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
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
  • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
• • 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
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57476—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncofetal proteins

Definitions

• Immunoassays involving serum-derived antibodies have included antibodies directed to the unique carboxy terminal portion of the ⁇ subunit of hCG (ibid.; and Birken, S. et al., Endocrinology, 110:1555 [1982]).
• This invention concerns an immunoassay for detecting hCG or measuring the level of hCG in a sample.
• the assay includes an antibody directed to the carboxy terminal portion of the 8 subunit of hCG and a monoclonal antibody directed to a determinant on hCG at a locus sufficiently remote from the carboxy terminal portion of the 8 subunit of hCG that both antibodies can simultaneously bind to hCG, wherein at least one of the antibodies is detectable when both are bound to hCG.
• the immunoassay can detect or measure levels of hCG or hCG ⁇ in a urine sample and includes a detectable, purified, serum-derived antibody directed to the carboxy terminal portion of the 3 subunit of hCG and a matrix-bound monoclonal antibody directed to a locus on the S subunit sufficiently remote from the carboxy terminal portion that both antibodies can simultaneously bind to hCG.
• the assay may be used to detect hCG or to measure hCG levels in urine samples. In this way the presence of hCG- producing neoplasms, subclinical spontaneous abortions and ectopic pregnancies can be diagnosed.
• Fig. 1 is a standard curve showing use of the assay to measure hCG in buffer ( ⁇ --- ⁇ ) and in urine ( ⁇ --- ⁇ ) and to show extent of cross-reactivity with hLH in urine ( ⁇ -- ⁇ ).
• Fig. 2 illustrates the sensitivity of a conventional radioimmunoassay (Con A Assay, ⁇ ⁇ ) as compared with the newly developed sandwich assay ( ⁇ --- ⁇ ) in the measurement of hCG levels in an artificially inseminated female patient. Also depicted is the hypothetical pattern of hCG levels occurring as a result of subclinical spontaneous abortion (----).
• this invention provides an immunoassay for detecting hCG or measuring levels of hCG, or both, in a sample.
• the immunoassay includes an antibody directed to the carboxy terminal portion of the ⁇ subunit of hCG which is a unique peptide sequence not found in hLH.
• the immunoassay also includes a monoclonal antibody directed to a determinant on hCG at a locus sufficiently remote from the carboxy terminal portion of the ⁇ subunit that both antibodies can simultaneously bind to hCG.
• at least one of the antibodies is capable of detection when both antibodies are bound to hCG.
• any antibody directed to the carboxy terminal portion of the ⁇ subunit of hCG would be effective, the presently available and preferred antibodies are purified, serum-derived antibodies. Examples include R525 and R529 as described more fully hereinafter.
• the present invention is not limited to serum- derived antibodies and also encompasses monoclonal antibodies directed to the carboxy terminal portion of the ⁇ subunit if and when efforts to produce such monoclonal antibodies are successful. Either antibody or both antibodies are capable of detection when the antibodies are bound to hCG. Various means for rendering an antibody detectable are known to those skilled in the art.
• suitable means include radioactive labeling, e.g., 125 I labeling, fluorescent labeling or linkage to an enzyme which catalyzes a detectable reaction, that is, an ELISA approach, e.g., linkage of an antibody to horseradish peroxidase.
• An additional approach involves the use of a third anti- body directed to one of the antibodies of the assay where the third antibody, either serum-derived or monoclonal, is detectable, e.g., labeled with a radioactive isotope or a fluorescent moiety.
• the immunoassay of the present invention may be carried out totally in liquid phase, totally in solid phase or in a mixed liquid/solid system.
• one of the antibodies, preferably the monoclonal antibody directed to a locus remote from the carboxy terminal portion of the ⁇ subunit be attached to a solid matrix, e.g., agarose or Sepharose.
• the immunoassay may be used with either urine, plasma, serum or other samples, it is particularly desirable to employ the immunoassay on urine samples because of the greater ease and convenience of obtaining such samples. This is, in fact, one of the substantial advantages provided by the present invention since most presently available immunoassays for hCG are not suitable for use with urine samples.
• the presently preferred embodiment of the invention involves an immunoassay for detecting hCG ⁇ or measuring levels of hCG ⁇ , and thereby detecting or measuring hCG.
• This immunoassay includes a purified, serum-derived anti- body directed to the carboxy terminal portion of the ⁇ subunit of hCG and a monoclonal antibody directed to a locus on the ⁇ subunit sufficiently remote from the carboxy terminal portion that both antibodies can simultaneously bind to hCG.
• the purified, serum-derived antibody is detectable when both antibodies are bound to hCG ⁇ , alone or as hCG, and the monoclonal antibody is attached to a solid matrix.
• This invention also concerns a purified antibody to the carboxy terminal portion of the ⁇ subunit of hCG, preferably labeled or otherwise detectable, e.g., 125 I-labeled.
• the immunoassay of this invention provides a method of detecting hCG or measuring the level of hCG, or both, in a urine sample.
• the sample to be tested is placed in the immunoassay under suitable conditions permitting formation of a detectable or measurable complex with hCG.
• the resulting complex may then be detected if present or the level of hCG present determined by reference to a standard containing a known amount of hCG.
• Measurements of hCG levels in turn can be used in the diagnosis or identification of disorders which involve production, or elevated levels, of hCG.
• disorders include certain neoplasms, e.g., male testicular cancer, subclinical spontaneous abortions and ectopic pregnancies.
• the antibodies used in this experiment were R525 and R529, Birken, S. et al., Endocrinology, 110:1555 (1982), rabbit antisera directed against the hCG ⁇ - CTP determinants, and B101, Ehrlich, P.H. et al.. Journal of Immunology, 128:2709 (1982), a mouse monoclonal antibody directed against an hCG ⁇ ⁇ onformational determinant.
• the methods of preparation and characteristics of these antibodies were as published in the preceding two cited references.
• Cyanogen bromide (CnBr) activated Sepharose 4B was obtained from Pharmacia Fine Chemicals and DEAE Affi-Gel Blue from Bio-Rad Laboratories. The preceding purchased materials were used in accordance with the manufacturers' instructions.
• Electrophoresis of the preparation on nonreducing SDS polyacrylamide gels Weber, K. and Osborn, M., J. Biol. Chem., 244:4406 (1969), indicated that at least 80% of the protein content was ⁇ -globulin.
• hCG ⁇ Ten mgs of hCG ⁇ was coupled to two grams of CnBr activated Sepharose 4B.
• the hCG ⁇ coupled Sepharose 4B was used to form an 0.9 x 10 cm column which was used for affinity purification of R525.
• a 54 ml pool of R525 from several different bleeds between boostings was recycled over the column continuously for 30 hours at 4°C using a peristaltic pump.
• the column was washed extensively with saline to remove loosely adhering material.
• the column was then eluted successively with 20 ml of 3M guanidine-HCl, pH 3.0, and 20 ml of 6 M guanidine-HCl, pH 3.0.
• the effluent was collected in one ml fractions and the absorbance at 280 nm determined.
• the absorbance profile had two discrete protein peaks corresponding in position to the areas of elution with 3 M and 6 M guanidine-HCl.
• the fractions having the peak absorbance at 280 nm were pooled and then dialysed extensively against 10 mM sodium acetate, pH 5.5, followed by 0.3 M NaPO 4 , pH 7.5, at 4°C.
• the dialysed preparations, an aliquot of the unpurified R525 pool and also the R525 pool which had been circulated over the hCG ⁇ affinity column were tested for their ability to bind 125 I-hCG.
• Buffer B containing 50,000 CPM 125 I-R525 was added to each tube.
• the samples were incubated vertically for 48-72 hours at 4°C with shaking on the Labquake Shaker.
• the tubes were then washed three times with 2 ml of Buffer B + 1% Tween 20 to reduce nonspecific binding.
• the hCG radioimmunoassay was conducted using the R529 antiserum to the hCG ⁇ CTP determinants to measure hCG extracted from urine with ⁇ oncanavalin A as described by Wehmann,et al. Wehmann, R.E., et al.. Clinical Chemistry, 27:1997 (1981).
• urinary hCG concentration was normalized to creatinine concentration.
• FIG. 1 A typical standard curve generated for hCG binding in the sandwich assay is shown in Fig. 1.
• the hCG dosages which give binding equivalent to 10% and 90% of Bmax (ED 10 and ED 90 ) have been tentatively accepted as the limits of the usable range of the standard curve.
• EDio and ED 90 correspond to 0.01 and 0.50 ng hCG/ml, giving a fiftyfold usable range.
• the Nonspecific Binding (NSB) in the assay is reduced to an acceptable level by extensive washing of the pellet with buffer containing Tween 20 and bovine ⁇ -globulin. In the assay represented NSB was 2.5% of the total counts added.
• Fig. 1 also shows the dose response curve of hCG extracted from normal male urine which had been spiked with dosages identical to those used in the standards.
• the dose response curve for hCG in urine is essentially identical to that for hCG in Buffer B.
• the slope and ED 50 of the dose response curve were 1.173 and 0.066 ng/ml for hCG in urine, compared to 1.098 and 0.072 ng/ml for hCG in buffer.
• the dose response curve for hLH added to normal male urine is also shown in Fig. 1. It should be noted that the units for hLH concentration are ⁇ g/ml as opposed to ng/ml for hCG concentration. A concentration of 2 ⁇ g hLH/ml is required to obtain a dose response equivalent to 0.01 ng hCG/ml. Thus, the cross-reactivity of this concentration of hLH in the sandwich assay is 0.0005% on the basis of mass. This level of hLH is approximately fifty fold higher than those present in urine from women at the mid-cycle hLH surge. Saxena, B.B. et al.. Fertility and Sterility, 28:163 (1977). Therefore, physiologic concentrations of hLH are incapable of interfering in the sandwich assay.
• Fig. 1 shows that the assay is equally effective for the measurement of hCG in buffer and in urine, highly sensitive, i.e., capable of detecting hCG at levels down to 0.004 ng/ml and that cross-reactivity with hLH is exceptionally low, i.e., about 0.0005%, thereby rendering the assay absolutely specific for hCG.
• Fig. 2 shows the use of the assay to measure hCG levels in the urine of an artificially inseminated female patient at various days after insemination.
• Fig. 2 also shows the use of a standard radioimmunoassay to measure hCG levels in the same patient's urine.
• the assay of the present invention is capable of detecting elevated hCG levels after the sixth day following insemination.
• the standard RIA is less sensitive and cannot detect hCG until the tenth day following insemination.
• Fig. 2 also illustrates the application of the assay in the diagnosis of subclinical spontaneous abortion.
• the hypothetical pattern illustrated in Fig. 2 shows that the standard RIA is incapable of detecting either the rise or the fall of hCG as the result of subclinical spontaneous abortion while the assay of the present invention will be able to do so because of its much greater sensitivity.

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• 1984
  • 1984-04-13 JP JP59502093A patent/JPS60501271A/ja active Pending
  • 1984-04-13 EP EP19840901866 patent/EP0146563A4/de not_active Ceased
  • 1984-04-13 WO PCT/US1984/000572 patent/WO1984004598A1/en not_active Application Discontinuation
  • 1984-04-18 CA CA000452265A patent/CA1229549A/en not_active Expired

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