EP0207979A1 - Immunological method of measuring unstable analytes using cross-reactive antibodies - Google Patents
Immunological method of measuring unstable analytes using cross-reactive antibodiesInfo
- Publication number
- EP0207979A1 EP0207979A1 EP86900699A EP86900699A EP0207979A1 EP 0207979 A1 EP0207979 A1 EP 0207979A1 EP 86900699 A EP86900699 A EP 86900699A EP 86900699 A EP86900699 A EP 86900699A EP 0207979 A1 EP0207979 A1 EP 0207979A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- analyte
- penicillin
- test
- fluid
- interest
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
Definitions
- the present invention relates generally to immunological assay techniques for determining the presence of an analyte in fluids and particularly to an immunoassay testing method utilizing cross-reactive antibodies which has particular application in the qualitative and quantitative determination of the level of unstable analytes such as beta-lactam antibiotics in fluids with particular emphasis on penicillin detection and measurement.
- immunoassay methods are extensively described in literature, for example as in Methods in Enzymology, Vol. 92, Pages 377-543, (1983) Academic Press, New York.
- immunoassay methods require (i) specific antibodies raised against the analyte of interest, and (ii) a signal generating molecule which is either derived from the analyte of interest or from the antibody raised specifically to the analyte of interest, or another molecule which reacts with the said antigen or antibody.
- the antibody, the analyte of interest and the signal generating molecule may be reacted in one-phase or two-phase systems. Thereafter, the amount of signal generating molecules that are bound directly or indirectly to the specific antibody are differentiated from the unbound signal generating molecule. The proportion of bound or free molecule gives a measure of analyte present in the sample.
- B highly purified analyte conjugated to a high molecular weight substance, or.
- C a highly purified fragment of the analyte of interest as such or as a conjugated molecule
- the living system responds to this "invasion" of a foreign molecule and produces specific antibodies to the analyte of interest.
- cross-reacting antibodies which not only partly react to the analyte of interest but also react with other constituents which may be commonly present in the sample to be assayed. These cross-reactive antibodies may interfere in the test procedure and prevent specific measurement of the analyte. It is because of this that the prior art has generally required the use of specific antibodies .
- the prior art also describes ways of minimizing the harmful effect of cross-reactive antibodies. This could be achieved by the purification of antibodies by affinity chromatography, the use of hybridoma techniques to make specific antibodies or the use of so called “sandwich” techniques.
- the "sandwich” approach relies on redundancy to eliminate harmful effects of cross-reactive antibodies.
- specific antibodies are raised against the analyte in two different living systems. In the actual test, the analyte is sandwiched between these two different but specific antibodies. If the cross-reactivity was resulting from the living system used to make the antibody, such an approach will eliminate cross- reactivity.
- Another common “sandwich” technique involves making two pure fragments of the analyte and making specific antibodies against each fragment. In the actual assay, the intact analyte is sandwiched between the antibodies made against its fragments. Such an approach will eliminate cross-reactivity problems which occur due to the presence of antibodies against a portion of the analyte.
- Beta-lactam antibiotics for example react very easily with proteins. One often needs to form a conjugate of beta-lactam antibiotics with a protein to render it immunogenic. However, the beta-lactam ring is opened during such conjugation and antibodies so raised will only cross-react with the intact antibiotic molecule and are not specific to the native antibiotic molecule.
- the prior art fails to disclose methods of using cross-reactive antibodies to measure analytes which fall in the Categories a and b above.
- the analyte is reactive and changes from its native analyte form (NAN) to an altered analyte form (abbreviated as AAN) when conjugated for immunization.
- NAN native analyte form
- AAN altered analyte form
- the antibody thus formed is not specific to the native analyte NAN; however, the antibody would have some cross-reactivity to that molecule. Since such analytes are very reactive, they lend themselves to easy alteration.
- a sample containing the analyte of interest often contains both the native (NAN) and altered (AAN) forms of the analyte, and the antibody would react with differing intensities with both forms and would not be suitable for measuring these forms.
- Roberts et al . (Lancet, Vol. II, Pages 319-321, August 1977) have described the use of cross-reactive antibodies. They use antibodies raised against CK-BB (creatine kinase, BB isomer) to measure CK-MB (creatine kinase, MB isomer) . Antibodies to CK-BB are not raised against CK-MB, they cross-react with CK-MB.
- the difference between teachings of Roberts and the present invention lies in the fact that two analytes, for example CK-BB and CK-MB, one specific to the antibody and one cross-reactive with the antibody are left unaltered by Roberts and both of them are measured.
- beta-lactam ring of penicillin and other such antibiotics is very reactive.
- the native penicillin may undergo changes, particularity through opening of the beta-lactam ring.
- Such conversion depends upon the environmental conditions such as pH, temperature, presence of sugars, proteins etc. to which the antibiotic is exposed.
- the degree of conversion may be different when it is injected into animals to treat infection, from when it is present in milk or from when it is present in fermentation broth.
- the open bata-lactam ring form e.g. penicilloyl group
- Antibodies used to immunologically measure antibiotics such as that described in U.S. patent #4,347,312, are usually formed in such a way that they are specific to the open beta ⁇ lactam ring (e.g. penicilloyl group) rather than native molecule (e. g. penicillin) .
- Authors such as Kitagawa et al.
- the opening of the beta-lactam ring causes the antibiotic (e.g. penicillin) molecule to loose its antibacterial activity.
- antibiotics e.g. penicillin
- conventional microbiological methods measure native antibiotics (e.g. penicillin) rather than the open beta-lactam ring (e.g. penicilloyl) derivatives.
- the native antibiotic e.g. penicillin
- the native antibiotic still has a very reactive beta-lactam ring and is capable of forming allergenic antigens when it could combine with other molecules to form open beta-lactam derivatives.
- microbiological methods for measuring antibiotics contamination give only a partial and an indirect measure of allergenicity of such products.
- the allergic reaction in humans which could be caused by food or drug products contaminated with penicillin or other beta-lactam antibiotics, may result from both native form as well as the altered derivatives. This is because the human body will convert some of the native antibiotic into allergenic derivatives in-vivo. It would be useful therefore to simultaneously measure both the altered form which is allergenic and the intact form which has potential allergenicity.
- the prior art does not describe a method capable of measuring simultaneously both forms of antibiotics, namely those containing an intact beta-lactam ring and those containing open beta ⁇ lactam ring derivatives.
- the present invention provides means of immunologically measuring both the closed ring antibiotics and the open beta-lactam ring derivative. This is achieved by converting the closed ring antibiotic molecule in the sample into its open beta ⁇ lactam ring form. Such a measurement of both forms would provide a more reliable estimate of allergenicity of penicillin and similar beta-lactam ring antibiotics when present as contaminants in foods and drugs.
- a reactive analyte such as an antibiotic molecule containing a beta-lactam ring
- the test method of the present invention is useful when the analyte of interest in sample exists in two related molecular • structures and it is desired to measure all of the analyte, regardless of structure.
- the method includes utilizing an antibody which is cross-reactive with one structure and specific to the other structure.
- the analyte of the cross-reactive structure is converted through conjugation such that the form of the analyte structure which is specific to the antibody is created in the test sample.
- the actual procedure may utilize competitive, sequential immunological or sandwich techniques and be conducted with a variety of tags.
- the test is particularly suited for beta-lactam ring antibiotics which may exist in either open ring or closed ring molecular structures.
- the practiced test procedure converts all molecular structures to open ring structures and uses a RIA tagged antibody.
- Particular examples describe a test procedure for measuring penicillin contaminants in milk.
- Figure 1 depicts various reactions between native analytes, altered analytes and the cross-reactive antibodies.
- FIG. 1 depicts various immunoassay techniques that may be used with the present invention.
- the present invention teaches a novel method of using cross-reactive antibodies to immunologically measure a native analyte (NAN) , where such native analyte (NAN) (i) also typically exists in an altered analyte (AAN) form in the sample environment in which it is commonly found, and (ii) the altered (AAN) form of such analyte is immunologically distinct from its native (NAN) form, and (iii) the AAN form is immunologically similar or identical to immunogens (abbreviated as CAAN) prepared from the NAN analyte, and (iv) the NAN analyte can be converted to its AAN form in a reaction matrix which enhances the conversion of the NAN analyte to its AAN form.
- NAN native analyte
- CAAN immunogenic form
- CAAN immunogenic form
- AAN altered form of the native analyte
- NAN native analyte
- Sequential reaction See Fig 2b in which sample is reacted with cross-reactive antibodies and then signal generating molecule made from altered analyte.
- Two Site Immunometric reaction (See Fig 2d) in which a second site M is created by using a competitive reaction followed by the use of signal generating molecule made from antibodies to M.
- the obvious extension of this invention will involve separate estimation of NAN and AAN by (i) measuring both together as described by the present invention and, (ii) measuring AAN above, in the absence of the matrix which converts NAN to AAN.
- the process of this invention can be practiced with a variety of analytes, a variety of monoclonal and polyclonal antibodies cross-reactive to such analytes, labels such as radioactive, enzyme, fluorescence etc., a variety of matrices, and a variety of solid-phases.
- the process of this invention can be practiced to measure a family of analytes which contain a common labile group which gets similarly altered in the sample environment and on conjugation. Such is the case with * the family of beta-lactam antibiotics.
- PEN Native Analyte - Benzylpenicillin (Penicillin-G) (PEN) was covalently conjugated to rabbit serum albumin (RSA) through the beta-lactam ring of the penicillin molecule and the epsilon-amino groups of proteins according to published procedures (Brown et al, U.S. Patent #4,347,312 (1982) and Wal, J.M. et al, FEBS Letters _57_, pages 9-13 (1978)) and described below.
- RSA rabbit serum albumin
- hydrochloride was added to give a concentration of 0.1 molar and the pH was adjusted to 7.5. The mixture was then incubated at 37°C for 1 hour. Thereafter, the mixture was again eluted through a Sephadex G-200 column, washed with water and the resulting PEN-RSA conjugate lyophilized for storage until ready for further use. An alternative method is next described.
- Bovine Gamma Globulin BgG
- the immunogen is obtained by coupling native analyte penicillin to bovine gammaglobulins (BGG) , a penicillin-protein conjugate being formed.
- BGG bovine gammaglobulins
- the conjugation reaction involving the formation of covalent bond between penicilloyl groups and available epsilon-a ino groups of BGG (Wal , J.M. et al, FEBS Letters 57, pages 9-13 (1975) as described below.
- reaction mixture is put on a Sephadex column G25 (K26/40 - Pharmacia) equilibrated with 0.15M NaCl in 0.01 M phosphate buffer (pH7.4). Elution is done with the same buffer at laboratory temperature. Fractions of 0.5 ml are recovered and tested by ultraviolet spectroscopy at 280 nm. The penicilloyl-BGG conjugate is thus entirely separated from the excess free penicillin. An alternative method is next described.
- bovine gamma globulin was placed in a minimum volume sodium carbonate buffer at a ph of 10.4 and temperature of 4°C to which 2.0 g of penicillin-G
- PEN PEN
- pH 9.6
- an additional 0.75 g PEN was added, the temperature being maintained at 4°C.
- another 0.75 g PEN was added. Twelve hours later the mixture was eluted through a Sephadex G-200 column. After elution, cysteine hydrochloride was added to give a concentration of 0.1 molar and the pH was adjusted to 7.5. The mixture was the incubated at 37° C for 1 hour. Thereafter, the mixture was again eluted through a Sephadex G-200 column, washed with water and the resulting PEN-BGG conjugate lyophilized for storage until ready for further use.
- Antibodies to penicillin were raised in rabbits by injecting immunogen (PEN-BGG) , dissolved in saline and emulsified 50:50 with Freund's complete adjuvant, subcutaneously at ten day intervals. Serum was collected and held at -10 C until needed.
- Antibodies to Pen.BgG raised in goat were purchased from Biolac, Logan, Utah.
- BGG 250 mg was dissolved in 5 ml of 0 1 M phosphate buffer pH 7. While the solution was stirred at room temperature, 1 ml of a 2.5 per cent aqueous solution of glutaraldehyde was added dropwise. A gel was formed almost instantaneously, and this was allowed to stand for 3 hours at room temperature.
- the immunoabsorbent To employ* the immunoabsorbent, appropriate volumes of whole immune serum were mixed in centrifuge tubes with the insoluble protein. The mixture was stirred gently for 30 min at room temperature and then centrifuged (3,000 rpm, 15 min, + 4°C) . The supernatant was kept in order to measure non-absorbed antibodies. The precipitate was suspended in buffered physiological saline (PBS) 3-4 times the volume of the antiserum added, and centrifuged as above. Washing with PBS was continued (about 3-4 times) until the spernatant had an optical density of less than 0.040 at 280 nm. To elute absorbed proteins 0.1 M glycine-HC buffer pH 2.8 was used.
- PBS buffered physiological saline
- the i munosorbent was suspended in a small volume, stirred with a magnetic stirrer for 5 min at room temperature and then centrifuged at 10,000 rpm for 15 min. This step was repeated twice. The supernatants were filtered and dialysed against several changes of cold saline.
- Glass beads having a suitable surface for bonding the conjugate may be prepared by:
- succinamidopropyl-surface to the acyl chloride derivative by treatment under anhydrous conditions.
- silanizing and succinylating is carried out according to known procedures.
- silanizing is accomplished generally by treating the inorganic metal oxide material with an aqeous solution (e.g., about 10%) of the triethoxyaminopropylsilane at a pH of about 4.
- succinylating is accomplished by treating the material with either an aqueous solution of succinic anhydride at a pH of about 6, or an aqueous solution of succinic acid and carbodiimide.
- acyl derivative is accomplished by reaction with thionyl chloride dissolved in an inert organic solvent (e.g., methylene chloride) in the absence of water, i.e., under anhydrous conditions. At this point, the acyl derivative is reacted with either mercaptopropionic acid or mercaptoacetic acid dissolved in an inert organic solvent such as methylene chloride in the absence of water. Upon completion of the reaction, the material is dried, in vacuo, and stored until ready for use.
- an inert organic solvent e.g., methylene chloride
- Immobilization of the Pen.RSA conjugate is accomplished by simply adding a solution of the conjugate at a pH around neutrality (e.g., 5 to 8) to a solid matrix (e.g., dry glass beads). The antiserum was then purified by passing through columns of these beads and the columns thoroughly washed with physiologically buffered saline solution(PBS) then eluted with 0.1 M acetic acid. The eluent was mixed immediately with pH 10, 0.1M sodium bicarbonate to neutralize the acid.
- a pH around neutrality e.g., 5 to 8
- PBS physiologically buffered saline solution
- 1251 penicillin RSA was prepared by reacting 20 ul of 1 mg/ml penicillin RSA with 1 mCi of 125INa and 10 ul of chloramine T. The reaction was stopped by adding sodium metabisulfite. The reaction product was separated on a column and active fractions were used.
- Penicillin was linked to HRP by using an amide linkage using a carbodiimide carboylactivating reagent.
- Antibodies purified according to Step VI were conjugated to alkaline phosphatase by the gluteraldehyde method. Similarly, purified antibodies can also be conjugated to horseradish peroxidase.
- Antibody raised against penicillin BGG was used to study the conversion of penicillin to penicilloyl derivative under alkaline conditions in milk and in wate .
- Example II The same method as Example I was repeated with differing amounts of penicillin being added to milk at various pH levels.
- penicillin HRP conjugate was diluted 1:25 in Tris buffer with 10% polyethylene glycol. Milk containing various amounts of penicillin (300 ul) was placed with 100 ul of 0.2N NaOH containing 3% polyethylene glycol in contact with anti-penicillin BGG coated 8mm polystyrene beads in 12x75 antibody coated plastic tubes. The reaction was carried out for 10 minutes. The tubes were washed three times and 200 ul ortho phenylenediamine-H202 mixture was added. The intensity at 490 nm of color was as follows after 5 minutes .
- test data shows that Pen.RSA conjugate could be used instead of penicillin conjugation.
- Polystyrene beads were coated with Pen.RSA. Alkaline phosphatase anti-penicillin antibody conjugate made per Step X was used. Tube containing coated bead was incubated with 300ul milk, 100ul of 0.2n NaOH and 100ul of conjugate (1:10 dilution) for 10 minutes. Tubes were washed three times with water and 150ul of P-nitrophenyl phosphate was added and color density was read after 15 minutes.
- test data shows that a labelled antibody could be used for the test.
- test results show the use of the sequential assay technique.
- Polystyrene beads were coated with Pen.RSA. HRP anti-penicillin antibody conjugate made per Step X was used. 205ul of milk containing various amounts of penicillin was incubated for 10 minutes in a tube containing a coated bead and 150ul of solution containing enzyme (lactamase) , 0.2m EDTA, 10% peg and HRP conjugated antibodies. Tubes were washed three times with buffer after the incubation period. 250ul of OPD-H202 was added and color density was read after 5 minutes.
- test results show the use of a biochemical agent for the detection of penicillin.
Abstract
Dans un procédé d'essais immunologiques d'analytes instables, on utilise des anticorps à réaction croisée, la structure moléculaire de l'analyte étant modifiée pour devenir spécifique à l'anticorps. Le procédé peut être utilisé dans des essais immunologiques compétitifs, séquentiels, immunométriques à deux sites. Une application particulière concerne le dépistage qualitatif et quantitatif d'antibiotiques bêta-lactam dans des fluides, notamment le dépistage et la mesure de la penicilline.In a method of immunoassaying unstable analytes, cross-reacting antibodies are used, the molecular structure of the analyte being modified to become specific for the antibody. The method can be used in competitive, sequential, immunometric two-site immunoassays. One particular application relates to the qualitative and quantitative screening of beta-lactam antibiotics in fluids, in particular the screening and measurement of penicillin.
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US68434184A | 1984-12-20 | 1984-12-20 | |
US684341 | 1984-12-20 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0207979A1 true EP0207979A1 (en) | 1987-01-14 |
EP0207979A4 EP0207979A4 (en) | 1989-04-12 |
Family
ID=24747658
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19860900699 Ceased EP0207979A4 (en) | 1984-12-20 | 1985-12-20 | Immunological method of measuring unstable analytes using cross-reactive antibodies. |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP0207979A4 (en) |
AU (1) | AU5357886A (en) |
WO (1) | WO1986003841A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5166078A (en) * | 1986-08-19 | 1992-11-24 | Idexx Laboratories, Inc. | Hapten-macromolecule conjugates useful in hapten assays |
GB8722470D0 (en) * | 1987-09-24 | 1987-10-28 | Blackmore D J | Production of anti-genic protein-hapten conjugates |
US5273909A (en) * | 1991-06-03 | 1993-12-28 | Quantix Systems, L.P. | Immunoassay for the detection of small aliphatic organic compounds |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4288538A (en) * | 1979-01-31 | 1981-09-08 | Baxter Travenol Laboratories, Inc. | Test method and composition therefor |
US4347312A (en) * | 1980-03-20 | 1982-08-31 | Research Triangle Institute | Detection of antibiotics in milk |
JPS5811857A (en) * | 1981-07-15 | 1983-01-22 | Yamasa Shoyu Co Ltd | Determination of adenosine |
-
1985
- 1985-12-20 AU AU53578/86A patent/AU5357886A/en not_active Abandoned
- 1985-12-20 EP EP19860900699 patent/EP0207979A4/en not_active Ceased
- 1985-12-20 WO PCT/US1985/002561 patent/WO1986003841A1/en not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
See references of WO8603841A1 * |
Also Published As
Publication number | Publication date |
---|---|
EP0207979A4 (en) | 1989-04-12 |
WO1986003841A1 (en) | 1986-07-03 |
AU5357886A (en) | 1986-07-22 |
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