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Nursing point determination method

A point-of-care, immunoassay technology, applied in measuring devices, instruments, disease diagnosis, etc., can solve problems such as misdiagnosis of liver diseases and elevated enzyme activity levels

Inactive Publication Date: 2018-04-06
NANJING BIOPOINT DIAGNOSTIC TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the main limitation of these methods is the general need for instrumentation for two purposes; (a) to perform the enzymatic reaction at a fixed temperature, since enzyme activity is highly temperature sensitive, and (b) at the exact time or at the Measuring enzymatic products under kinetic conditions that provide precise measurements
[0008] A further limitation in the case of ALT is the presence in humans of a second isoform, ALT2, which is more abundant in muscle tissue but is also released from damaged cells, for example after physical exercise or heart failure, resulting in blood, plasma or Elevated levels of enzyme activity in serum, which can lead to misdiagnosis of liver disease

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0130] Example 1 - Human plasma strongly interferes with the ability of a series of mouse antibodies to bind ALT

[0131] A commercial ELISA for measuring ALT in human plasma also exhibits very poor correlation with enzymatic ALT levels and, surprisingly, yields results that are highly dependent on sample dilution for serum or plasma (see figure 1 ).

[0132] As shown in Figure 2, preliminary experiments disclosed herein by the inventors provided similar results. Using a series of MAbs generated in mice using ALT1 protein as an immunogen (Figure 2), the results were highly dependent on sample dilution for serum or plasma. However, the inventors reasoned that one or more factors present in human plasma could interfere with the antigenic reactivity of ALT with antibodies, thus preventing the development of a useful assay for ALT mass concentration using this method.

[0133] The surprising and novel observation of strong interference by human plasma at the level of ALT antigen...

Embodiment 2

[0134] Example 2 - Rabbits generate potent antibodies against human ALT1

[0135] The inventors compared the amino acid sequence of ALT1 in humans and mice (see Figure 3A ), and identified proteins with a high level of identity. Thus, only certain parts of the protein surface can be expected to be sufficiently different between humans (antigens) and mice (host animals) to be able to function as exogenous antigens to induce an immune response. Furthermore, a limited number of highly antigenic epitopes can overlap with sites that bind factors present in human plasma, preventing correct recognition of ALT proteins when assayed in human plasma using antibodies directed against such sites.

[0136] The inventors devised a solution to this problem by exploiting the known fact that rabbits have no homologues of the human ALT1 protein or gene, but only have homologues of the ALT2 protein, which appear to provide relevant biochemical function. Thus, it would be expected that substa...

Embodiment 3

[0138] Example 3 - Lack of Interference from Human Plasma in Results of Rabbit Antibody ELISA

[0139] After purification of specific anti-ALT antibodies from total rabbit IgG by antigen affinity chromatography, rabbit anti-ALT was used to capture ALT in human plasma, and biotin-labeled rabbit anti-ALT and horseradish peroxidase-labeled chains An ELISA was established for the detection of captured ALT by mycovidin.

[0140] like Figure 5 As shown, this ELISA is not adversely affected by the presence of human serum / plasma, making it suitable for analytical determination of ALT mass concentrations in human clinical specimens. Image 6 A strategy for converting the tested ELISA format into a colloidal gold detection method suitable for lateral flow immunochromatography is described.

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Abstract

The invention relates to an immunoassay for evaluating liver diseases or functions by virtue of a nursing point. The immunoassay comprises the following steps: (i) enabling a blood sample coming froma human subject to be contacted with a specific binding agent used for identifying an epitope of a liver enzyme or metabolite, so as to form an antigen-binding agent composite, and detecting the composite by using a second binding agent connected with a detectable reporter, wherein a rodent antibody can not identify the epitope in presence of human blood plasma; and (ii) detecting the liver diseases or functions in the subject according to mass concentration of the liver enzyme or metabolite in the sample.

Description

technical field [0001] The field of the specification is point-of-care testing, and more particularly assays and kits for identifying abnormal enzyme levels or abnormal liver function in a subject. Screening assays are also provided. Background technique [0002] Bibliographic details of references in this specification are also listed at the end of the specification. [0003] Reference to any prior art in this specification is not and should not be construed as an admission or any form of suggestion that such prior art forms common general knowledge in any country. [0004] Liver disease or hepatitis is a major health care burden globally. Liver disease can be caused by viruses (including hepatitis A, B, C, D, and E) or other infections, or by drug toxicity (including excessive alcohol consumption, and drugs used to treat conditions such as human immunodeficiency virus or tuberculosis infection) , or via abnormal metabolic processes including non-alcoholic fatty liver di...

Claims

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Application Information

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IPC IPC(8): G01N33/558G01N33/573G01N33/576
CPCG01N33/558G01N33/573G01N33/576G01N2800/08G01N2333/91188G01N2800/085G01N33/54388
Inventor D·A·安德森M·L·加西亚H·范张志美
Owner NANJING BIOPOINT DIAGNOSTIC TECH CO LTD
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