The literature describes classes of compounds that give off light or "luminesce" by reaction through chemical treatment. The compounds that have this capability are termed chemiluminescent materials. Their dissociation is typically caused by treatment with peroxide or molecular oxygen at high pH. Light is produced by the decay of the transient ("intermediate") structure formed by peroxide or molecular oxygen reaction at an sp.sup.2 or sp.sup.3 hybridized carbon in the compound that is part of a chain or a ring or ring system.
As the literature indicates, any series of reactions which produce the intermediate: ##STR1## will lead to moderate to strong chemiluminescence. F is a structure such that the product carbonyl derivative ##STR2## is fluorescent and X is a good leaving group, usually with XH, for efficient chemiluminescence, having a pK.sub.a of about .ltoreq.11, preferably &lt;11, and most preferably, from about 5 to about 8. The reaction may require base catalysis. The intermediate can be prepared (in isolable or transient form, depending on F) from species such as: ##STR3## and H.sub.2 O.sub.2 (Y is halogen, --OSO.sub.2 R, and the like) or ##STR4## and base/O.sub.2. See Endeavour, 23, No. 117 (1973) p. 140, The Chemistry of Bioluminescence in "Bioluminescence in Action" (P. J. Herring, ed.), Academic Press, London, 1978 (pp. 64-5), Proc. R. Soc. Lond., B 215, p. 256 (1982), Progress in Organic Chemistry, (W. Carruthers and J. K. Sutherland, eds.), Butterworth, London (1973), p. 261, all authored by F. McCapra.
For example, chemiluminescent aryl esters that contain such hybridized carbon, termed a labeling compound, react according to the following general reaction: ##STR5## where A is an aryl ring or ring system and B is a heterocyclic ring or ring system. In this reaction, --O--A, the leaving group, is cleaved by perhydrolysis resulting in steps leading to the transient intermediate, B.dbd.O, that proceeds to decay generating luminescence.
The characteristics of some of these chemiluminescent compounds, their chemistry of manufacture, and other factors relating to them, are more fully described by McCapra, "Chemiluminescence of Organic Compounds," in Progress in Organic Chemistry, vol. 8, Carruthers and Sutherland ed., Wiley & Sons (1973); Kohen, Bayer, Wilechek, Barnard, Kim, Colleins, Beheshti, Richardson and McCapra, "Development Of Luminescence-Based Immunoassays For Haptens And For Peptide Hormones," pp. 149-158, in Analytical Applications Of Bioluminescence and Chemiluminescence, Academic Press, Inc. (1984); Richardson, Kim, Barnard, Collins and McCapra, Clinical Chemistry, vol. 31, no. 10, pp. 1664-1668 (1985); McCapra, "The Application of Chemiluminescence in Diagnostics," 40.sup.th Conference of the American Association of Clinical Chemists, New Orleans, La., Jul. 28, 1988; McCapra, "The Chemiluminescence Of Organic Compounds," Quarterly Reviews, vol. 20, pp. 485-510 (1966); McCapra, "The Chemiluminescence Of Organic Compounds," Pure and Applied Chemistry, vol. 24, pp. 611-629 (1970); McCapra, "The chemistry of bioluminescence," Proceedings Of Royal Society, vol. B215, pp. 247-278 (1982); McCapra and Beheshti, "Selected Chemical Reactions That Produce Light," Bioluminescence and Chemiluminescence: Instruments and Applications, CRC Press, vol. 1, Chapter 2, pp. 9-37 (1985); McCapra, "Chemiluminescent Reactions of Acridines," Chapt. IX, Acridines, R. M. Acheson, Ed., pp. 615-630, John Wiley & Sons, Inc. (1973); McCapra, "Chemical Mechanisms in Bioluminescence," Accounts Of Chemical Research, vol. 9, no. 6, pp. 201-208 (June 1976); and in many other publications and presentations on the subject.
As noted in the above literature, chemiluminescent compounds of a variety of structures have been projected as labels for a variety of assays including immunoassays (in this respect, see U.S. Pat. Nos. 4,383,031, 4,380,580 and 4,226,993). The esters, thiolesters and amides, alone or conjugated (i.e., chemically coupled to another material), are especially desirable forms of chemiluminescent labels. However, they lose their luminescence capability over time in an aqueous system because they hydrolyze to products that are not available to the assay. Until recently, these compounds have not been used in commercial assays. Until this invention, the ester, thiolester and amide forms of this class of materials lacked sufficient hydrolytic stability to be stored in the most convenient form over an extended period of time, which is as a component of an aqueous system.
The sensitivity of an assay is a measure of its ability to find a component present at low concentrations in the sample. The smaller the concentration in the sample being analyzed, the more sensitive is the assay if it is capable of measuring to that concentration. As pointed out by Spencer et al., Thyroid Today, vol. XIII, no. 4, 4th Q. 1990, a new category of sensitivity in immunometric assay (IMA) of TSH has been created as a result of the introduction of the chemiluminescent labels, conjugates, assays and kits of this invention. Chemiluminescent labels of this invention have been on sale as of about August 1988, and they have received critical acclaim by those in the art for unique sensitivity. Prior to this invention, sensitivity of TSH IMA was rated as "sensitive," "ultrasensitive" and "supersensitive." There was thereafter developed a generational classification, such as 1st generation assays, 2nd generation assays and 3rd generation assays, leaving open the possibility for later generations. The only IMA that is rated as a third generation IMA is the assay encompassed by this invention. As the authors stated:
"Appropriately, third generation TSH IMAs,.sup.4,5 display a further tenfold improvement in functional sensitivity, with an assay limit of 0.01 to 0.02 mU/L." PA0 "Most s-TSH immunometric assays (IMA) are excellent at discriminating TSH levels that are normal from those consistent with hyperthyroidism. However, the detection limit of these assays has generally been only slightly less than the lower limit of the euthyroid range, so that the measurement of subnormal but detectable TSH concentrations has been limited. PA0 "A new commercially available chemiluminescent assay (London Diagnostics) has an eight- to 10-fold increase in sensitivity over commercially available IMAs. The lower detection limits of so-called third-generation assays such as this one will allow partial suppression to be discriminated from more complete thyrotrope suppression. For example, a recent study found that half of the patients taking suppressive doses of levothyroxine who had an undetectable value in an s-TSH IMA had a detectable value in the more sensitive chemiluminescent assay." PA0 "Another example of steric hindrance is found in 2,6-disubstituted benzoic acids, which are difficult to esterify no matter what the resonance or field effects of the groups in the 2 or the 6 position. Similarly, once the 2,6-disubstituted benzoic acids are esterified, the esters are difficult to hydrolyze." (Emphasis in the original)
Ross, Clinical Chemistry News, Vol. 15, No. 11, entitled "New Assays Reveal TSH Deviations" states:
The uniqueness of the chemiluminescent labels of the invention to achieve more sensitive IMA or immunochemiluminometric assay (ICMA) is discussed by Spencer et al., "Application of a New chemiluminometric Thyrotropin Assay to Subnormal Measurements," Journal of Clinical Endocrinology and Metabolism, Vol. 70, No. 2, pp. 453-460 (1990).
It is well understood in chemistry that carboxylic acid esters, thiolesters and amides are susceptable to hydrolytic attack under acidic or basic conditions resulting in the formation of the carboxylic acid and the hydroxy, mercapto or amino component that is the theoretical or actual precursor to the ester, thiolester or amide. Hydrolysis is more pronounced under greater acidity or basicity. It is also recognized in chemistry that certain levels of hydrolysis can be inhibited by the inclusion of bulky groups that chemically sterically hinder those linkages, see Nishioka, et al., J. Org. Chem., vol. 40, no. 17, pp. 2520-2525 (1975), Fujita et al., "The Analysis of the Ortho Effect," Progress in Physical Organic Chemistry, 8, pp. 49-89 (1976), and Morrison and Boyd, Organic Chemistry, 5.sup.th Ed., pp. 842-843 (1987) and March, Advanced Organic Chemistry, 3rd Ed., page 240 (1985). According to March:
The difficulty in esterification is not the same in making esters from 2,6-substituted phenols, but the general principles described by March are applicable to enhancing the hydrolytic stability of the resultant ester so long as the ortho substitutions are electron donating. As this invention demonstrates, effective levels of hydrolytic stability require the presence of a select level of electron withdrawing chemical effect in conjunction with (and in addition to) traditional chemical steric hindrance factors.
As this invention demonstrates, effective levels of hydrolytic stability and sensitivity of chemiluminescent labeled conjugates utilized for ICMA are materially enhanced by the presence of a halosulfonyl in the label compound and sulfonamide in the conjugate utilized in the ICMA.
The functional electron withdrawing or electron donating characteristics of a group in an organic compound is conventionally measured relative to hydrogen. This relative ranking accepts that all groups on a molecule will provide some electron withdrawing effect, and distinquishes them by the nature of impact the group has on the molecule's performance. An electron withdrawing functional group, characterized by a positive number, will draw electrons to itself more than hydrogen would if it occupied the same position in the molecule. The opposite occurs with an "electron donating group," a lesser electron withdrawing group which chemical convention characterizes by a negative number. Sigma para values (.sigma..sub.p) are the relative measurement of electron withdrawing or electron donating qualities of a functional group in the para position on benzoic acid. See March, Advanced Organic Chemistry, 3rd Edition, Publ. by John Wiley & Sons, New York, N.Y. (1985) at pp. 242-250 and 617-8. Tables of .sigma..sub.p values for various groups can be found in Hansch et al., J. Med. Chem. 16(11): 1209-1213 (1973) and Hansch and Leo, "Substituent Constants for Correlation Analysis in Chemistry and Biology," Ch. 6, pp. 49-52 (John Wiley & Sons, New York 1979). The .sigma..sub.p values reported in the Hansch articles are relied on herein in characterizing relative values for groups both in the meta and para position.
The function of chemiluminescent labels in assay applications involves the coupling of the label compound to a substrate molecule. Such coupling can be achieved by solvent interraction (e.g., molecular compatibility), any heterolytic or homolytic mechanism induced by chemical means and influenced by physical effects, such as time, temperature and/or mass action. For example, the reaction can be nucleophilic or electrophilic, or it can involve free radical mechanisms. In the broadest perspective, the coupling can be viewed as achievable via strong to weak bonding forces.
A chemiluminescent label in assays is an associated moiety of a binding material. The moiety is derived from a chemical compound which, as such, possesses chemiluminescent capabilities. Hereinafter, the term moiety as applied to the label as such, is a reference to the compound prior to being associated with a binding material. The term associated is intended to include all or any of the mechanisms for coupling the label to the substrate molecule.
The term "functional" in chemistry typically refers to a group that influences the performance of a chemical or constitutes the site for homolytic or heterolytic reactions. For example, a functional alkyl substituent, used in the context of interreactions through that substituent, means an alkyl group substituted so that it can effect that reaction. But an alkyl group termed functional for the electronic effects it induces in the molecule is a reference to the alkyl group per se.