DE2729555A1 - PROCEDURE FOR APPLYING A RECIPE TO A PLASTIC SUBSTRATE, THE PRODUCT CONTAINED AND ITS USES - Google Patents

Description

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BECTON, DICKINSON and COMPANY, Rutherford, "New Jersey / USA

Process for applying a receptor to a plastic substrate, the product obtained thereby and its use

709886/0604

description

The invention relates to the examination or analysis of ligands and, more particularly, to an improved method for providing a receptor which is applied to a plastic substrate is coated and is suitable for the analysis of ligands, as well as the product obtained thereby.

The analysis of ligands is based on the competition between a labeled and an unlabeled ligand for a limited number of sites on a receptor for the ligand. In short, there will be a known amount of a labeled ligand, a sample containing an unknown amount of the unlabeled ligand and a known amount of receptor for the Contains ligand, combined, and the percentage of labeled ligand that is bound to the receptor depends on the amount of unlabeled ligand in the sample. After Separation of the receptor, including the bound ligand, from the sample, the amount of labeled ligand, that is bound to the receptor or remains in the sample is determined and compared with a standard curve to determine the amount of unlabeled ligand that was present in the sample

determine.

To facilitate the separation of the receptor from the sample, the receptor is in many cases attached to a plastic substrate, such as a tube, thereby easily separating the receptor including the bound ligand from a sample can be used to determine the amount of labeled ligand either bound to the receptor or remaining free in the sample.

The invention relates to improvements in the manufacture of a receptor coated plastic substrate for analysis of a ligand.

According to the invention, a receptor is applied to a plastic substrate in the presence of a disaggregating agent for the receptor and a blocking agent for the receptor sites for the ligand coated to prevent the receptor from sticking to the To effect plastic substrate at the points different from the receptor sites.

In particular, the blocking agent is a substance that has limited crosslinking reactivity with the receptor in order to cause a blockage of the receptor parts for the ligand and thus the adhesion or to prevent the sticking of the receptor to the plastic at the receptor sites for the ligand, however, also a later binding of a ligand to the receptor becomes possible. Generally used as a blocking agent for the Ligand receptor sites used substance have a crosslinking reactivity with the receptor from about 0.0001 to about 5 # and preferably from about 0.001 to about 1 #. The upper The reactivity limit for the cross-links ensures that in a subsequent investigation the blocking agent will be replaced by the ligand to be investigated can, whereby a competitive binding can take place at the receptor sites. It goes without saying that the substance used as a blocking agent should have an affinity for the plastic surface.

109886/0604

Zeptors for the plastic surface is to allow orientation of the receptor on the plastic. As The substance used as a blocking agent is preferably hydrophilic, but it goes without saying that neither are hydrophilic substances can be used.

Examples of suitable blocking agents can be mentioned

be: cardiac glycosides such as ouabain, gitoxin, gitaloxin,

Acetylstrophanthidine, etc., steroids such as progesterone, testosterone, cortisol, etc.

The choice of an appropriate means of blocking the receptor sites is within the skill of the art, given the teachings of this specification.

The coating of antibodies on the plastic substrate is also carried out in the presence of a disaggregating agent for the receptor in order to keep the antibody monomeric and to prevent sticking or sticking in the form of dimers or aggregates to the plastic substrate, thereby increasing the accessibility or availability of the receptor sites would be restricted during the later analysis. The disaggregation agent can be one of the many commonly used and examples of such agents can be: Amino acids such as glycine, lysine, etc .; Urea; Guanidine; soluble inorganic salts; especially sodium and calcium salts and the same. The choice of a suitable deaggregation agent is based on the present invention Description given teaching in the field of technical ability.

The blocking agent is used in an amount effective to block the receptor sites of the receptor for the Ligand is and varies with the amount of receptor and the particular receptor used. Generally one uses this Blocking agents in an amount from 0.0001 to 0.1 #, preferably from 0.0005 to 0.005 #, each based on the weight (e.g. of the solution). In the same way, the amount of the used disaggregating agents from the receptor and its

Amount from, the disaggregating agent generally in an amount of 0.01 m to 0.9 m, preferably 0.25 m to 0.35 m (or 0.01 mol to 0.9 mol, preferably from 0.25 mole to 0.35 mole) is used. The amount of blocking agent and disaggregating agent to be used is within the range while considering the teachings of the present specification within the range of one skilled in the art.

It has been found that when coating the plastic substrate with antibodies in the presence of both a blocking agent and a disaggregating agent, the amount of Receptor required for coating formation is significantly lower than that required in known processes.

The plastic substrate is coated with the receptor at temperatures as are generally used in this field, for example at temperatures of the order of magnitude from 10 ^° C. to 37.8 or about 38 ^° C. (50 ^° F. to 100 ^° F. ) and for a time sufficient to ensure that the receptor adheres to the plastic substrate, for example a time of the order of 15 minutes to 24 hours. The antibody-coated or lined tube is generally further treated with a protein-containing buffer in order to coat any uncoated portions and to reduce or eliminate non-specific binding in a later analysis. After the plastic substrate has dried, the plastic substrate coated with the receptor can be used for an examination or analysis according to known procedures.

The receptor applied to the plastic substrate can either be a developed or produced antibody or a naturally occurring receptor; such receptors are known. As is known, the antibodies are produced by injection of an antigen or hapten which is attached to an antigenic carrier ger is bound, developed in the bloodstream of a vertebrate animal, such an antibody being a specific receptor for such an antigen or hapten let. That with the

10988 ^ / 0804

Receptor-coated plastic substrate can then be used for the analysis of a wide variety of ligands, such as 1) antigens that, when introduced into the bloodstream of a vertebrate, lead to the formation of antibodies; 2) Heptenes, the when bound to an antigenic carrier and introduced into the bloodstream of a vertebrate animal, antibodies specific for the Form haptens or 3) ligands that have naturally occurring receptors that are specific for the ligand Shape can be isolated. It will be understood that a ligand can have naturally occurring receptors and also when bound to a protein can act as a hapten.

Examples of the ligand to which the invention can be applied include: polypeptides, nucleotides, nucleosides and proteins such as ACTH, oxytocin, luteinizing hormone, insulin, proinsulin, Bence Jones protein, chorionic gonadotropin, Pituitary gonadotropin, growth hormone, renin, thyroxine binding globulin, bradykinin, angiotensin, follicles stimulating hormone, cyclic AMP, cholylglycine, cyclic GMP, etc .; Steroids, including estrogens, gestrogens, androgens, adrenocortical hormones, bile acids, cardiotonic Glycosides, aglycones and saponins. The following can be mentioned as specific examples: thyroxine, triiodothyronine, Testosterone, androsterone, estrone, estriol, progesterone, pregnenols or pregnenolone, 17 ~ hydroxy-dioxycorticosterone (compound S), deoxycorticosterone, cortisone, corticosterone, cortisol, Aldosterone, digoxin, digitoxin, etc., vitamins such as vitamin A, the vitamin B group, vitamin C, the D vitamins, Folic acid and vitamins E and K, as well as various ligands, such as antigens for viral hepatitis A and B, rubella, Herpes simplex, α-beto-protein or α-feto-protein, etc.

The present substances are only examples, and it goes without saying that such substances can be given in the form of more suitable ones Analog can be used.

The analysis using the coated with the receptor Plastic substrate can be done by using a labeled ligand, e.g. labeled with a radioisotope, enzyme,

109888/0604

a fluorescent substance, etc. Such procedures are known and therefore need no further explanation.

The plastic substrate or polymer substrate can be in particulate form, in the form of a tube, in the form of an arch or plate, etc., the tubular or tubular form being preferred is. The plastic can be any plastic made from a wide variety of polymers, commonly known as bus rates for ei ^ NEN receptor can be used including, for example, polystyrene, polyethylene, polypropylene, polyamides, polyacrylamides, etc. The choice of a particular plastic substrate will be apparent to those skilled in the art from the teachings herein Description.

The following examples serve to further illustrate the invention without restricting it.

example 1

Digoxin antibodies generated in sheep with a digoxin human serum albumin conjugate in an aqueous buffer solution (0.05 M carbonate / bicarbonate, pH 9.6, 0.3 o glycine, 0.001j6 ouabain and 0.005 # chloramphenicol) (1 , 0 ml) is filled into a polystyrene tube at room temperature and kept for two hours at room temperature, after which the liquid is sucked out of the tube. A buffer solution (0.1 M phosphate, pH = 7.0, with 0.9 # NaCl and 0.1 # NaN,) including 1 g of lysozyme per 1 is fitted into the tube and en is maintained for about 15 minutes at room temperature. The liquid is sucked out of the tube and the tubes are dried in vacuo at room temperature.

Example 2

The pipes coated or lined with antibody were produced as in Example 1, are used to study digoxin as follows:

1. At room temperature, 50 µl of a digoxin standard or a sample from a patient, 100 μl 3-0-succinyl-digoxi-

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Pipe no. 2 1. 4th 3, 6th 5, 8th 7, 10 9, , 12 11 , 14 ' 13th , 16, etc. 15th

Genin-tyrosine (' ^P I) and 900 μl 0.1 M phosphate buffered saline (PBS) are pipetted into the tubes as follows:

Digoxin in standard (ng / ml ) 0.0 0.5 1.0 1.5 2.0 3.0 5.0 patient sample.

^{The tubes are incubated at 37 °} C. for 60 minutes in a water bath.

2. The liquid is sucked out of the tubes, whereupon 1.0 to 1.5 ml PBS are added and sucked off (decanted). The tubes now contain labeled and unlabeled digoxin bound to antibodies.

3. The tubes are gamma-counter measured in a minimum of 0.5 minute runs and a standard curve is made comprising the range of 0.5-5.0 ng digoxin per ml of patient sample.

4. The digoxin concentration in the sample is determined on the standard curve.

A particular advantage of the present invention is that high amounts of receptor can be used to coat the plastic substrate, e.g. the antibody concentration can be 1/10 to 1/5 of the previous antibody concentration, which is a five to ten fold increase of the antibody titer.

Obviously, numerous variations of the foregoing embodiments are possible and fall within the scope of the present invention.

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Claims (13)

Claims 1. Method of coating a receptor for one to investigating ligands on a plastic substrate, characterized in that this coating with a Forms solution of the receptor, which contains a blocking agent for the receptor sites, in order to adhere or stick to prevent the receptor sites on the plastic substrate, as well as in the presence of a disaggregating agent for the receptor performs to stick the receptor to the plastic substrate in the form of aggregates to prevent the blocking agent having a crosslinking reactivity or binding reactivity the receptor has from 0.0001 to 5 # and by the to analyzing ligands can be replaced. 2. The method according to claim 1, characterized in that the binding reactivity of the blocking agent Is £ 0.001 to £ 1. 3. The method according to any one of the preceding claims, characterized in that the blocking agent in one Amount of 0.0001 to 0.01 # based on the weight of the solution is used. 4 · Method according to one of the preceding claims, characterized in characterized in that the disaggregating agent in a concentration of 0.01 m to 0.9 m is used. 5. The method according to any one of the preceding claims, characterized in that the blocking agent is selected from the group of cardiac glycosides and steroids. 6. The method according to any one of the preceding claims, characterized in characterized in that one uses a plastic substrate in the form of a tube. 7Q98ÖB / 06CU ORIGINAL INSPECTED 7. The method according to any one of the preceding claims, characterized characterized in that the plastic used is polystyrene. 8. The method according to any one of the preceding claims, characterized in that a digoxin antibody is used as the receptor used. 9. The method according to any one of the preceding claims, characterized in that the blocking agent Uses ouabain. 10. The method according to any one of the preceding claims, characterized characterized in that glycine is used as a disaggregating agent. 11. The method according to any one of the preceding claims, characterized characterized in that the plastic coated with receptor is treated with a buffer containing protein, to coat any uncoated areas on the plastic substrate with protein. 12. Plastic substrate with a coated receptor for a ligand prepared according to the process of any one of the preceding claims. 13. Use of the plastic substrates according to claim 12 for Analysis of ligands. 70,988 $ / 06tU