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Methods and compositions for diagnostic assays for measuring cell mediated immune response

一种免疫应答、细胞的技术，应用在诊断试验领域，达到降低试验成本、增加特异性的效果

Inactive Publication Date: 2012-07-04
VACCINE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, none of these assays measure T-cell responses to pathogenic infection

Method used

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  • Methods and compositions for diagnostic assays for measuring cell mediated immune response
  • Methods and compositions for diagnostic assays for measuring cell mediated immune response
  • Methods and compositions for diagnostic assays for measuring cell mediated immune response

Examples

Experimental program
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Effect test

Embodiment approach

[0380] 1. A method of measuring a cell-mediated immune response (CMI) to a target antigen in a subject, said method comprising the steps of:

[0381] a. Incubate a biological sample from said subject with at least one fusion polypeptide comprising

[0382] A part of the LF polypeptide that lacks LF enzyme activity but is sufficient to facilitate transmembrane delivery of said fusion polypeptide to cells,

[0383] said portion is fused to a target antigen polypeptide or fragment thereof, wherein said biological sample comprises cells of the immune system that release at least one cytokine in response to the antigen;

[0384] b. measuring the level of at least one cytokine released in said biological sample; and

[0385] c. comparing the level of said cytokine in said biological sample to a reference level of the same cytokine,

[0386] wherein an increase in the level of said cytokine in said biological sample from a subject relative to a reference level of cytokine is indica...

Embodiment 1

[0540] IFN-γ ELISA protocol. As an example, a 1 plate (96 well) protocol is described below, with calculations based on 100 wells.

[0541] Coating: 10 μl of capture antibody (stock solution 1 mg / ml) was added to 10 ml of coating solution to dilute the capture antibody to 1 ug / ml. 100 μl of capture antibody at 1 μg / ml was added to each well of a 96-well plate and incubated overnight at 4°C. After this incubation at 4°C, the capture antibody is aspirated from the wells and the wells are washed at least 3 times, preferably 5 times with 250 μl PBST / well. After the washing step, the wells were blocked with 250 μl of blocking buffer per well for 2 hours at 37°C.

[0542] Wash step: Blocking solution was aspirated from the wells, followed by the addition of 250 μl PBST / well. This process is repeated at least 3 times, preferably 5 times.

[0543] Sampling procedure: 100 μl of IFN-γ for each standard curve dilution (ie from 300 pg / ml to 4.7 pg / ml, see “Standard Curve Preparation (...

Embodiment 2

[0551] Confirm the TB smear test using an in vitro CMI response test. Samples from 13 patients identified as positive for TB in the TB smear test were assayed for CMI response to TB using the diagnostic assay described herein. The 13 samples were also determined to be positive for TB using a culture assay. After incubation with LFn-TB antigen, INF-γ levels were determined using ELISA. The results are shown in Tables 3 and 4.

[0552] Table 3 shows an example of the results of the CMI assay disclosed herein as a diagnostic and prognostic identification of subjects with TB in which INF-γ levels were measured as follows: Using an ELISA assay, INF-γ protein level.

[0553]

[0554] Samples in bold were identified as TB positive by TB smear test and TB culture test. In this example, the assay was performed as follows: using reblock plates, Lot No. 20081217, GMP INF-γ (Lot No. 20081001) for standard curve - 1 mg / ml, detection Ab Pierce, Lot No. IJ116994. HRP: Biosource, Lot N...

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Abstract

Described are compositions and methods for detecting or monitoring the ability of an individual to mount a cell mediated immune response to a target antigen. Methods rely in part upon the physical association, e.g., by fusion, of a Lethal Factor (LF) polypeptide with a target antigen. The LF polypeptide moiety, including, for example, an LFn polypeptide moiety, serves as a transport factor to deliver target antigens, including full length target polypeptides, to the cytosol of an intact, living immune cell from an individual. Measurement of a cytokine response by the immune cell from the individual provides a read out of a cell mediated immune response. The methods and compositions described provide diagnostic as well as prognostic information and can guide the direction of therapy.

Description

technical field [0001] The present application relates generally to diagnostic assays and, more specifically, to assays for measuring cell-mediated immune (CMI) reactivity in biological samples. More specifically, the present invention provides assays and kits for measuring cell-mediated responses to target antigens in a biological sample, wherein the target antigen is delivered to the cells in the biological sample using a transport factor. [0002] Cross References to Related Applications [0003] This application claims the benefit under 35 U.S.C. §119(e) of US Provisional Application 61 / 186,437, filed June 12, 2009, the entire contents of which are hereby incorporated by reference. Background technique [0004] Epidemiological surveys suggest that 1 / 3 of the world's population is infected by Mycobacterium tuberculosis. Primary infection results in active tuberculosis (A-TB) in a minority (approximately 10%) of infected individuals, usually within 2 years. In other cas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50G01N33/569
CPCG01N33/56972G01N33/5091G01N33/5047G01N33/505G01N2800/24G01N33/6863C40B40/10
Inventor 吕亦晨N·托斯简H·郭W·郑
Owner VACCINE TECH
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