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Method detecting content of Hib (haemophilus influenzae type b) polysaccharide antibodies in serum through adopting ELISA method

A serum and antibody technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve problems such as the difficulty of clinical serum determination of Haemophilus influenzae type b, the shortage of human serum albumin prices, and the difficulty in handling serum samples, etc., to achieve easy Obtain, reduce differences between boards, and reduce costs

Inactive Publication Date: 2012-10-31
CHENGDU OLYMVAX BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, human serum albumin is usually used as a blocking agent when measuring the content of Hib polysaccharide antibody in serum. The clinical serum determination of the
In addition, because the ELISA method is very sensitive, even if the same serum is completed on different microtiter plates or at different times, the measured values ​​will vary greatly. Due to the large amount of clinical serum samples, the existing serum The reaction time is 1.5h at 37°C, which has strict requirements on the sample addition time, and it is difficult to process a large number of serum samples in a short period of time

Method used

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  • Method detecting content of Hib (haemophilus influenzae type b) polysaccharide antibodies in serum through adopting ELISA method

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Experimental program
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Effect test

Embodiment 1

[0045] The method for detecting Hib polysaccharide antibody content in serum by ELISA method mainly comprises the following steps:

[0046] (I) Coating: After diluting the tyrosidated derivative of Haemophilus influenzae type b capsular polysaccharide (PRP-Ty) with carbonate buffer solution of pH 9.6, add 100 μl / well to the microwell In the plate, overnight at 2-8°C, wash 8 times the next day, and block with bovine serum albumin at 37°C for 3 hours;

[0047] (II) Adding samples: add diluted serum to be tested and standard serum, seal the plate with plastic wrap to 2-8°C, wash 8 times after 16 hours;

[0048] (Ⅲ) Add enzyme-labeled antibody: add horseradish peroxidase-labeled antibody IgG, incubate in a 37°C incubator for 2 hours, and then wash 8 times;

[0049] (Ⅳ) Add substrate for color development: Add o-phenylenediamine (OPD) substrate for color development in each reaction well, and develop color at 37°C in the dark for 30 minutes;

[0050] (Ⅴ) Stopping the reaction: ad...

Embodiment 2

[0053] The method for detecting Hib polysaccharide antibody content in serum by ELISA method mainly comprises the following steps:

[0054] (I) Coating: After diluting the tyrosidated derivative of Haemophilus influenzae type b capsular polysaccharide (PRP-Ty) with carbonate buffer solution of pH 9.6, add 100 μl / well to the microwell In the plate, overnight at 2-8°C, wash 7 times the next day, and block with bovine serum albumin at 37°C for 2.5h;

[0055] (II) Adding samples: add diluted serum to be tested and standard serum, seal the plate with plastic wrap to 2-8°C, wash 7 times after 16 hours;

[0056] (Ⅲ) Add enzyme-labeled antibody: add horseradish peroxidase-labeled antibody IgG, incubate in a 37°C incubator for 1.5h and wash 7 times;

[0057](Ⅳ) Add substrate for color development: Add o-phenylenediamine (OPD) substrate for color development in each reaction well, and develop color at 37°C in the dark for 25 minutes;

[0058] (Ⅴ) Stopping the reaction: adding 2M sulfu...

Embodiment 3

[0061] The method for detecting Hib polysaccharide antibody content in serum by ELISA method mainly comprises the following steps:

[0062] (I) Coating: After diluting the tyrosidated derivative of Haemophilus influenzae type b capsular polysaccharide (PRP-Ty) with carbonate buffer solution of pH 9.6, add 100 μl / well to the microwell In the plate, overnight at 2-8°C, wash 6 times the next day, and block with bovine serum albumin at 37°C for 2 hours;

[0063] (II) Adding samples: add diluted serum to be tested and standard serum, seal the plate with plastic wrap to 2-8°C, wash 6 times after 16 hours;

[0064] (Ⅲ) Add enzyme-labeled antibody: add horseradish peroxidase-labeled antibody IgG, incubate in a 37°C incubator for 1 hour and wash 6 times;

[0065] (Ⅳ) Add substrate for color development: Add o-phenylenediamine (OPD) substrate for color development in each reaction well, and develop color at 37°C in the dark for 20 minutes;

[0066] (Ⅴ) Stopping the reaction: adding 2M...

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Abstract

The invention discloses a method detecting content of Hib (haemophilus influenzae type b) polysaccharide antibodies in serum through adopting the ELISA method. The method provided by the invention mainly comprises the following steps: I, coating; II, applying sample; III, adding enzyme labeled antibodies; IV, substrate adding and coloration; V, reaction termination; and VI, color comparison. The method has the benefits that bovine serum albumin is adopted to substitute human serum albumin and serves as a sealing agent during the content detection of Hib polysaccharide antibodies in serum, so that the difficult problem that the sealing agent in an experiment needs a lot, yet the human serum albumin is short and high in price is solved, cost is greatly reduced, and the bovine serum albumin is easy to obtain, as a result, the popularization of the method can be facilitated; and the reaction time is changed into 16 hours at 2 to 8 DEG C, so that more serum can be detected at one time, the reaction time is sufficient, difference among plates is reduced, and the improvement on the detection accuracy is facilitated.

Description

technical field [0001] The invention relates to the technical field of methods for detecting the content of Hib polysaccharide antibodies in serum, in particular to a method for detecting the content of Hib polysaccharide antibodies in serum by ELISA. Background technique [0002] Haemophilus influenzae type b (Hib) is one of the main causes of severe disease in infants under 5 years old. Meningitis and pneumonia are the two main diseases of Hib infection, the most common being meningitis, The mortality rate of its children is 5%. An effective way to prevent infants from getting sick is early immunization. The currently used vaccine is a polysaccharide-protein conjugated vaccine. [0003] At present, human serum albumin is usually used as a blocking agent when measuring the content of Hib polysaccharide antibody in serum. The clinical serum determination brings great difficulties. In addition, because the ELISA method is very sensitive, even if the same serum is completed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543
Inventor 陈庚关晓峰李洪光
Owner CHENGDU OLYMVAX BIOPHARM
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