Assay method

A technique for measuring method and haptoglobin, applied in the field of measuring

Inactive Publication Date: 2014-01-01
REACTIVLAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Not only does this limit the utility of the assay, but it also makes the assay unsuitable for adaptation to dry chemical formats such as "dip-stick" arrangements for visual or instrument-based evaluation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1: On a microtiter plate

[0098] Reagent A

[0099] A 50 μl aliquot of the equine hemoglobin stock solution was added to 25 ml of phosphate buffered saline. Add 1.25 ml of glucose oxidase stock solution to 10 ml of diluted hemoglobin solution.

[0100] Reagent Bi

[0101] Add 0.01ml AAP; 0.02ml phenol; 0.03ml ANS; 0.006ml DTT to 1.0ml citrate buffer pH3.8 containing 1% Tween 20.

[0102] Reagent Bii

[0103]To 1.0 ml phosphate buffer pH 7.4 was added 0.01 ml AAP; 0.02 ml phenol; 0.03 ml ANS; 0.006 ml DTT.

[0104] Working Reagent B

[0105] Mix 1 ml of reagent Bi with 1 ml of reagent Bii and add 0.56 ml of phosphate buffered saline containing 0.5M glucose.

[0106] Samples (6 μl) were placed in wells followed by addition of 100 μl of hemoglobin / glucose oxidase reagent A and 100 μl of chromogen reagent B, incubation at room temperature for 10 minutes and absorbance measured at 595 nm on an ELISA reader.

[0107] The results obtained are shown in Table 1...

Embodiment 2

[0111] Example 2 Comparison of DTT and Cys as reducing agent

[0112] DTT is known to be the least stable of the reagent mixture, and an alternative reagent capable of reducing the SS double bond would enhance the stability of the haptoglobin assay.

[0113] In experiments comparing the effectiveness of cysteine ​​and DTT in inhibiting background peroxidase activity, samples (4 μl) were placed in wells followed by the addition of 75 μl of hemoglobin / glucose oxidase reagent A and 75 μl of chromogens Bi DTT or Bii For Cys, incubate at room temperature for 30 min and measure absorbance at 600 nm on an ELISA reader.

[0114] working solution

[0115] Working Reagent A

[0116] Add 5 μl of hemoglobin and 30 μl of glucose oxidase solution to 2.5 ml of phosphate buffered saline.

[0117] Working Reagent Bi DTT

[0118] 10ml citrate buffer + 10ml phosphate buffer (pH 4.1)

[0119] Then add per 1ml of mixed buffer:

[0120] 0.01mlAAP

[0121] 0.03 mlANS

[0122] 0.02ml phenol...

Embodiment 3

[0138] Example 3: pH optimization

[0139] (a) pH 3.9-4.5

[0140] The effect of varying pH on the haptoglobin response was determined in a microtiter plate assay. Buffers for Reagent B were prepared by mixing citrate buffer and phosphate buffer as follows (Table 3) and adding other chemicals such as Bi DTT in Example 2 using DTT as reducing agent.

[0141] The pH of the buffer mix (B2-B5) was determined after all chemicals were added. Samples (5 μl) were placed in wells followed by addition of 90 μl hemoglobin / glucose oxidase reagent A and 90 μl chromogens B3-B6, incubated at room temperature for 20 minutes and absorbance measured at 600 nm on an ELISA reader.

[0142] Table 3: pH of the reaction buffer

[0143] Reagent citrate buffer volume ml Phosphate buffer volume ml Measured pH B2 10 0 3.95 B3 7.5 2.5 4.01 B4 5 5 4.10 B5 2.5 7.5 4.44

[0144] The results obtained are shown in Table 4 below.

[0145] Table 4: Absorban...

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PUM

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Abstract

The invention provides an assay method for determining a level of haptoglobin in a sample comprising the steps of: (i) mixing haemoglobin with the sample to be assayed so as to form a haptoglobin-haemoglobin complex with haptoglobin present in the sample; (ii) contacting the product of step (i) with reagents for generating hydrogen peroxide and one or more chromogens which undergo a spectroscopically detectable change when peroxidase activity is present, in the presence of a buffer, under conditions in which hydrogen peroxide is generated from said reagents and forms a substrate for the peroxidise activity of the haptoglobin-haemoglobin complex present, and wherein the pH of the buffer is within a range which is sufficiently low that the peroxidise activity of any uncomplexed haemoglobin is substantially suppressed but sufficiently high that hydrogen peroxide generation occurs; (iii) determining the peroxidase activity of the haptoglobin-haemoglobin complex by measuring the change in an optical property of the reaction mixture; and (iv) correlating the level of peroxidise activity of the haptoglobin-haemoglobin complex with the amount of haptoglobin in the sample. A kit for use in such a method is also provided.

Description

field of invention [0001] The present invention relates to assays and kits for determining the level of haptoglobin in a sample. In particular, the present invention relates to assays for determining haptoglobin levels that are readily adaptable to be run in a dry format in which reagents are dried on a surface prior to running the assay. Background of the invention [0002] Haptoglobin is one of a group of proteins known as acute phase proteins, whose concentrations rise dramatically following infection, inflammation or trauma. Measuring the concentration of haptoglobin in plasma thus provides valuable diagnostic information on the health status of the human or animal from which the sample was obtained, and it is in the art to develop assays for haptoglobin in plasma, serum and other biological fluids There has been considerable interest. [0003] Current assays for determining the concentration of haptoglobin in a sample are generally based on immunoassays or on hem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/725G01N21/78C12Q1/28
Inventor P.D.埃科萨尔E.麦库洛奇S.多彻尔蒂
Owner REACTIVLAB
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