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Immunogenicity Assay

a technology of immunogenicity and assay, which is applied in the field of immunogenicity assay, can solve the problems of reducing the ability of conventional methods to detect low titers of antibodies of interest, such as ada, and achieve the effects of improving the likelihood of detecting antibodies, increasing the availability of antigenic epitopes, and facilitating the detection of such antibodies

Inactive Publication Date: 2012-08-30
ANP TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009]Advantageously, the instant assays do not require sample dilution, which reduces the ability of conventional methods to detect low titers of the antibodies of interest, such as, ADA. Also, the instant assays do not require one or more wash steps. That enhances the likelihood of detecting antibodies with a wide range of affinities, particularly low affinity or low avidity ADA.
[0010]The instant assays also can employ certain dendrimers, dendrigrafts, hyperbranched polymers, dendritic polymers, star-shaped polymers, comb-shaped polymers, and randomly branched polymers as a component of a reagent of interest. All of these polymers are generally referred to as branched polymers in this invention. Such a manipulation often increases the availability of antigenic epitopes on the drug of interest, enabling more efficient detection of such antibodies and reduces or removes the high dose hook effect that challenges other assays and assay formats.
[0011]In a similar manner, the performance of immunogenicity assays of various designs in conventional ELISA microplate and bead assay formats can also be enhanced by the use of the same branched polymers as a component on the reagents employed in such assays for the detection of such antibodies.

Problems solved by technology

Advantageously, the instant assays do not require sample dilution, which reduces the ability of conventional methods to detect low titers of the antibodies of interest, such as, ADA.

Method used

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Examples

Experimental program
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example 1

Biotin and Hapten Conjugation Methods

[0156]The PEG and IgG molecules are used as a generic example for the conjugation of PEG or drug molecules with biotin and haptens such as DNP, digoxigenin, digoxin, fluorescein, thyroxine, THC, morphine, amphetamine, heroin, barbiturates or other tags. Other conjugations of peptide, protein, and DNA / RNA drugs were prepared in a similar manner.

[0157]Method 1 Conjugation of 40 kDa PEG with a biotin (without a linker). PEG-NH2 (MW 40,000, NOF Corporation) (1.33 mg) was dissolved in 1 mL of PBS (BupH phosphate buffered saline from Thermo). N-hydroxysuccinimidobiotin EZ-Link NHS-Biotin from Thermo) (113 μg) was dissolved in 33.3 uL of dimethyl sulfoxide (DMSO). The solution was then added to the PEG solution. The reaction was incubated for 1 hour at room temperature. The product was purified on a desalting column, by ultrafiltration, or dialysis.

[0158]Method 2. Conjugation of 40 kDa PEG with a digoxigenin (without a linker). PEG-NH2 (MW 40,000, NOF C...

example 3

Preparation of Gold-Ab Conjugates

[0187]To a 125 ml flask were added 60 ml of colloidal gold solution (20-80 nm in diameter, O.D. 1.078). The pH of the solution was adjusted to 8-11 by addition of a 0.2 M potassium carbonate solution. To that solution were added 600 ml of conjugated antibody / streptavidin solution (O.D. 0.1-1.5 in sodium borate buffer) while stirring, followed by subsequent addition of 600 ml of bovine serum albumin (20% with sodium azide stabilizer). The mixture was stirred at 20° C. for 20-60 more minutes. The solution remained purple in color and some foaminess was observed. On completion, the stir bar was removed and the reaction mixture was transferred to two 50 ml conical tubes. The material was centrifuged until very little color was observed in the supernatant. The supernatant was removed and 400 ml of 25 mM sodium borate buffer were added to each tube. The contents were mixed thoroughly and the two tubes of material were combined and characterized by UV-Vis.

example 4

Immunogenicity Assay for the Detection of Anti-mouse IgG

[0188]A typical result obtained from a lateral flow assay design is shown in FIG. 3 wherein the drug is a mouse IgG, and the host is a goat that was exposed to the mouse IgG. The LFA format depicted in FIG. 2 was used to generate the dose response curve of FIG. 3 with serial dilutions of the goat antibodies, using an LFA test strip reader (LUR reader, ANP Technologies]. The limit of detection for that assay is 25 ng / mL ADA. The assay design can be used to detect ADA's to any type of therapeutic antibody.

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Abstract

Assays for detecting antibodies to pharmaceutical preparations, food allergens and environmental allergens are described.

Description

FIELD OF THE INVENTION[0001]The present invention relates in part to the measurement of drugs and antibodies to drugs, food antigens and other environmental antigen, such as those which are unmodified and modified biological molecules, in host animals and patients. The use of, for example, proteins and nucleic acids as drugs, such as recombinant proteins, peptides, antibodies, binding receptors, DNA, small interfering RNA (siRNA), micro RNA (miRNA), and RNA interference (RNAi), has proliferated in the last two decades. Because of size, charge and content, for example, such molecules are commonly immunogenic eliciting antibodies thereto in a host animal or a human patient. Many such large molecules are manipulated to contain substituents in an effort to reduce immunogenicity. Hence, many such large molecule drugs are composed of multiple elements, the drug per se and other substituents. However, those substituents also may be immunogenic. In addition, this invention disclosure also c...

Claims

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Application Information

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IPC IPC(8): G01N33/566G01N33/558B82Y15/00
CPCG01N33/558G01N33/54388G01N33/6854G01N33/54306
Inventor YIN, RAYPANG, JINGVALLEJO, JR., YLI REMOSMALL, THOMASQIN, DUJIECHEN, DE
Owner ANP TECH INC
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