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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • 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
  • G01N33/57469—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving tumor associated glycolinkage, i.e. TAG
• • 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

Definitions

• hCG Human chorionic gonadotropin purified from urine of pregnant women has been shown to have approximately 30% carbohydrate content, consisting of two Nasparagine linked carbohydrate side chains on each of its and subunits (1) and four O-serine linked side chains attached to the COOH-terminal peptide region of its subunit (2,3).
• Nishimura et al. (4) observed that hCG purified from the urine of a patient with choriocarcinoma had a reduced carbohydrate content and that this difference was due to a low sialic acid content.
• lectins These carbohydrate binding proteins, have proved to be useful reagents for probing structural features of cell surface glycoproteins and for isolating glycoproteins.
• carbohydrate binding properties of two lectins peanut lectin, (Arachis hypogea agglutinin, PNA), and castor bean lectin, (Ricinus communis agglutinin, RCA), to develop highly specific methods for the detection of ashCG in urine.
• PNA and RCA specifically bind the desialylated O-serine (9,10) and N-asparagine (9) linked oligosaccharides of hCG respectively.
• Solid phase lectin is utilized to extract ashCG from urine, and lectin bound ashCG is then measured utilizing a purified and radiolabeled monoclonal antibody or rabbit antiserum.
• the form of desialylated hCG detected is dependent not only on the carbohydrate specificity of the lectin but the peptide specificity of the monoclonal antibody or antiserum employed.
• This unique type of assay has been described as a Lectin-Immunoradiometric Assay (LIRMA) and may be extended to the measurement of a variety of soluble glycoproteins by using combinations of lectins and antibodies with different specifications.
• LIRMA Lectin-Immunoradiometric Assay
• Lectins are proteins or glycoproteins of non-immune origin long known to be useful for agglutinating erythrocytes and other types of cells and useful for studying cell surface properties.
• a lectin to a glycoprotein, particularly a relatively small glycoprotein such as hCG, would prevent subsequent or contemporaneous binding of an antibody directed to an antigenic determinant on the glycoprotein.
• a lectin which selectively binds to a specific sugar moiety and of an antibody provides a highly sensitive and specific method for qualitatively detecting or quantitatively determining a desialylated glycoprotein.
• the method provided permits exploitation of the dual nature of glycoproteins.
• the lectin component binds specifically to the carbohydrate moiety while the antibody component binds specifically to the peptide moiety.
• An example of the importance of the invention is the ability to distinguish desialylated hCG from sialylated hCG and, thereby, diagnose gestational trophoblastic disease.
• a method for determining the presence of a soluble desialylated glycoprotein such as human chorionic gonadotropin in a biological fluid such as urine or blood comprises contacting a sample of the biological fluid with a suitable amount of an appropriate lectin capable of selectively binding to the desialylated glycoprotein to produce a complex.
• an appropriate lectin capable of selectively binding to the desialylated glycoprotein to produce a complex.
• lectins include peanut lectin and castor bean lectin.
• the resulting complex is separately recovered from the biological fluid.
• the recovered complex is contacted under appropriate conditions with at least one detectable antibody such as radiolabeled or fluorescently labeled monoclonal antibody directed to an antigenic determinant on the desialylated glycoprotein and capable of selectively binding to glycoprotein present in the complex.
• the presence of antibody so bound is detected and, thereby, the presence of desialylated glycoprotein in the biological fluid determined.
• a method for quantitatively determining a soluble desialylated glycoprotein in a biological fluid comprises contacting a sample of the biological fluid with a suitable amount of an appropriate lectin capable of selectively binding to the desialylated glycoprotein to produce a complex.
• the resulting complex is separately recovered from the biological fluid.
• the complex so recovered is contacted under appropriate conditions with a predetermined amount of at least one detectable antibody directed to an antigenic determinant on the desialylated glycoprotein and capable of selectively binding to glycoprotein present in the complex.
• the amount of antibody so bound is determined and, thereby, the amount of desialylated glycoprotein in the biological fluid determined.
• this invention provides both qualtitative methods for detecting and quantitative methods for determining the presence in biological fluids of soluble glycoproteins. As will be clear to those skilled in the art to which this invention relates, these methods may be readily modified in several respects.
• the lectin rather than the antibody may be detectably labeled; the antibody rather than the lectin may be employed to initially contact the sample or the sample may be simultaneously contacted with the antibody and the lectin.
• the extraction method uses a solid phase coupled monoclonal antibody to hCG ⁇ subunit for extraction of hCG or ashCG from buffer or urine.
• FIG. 2 Characteristics of four lectin immunoradiometric assays (LIRMA) for measurement of ashCG in buffer or urine. Comparisons of dose responses were made for the following hormones or fragments: AshCG (o----o), ashCG ⁇ (o----o), as ⁇ -CTP
• Inserts on upper right corners of panels A and D illustrate measurement of ashCG in buffer (o----o ) and normal male urine (o----o ) using PNA- 1 25 -R525 (A) and RCA- 125 -I R525 (D) .
• FIG. 3 Sephadex G-100 gel filtration of a urine concentrate of a patient with choriocarcinoma (panel A) and of a normal pregnant woman (panel B) .
• 10 ml of the urine concentrate was applied to a col umn (2.5 x 196 cm) of Sephadex G-100 , and eluted with 0.05 M Tris-HCl buffer containing 0.1 H NaCl (pH 7.4) .
• Fractions of 6 ml were collected and al iquots were assayed in various assay systems.
• Total hCG was determined by R529 RIA ( ⁇ ), and ashCG by R141 RIA (direct method) (o) and PNA 125 I-R525 ( ⁇ ) LIRMA. 100,000 cpro of 125 I-hCG in 10 ml of the same buffer was eluted in separate run as a marker.
• the insert in panel B illustrates the amounts of asialo hCG immunoreactivity detected using the three methods on an
• a method for determining the presence of a soluble desialylated glycoprotein in a biological fluid which comprises contacting a sample of the biological fluid with a suitable amount of an appropriate lectin capable of selectively binding to the desialylated glycoprotein to produce a complex.
• the resulting complex is separately recovered from the biological fluid.
• the so recovered complex is contacted under appropriate conditions with at least one detectable antibody directed to an antigenic determinant on the desialylated glycoprotein and capable of selectively binding to glycoprotein present in the complex.
• the presence of antibody so bound is detected and, thereby, the presence of desialylated glycoprotein in the biological fluid is determined.
• the soluble desialylated glycoprotein may be hCG, thyroglobulin (11), carcinoembryonic antigen or CA19-9 (12), or any other desialylated glycoprotein or subunit thereof, e.g. desialylated hCG.
• the biological fluid may be urine, blood, semen, saliva, pus or any other biological fluid, a sample of which one wishes to examine.
• biological fluid will be obtained from a subject, e.g. a human patient.
• desialylated hCG may be detected in human urine.
• a sample of the biological fluid is obtained and contacted with a suitable amount of an appropriate lectin to produce a complex.
• the contact typically involves simply adding the lectin to the sample under ambient conditions.
• the amount and type of lectin added may vary widely depending upon factors well known in the art such as the concentration of soluble glycoprotein normally present in the sample and the nature of the glycoprotein. Typcially, the amount of lectin added will be in molar excess of the amount of glycoprotein present in the sample.
• Appropriate lectins are lectins capable of selectively binding to the desialylated glycoprotein.
• Numerous lectins are known which selectively bind to specific sugar moieties and which may therefor be employed in this invention.
• peanut lectin may be derived from Arachis hypo ⁇ ea. Such lectin selectively binds to the carbohydrate structure Gal ⁇ 1 ⁇ 3GalNAc.
• castor bean lectin derived f rom Ri cinus commu ⁇ is which selectively binds to the carbohydrate structure Gal ⁇ l ⁇ 4GlcNAc.
• the complex which results from contacting the sample of biological fluid with the lectin is then separated from the biological fluid, e.g. by centrifugation followed by decantation of the supernatant.
• centrifugation followed by decantation of the supernatant.
• other methods of recovery may be employed such as filtration.
• the separately recovered complex is contacted with at least one detectable antibody directed to an antigenic determinant on the desialylated glycoprotein and capable of selectively binding to glycoprotein present in the complex.
• Appropriate conditions for effecting such contact are well known to those skilled in the art, e.g. the complex may be redissolved in a suitable buffer and the antibody, dissolved in the same or a compatable buffer added at a temperature, e.g. 37°C, at which activity of the antibody is retainee.
• the antibody used in this method may be either a polyclonal or a monoclonal antibody which is detectable e.g. because an identifiable label such as a radiolabel or a fluorescent label has been attached to it.
• the antibody may be detected by use of a second antibody directed it, the second antibody being labeled or having an enzyme substrate bound to it.
• the antibody may be a serum or monoclonal antibody directed to a determinant on the subunit such as an antibody directed to the carboxy terminal region of the subunit.
• a type of desialylated hCG has been detected. It is to be understood that desialylated is intended to encompass both glycoproteins which do not naturally include sialo groups and these which naturally include sialo groups, but from which they have been removed. The latter situation is believed to be a general indicator of disease and has been so implicated in choriocarcinoma and hydatiform mole.
• the presence of antibody bound to the lectinglycoprotein complex may be readily detected using well known techniques.
• the antibody is fluorescently labeled with a moiety such as a fluorescent dye covalently bound to the antibody the fluorescent emission of the dye upon excitation with appropriate electromagnetic radiation such as ultraviolet radiation may be measured or detected using a conventional fluorimeter.
• a radioactive isotope such as I 125 bound to the antibody may be detected using a coventional scintillation spectrometer.
• a method for quantitatively determining the amount of a soluble desialylated glycoprotein in a sample of a biological fluid comprises contacting the biological fluid with a suitable amount of an appropriate lectin capable of selectively binding to the desialylated glycoprotein to produce a complex.
• the resulting complex is separately recovered from the biological fluid.
• the complex so recovered is contacted under appropriate conditions with a predetermined amount of at least one detectable antibody directed to an antigenic determinant on the desialylated glycoprotein and capable of selectively binding to glycoprotein present in the complex.
• the amount of antibody so bound is determined and, thereby, the amount of desialylated glycoprotein in the biological fluid determined.
• the desialylated glycoprotein to be quantitatively determined is hCG.
• One application of the methodology for quantitatively determining soluble desialylated glycoprotein involves the diagnosis of a disease associated elevated levels of desialylated hCG such as choriocarcinoma or hydatiform mole.
• a disease associated elevated levels of desialylated hCG such as choriocarcinoma or hydatiform mole.
• diseases such as choriocarcinoma or hydatiform mole may be diagnosed.
• a method for determining the presence of soluble desialylated glycoprotein in a biological fluid comprises contacting a sample of the biological fluid with at least one antibody directed to an antigenic determinant on the desialylated glycoprotein and capable of binding to the glycoprotein to produce a complex.
• the resulting complex is separately recovered from the biological fluid.
• the so recovered complex is contacted with a suitable amount of an appropriate detectable lectin.
• the presence of the lectin so bound is detected and, thereby, the presence of desialylated glycoprotein in the biological fluid is determined.
• the detectable lectin is radiolabeled or fluorescently labeled.
• the preceding method may be rendered quantitative as may the various related methods which follow.
• the invention also provides a method for determining the presence of a soluble desialylated glycoprotein in a biological fluid which comprises contacting a sample of the biological fluid with a suitable amount of an appropriate detectable lectin capable of selectively binding to the desialylated glycoprotein to produce a complex.
• the complex under appropriate conditions, is contacted with at least one antibody directed to an antigenic determinant on the desialylated glycoprotein and capable of selectively binding to glycoprotein in the complex.
• the resulting complex is separately recovered from the biological fluid.
• the presence of lectin bound to the recovered complex is detected and, thereby, the presence of desialylated glycoprotein in the biological fluid is determined.
• the preceding method may be carried out in a different sequence.
• a method for determining the presence of soluble desialylated glycoprotein in a biological fluid comprises contacting a sample of the biological fluid with at least one detectable antibody directed to an antigenic determinant on the desialylated glycoprotein and capable of binding to the glycoprotein to produce a complex.
• the complex is contacted with a suitable amount of an appropriate lectin capable of selectively binding to glycoprotein present in the complex.
• the resulting complex is separately recovered from the biological fluid.
• the presence of antibody bound to the recovered complex is detected and, thereby, the presence of desialylated glycoprotein in the biological fluid is determined.
• the invention provdies a method for determining the presence of a soluble desialylated glycoprotein in a biological fluid which comprises subtantially concurrently contacting a sample of the biological fluid under appropriate conditions with both a suitable amount of an appropriate lectin capable of selectively binding to the desialylated glycoprotein to produce a recoverable complex and at least one detectable antibody directed to an antigenic determinant on the desialylated glycoprotein capable of selectively binding to the glycoprotein as well as to the recoverable complex.
• the resulting complex is separately recovered from the biological fluid.
• the presence of antibody bound to the recovered complex is detected and, thereby, the presence of desialylated glycoprotein in the biological fluid is determined.
• a method for determining the presence of soluble desialylated glycoprotein in a biological fluid comprises contacting a sample of the biological fluid under appropriate conditions with both an antibody directed to an antigenic determinant on the desialylated glycoprotein to produce a recoverable complex and a suitable amount of an appropriate detectable lectin capable of selectively binding to the desialylated glycoprotein as well as to the recoverable complex.
• the resulting complex is separately recovered from the biological fluid.
• the presence of lectin bound to the recovered complex is detected and, thereby, the presence of desialylated glycoprotein in the biological fluid is determined.
• HCG HCG
• CR121 biopotency, 13,450 I ⁇ /mg Second International Standard
• hCG ⁇ CR123
• ⁇ -CTP hCG 123145
• ashCG ⁇ asS-CTP
• ashCG ⁇ ashCG ⁇ 123-145
• HLH HCG-I-1 AFP-4345B
• biopotency 6000 IU/mg WHO International Standard of urinary FSH/LH70
• Antisera to ⁇ -CTP R525 and R529) and as B-CTP (R141) were characterized previously (8,16).
• Monoclonal antibody B101 has an equilibrium association constant (Ka) of 7 x 108 M-1 for hCG, and cross-reactivities of 9 and 2% for hCG ⁇ and hLH, respectively.
• Ka equilibrium association constant
• the Ka of B105 for hCG is 1.5 x 1011 M- 1 and it cross-reacts 100% with both hCG ⁇ and hLH.
• the Ka of B107 for hCG is 4 x 10 10 M -1 and it has only 0.1% cross-reactivity with hCG ⁇ and less than 0.5% cross-reactivity with hLH.
• Monoclonal antibody, B101 was conjugated to CNBr activated Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, NJ) according to the manufacturer's instructions.
• Iodinated ashCG, hCG and antibodies were prepared using Na 125 -I (Amersham Corp., Arlington Heights, IL) with Iodogen (Pierce Chemcial Co., Rockford, IL) as an oxidizing agent as described by Fraker and Speck (18).
• Peanut lectin, Arachis hypogea agglutinin, (PNA) and castor bean lectin, Ricinus communis agglutinin, (RCA) were covalently linked to Agarose were obtained from E-Y Laboratories Inc. (San Hateo, Ca.).
• RIAs of urine specimens processed by gel filtration were performed using antisera R529 (14) and R141 (8) to determine total hCG, ⁇ CTP and ashCG CTP immunoreactivities, respectively, as described by Amr et al. (6).
• An extration step was added to the RIA using R141 as antiserum (R141 RIA) in order to improve its sensitivity and to apply it to the measurement of ashCG in urine specimens without gel filtration.
• R141 RIA antiserum
• LIRMA Lectin-immunoradiometric assays
• the conditions for the LIRMA's were optimized as follows: The amounts of PNA-Agarose and RCA-Agarose and time of incubation at room temperature required to give maximum adsorption of ashCG from buffer A were determined. The amount of iodinated antibody added to the assay was chosen by determining the amount of tracer which gave the highest ratio of specific counts bound over non-specific binding (NSB: binding of tracer in absence of ashCG). Time studies of radiolabeled antibody binding indicated that equilibrium was not attained until 96 hours of incubation at 4°C However, since an incubation period of 48 to 72 hours was sufficient to obtain high assay sensitivity, it was the chosen time of incubation.
• urine samples Prior to assay, urine samples were adjusted to pH 7.4 with NaOH and centrifuged at 3,000 xg for 15 min. Duplicate or triplicate 4 ml aliquots of urine (standards containing appropriate concentration of ashCG in buffer A or buffer A alone for determination of NSB) were pipetted into 12 x 75mm polystyrene tubes. Two-hundred microliters of 10% suspension of PNAAgarose or RCA-Agarose in buffer A were pipetted into each tube. The tubes were capped, placed horizontally on a Labquake Shaker and incubated for 2 hours at room temperature in order to extract ashCG from the samples. The tubes were centrifuged for 30 min at 3,000 xg.
• the supernatants were removed by aspi ration and the pellets were washed with 2 ml of wash buffer (buffer B: buffer A containing 1% Tween 20).
• buffer B buffer A containing 1% Tween 20
• One hundred microliters buffer A containing approximately 50,000 CPM tracer ( 125 I-R525, 125 I-B105 or 125 I-B107) were added to each tube.
• the samples were incubated for 48-72 hours at 4°C with shaking on a Bellco Shaker.
• the tubes were then washed two times with 2 ml of buffer B to reduce NSB.
• the radioactivity remaining after washing was determined in a Packard Auto-Gamma Scintillation Spectrometer. Data reduction for the generation of standard curves and ashCG concentrations of urine specimens was accomplished using a four parameter logistic fit (19).
• the IRMA of hCG was conducted using BlOl-Sepharose 4B to extract hCG and radiolabeled purified antiserum to ⁇ -CTP (R525) to measure urinary hCG as described by Armstrong et al. (20).
• Typical standard curves generated for the RIA with and without an extraction step are shown in Figure 1.
• the ashCG dosages which resulted in binding equivalent to 90% (ED 90 ) and 10% (ED 10 ) of Bo (binding in the absence of ashCG) were used as the limits of the usable range for the standard curves.
• the ED 90 and ED 10 for the direct method, which does not employ an extraction step corresponded to 0.8 and 20 pmoles ashCG/ml respectively.
• the ED 90 and ED 10 of the RIA utilizing an extraction step were 0.05 and 1.0 pmoles ashCG/ml, respectively.
• the use of an extraction step resulted in approximately a 16 fold improvement in assay sensitivity.
• the crossreactivities of hCG in the assays with and without extraction step were: 0.69 and 0.15% at ED 50 , 7.20 and 6.15% at ED 90 and 0.17 and 0.03% at ED 10 , respectively.
• Figure 1 also shows the dose response curve of ashCG extracted from a pool of normal male urine for which portions had been augmented with dosages identical to those employed in the standard curve.
• ED 50 of the dose response curve in the extraction method were -1.401 and 0.250 pmoles/ml for ashCG in urine, compared to -1.318 and 0.238 pmoles/ml for ashCG in buffer. These differences were not statistically significant.
• Fig. 2 A, B, C, D Typi cal standard curves for ashCG binding in the LIRMA ' s using PNA and RCA as extraction reagents and R525 , B105 or B107 as antibodies are shown in Fig. 2 A, B, C, D.
• the ashCG dosages which gave binding equivalent to 10% (ED 10 ) and 90% (ED 90 ) of the maximum binding (Bmax) are desginated as the l imits of the usable range of the standard curves.
• ED 10 and ED 90 corresponded to 0 .01 and 2.5 pmoles ashCG/ml in PNA 125 I-R525 system , 0 .0002 and 0 .05 pmoles ashCG/ml in PNA- 1 25 I-B105 system , 0.005 and 0.6 pmoles ashCG/ml in
• the insert in Fig. 2A shows the dose reponse curves of ashCG extracted wi th PNA from normal male urine whi ch had been augmented with dosages identical to those used in the standards.
• the dose response curve for ashCG in urine was essentially identical to that for ashCG in buffer A in the PNA- 125 I-R525 system.
• the slope and ED 50 were 1.141 and 0.136 pmoles/ml for ashCG in urine compared to 1 .051 and 0.122 pmoles/ml for ashCG in buffer A in PNA- 125 l-R525 system .
• Fi g shows that shows that for ashCG in urine compared to 1 .051 and 0.122 pmoles/ml for ashCG in buffer A in PNA- 125 l-R525 system .
• the PNA 125 I-R525 system can detect ashCG, ashCG ⁇ and free as3CTP
• the PNA- 125 I-B105 system can only detect ashCG and ashCG ⁇
• the PNA- 125 I-B107 system detects only dimeric ashCG .
• RCA does not recognize asi alo O-serine linked carbohydrate side chains in the ⁇ -CTP region
• RCA- 125 I-R525 system detects only ashCG and ⁇ ashCG .
• the cross-reactivi ties of hCG, hCG and hLH were low in all the assay systems . Intra- and inter assay variance for these LIRMA' s have been established util izing normal male urine containing the dosages of ashCG coinciding approximately wi th ED 50 . These are also shown in Table 1 .
• the gel f iltration profiles of the concentrated urine from a choriocarcinoma patient and a normal pregnant woman are shown in Fi g. 3.
• the el ution prof ile of the choriocarcinoma urine concentrate had a maj or peak of hCG immunoreactivi ty as determined by an RIA using an antiserum to ⁇ -CTP (R529) which coincided with the elution vol ume (Ve) of 125 I-hCG.
• a peak of ashCG whi ch eluted under the peak of hCG was detected using the R141 RIA and the PNA- 125 I-R525 and RCA- 125 I-R525 LIRMA' s .
• the proportion of ashCG to toal hCG in this fi rst peak was 17% using PNA- 125 I-R525 , 16% using RCA 125 I-R525 and 16% using the R141 RIA.
• the PNA- 125 I-R525 LIRMA, RCA- 125 I-R525 LIRMA and R141 RIA wi th an extraction step were util ized to measure ashCG in urine specimens from patients with gestational trophoblastic tumors and women during normal pregnancy. The total concentrations of hCG in these specimens were determined util izing the B101-R525 IRMA (20) .
• the means of the percentages of asi alo hCG concentrations over toal hCG concentrations obtained using the three different methods are shown in Table 2.
• the Newman-Keuls Mul tiple Range Test ( 21) was used to determine if the mean values for the three groups of subjects were signifi cantly different from each other .
• the mean percentages of asialo hCG over total hCG concentration for specimens from choriocarcinoma patients obtained using all three assays were signifi cantly different from those for hydatidiform n
• the extreme selectivity of the LIRMA for asialo forms of hCG can be attributed to the carbohydrate specificity at the lectins employed.
• Peanut lectin, (PNA) has high specificity for the terminal carbohydrate structure Gal ⁇ l ⁇ 3GalNAc (9,10); and castor bean lectin (RCA), is highly specific for the terminal structure of Gal ⁇ l ⁇ 4GlcNAc (9).
• PNA Peanut lectin
• RCA castor bean lectin
• sialic acid When sialic acid is removed from hCG, its unique ⁇ -CTP region has four structures of O-serine linked Gal ⁇ l ⁇ 3GalNAc (2), and each of its and subunits has two structures of Nasparagine linked Gal ⁇ l ⁇ 4GlcNAc (1).
• PNA and RCA recognize asialo O-serine and N-asparagine linked carbohydrate side chains, respectively.
• the LIRMAs have the abili ty to distinguish between various forms of asialo hCG based on the specificities of the lectins and antibodies employed.
• the PNA- 125 l-R525 system detects ashCG, ashCG ⁇ and f ree as ⁇ CTP, indicating simul taneous binding of the lectin and antibody to the ⁇ CTP region. This finding was unexpected considering the potential for steric hindrance of binding two large molecules to this relatively small peptide.
• the PNA- 125 I-B105 system which uses a monoclonal antibody to hCG ⁇ , detects ashCG and ashCG ⁇ . Since B107 recognizes only dimeric hCG , PNA- 1 25 I-B107 detects only ashCG .
• PNA recognizes asialo O-serine l inked carbohydrate moieties which are local ized in ⁇ -CTP region (2 ,10) and RCA recognizes N-asparagine linked carbohydrate moieties which are in ⁇ and ⁇ subuni ts (1) . This difference in demonstrated by the inabili ty of the RCA- 125 I-R525 LIRMA to detect f ree as ⁇ -CTP. It should be possible to construct similar LIRMA systems to measure a variety of soluble glycoproteins by employing lectins and antibodies with different carbohydrate and peptide binding specificiti es.
• the PNA-R525 and RCA-R525 LIRMA' s and the R141 RIA with an extraction step have been util ized in conjunction with an IRMA for hCG to determine the concentrations of asialo forms of hCG relative to those of total hCG in three different subject groups (Table 2) .
• the mean percentages of asialo hCG over hCG obtained using all three assays were significantly higher for urines from choriocarcinoma patients than those hydatidiform mole patients and normal pregnant women. Although the mean val ues for hydatidiform mole patients were also higher than those for normal pregnant women, the differences were not statistically significant. This appears to be due to the high variance wi thin subject groups. Future studies with serially collected specimens from individuals within each subject group will be conducted in order to obtain a better understanding of the utili ty of asialo hCG measurements in discriminating between gestational trophoblasti c disease and normal pregnancy.
• Kessler MJ Reddy MS, Shah RH, Bahl OP, Structures of N-glycosidic carbohydrate units of human chorionic gonadotropin. J Biol Chem 1979; 254:
• Kessler MJ Mise T, Ghai RD, Bahl OP, Structure and location of the O-glycosidic carbohydrate units of human chorionic gonadotropin. J Biol Chem 1979; 254: 7909-7914.
• Mizucchi T Nishimura R, Derappe C, Taniguchi T, Hamamoto T, Machizuki M, Kobata A, Structures of the sugar chains of human chorionic gonadotropin produced in choriocarcinoma. J Biol Chem 1983; 258: 14126-14129. 6. Amr S, Wehmann RE, Birken S, Canfield RE, Nisula BC, Characterization of a carboxtyterminal peptide fragment of the human chorionic gonadotropin betasubunit excreted in the urine of a woman with choriocarcinoma. J Clin Invest 1983; 71:329-339.

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