum obtained by treating the serum with the 14 kEa protein of the liver of a mammal is used.
12. A set or diagnostic set for use in the methods of claims 1-11, characterized in that it comprises a suspension of whole, dead tumor cells, suitable buffer solutions and reagents for cytofluorometric analysis.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| MIMI96A000943 | 1996-05-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| MXPA98009346A true MXPA98009346A (en) | 1999-07-06 |
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