JPH025895A - Detection of trisomy 21 syndrome of embryo - Google Patents

Abstract

PURPOSE: To increase the detection accuracy of a fetal Trisomy 21 Down's syndrome, etc., by determining whether the concentration of SOD-1 (superoxide dismutase-1) in a maternal extracellular body fluid exceeds a threshold or not.

CONSTITUTION: The concentration of SOD-1 in a maternal extracellular body fluid such as serum is measured. When a woman in the 12-20 week of gestation has a concentration of SOD-1 exceeding 113 ng/mL, the test result is judged to be positive. Consequently, the detection accuracy of Trisomy 21 Down's syndrome, etc., can be raised.

COPYRIGHT: (C)1990,JPO

Description

[Detailed Description of the Invention] [Industrial Application Field] This invention detects twins, spontaneous miscarriage, premature birth, or decreased latency in the fetus based on the concentration of SOD-1 (peroxide molecule displacing coenzyme) in the mother's body fluid. The present invention relates to a method for detecting abnormalities such as syndrome (Trimiso 21). In addition, this invention
The present invention relates to a method for predicting latent Down's syndrome in a fetus based on the concentration of a plurality of substances such as the following in addition to the concentration of D-1.

11 SOD-1, hemoglobin2) SOD-1, human chorionic gonadotropin (hCG), unconjugated estriol and alpha-fetoprotein (AFP)
) 3) SOD-1, hemoglobin, hCG, unconjugated estriol, and one or more of AFP. Factors such as length are also useful.

[Conventional technology] Trisomy 21 (i.e. one extra 21st chromosome)
accounts for 95% of all cases of Down syndrome. This disease is one of the most common genetic defects,
It occurs in approximately 1 in every 800 live births. Patients with this syndrome have a unique facial appearance, mental retardation, and at least 30% have congenital heart disease.

In trisomi 21, homologous chromosomes do not separate in anaphase,
It is formed when non-disjunction occurs resulting in one monosomic daughter nucleus and one trisomic daughter nucleus. The frequency of nondisjunction in chromosomally normal women is related to age. For women between the ages of 20 and 30, the incidence increases linearly from about 0.3 per 1000 live births to about 2 per 1000 live births. However, after the age of 30, the incidence increases exponentially at a rate of about 30% per year. Additionally, women over the age of 35 give birth to 35% of all children with Trisomy 21 Down syndrome.

For women under the age of 20, the incidence of Trisomy 21 Down syndrome also increases with age. Therefore, a 15-year-old woman has about the same chance of having a child with Down syndrome as a woman between 30 and 35 years of age.

A woman under the age of 30 has a 1.4% risk of having a second child with Trisomy 21 Down syndrome. The risk this woman has after age 30 is the risk for women of that age plus 1%.

The nondisjunction that results in trisomy 21 Down syndrome also occurs in men. Nondisjunction in males accounts for 20% to 25% of children with this syndrome. For fathers aged 55 and older, the risk of having a child with Dashin syndrome is about twice the risk expected when adjusting for the mother's age.

Genetic defects in the fetus (including! ~ Lisomi 21) are
It can be detected through amniotic fluid diagnosis before birth. In amniotic fluid diagnosis, 10-20 amniotic fluid is removed from the amniotic cavity between the 15th day and 16th week of pregnancy. The amniotic fluid cells are then cultured in tissue culture for about two weeks.

The cells are then stained and genetic abnormalities, such as trisomy 21, identified by counting the chromosomes.

However, cell I8 cultivation is a skilled, labor-intensive, time-consuming process and is therefore expensive, often costing as much as $1200. Furthermore, the culture is easily destroyed if foreign matter enters the culture. For this reason, screening for genetic markers (proteins indicative of the presence of defects in the fetus) contained in the amniotic fluid is often performed before culturing the It cysts. If the screening results are positive, cell culture can be performed to confirm the presence of the defect. If the screening results are negative, expensive cultures can be postponed. Now 1],
Most pregnant women have amniotic fluid removed to detect the genetic marker trait furfaff 11-protein ("AFP").
) screening has become commonplace.

High levels of AFP are associated with open neural tube defects such as anencephaly and bifid vertebrae.
ube defects) or other fetal abnormalities. However, the method of monitoring only AFP lacks reliability as a screening test (preliminary test method) for trisomy 21.

Current screening tests for trisomi 21 are inadequate. Furthermore, accurate cell culture methods are expensive, so patients often postpone this test. Considering the severity of trisomi 21, it is desirable to develop a marker trait (and a screening test based on this marker trait) that more accurately indicates the presence of trisomi 21.

This is especially desirable for women who belong to high-risk groups.

By 1981, it was recognized that patients with trisomy 21 have increased concentrations of SOD-1 in their red blood cell lysates. Regarding this, B.C. Del Villano and J.A. Tischfield (J.A., Ti5chfield) have described “Immunoassay Methods
), 366-67 (1981), ``Quantification of human cuprozinc SOD-1 by radioimmunoassay and its usefulness (Quantitat
ion or HumanCuprozinc SOD
-1bylladioimmunoassay an
d ItsPossible 51gn1ficanc
e inlet 1 sea) is described in J. However, this paper does not propose detecting SOD-1 in maternal body fluids as a screening test for fetal trisomy 21. The authors only state that this discovery could shed light on the role of SOD-1 in normal metabolism, or the consequences of having excess SOD-1. The authors also note that further experiments may be able to relate the SOD level to the degree of retardation.

A 1985 study found that SOD-1 in fibroblasts, amniotic fluid cells, and amniotic fluid was found in trisomy 21 fetuses.
It was investigated whether the concentration of Regarding this, M.N. Bay 1-man (H, ^, Batema
n), M.G.
), Abret (A), Gan+erre (H) and G.F.
Acta Poediatr Skand (J, R, Hattei)
5cand,)”, 74:697-700 (
1985) [Immunoreactivity SOD-1 of amniotic fluid, amniotic llI cysts and fibroblasts of fetus 21].
(Immunorcactive SO Low 1in A
II+n1ojic Fluid, Aa+n1otic
cells and Fibroblasts fro
m Triso+Iv21 Fetus) J. He concludes that it is impossible to establish a relationship between SOD-Lubel in amniotic fluid and 1-Lisomi21 in the fetus (see 699). Therefore,
If a correlation is found between high SOD-Lubel levels in the body fluids of Lu parents and Trit21 in the fetus, the conventional conclusions will be completely overturned.

In addition, in the conventional technology, SO in the body fluid of ffl parents
There is no suggestion that detecting D-1 or hemoglobin levels could be used as a screening test for Trit-21. Generally speaking, in the conventional technology, 4.
There is no suggestion that detecting the concentrations of SOD1, hemoglobin, and one or more of 110G1 unconjugated estriol and AFP in the mother's body fluids could be used as a screening test for Trit-21. .

To date, no screening tests have been developed for abnormalities such as twins, spontaneous abortion, or premature birth.

If such a test were developed, it would be convenient for parents and doctors to detect all of the above abnormalities.

[Summary of the Invention 1 This invention is directed to the use of female bodily fluids in J3 S01)1.
A screening test to detect a high possibility of twins and/or spontaneous miscarriage or premature birth by detecting an increase in i1m degree, or to detect fetal trisomy 21 Down syndrome by detecting SOD concentration in extracellular body fluids. Concerning screening tests to identify children.

The invention also relates to a method for detecting trisomy 21 Down syndrome in a fetus based on increased concentrations of SOD-11 or both SOD-1 and hemoglobin in the serum of a pregnant woman. The invention further provides an increase in the concentration of SOD-1, or both SOD-1 and hemoglobin in the pregnant woman's serum, and one or more of the following: an increase in hcGf1 degree, an increase in unconjugated The present invention relates to a method for detecting trisomy 21 Down syndrome in a fetus based on one or more of a decrease in triol levels and a decrease in A F P levels.

To screen for SOD-1, or SOD-1 and hemoglobin, an assay is first performed on a body fluid sample known to be positive or negative for the fetal conditions described above. Next, the minimum threshold concentration is arbitrarily set. The minimum threshold, I If, is the concentration above which most positive samples have values and below which most negative samples have values, but this concentration can be adjusted. can.

Although the number of samples that are erroneously determined to be positive can be reduced by increasing the value of the minimum threshold concentration, in this case, the number of samples that are erroneously determined to be negative increases. Conversely, if the value of the minimum threshold concentration is lowered, the number of samples that are erroneously determined to be negative decreases, but the number of samples that are erroneously determined to be positive increases.

Hemoglobin and/or SOD-1 assays are then performed on the unknown sample and the indicative
ator) is greater than or equal to a threshold value.

High levels indicate a positive test.

Screening test 1- can thus be performed relatively quickly and easily. If you are investigating a fetus for Down syndrome and the test results indicate that SOD-1 levels (or SOD-1 and hemoglobin levels) are above the threshold, then check the results. Karyotype analysis (karVOtVI) in (1) can be performed to confirm.

Essentially the same techniques can be used to detect hCG levels, unconjugated Varnish 1 to Liol levels, or AFP levels. If hCG levels are high, or unconjugated estriol levels or A
If the FP level is low, it must be assumed that either the hemoglobin level or the SOD-Level, or both, are increased. If some or all of these indicators together show a positive result, the detection accuracy of fetal Trit21 is higher than when monitoring hemoglobin and/or SOD-1 alone. It would be nice.

Also,! 11 It is desirable to monitor parameters such as the age of the female and the length of the fetus's femur.

These signs of fetal trisomy 21 Down syndrome also increase the accuracy of trisomy 21 detection.

A screening test for Trit 21 could be performed relatively early in pregnancy, perhaps around 9 weeks 1. SOD-1 (and hemoglobin) levels are high enough by this time that trisomy 2
1 serves as a reliable indicator for detection. hCG
If this were added to the determining factors, it would be possible to advance the screening test to the 8th week. It is believed that most mothers carrying a Trisomy 21 fetus will have sufficiently high hCG levels by the 8th week.

By detecting an increase in SOD1m in the body fluids of pregnant women, this invention can be used to screen for people with mental illness, rheumatoid arthritis, or a predisposition to high alcohol consumption. Alzheimer's disease and S, which are known to be related
It relates to numbering in relation to OD-rubell.

[Example] General procedure The present invention provides a screening test for detecting an increased likelihood of twins and/or spontaneous miscarriage or premature birth by detecting an increased concentration of SOD in the body fluids of a pregnant woman. The present invention relates to screening tests for trisomy 21 Down syndrome in fetuses by detecting the SOD-1 concentration contained in the extracellular body fluid of pregnant women. The invention also relates to detecting fetal 1-lisomi21 Down syndrome from increased concentrations of SOD-1 or both SOD-1 and hemoglobin in the serum of a pregnant woman. This invention further provides SOD-1 or SOD in the serum of pregnant women.
-1 and hemoglobin concentrations, and hcG1
Relating to a method for detecting that a fetus is likely to have Trisomy 21 Down syndrome from one or more of the following: an increase in 1i1 degree, a decrease in unconjugated L-striol concentration, and a decrease in A F P il'J degree. . Parameters such as the pregnant woman's age and the fetal femoral attachment are also useful in detecting Down syndrome.

The concentration is determined to be higher or lower than normal when it exceeds or falls below an arbitrarily selected threshold value, respectively. The threshold level is set at a concentration such that most positive samples will be positive and most negative samples will be negative. However, this threshold concentration can be adjusted. By changing the threshold concentration, the number of samples that are later determined to be positive can be reduced, but in this case, the number of samples that are erroneously determined to be negative increases.

It is also possible to change the threshold concentration so that the number of samples that are erroneously determined to be negative is reduced, but in this case, the number of samples that are erroneously determined to be positive increases. The particular threshold selected will vary depending on the type of sample (ie, serum or amniotic fluid) on which the array is performed and the gestational age of the woman from whom the sample was taken.

Rather than setting a threshold concentration, SOD-i, hemoglobin, and hCG1 contained in the body fluids of pregnant women of a certain age
It is also possible to analyze the probability that a fetus has Down's syndrome for a particular strength of unconjugated Nistria and AFP. This analysis is performed by multistage statistical analysis.

Finally, this analysis shows that SO for a specific maternal age
Fetal 1 to Lysomi 2 for specific intensities of D-1, hemoglobin, hCG, unconjugated estriol, and AFP.
A 1 down is generated. Details of this procedure are described below, and Cuckle et al.'s ``Method for assessing a woman's risk of having a Down syndrome-related pregnancy using age and serum alpha-fetoprotein levels'' (Es.
timing a Wasan”s R15k o
rllloving a pragrance A
s5ociated w eye h rown's synd
rome Usino her Aqe and Se
rumAIpl+a-Fctoprotein Lev
el) ”, British The 1?-Naru of.
Obstetrics and China 110ji (Briti
sh Journal of 0bstetricsa
nd cynaeco+ogy,), Volume 94,
387-402 (May 1987) and Wald et al.'s Screening of Pregnant Serum for Down Syndrome in Early Pregnancy (Maternal
Serum Screening for Down
'sSyndrome in Early Prcgna
ncy”, British Medical Journal
), No. 297, 883-887 (October 8, 1988) (hereinafter referred to as "Wald ■").

Quantification of SOD-1, hemoglobin, hCG, unconjugated estriol, and AFP can be performed using any of the standard assay methods, either by monoclonal antibody assays or by voloxol antibody assays. can. For example, enzyme linked immunoassay (II)
y) (“ELISA”) and radioimmunoassay (“
RIA”) or luminescence assays can be used. All of these assays are of single or tandem (TM) antibody type and can be performed in solid or liquid phase. For a general description of luminescence assays, see , Methods in Enzymology
y) Volume 74 of Part C for solid phase RIA and solid phase ELIS
For a general description of A, see T.E.

BtltlOr) Journal φ of Immunological φ Methods (Journal or I++v+uno
Logical Methods) Volume 83, 28
3-299 (1985), and for a general description of tandem (TM) type assays, see U.S. Pat.
See No. 376, 110.

Polyclonal antibodies are best defined as different antibodies that bind to many different epitopes. Polyclonal antibodies are produced by immunizing a large number of host animals with a particular immunogen and then collecting serum from each. Serum contains a variety of antigens, ie, antibodies to all antigens to which the animal has been exposed in addition to antibodies to the immunogen under test.

Sera collected from animals that produce polyclonal antibodies with the greatest specificity and affinity for the immunogen are
selected for use in the assay. This selection may be used, for example, in RIA, immunofluorescence assay, Western Blot analysis (14 Western Blot analysis),
analysis), ELISA, or histochemical staining.

Unlike polyclonal antibodies, monoclonal antibodies bind to one specific bitope. Monoclonal antibodies are all produced by hybridoma cells clonally generated from a single fused cell. All clones are identical to the parent, so hybridomas of the same clone produce identical antibodies that bind to the same epitope.

The method for producing monoclonal antibodies was developed by Kohler
It was first reported by Hler and Milstein. Milstein et al., 256 Nature, 495-97 (1975)
, and Köhler et al., 6. Euro Tea Imzul (
Eur J.

1Iaunol), 511-19 (1976). - In general, the host animal (usually a mouse) is immunized and then killed. Next Ba1
lllll is usually removed from the lung or other lymphoid tissue. The extracted BtIall is fused with bone wan to form a hybridoma. Hybridomas that produce antibodies against the immunizing antigen are isolated from other hybridomas and cells. The selected hybridomas are then used to produce the desired monoclonal antibodies.

Monoclonal antibodies have several advantages over polyclonal antibodies. The main advantage is that all monoclonal antibodies bind to specific epitopes and are therefore more specific. However, it must be noted that polyclonal antibodies often perform as well as monoclonal antibodies in assays. Another advantage of monoclonal antibodies is that monoclonal antibodies can be easily produced. Because hybridomas are fused with bone VI cells, they are essentially immortal if properly preserved and can regenerate almost endlessly.

Monoclonal antibodies can be produced by a number of conventional methods. - Phos inside the shell] - After immunizing an animal (usually a mouse), boss 1~
The animal is killed and B cells removed from the lungs are fused with bone marrow cells. Fusion is performed using a fusion agent such as polyethylene glycol 4000. The hybridomas generated by the fusion are then resuspended and transferred to a 96-well plate for growth.

Many variations are possible to the immunization, fusion, and screening steps and methods of antibody production described above. First, host animals other than mice can be used.

In addition, blood and tissues can be extracted and placed in a test tube (in vitro).
Human hybridomas can also be formed by immunization with o).

Also, reagents other than polyethylene glycol 4000 can be used for fusion. Furthermore, instead of chemical fusion as described in this invention, electrical fusion can be performed. This technique is well established.

Instead of fusion, e.g. Epstein-Barr virus (t
:pstein Barrv+rus) can also be used to transform B cells to immortalize Bm follicles. (For information on how to convert B cells, please refer to R.H. Kennet 1-(
cnnett, R, lI,) Other line ``Monoclonal Antibodies'' (Plcnun+ Press
), N.Y., 1980. Buoy inside PP19-33)
R Zurawski, V, IL
'Continuous propagation of human somatic cell lines that synthesize antibodies with predetermined specificity' by et al.
lyProrifcrating lluman Ce
1l Lines Synthesizing Anti
body or Predetermined 5pe
In this invention, hybridomas that produced monoclonal antibodies against SOD-1 were isolated by a series of screening procedures. , remove the supernatant from each cell containing a large number of clones.
1 to each well and measuring the combined suspension. Isolation can be performed using a variety of techniques, such as ELISA, immunofluorescence assay, Western blot analysis, or immunohistochemical staining. The only requirement is that this procedure selects hybridomas that secrete monoclonal antibodies with specificity for SOD1.

Selected cells are then limited diluted and grown prior to RIA.
The procedure was characterized for specificity, sensitivity, and affinity.

A rapid and sensitive enzyme immunoassay for Cl/Zn peroxide molecular displacement coenzyme using polyclonal and monoclonal antibodies
dSensitive [:nzyme Immuno
assay for Cu/Zn5uperoxida
Dismutase with Polyclona
l andH, onoclonal Antibody
es) ” (Clinica Chemiriki Acta (C1in
ica C1inica Acta), Volume 171, 1
-10) describes assays for SOD-1 based on monoclonal and polyclonal antibodies. These methods can also be used for SOD-1 detection according to the present invention.

Polyclonal and monoclonal antibodies to hemoglobin that can be used to detect fetal trisomy 21 can also be produced using essentially the same techniques as described above.

Regarding monoclonal antibodies against hCG, please refer to M.
H. Il. Boaart et al.
Abnormal serum chorionic gobdotropin levels of pregnant women in pregnancies in which the fetus has chromosomal abnormalities (^bnorma1Mat
ernal 5erua+ Chorionic Go
nadotropin Levelsin Preg
nancies with Fetal Chr
omosome Abn Orllality S” (Prenatal Di Acnosis)
agrosis), 623-630 < 1987
) ) is described. The monoclonal antibodies used by Bogart et al. consist of two types of wood. That is, one that detects alpha LI CG and one that detects complete (
It detects both intact tlCG and free beta-HCG. When the latter antibody was used to detect elevated hCG levels (MOM), the correlation with trisomy 21 fetuses was greater than with anti-alpha l-1CG antibodies (using 2.5M OM as a threshold). . From these results, free Beta I
Antibodies to cG and complete hCG are actually directed against complete hCG.
It is thought to bind to the beta epitope on CG. The alpha epitope on intact hCG does not seem to contribute to the Nagika reaction, and this
This is probably the reason why CG cannot detect trisomi 21 with high reliability.

Unconjugated estriol was tested as a prenatal screening test for pregnant women's serum by Wald et al.
5eru+++ Unconjugated 0est
riol as an^ntenital Scree
ning Te5t for Down's Syndr
ome) ''' (British Journal of Obstetrics and Gibecology).

334-41 (April 1988), hereinafter "Wald I
The assay can be carried out by the method described in Section II). That is, a direct non-extractive radioimmunoassay (Amerlex oestriol-RIA kit (Amerlex oestriol R1-A)
kit), Amersham). AFP b of pregnant woman serum,
The technique described in Wald ■, i.e., an immunoradiometric assay (Boots-Celtic Diagnostics Ltd.)
7 tests can be performed by I techOiaQnO3tiC3ttd,). Wald ■ represents the levels of AFP and unconjugated estriol in multiples of median (MOM).

After the desired monoclonal and polyclonal antibodies have been isolated, the final step is to use them in an assay against the antigen under test. RIA was used to detect both concentrations of 500-1 and globin. Bogart et al. used RIA to detect hCG levels. As mentioned above, assays for unconjugated nistriol and AFP are well known.

RIA is a so-called “competitive (COlllpeLitiVO)” assay,
Labeled and unlabeled antigens are added to the antibody-containing supernatant and the compounds are precipitated. Unlabeled antigen displaces some of the labeled antigen from the antibody binding region. The degree of substitution is determined by measuring the m of bound labeled antigen or free labeled antigen.

a! Concentrations of labeled antigen vs. A standard curve is obtained. When an unlabeled antigen sample of unknown m + i is added to a system of bound labeled antigen and the bound (or displaced) labeled antigen is measured, the concentration of the system can be determined by using a standard curve. . It must be emphasized here that SOD-1 or hemoglobin concentrations cannot be determined by polyclonal (or monoclonal) antibodies using competitive assays or any of the other assays mentioned above other than RIA. The point is that it can be done.

Below, examples of methods for producing antibodies and assay methods of the present invention will be described in detail.

Production and selection of monoclonal piles Telegram 1: In order to investigate immunity,
8th edition B/C mice were selected. SOD-1 was diluted with normal saline (00 g%) and emulsified in Freund's complete adjuvant by injecting and removing the syringe. The final solution contains 100-200 μ9 SOD-1 per 100 μg. 100 μg of the solution was subcutaneously injected into different parts of each mouse. The same concentration and dose of SOD-1 was administered as a potency enhancer 4-6 weeks later. However, SOD-1 at this time was emulsified within 70% of India.

'SOD-1 (mixed in normal saline) in the same amount and at the same concentration 4-6 weeks after administering the potency enhancer.
was injected into the lungs for reimmunization.

On day 4 19, mice were sacrificed and their lungs removed for fusion.

(2) Form of pibridoma due to fusion: Single cell suspension of pneumocytes from the lungs (single cell suspension)
) was prepared. Lung tissue was grown in DMEM, i.e., Dulbecco's modification of Eagle's basal medium (D
ulbeccos hodifica) and centrifuge at 1000 g for 10 minutes or 1 hour.

Sp210 bone marrow 1 maintained in culture medium with supernatant removed.
Ifi cells were added to the cell pellet and resuspended.

The resulting suspension was centrifuged at 1000 rpm for 10 minutes and the supernatant was removed. Next, 1-polyethylene glycol 4000 was slowly added over 10 minutes along with 20d DMEM.

The fused cells were centrifuged and the supernatant was removed.

I cells were resuspended in 100 d of DMEM and 100 μm aliquots were added to each of 96 wells of the Linpro plate. Ten plates were prepared in this way. Fusion products were grown in plates for two weeks. See below for a description of the hybridoma production procedure. K Flurkey, M.B.
Bolger (H, B, Bolger) and Day Nis Linsinkum (D, S, Lint, former ncura)
J, Neuroimmune
115-127 (1985) [Formation of antiserum and monoclonal antibodies against serotonin and dopamine ligands and their properties (
Preparation andCharacteri
Sticks or Antisera and Hon
oclona 8 tibodi s to Ser
otonergic and dopaiiner
gicLigands).

(3) 1. Screening process: The first screening process is to select clones that produce SOD-1 antibodies from the wells.

100 μg of hybridoma supernatant (some of which should contain S Q L')-1 antibody) was transferred to a 96-well polyvinyl chloride plate. Play 1~ was pre-treated with A7G anti-mouse lQG (goat an G) at a concentration of 0.05 gig/ae.
ti 1otlse IQG) (This is Hiles Laboratories)
(can be purchased from ). The plates were then incubated at 37°C for 2 hours. After washing the plates, 125 ml of labeled SOD-1 (labeled as described in Villano and Tischfield, supra, 363) was added. Label SOD-1 is 1
00 μρ J3 about 5000 force und/min (CPM
) until it becomes Hou M salt! 2! solution, 0.1% [diluted in 3SA.

After incubation at 37° C. for 2 hours, the plate was washed twice, and the wells were cut with a Hola 1-nichrome wire, transferred to a plastic dupe, and subjected to gamma spectroscopy.

The cells corresponding to the wells that showed the largest CPM and thus produced the most antibodies were then limited diluted and screened again to ensure that only one clone was present. The cells were then grown in larger plates.

After growth, 1.000.000-3.000.000 cells were injected into the peritoneal cavity of mice that had been given Bristane for 1 week. After 8 to 10 days, fluid was removed from the peritoneal cavity and ascites was collected. 0.1% for p118.2-8.4
By diluting with a borate solution of BSA,
Ascitic fluids of various titres were made. SOD-
Titers with 30-50% binding of 1 (as determined by the RIA method described below) were selected for further evaluation by the RIA screening procedure.

(4) Second screening process (RIA screening process: The RIA screening procedure is performed to characterize the specificity, sensitivity and affinity of antibodies for 800-1.
of ascitic fluid to 100 μg of SOD-1 with various concentrations of 11
and i-labeled SOD-1 exhibiting 25.0 OOCP M of 100 μm were added to a polyethylene tube and incubated at room temperature for 4 hours. Goat anti-mouse IqG diluted 1:1 is added to Polynibrene Gelicol 6000, the tube is shaken, and then incubated for 15 minutes. The tube is 250
It lasted 15 minutes at 0g. The supernatant was removed and the pellets were evaluated for radioactivity.

This second screening process allows 30 nO/- of unlabeled SOD-1 to infiltrate for a reasonable period of time (4 hours is considered optimal) to remove enough MAall-labeled SOD-1 from the antibody binding site. This is to determine whether or not it has been replaced.

In other words, this process is for selecting monoclonal antibodies that have greater affinity for unlabeled antigen than for labeled antigen. To do this, the final CPM in the pellet is compared to the count in a reference pellet. Reference Berrett used goat anti-mouse IaG, antibodies and ! as described above. It is made by precipitating labeled SOD-1, but F! The difference is that there is no unrecognized SOD-1. Antibody samples that showed a much lower CPM than the reference one (thus binding much more unlabeled SOD-1) were selected for use in SOD-Level measurements in amniotic fluid. For a description of the RIA screening process, see K.F. Miller.
, D.G. Bolt (D, J, BOIt),
and R.A. Goldsby
by) is 59. ``The Imzuru Methods (J,
Immunol, Methods)”, 277-
280 (1983), ``Rapid liquid phase screening method for hybridoma culture supernatant using radioactively labeled antigen and immunoadsorbent'' (A Rapi)
d5solution-Phase Screeni
ng yechntquefor tlvbrido
la Culture 5uDernatantS
1 ain.

11diolabeled Antigen a
nd a 5 solid-PhaseIn+munoa
dsorbant) J.

As a result, a monoclonal antibody of any sensitivity was not obtained. Nevertheless, the monoclonal antibodies obtained were found to be present in the amniotic fluid or in the mother's body fluids.
- It must be emphasized that it performs the function of measuring the level accurately.

However, this function is demonstrated when a competitive test such as ELISA is performed. However, unlabeled SOD-i
A polyclonal antibody having high affinity for was isolated by the method described below. These antibodies were subjected to SOD-1 testing.

Since these tests have been performed, further experiments have led to the isolation of high affinity monoclonal antibodies that can perform in competitive tests. These monoclonal antibodies were essentially isolated by methods similar to those described above. These monoclonal antibodies are stored in an American-style culture collection (Culture Collection).
Co11ection) was placed on top of the sediment (
Access N (IIIB-9937).

Production and Selection of Polyclonal Antibodies (1) Twenty-eight BALB/C mice are immunized by the same procedure described for the monoclonals. The only difference is that a final intrapulmonary injection is not performed.

10-12 days after administering the potency enhancer, blood was collected from each mouse and centrifuged to collect serum. This serum containing polyclonal antibodies is then screened by essentially the same RIA screening process described above.

(2) RIA Screening: Serum collected from each mouse was diluted with 9118.2-8.4.0.1% BSA borate buffer to produce several titers. Next, a titer with 30-50% binding of SOD-1 (RIA
(measured by the method) was selected. Sera with a titer of 1:100O were selected from 2 of 8 mice.

These titers are 30 ng/le of unlabeled SO
It is checked to determine whether D-1 has sufficiently displaced labeled 800-1 from the antibody binding site within 4 hours. The results are satisfactory and it appears that these titers can be used to measure SOD-1 concentrations in amniotic fluid.

Since more serum is required for poly[1-flu] than for monoclonals, animals larger than mice are required to produce polyclonals of m for commercial use. The polyclonal immunization and selection procedures described above can be performed in a similar manner in larger animals such as goats.

An example of the use of polyclonals in radioimmunoassays: the determination of SOD-Level in amniotic fluid
:1000 using a polyclonal titer and RIA. RIA is performed essentially the same way as described above for RIA screening procedures. but,
This time some known SOD-1a degrees are 1:1000
Mixed with polyclonal titer and labeled SOD-1.

After adding goat anti-mouse IQG, the pellets were evaluated for radioactivity. Using samples of known SOD-in degrees, a standard curve can be constructed in 11 to be used to measure the SOD1 concentration in an unknown liquid sample. This procedure is explained in more detail below.

(1) Check procedure To ensure proper coupling, it is necessary to check the system. Labeled SOD-1 is added to the tube and the calibrated average CPM is calculated (this value is referred to below as "total").

To calculate all values, prepare two tubes to be tested. Calibrate the average CPM by averaging the CPM of both tubes and subtracting non-specific binding counts (or background counts) from the average CPM.
Calculate. Next, add SOD-1, antibody, and goat anti-mouse IqG to another tube and count the pellet's calibrated average CPM (this value is designated hereinafter as "BO"). B o / t - tal x 100 is 35% ± 3%
It should be. In the present case, it was found to be 34.8%.

The system is also checked to ensure that non-specific binding counts are within normal ranges. All assays are performed with unlabeled SOD-1 and metered with a calibrated average CPM of pellets. This value is represented by ``blank°''.

Blank/l-tal x 100 should be less than 10%. In the present case, it was found to be 6.8%.

(2) RIA Procedure Next, RIA was performed on seven standards containing known SOD-11 concentrations ranging from 30 ng/rd to 2500 nQ/ml. To calculate the calibrated average CPM of the seven pellets (denoted below as B II), B/Bo was plotted against the concentration of each sample to obtain a standard curve. This standard curve is shown in FIG.

RIA was then performed on 71 different samples from fetal amniotic fluid known to be negative for trisomy 21 and 15 samples known to be positive. All samples were diluted 1:1. Note that 60 of these 86 samples were collected from pregnant women in the 13th to 20th week of pregnancy. The gestation period of the test bacteria for the other 25 samples is unknown, but they are at least 12 weeks old.

The remaining one was collected from a pregnant woman with tFi28N. By using the standard curve, the obtained B/B
The concentration of SOD1 in amniotic fluid (nMId) is related to the O value.
It was determined. The results are shown in bar graphs in FIGS. 2 and 3.

(3) Class” Of the 71 samples known to be negative,
Only 9 (or 13%) are 314μg/d (this value of 314na/IR1 is trisomy 2 in amniotic fluid).
selected as the threshold level for indicating 1) or more SO
D-Lebel was shown. That is, 87% of samples known to be negative were below the threshold. On the other hand, of the 15 samples known to be positive, only 1 (or 7%) had an S of less than 314 nlJ/d.
OD-Lebel was shown. In other words, 93% of the 11 samples known to be positive were at or above the threshold level. The reason why the value of 314 μg/d was chosen as the threshold for detecting trisomy 21 in amniotic fluid was because of this 314 μg/d value.
This is because false positives and false negatives are least likely to occur at the value μg/Inl. From the above results, it can be seen that this □ test is suitable as a screening procedure for detecting patients requiring karyotype analysis.

The subjects' ages ranged from 20 to 42 years old.

The age of the subject is not believed to affect the reliability or threshold of the test as an indicator of trisomy 21.

Screening of pregnant women's serum for SOD-1 Mother pregnant with normal fetus 1! Serum collected from 176 pregnant women (pregnant women who were at least 12 weeks pregnant) and Trimiso 21
A similar screening procedure was performed using the RIA method on serum collected from 21 mothers carrying fetuses with . All gestations were between 15 and 18 weeks. The ages of all six mothers carrying Trimiso 21 fetuses were between 35 and 43 years old. Since the screening was performed using serum rather than amniotic fluid, the threshold had to be changed.

Of the known positive samples, 71% were 105r+o/-
Only 29% of the samples showed a SOD level of 105 no/ad! or lower, and were ``false negative'' or below this level. Of the negative samples, 85% were below 105 no/ad! ''That is, only 15% exceeded this level. 105 false positives and false negatives
Since it was the smallest at /d, this II IJi was selected as the minimum distance + fia degree. Although the threshold value is different from that for amniotic fluid, it can be seen that an increase in SOD-Level in pregnant women's serum is also a highly reliable indicator of the presence of trimiso21.

It is possible to adjust the threshold concentration according to the purpose.

The threshold concentration was first increased to 1150Md, and then 125Md.
0(+/Inl), the false positives drop to 10% and 8%, respectively. However, these two
In both cases, false negatives increase to 42%, making the test less reliable as an indicator for fetal trimiso21. Despite these results, the threshold may be adjusted somewhat to further improve the accuracy of the test.

SOD-1 and hemoglobin screening SOD-
Serum samples tested for hemoglobin concentration were also tested for hemoglobin concentration. Next, the performance of SOD-11f1 degree and hemoglobin concentration as indicators for fetal trimiso21 was investigated when both were combined and separately.

Hemoglobin concentration was measured using polyclonal antibody to R1. In this RIA, after the screening step, 0.1% BSA/PBS (0,
108) if the unlabeled hemoglobin concentration in 2.
A standard curve was created using samples of 10.50, 500.2500 μg/-.

Force fil: 200 rabbit polyclonal antibodies (
Accurate Chemical and Scientific Corporation (ACCuratf3Chemi)
cal and 5 scientific corp,)
(purchased from ) 100μm and 30,0OOCP
Twenty-five micrograms of each sample was added to a test tube containing 100 micrograms of I-labeled hemoglobin (labeled as described by Villano and Tischfield, supra, p. 363). After adding the sample,
Tubes were incubated for 2 hours at room temperature, precipitated with goat anti-rabbit 1aG and polyethylene glycol, centrifuged to remove the supernatant, and CPM was monitored. As described above for the RIA targeting SOD-1, all test wA tubes were prepared in pairs and the adjusted CPM was 61q.

The standard curve generated by this procedure was then used to determine the hemoglobin concentration in unknown pregnant serum samples by the RIA procedure described above. However, hemoglobin concentration can be measured using any assay or technique sensitive enough to quantify concentrations on the microgram scale.

After monitoring the results, 85μq for hemoglobin
/d and 113B for SOD-1.
/d threshold m degrees was chosen. Of the 176 known negative samples, only 5% were above these levels. This is an order of magnitude smaller than the false positive rate of 15% obtained when only SOD1 concentration was monitored.

When using the thresholds described above, false positives were also slightly reduced. S
When only OD-1 was monitored, 2 of the positive samples
9% were false positives, whereas hetglobin and SOD
-1, the false positive rate was slightly over 26%.

These results indicate that measuring both SOD-1 and hemoglobin concentrations in serum is a more reliable test than monitoring SOD1i1m alone.

Pregnant serum (or other body fluids) can be screened for hCG using the method described in Bogard et al., supra.

The antibody used is one that binds to both free beta hCG and intact CG as described above. minimum h
Employing a CG level of 2.5M OM (above which is considered high), this screening can improve the results of SOD-1 and/or hemoglobin screening tests. hC
G screening can be used in conjunction with a SOD-1 screening test and/or a hemoglobin screening test. In addition to hCG, SOD-1
If the concentration of SOD-1 or the concentration of both SOD-1 and hemoglobin increases, this indicates a high probability of the fetus having l.rimiso21. When hCG levels are monitored in this way, only SOD-1 or SOD-1 is detected.
This would likely reduce the number of "false positives" obtained when monitoring both OD-1 and hemoglobin.

Assays for unconjugated estriol and AFP in serum are performed using techniques described by Ningwald et al. and well known to those skilled in the art. The assay is performed on a series of serum samples, some known to be positive and some known to be negative. A table is created based on these results and those obtained from assays for hCG, SOD-1, and hemoglobin. The table shows various ``detection rates'' (positive It shows the ratio of false positive rate (proportion of healthy pregnancies with positive results) to the percentage of healthy pregnancies with a positive result.

Wald's method includes multistage analysis of variables consisting of maternal age, AFP, unconjugated estriol, and hCG. By performing a systematic analysis, a table like the one described above is finally obtained.

Table 2 shows the percentage of false positives (%) for age and the variables mentioned above (see Table 2).

To evaluate the results shown in Table 2, first determine the estimated risk of Down syndrome for each age group of pregnant women from 15 to 50 years of age. The results of eight studies were combined to obtain an estimated risk ratio for each age, and the method used by Cuckle et al. ``A method of evaluation using levels'', Applish Journal of Obstetrics and Ginaecology, Vol. 94, 387-402 (198
For iQ1, Kutskul et al. weighted the individual risk estimates from each study on a logarithmic scale. In this case, each weight is proportional to the reciprocal of the variance.In order to reduce the random error of the estimated risk at each age, Tackle et al. A regression analysis of the probability of birth with Down syndrome against age was performed using a model in which an exponential function of age was added to a constant proposed by Llook (1981).For this, see Tackle et al., p. 388. About.

To estimate the risk ratio for Down syndrome for a given age and AFP level, Kutskul et al.
multiplied by a factor known as o). This accuracy ratio is
It is the proportion of Down syndrome pregnancies for a given AFP level divided by the proportion of healthy pregnancies for the same AFP level. Kutskul et al. describe a method for estimating this ratio for cases in which the number of Down syndrome pregnancies investigated is less than 1,000. See pages 388-89.

The authors then arbitrarily assigned six different risk levels to indicate that an individual woman was carrying a fetus with Down syndrome.

Subsequently, Kutukul et al. determined AFP cutoff levels specified by the pregnant woman's age. This Kak1~Off level is
The risk ratio of Down syndrome for an individual woman with an AFP value equal to each cutoff level is 1:100,1:
150.1:200.1:300, and 1:350
It was decided that it would be one of the two. The AFP cut-off level for each risk rate is the accuracy ratio required to find a specific risk rate from the risk rate specified by age.
(For example, if the risk identified by age is 1:375, the risk rate is 1:200.
(IU ratio to 375/200). The accuracy ratio can be used to solve the equation shown in Appendix 1 (page 400) of Kutskul to determine the AFP cutoff level.

Kutskul et al. also provide a method for estimating the detection rate and false positive rate at each risk level for Down syndrome.

However, I gave the P cutoff level to A who was above.
With the V stage setting, these rates can be determined by examining the data without making any estimates.

In Wald I, in addition to monitoring the pregnant woman's age and AFP levels, unconjugated estriol is also monitored. Essentially, the method of Kutzkul et al. has been used to measure detection rate and false positive rate. However, the dispersion between long-term batch assays for unconjugated estriol (Iong-tern+between batch
(assay vartance). Furthermore, the detection rate and false positive rate for the three variables were evaluated using a fitted Gaussian distribution, rather than the 1AN4 distribution used by Kutsukuru et al. Table 1 below shows the false positive rate and detection rate when using all three variables.

Table 1 Age, AFP, and unconjugated Nistrimer 55 9, 1 50 7, 0 45 5, 3 35 2, 8 30 1.9 25 1.2 20 0, 7 Screening stage for Wald ■ 11hcG has been added. hCG screening was performed using an immunoradiometric assay (First International Calibration Model Book).
st 1 international reference
The MAIA-clone kit (manufactured by SOrOnO) was used to calibrate the MAIA-clone kit (produced by SOrOnO). The long-term batch assay variance was estimated and the commonly used standard deviation quasi-determined value was calculated in addition to the estimated measurement variance of simulated pregnant serum hCG.

In Wald ■, next hCGlAFP and non-conjugated varnish 1~
The detection rate and false positive rate are calculated using various combinations of SAR and the age of the pregnant woman. The results are shown in Table 2.

(continued on next page) Table 2 AFP U, 0. hCG 14 9.5 11 γ, 2 8.8 5.4 613.9 5.0 2.8 3.7 1.9 2.5 1.2 1.6 0.8 AFP AFP U, O,, AFP, U, 0. .

U, O, hCG hcG hcG8.8 8.
1 6.7 6.0 5.04゜4 3.7 3.2 2.7 2.3 1.916 1.3 1.0 0.8 0.7 0.5 0.4 *Pregnant woman's age and When the risk of Down syndrome estimated from the biochemical test results is high, the result is determined to be positive.

**U, O, represent non-conjugated estriol.

In this invention, the methods of Wald ■ and Wald ■ are used to determine the detection rate and false positive rate for the pregnant woman's age and one or a combination of the antigens hCG, unconjugated estriol, and AFP. I am using it. but,
In all cases, SOD1 and hemoglobin, or SOD
-1 is added as a variable.

A table of “detection rate versus “false positive rate” is created as described above. This table has 21 Jllll(colum+n) (3 times the number of Ill in Table 2).
1 or different combinations of SOD-1 and hemoglobin with one or more of the antigens hCG, unconjugated estriol, and AFP. Variable (SO
D-1, hemoglobin, hCG, unconjugated estriol, and AFP), detection rates and false positive rates in these columns are much better than those obtained in Wald ■. You should be able to get it.

As mentioned above, when only 800-1 in pregnant women's serum was used, the detection rate was 71%, and at the cutoff level of 10105n, the "false positive" rate was 15%. When monitoring both SOD-1 and hemoglobin in serum of pregnant women, using a cut-off level of 85 μ9/rd for hemoglobin and a cut-off level of 113 no/- for SOD-1, detection was achieved. There were only 5% false positives for a rate of 74%. In comparison, AFP, hCG.

When three variables, ie and unconjugated estriol, were monitored, the detection rate was 70%, but the false positive rate was 8.
It was 6%. Adding the aforementioned SOD-Level and hemoglobin levels to the multivariance analysis for the three variables monitored by Wald ■ should increase the detection rate and decrease the false positive rate. Combining these variables, we get
It should be a 100% reliable and highly effective screening test for fetal Down syndrome.

Wald ■ is also considering measuring fetal femoral length using ultrasound screening as an adjunct technique to serum screening tests.

This screening will further improve the results of the screening tests described above.

The assay was performed on amniotic fluid collected from two mothers who were pregnant with twins and had spontaneous abortions. These samples are S
OD-Lebel is 377nQ/d! and 4120g/I1
It was 1. These level values are much higher than the threshold of 314 ng/ml for Down syndrome. This means that if SOD-Level in body fluids is high, twins and/or
Or it indicates a high possibility of spontaneous miscarriage. However, elevated SOD-Level, which has been found to be associated with twins or spontaneous abortion, may also indicate fetal trimiso21, as discussed above. Since the relationship with the upper bound of SOD-Level is uncertain, it is preferable to perform karyotype analysis on mothers with high SOD-Level and R-Hiragi test for trimiso21.

SOD-1 Screening to Detect the Possibility of Preterm Birth The SOD-1 assay was also performed on serum collected from mothers who were pregnant with a fetus that was delivered prematurely.

SOD-Level in normal serum is quite high. This is because SOD-1 is contained in red blood cells, and SOD-1 is contained in red blood cells.
This is because i enters the serum from red blood cells. Therefore, SOD-Level in serum varies greatly depending on the number of red blood cells present in the sample. SOD-1 released from red blood cells
In order to correct for the effect of SOD-
An operation of dividing by the concentration (nM ae ) of 1 was performed.

Serum collected from &i parents carrying a normal fetus was 1/1. In contrast, serum collected from a mother who was pregnant with a prematurely born fetus showed a ratio of 1/2.7. This indicates that the upper limit of SOD-Level in serum is an indicator that there is a high possibility of premature birth. Once again, it is important to perform karyotype analysis on all mothers with elevated SOD-1 levels, as elevated SOD-Level may indicate trimiso21 in the fetus.

The embodiments described above are merely illustrative and do not limit the invention. Therefore, the method for detecting trisomy 21 Down syndrome according to the present invention can be implemented in any form without departing from the spirit and scope of the invention.

[Brief explanation of the drawing]

The drawings show examples of the present invention, with Fig. 1 showing the standard curve, Fig. 2 showing the measurement results of SOD-1i1 degree in the negative sample, and Fig. 3 showing the SOD-in of the positive sample.
It is a figure showing the measurement result of degree. Applicant Monoclonetakes International Inc.

Claims (1)

[Scope of Claims] (1) Measuring the SOD-1 concentration in the extracellular body fluid of a pregnant woman, and when the SOD-1 concentration exceeds a specific threshold set for women of the same gestational age. 1. A method for detecting trisomy 21 in a fetus, the method comprising the steps of: (2) A patent claim in which the extracellular body fluid is the serum of a pregnant woman, and the test result is positive when the SOD-1 concentration exceeds 113 ng/ml for a woman whose gestation period is between 12 weeks and 20 weeks. A method for detecting trisomy 21 in a fetus according to item 1. (3) The method for detecting trisomy 21 in a fetus according to claim 1, wherein the test result is positive when the extracellular body fluid is amniotic fluid and the SOD-1 concentration is greater than 314 ng/ml. (4) Measuring SOD-1 and hemoglobin concentrations in the pregnant woman's serum, and determining whether both of said concentrations exceed specific respective thresholds established for women at the same gestational age; A method for detecting trisomy 21 in a fetus having (5) S for women whose gestation period is 12 to 20 weeks
5. The method for detecting trisomy 21 in a fetus according to claim 4, wherein the OD-1 threshold is 113 ng/ml and the hemoglobin threshold is 85 μg/ml. (6) Human chorionic gonadotropin (hC), which is a determining factor
G) measuring the concentration of one or more of unconjugated estriol and alphafuetoprotein;
determining whether the G concentration is greater than the median value for women of the same age and gestational age and whether the unconjugated estriol and alfahuetoprotein concentrations are less than the median value for women of the same age and gestational age; The method for detecting fetal trisomi 21 according to claim 4, further comprising: (7) a spontaneous abortion comprising the steps of measuring the SOD-1 concentration in a pregnant woman's body fluids and determining whether the SOD-1 concentration is greater than a certain threshold for a normal fetus of the same gestational age; How to detect premature birth or the possibility of twins. (8) The method for detecting a high possibility of spontaneous abortion, premature birth, or twins according to claim 7, wherein the body fluid is amniotic fluid or serum. (9) The body fluid is amniotic fluid, the target is detection of a high possibility of spontaneous abortion or twins, and the minimum threshold concentration for SOD-1 is about 314 ng/ml. How to detect spontaneous miscarriage, premature birth, or likely twins. (10) The body fluid is serum, the detection of premature birth is targeted, and the hemoglobin concentration (μg/ml) in the serum and SOD
10. The method for detecting a high possibility of spontaneous abortion, premature birth, or twins according to claim 9, wherein the ratio of -1 concentration (ng/ml) is less than 1/1.13. (11)S
OD-1 concentration is the subject of the assay, and the assay is a homogeneous or heterogeneous, one-site or two-site immunoassay using monoclonal or polyclonal antibodies, or spontaneous abortion or premature birth according to claim 8. How to detect if you're likely having twins. (12) detecting the serum SOD-1 concentration and serum hemoglobin concentration of a pregnant woman; dividing the hemoglobin concentration by the serum SOD-1 concentration to obtain a ratio between the two; a screening method for detecting a high possibility of premature birth, comprising the step of determining whether the value is smaller than a threshold value. (13) The hemoglobin concentration is measured in μg/ml, the SOD-1 concentration is measured in ng/ml, and the threshold is 1/1.13×10^3. The screening method according to item 12. (14) hCG concentration in body fluids of pregnant women, SOD-1
measuring the hCG concentration or both the SOD-1 concentration and the hemoglobin concentration; and determining whether the hCG concentration is higher than a minimum threshold for a gestational age fetus;
determining whether the OD-1 concentration or both the SOD-1 concentration and the hemoglobin concentration are higher than a minimum threshold. (15) The body fluid is serum, and hCG is 2.5MoM
It is judged to be high if it exceeds the minimum threshold for SOD-1 and hemoglobin, which is 113 ng/ml and 8 ng/ml, respectively.
15. The method for detecting trisomy 21 in a fetus according to claim 14, wherein the concentration is 5 μg/ml. (16) Claim 1, wherein the levels of hCG, SOD-1 and hemoglobin are measured by an assay.
The method for detecting trisomy 21 in a fetus according to item 4. (17) Age is known in order to measure the variables SOD-1 or both SOD-1 and hemoglobin, along with one or more of the variables hCG, unconjugated estriol, and alpha afetoprotein. The steps involved performing the assay on a series of serum samples taken from pregnant women and determining the rate of positive Down syndrome pregnancies and positive results for a specific concentration of each variable assayed and for a specific maternal age. creating a table relating the indicated rates of normal pregnancy; and determining from said table the probability that the unknown sample is indicative of fetal trisomy 21 Down syndrome;
The assay detects the certainty of fetal trisomy 21 Down syndrome in which some of the serum samples are known to be positive for fetal Down syndrome and some are known to be negative for fetal Down syndrome. how to. (18) The pregnancy rate of Trisomy 21 Down syndrome is 20
18. The method of claim 17, wherein the specific concentration of the variable for which the assay is performed is adjusted to give a positive result in 5% increments from % to 80%. How to detect accuracy. (19) The variable on which the assay is performed is SOD-1
, hemoglobin, alpha fetoprotein, hCG,
and unconjugated estriol, the method for detecting the certainty of trisomy 21 Down syndrome in a fetus according to claim 17. (20) The variable on which the assay is performed is SOD-1
, hemoglobin, alpha afetoprotein, the method for detecting the certainty of Trisomy 21 Down syndrome in a fetus according to claim 17. (21) The variable on which the assay is performed is SOD-1
, hemoglobin, alphafuetoprotein, and hC
18. The method for detecting the certainty of Trisomy 21 Down syndrome in a fetus according to claim 17, wherein the fetus is G. (22) The variable on which the assay is performed is SOD-1
18. The method for detecting the certainty of trisomy 21 Down's syndrome in a fetus according to claim 17, which is hemoglobin, alphafuetoprotein, and unconjugated estriol. (23) The variable on which the assay is performed is SOD-1
, hemoglobin, and hCG.
7. A method for detecting trisomy 21 Down syndrome in a fetus according to item 7. (24) The variable on which the assay is performed is SOD-1
18. The method for detecting the certainty of trisomy 21 Down syndrome in a fetus according to claim 17, wherein the fetus is hemoglobin, hCG, and unconjugated estriol. (25) The variable on which the assay is performed is SOD-1
18. The method for detecting the certainty of trisomy 21 Down syndrome in a fetus according to claim 17, wherein the method is hemoglobin, hemoglobin, and unconjugated estriol. (26) The variable on which the assay is performed is SOD-1
18. The method for detecting the certainty of trisomy 21 Down syndrome in a fetus according to claim 17, which is alphafuetoprotein, hCG, and unconjugated estriol. (27) The variable on which the assay is performed is SOD-1
18. The method for detecting the certainty of trisomy 21 Down syndrome in a fetus according to claim 17, which is alphafuetoprotein. (28) The variable on which the assay is performed is SOD-1
18. The method for detecting the accuracy of trisomy 21 Down syndrome in a fetus according to claim 17, wherein the method is alpha fetoprotein, and hCG. (29) The variable on which the assay is performed is SOD-1
, alphafuetoprotein, and non-conjugated estriol.
1. How to accurately detect Down syndrome. (30) The variable on which the assay is performed is SOD-1
and hCG, the method for detecting the accuracy of trisomy 21 Down syndrome in a fetus according to claim 17. (31) The variable on which the assay is performed is SOD-1
, hCG, and unconjugated estriol, the method for detecting the certainty of trisomy 21 Down syndrome in a fetus according to claim 17. (32) The variable on which the assay is performed is SOD-1
and unconjugated estriol, the method for detecting the certainty of trisomy 21 Down syndrome in a fetus according to claim 17. (33) Determining the risk of fetal trisomy 21 Down syndrome for many different maternal ages and with one or more of the variables hCG, unconjugated estriol, and alpha-fetoprotein. performing the assay on a series of serum samples taken from pregnant women of known age to measure SOD-1 or both SOD-1 and hemoglobin, and the level of the variable for which the age and assay are performed; The stages of assessing the risk of Down syndrome for each cut-off level and the age of the pregnant woman for which variables are assayed such that the risk of Down syndrome for an individual pregnant woman at each cut-off level is a specific rate. Steps in determining a series of cut-off levels and determining the rate of Down's syndrome pregnancies with a positive result and the rate of normal pregnancies with a positive result for a given concentration of each variable assayed and a given maternal age. and a table relating the rate of Down's syndrome pregnancies with a positive result to the rate of normal pregnancies with a positive result for a particular concentration of each variable assayed and a particular maternal age. and producing a variable SOD-1, or SO with one or more of the variables hCG, unconjugated estriol, and alphafuetoprotein in the unknown pregnant serum sample.
measuring the concentration of D-1 and hemoglobin; and determining from said table the probability that the unknown sample is indicative of Trisomy 21 Down syndrome; Fetal trisomy 21 that is known to be positive for fetal Down syndrome and some are known to be negative.
How to detect the accuracy of. (34) The pregnancy rate of Trisomy 21 Down syndrome is 20
34. The method according to claim 33, wherein the specific concentration of the variable on which the assay is performed is adjusted to give a positive result in 5% increments from % to 80%. How to detect. (35) The variable on which the assay is performed is SOD-1
34. The method for detecting the accuracy of trisomy 21 in a fetus according to claim 33, wherein the method is hemoglobin, alphafuetoprotein, hCG and unconjugated estriol. (36) The variable on which the assay is performed is SOD-1
34. The method for detecting the accuracy of trisomy 21 in a fetus according to claim 33, which is alpha fetoprotein, hCG and unconjugated estriol. (37) Claim 33, further comprising the step of measuring the fetal femoral length as another variable in screening for fetal trisomy 21 Down syndrome, and determining whether the femoral length is less than or equal to the median value. A method for accurately detecting trisomy 21 in a fetus as described in Section 1.