# US 2014/0193838 A1 · Method for measuring sialic acid in immunoglobulin g and immunoglobulin g anti-double-stranded dna antibodies

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- Number: US 2014/0193838 A1
- Document ID: US20140193838A1
- Status: Published application
- Kind: A1
- Application number: 13/735,046
- Filed: 2013-01-07
- Published: 2014-07-10
- Earliest claimed: 2013-01-07
- Assignees: Chang Gung Medical Foundation, Linkou Branch
- Inventors: Lieh-bang Liou
- Web page: https://patentgrep.com/patent/US20140193838A1

## CPC

- G01N 33/6854: {Immunoglobulins}
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- G01N 33/54306: {Solid-phase reaction mechanisms}
- G01N 2400/00: Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N 2440/38: addition of carbohydrates, e.g. glycosylation, glycation
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

A method for measuring the amount of sialic acid in immunoglobulin G and immunoglobulin G anti-ds DNA antibodies is disclosed. The method for measuring the amount of sialic acid in immunoglobulin G in the present invention uses culture fluid, blood, plasma, or serum to directly measure the amount of sialic acid in immunoglobulin G. Also, using a mouse monoclonal antibody immunoglobulin G as a standard, which is diluted from 1000 ng/ml to 15.625 ng/ml in phosphate buffered saline (PBS), produces good results. The method for measuring the amount of sialic acid in immunoglobulin G anti-ds DNA antibodies has never been done and the present invention produces good results as well.

## Claims

### Claim 1

A method for measuring the amount of sialic acid in immunoglobulin G comprising steps of:
Step 1: in an enzyme immunoassay petridish, adding 200 ng of sambucus nigra agglutinin lectin dissolved in 100 μl PBS (pH=7.2) in each grid, and placing said petridish in a refrigerator at 4 overnight;
Step 2: washing four times with PBS-0.5% Tween 20 as a rinse solution, adding 300 μl of 1% bovine serum albumin (BSA) dissolved in phosphate buffered saline in each grid, and placing said petridish at room temperature for 2 hours in order to block non-specific adhesion;
Step 3: washing (four times) the enzyme immunoassay petridish with PBS-0.5% Tween 20, adding plasma, serum, blood, or the separated immunoglobulin, and placing said petridish at room temperature for 2 hours;
Step 4: washing (four times) the enzyme immunoassay petridish as in Step 2 with the PBS-0.5% Tween 20, adding 300 μl of 1% bovine serum albumin dissolved in PBS in each grid, and placing said petridish in room temperature for 1 hour;
Step 5: using PBS-0.5% Tween 20 in Step 2 to wash (four times) the petridish, adding 100 μl of horseradish peroxidase-linked goat anti-mouse immunoglobulin G (diluted 8000 times) dissolved in phosphate buffered saline, and placing said petridish in a room temperature for 1 hour;
Step 6: using PBS-0.5% Tween 20 as in Step 2 to wash (four times) the petridish, adding tetramethylbiphenyl ammonia (tetramethyl benzidine, TMB) solution and hydrogen peroxide solution (same volume) 100 μl in each grid, and placing said petridish at room temperature for 10 minutes, and adding 100 μl of 0.5N H2 SO4 to stop the reaction; and
Step 7: measuring the absorption value at 450 nm in each grid in an enzyme immunoassay analyzer.

### Claim 2 (depends on 1)

The method for measuring the amount of sialic acid in immunoglobulin G of claim 1, wherein a mouse monoclonal antibody immunoglobulin G is used as a standard, which is diluted from 1000 ng/ml to 15.625 ng/ml and placed at room temperature for two hours.

### Claim 3 (depends on 1)

The method for measuring the amount of sialic acid in immunoglobulin G of claim 1, wherein said enzyme immunoassay petridish has ninety-six (96) grids.

### Claim 4

A method for measuring the absolute amount of sialic acid in immunoglobulin G anti-double-stranded DNA antibodies comprises steps of:
Step 1: in an enzyme immunoassay petridish, adding 150 μl of 0.5 mg/ml of protamine chloride in each grid and placing said petridish at room temperature for 2 hours;
Step 2: washing (three times) with PBS (pH=7.2) and adding 100 μl of 50 μg/ml calf thymus double-stranded DNA overnight at 4° C.;
Step 3: preparing oxidized bovine serum albumin, wherein the bovine serum albumin is dissolved in 20 mM potassium periodate (PBS, pH=7.2) and 50 mM sodium acetate (final pH=4.0 of the mixture of sodium periodate and sodium acetate) at 4° C. (for 30 minutes) to obtain a mixture, and the mixture is dialyzed with tris-buffered saline (pH=7.4), and adding (0.1% by volume) of polyoxyethylene (20) sorbitan monolaurate solution to make 1% oxidized bovine serum albumin;
Step 4: using PBS-0.5% Tween 20 as a flushing fluid to flush the enzyme immunoassay petridish (four times), adding 300 μl of 1% of oxidation bovine serum albumin into each grid, and placing said petridish at room temperature for 2 hours in order to block non-specific adhesion;
Step 5: washing (four times) the enzyme immunoassay petridish as in Step 4 with PBS-0.5% Tween 20, adding 100 μl of immunoglobulin G isolated from protein G, and placing said petridish at room temperature for two hours;
Step 6: washing (four times) the enzyme immunoassay petridish as in Step 4 with PBS-0.5% Tween 20, and adding diluted (500 times) horseradish peroxidase-linked sambucus nigra agglutinin lectin (SNA) 100 μl to each grid;
Step 7: washing (four times) the enzyme immunoassay petridish as in Step 4 with PBS-0.5% Tween 20, adding mixture of tetramethyl benzidine solution 50 μl and peroxide hydrogen solution 50 μl at room temperature for 5 minutes, and adding 100 μl of 0.5N H2SO4 into each grid to prevent the reaction; and
Step 8: measuring the absorption value at 450 nm in each grid in the enzyme immunoassay analyzer.

### Claim 5 (depends on 4)

The method for measuring the amount of sialic acid in immunoglobulin G anti-double-stranded DNA antibodies of claim 4, wherein a mouse monoclonal antibody immunoglobulin G anti-double-stranded DNA is used as a standard, which is diluted from 1000 ng/ml to 15.625 ng/ml and placed at room temperature for two hours.

### Claim 6 (depends on 4)

The method for measuring the amount of sialic acid in immunoglobulin G anti-double-stranded DNA antibodies of claim 4, wherein said enzyme immunoassay petridish has ninety-six (96) grids.