Methods for quantifying inter-alpha inhibitor proteins

a technology of interalpha inhibitors and proteins, applied in the field of methods for quantifying interalpha inhibitor proteins, can solve the problems of limitations of competitive iaip immunoassays in the assessment of active iaip in patient samples, and achieve the effect of reducing or ameliorating disorders and/or symptoms associated with them

Inactive Publication Date: 2020-02-20
POROTHERA BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0068]As used herein, the term “treating” refers to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated ...

Problems solved by technology

Thus, the competitive IAIP immunoassay has limita...

Method used

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  • Methods for quantifying inter-alpha inhibitor proteins
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  • Methods for quantifying inter-alpha inhibitor proteins

Examples

Experimental program
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Effect test

example 1

AIP Assay

[0203]Preparation of Biotinylated Heparin

[0204]Heparin (Heparin Sodium Injection USP, Sagent Pharmaceuticals, Cat # NDC 25021-400-30) was conjugated with biotin using the Biotin Hydrazide reagent (ApExBIO, Cat # A87007) according to the manufacturer's instructions. Briefly, 1000 IU heparin solution was mixed with 0.25 mg crosslinker reagent EDC (1-(3-Dimethylaminopropyl)-3-3ethylcarbodiimide hydrochloride, Alfa Aesar Cat # A10807) and 0.5 mM Biotin hydrazide that had been previously dissolved in DMSO in 0.1 M MES buffer pH 4.7 with gentle mixing at room temperature for 3 hrs. The unconjugated biotin and buffer exchange was carried out by ultrafiltration on an Amicon Ultra centrifugal filter device with 5 kDa cut off filter membrane (Millipore). Following dilution in d-H2O, the biotinylated Heparin was ready for use in the assay.

[0205]“Sandwich Type” Heparin-IAIP ELISA

[0206]Purified mouse monoclonal antibody against the light chain of human IAIP (MAb 69.26) was immobilized o...

example 2

Assay

[0207]Preparation of Biotinylated Endotoxin / LPS (Lipopolysaccharide):

[0208]Lipopolysaccharide (LPS / endotoxin) from Escherichia coli 055:B5 (Sigma Catalog # L2280) was labeled with biotin using Biotin Hydrazide reagent (ApExBIO, Cat # A87007) according to the manufacturer's instructions and similarly to the protocol used for heparin. 10 mg LPS was reconstituted in 0.1 M MES buffer and 2.5 mM Biotin-Hydrazide and 2.5 mg EDC (1-(3-Dimethylaminopropyl)-3-3ethylcarbodiimide hydrochloride, Alfa Aesar Cat # A10807) were gently mixed for 3 hrs at room temperature. The removal of unconjugated LPS and buffer exchange were carried out by ultrafiltration on Amicon Ultra centrifugal filter device with a 5 kDa cut off filter membrane (Millipore). Following dilution in d-H2O, the biotinylated LPS was ready for use in the assay.

[0209]“Sandwich Type” LPS-IAIP ELISA

[0210]Similar to the heparin-IAIP protocol described above, purified mouse monoclonal antibody against the light chain of human IAIP...

example 3

of Blood Samples from Patients Diagnosed with Severe Community Acquired Pneumonia (sCAP)

[0211]Serial blood samples were collected from patients with a confirmed diagnosis of sCAP who were hospitalized in the Intensive Care Unit at Rhode Island Hospital. 16 patients were enrolled in the study and plasma was collected on days 0 (time of admission to the ICU), 1, 3 and 7. The level of IAIP was determined using the established competitive ELISA (FIG. 2A) and both sandwich-type ELISAs using biotinylated heparin or biotinylated LPS as detecting molecules (FIG. 3A). Blood samples from 95 healthy controls aged between 17 to 71 years old (obtained from healthy blood donors and purchased from Rhode Island Blood Center) were included in this study to compare IAIP levels in health controls to the levels measured in sCAP patients. The results are shown in FIGS. 4, 5, and 6.

[0212]The results indicate that IAIP levels were significantly lower in sCAP patients compared to healthy controls at the ti...

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Abstract

Described herein are methods for quantifying lAIP levels in a sample (e.g., from a subject) using lAIP ligand-based assays. Also disclosed are methods for measuring lAIP-IAIP ligand complexes, and methods of evaluating, monitoring, and treating subjects using the aforementioned lAIP quantification methods.

Description

BACKGROUND[0001]IAIP (Inter alpha Inhibitor Proteins) are a family of naturally occurring, immunomodulatory plasma proteins that circulate in high concentrations in the blood of all mammals. IAIP are primarily produced in the liver, released into the blood and a subunit (bikunin) is excreted in the urine. IAIP have an important role in modulating inflammation. They have extensive protective effects toward the serious inflammation caused by infection, trauma, and injury and importantly, the protective effects of IAIP are independent from the causative microbial agents or triggers. Members of this family are composed of heavy and light polypeptide subunits that are covalently linked by glycosaminoglycan. IAIP can be found in vivo as Inter-alpha-Inhibitor (IαI), a 250 kDa molecule composed of two heavy chains (H1 & H2) and a single light chain (L), and Pre-alpha-Inhibitor (PαI), a 125 kDa molecule composed of one heavy (H3) and one light chain (L).[0002]When the body generates inflamma...

Claims

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Application Information

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IPC IPC(8): G01N33/68A61K38/57A61K45/06
CPCA61K38/57G01N2800/7095G01N33/6893G01N2333/4704A61K45/06C07K14/81C07K14/8114A61P31/00G01N33/543C07K16/38A61P29/00A61K38/00G01N2800/50G01N2800/52G01N2800/56
Inventor LIM, YOW-PINSPERO, DENICE
Owner POROTHERA BIOLOGY
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