# US 9,518,992 B2 · Kits for the diagnosis of pre-eclampsia or eclampsia

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- Number: US 9,518,992 B2
- Document ID: US9518992B2
- Status: Granted
- Kind: B2
- Application number: 12/220,245
- Filed: 2008-07-23
- Published: 2009-05-07
- Granted: 2016-12-13
- Earliest claimed: 2002-07-19
- Assignees: Beth Israel Deaconess Medical Center, Inc.
- Applicants: S. Ananth Karumanchi; Vikas P. Sukhatme
- Inventors: S. Ananth Karumanchi; Vikas P. Sukhatme
- Web page: https://patentgrep.com/patent/US9518992B2

## CPC

- G01N 33/689: {related to pregnancy or the gonads}
- A61K 31/522: having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
- A61P 15/00: Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives
- C07K 16/1217: Neisseriaceae (F)
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C12Q 1/485: {involving kinase}
- G01N 33/6893: {related to diseases not provided for elsewhere}
- G01N 33/74: involving hormones {or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors}
- A61K 2039/505: {comprising antibodies}
- G01N 2500/00: Screening for compounds of potential therapeutic value
- G01N 2800/368: Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Disclosed herein are kits for diagnosing pre-eclampsia and eclampsia or a propensity to develop pre-eclampsia or eclampsia that include agents for the detection of levels of free placental growth factor in a subject.

## Claims

### Claim 1

A diagnostic kit for use in diagnosing pre-eclampsia or eclampsia or a propensity to develop pre-eclampsia or eclampsia in a subject, said kit comprising:
(a) a first antibody, or antigen-binding fragment thereof, which specifically binds free placental growth factor (PlGF), wherein said first antibody, or antigen-binding fragment thereof, is detectably labeled or coupled to an enzyme;
(b) a normal reference sample for PlGF, wherein said normal reference sample for PlGF is a sample containing a level of PlGF found in a pregnant subject not having pre-eclampsia or eclampsia or a propensity to develop pre-eclampsia or eclampsia; a subject not having early-onset pre-eclampsia or eclampsia; or a subject that is not pregnant, wherein the normal reference sample for PlGF comprises purified or recombinant PlGF at a concentration of 400 to 800 pg/ml;
(c) a second antibody, or antigen-binding fragment thereof, which binds soluble Flt-1 (sFlt-1), wherein said second antibody or antigen-binding fragment thereof is detectably labeled or coupled to an enzyme;
(d) a normal reference sample for sFlt-1, wherein said normal reference for sFlt-1 sample is a sample containing a level of sFlt-1 found in a pregnant subject not having pre-eclampsia or eclampsia or a propensity to develop pre-eclampsia or eclampsia; a subject not having early onset pre-eclampsia or eclampsia; or a subject that is not pregnant, and wherein said normal reference sample comprises purified or recombinant sFlt-1 at a concentration of 0.9-2 ng/ml; and
(e) instructions for use of the kit for the diagnosis of pre-eclampsia or eclampsia or the propensity to develop pre-eclampsia or eclampsia in a subject, wherein said instructions comprise a step for comparison between a subject sample and the normal reference sample of (b) and (d), wherein a subject sample with a level of free PlGF that is less than the normal reference sample of step (b) and a level of sFlt-1 that is greater than the normal reference sample of step (d) is diagnostic of pre-eclampsia or eclampsia or the propensity to develop pre-eclampsia or eclampsia.

### Claim 2

A diagnostic kit for use in diagnosing pre-eclampsia or eclampsia or a propensity to develop pre-eclampsia or eclampsia in a subject, said kit comprising:
(a) a first antibody, or antigen-binding fragment thereof, which specifically binds free placental growth factor (PlGF), wherein said first antibody or antigen-binding fragment thereof is detectably labeled or coupled to an enzyme;
(b) a positive reference sample for PlGF, wherein said positive reference sample for PlGF is a sample containing a level of PlGF found in a pregnant subject having pre-eclampsia, eclampsia, or early-onset pre-eclampsia or a propensity to develop pre-eclampsia, eclampsia, or early-onset pre-eclampsia, wherein the positive reference sample for PlGF comprises purified or recombinant PlGF at a concentration of less than 400 pg/ml;
(c) a second antibody, or antigen-binding fragment thereof, which binds soluble Flt-1 (sFlt-1), wherein said second antibody or antigen-binding fragment thereof is detectably labeled or coupled to an enzyme;
(d) a positive reference sample for sFlt-1, wherein said positive reference sample for sFlt-1 is a sample containing a level of sFlt-1 found in a pregnant subject having pre-eclampsia, eclampsia, or early-onset pre-eclampsia or a propensity to develop pre-eclampsia, eclampsia, or early-onset pre-eclampsia, and wherein said positive reference sample of comprises purified or recombinant sFlt-1 at a concentration of greater than 2 ng/ml; and
(e) instructions for use of the kit for the diagnosis of pre-eclampsia or eclampsia or the propensity to develop pre-eclampsia or eclampsia in a subject, wherein said instructions comprise a step for comparison between the subject sample and positive reference sample of (b) and (d), wherein a subject sample with a level of free PlGF that is less than or equal to the positive reference sample of step (b) and a level of sFlt-1 that is greater than or equal to the positive reference sample of step (d) is diagnostic of pre-eclampsia or eclampsia or the propensity to develop pre-eclampsia or eclampsia.

### Claim 3 (depends on 1)

The kit of claim 1, wherein said recombinant PlGF comprises the sFlt-1 or Flt-1 binding region of PlGF.

### Claim 4 (depends on 1, 2)

The kit of claim 1 or 2, wherein said antibody or antigen-binding fragment thereof, of (a) specifically binds the sFlt-1 binding domain of PlGF.

### Claim 5 (depends on 1, 2)

The kit of claim 1 or 2, wherein said antibody, or antigen-binding fragment thereof, of (a) or (c) is detectably labeled.

### Claim 6 (depends on 5)

The kit of claim 5, wherein said detectable label is a colorimetric label or a fluorescent label.

### Claim 7 (depends on 1, 2)

The kit of claim 1 or 2, wherein said antibody or antigen-binding fragment thereof of (a) or (c) is coupled to an enzyme.

### Claim 8 (depends on 7)

The kit of claim 7, wherein said enzyme is horseradish peroxidase, 3-galactosidase, or alkaline phosphatase.

### Claim 9 (depends on 1, 2)

The kit of claim 1 or 2, further comprising a second antibody, or antigen-binding fragment thereof, which specifically binds to said free PlGF.

### Claim 10 (depends on 9)

The kit of claim 9, wherein said second PlGF antibody, or antigen-binding fragment thereof, is coupled to an enzyme and the kit further comprises a preparation of a substrate for the enzyme.

### Claim 11 (depends on 10)

The kit of claim 10, wherein said enzyme is horseradish peroxidase, galactosidase, or alkaline phosphatase.

### Claim 12 (depends on 1)

The kit of claim 1, wherein said instructions comprise a step of determining the pre-eclampsia anti-angiogenic index (PAAI) of the subject and said kit further comprises a normal PAAI reference, wherein a subject sample with a PAAI that is greater than the normal PAAI reference is diagnostic of pre-eclampsia or eclampsia or the propensity to develop pre-eclampsia or eclampsia.

### Claim 13 (depends on 1)

The kit of claim 1, wherein said instructions comprise a step of determining the sFlt-1/PlGF of the subject and said kit further comprises a normal sFlt-1/PlGF reference value, wherein a subject sample with a sFlt-1/PlGF that is greater than the normal sFlt-1/PlGF reference is diagnostic of pre-eclampsia or eclampsia or the propensity to develop pre-eclampsia or eclampsia.

### Claim 14 (depends on 1, 2)

The kit of claim 1 or 2, further comprising a membrane or a solid phase wherein said antibody or antigen-binding fragment thereof of (a) or (c) or both are immobilized on said membrane or said solid phase.

### Claim 15 (depends on 1, 2)

The kit of claim 1 or 2, wherein said kit comprises components for an immunological assay, an enzymatic assay, a fluorescence polarization assay, or a colorimetric assay.

### Claim 16 (depends on 1, 2)

The kit of claim 1 or 2, wherein said free PlGF of step (a) is an alternatively spliced isoform of PlGF.

### Claim 17 (depends on 1, 2)

The kit of claim 1 or 2, wherein said sFlt-1 of step (c) is an isoform of sFlt-1.

### Claim 18 (depends on 1, 2)

The kit of claim 1 or 2, wherein said sFlt-1 of step (c) is free or total sFlt-1.

### Claim 19 (depends on 1, 2)

The kit of claim 1 or 2, wherein said sFlt-1 of step (c) is bound sFlt-1.

### Claim 20 (depends on 1)

The kit of claim 1, wherein said antibody, or antigen-binding fragment thereof, of (c) specifically binds sFlt-1.