CN1141396C

CN1141396C - Encapsulation method of bacterial lipopolysaccharide, reagent kit and method of detecting specific lipopolysaccharide

Info

Publication number: CN1141396C
Application number: CNB021150400A
Authority: CN
Inventors: 陈晓燕; 胡超群; 任春华
Original assignee: South China Sea Institute of Oceanology of CAS
Current assignee: South China Sea Institute of Oceanology of CAS
Priority date: 2002-04-05
Filing date: 2002-04-05
Publication date: 2004-03-10
Anticipated expiration: 2022-04-05
Also published as: CN1374526A

Abstract

The present invention relates to a coating method of bacterium lipopolysaccharide, which comprises the steps that LPS is dissolved in 0.1 mol/L of bicarbonate buffer solution (coating solution) containing 0.02 to 2% of trichloroacetic acid with the pH value of 9.6, and the LPS and the bicarbonate buffer solution are added to an enzyme labelling detecting hole illuminated by a sterilization ultraviolet lamp in advance; the LPS and the bicarbonate buffer solution are in warm bath at the temperature of 35 to 39DEG C and are kept at the temperature of 4 DEG C; the solution is dried, and defatted milk powder or albumin confining liquid is added and is enclosed in warm bath at the temperature of 35 to 39DEG C; the solution is dried and is washed by a washing solution, and the solution is preserved at the temperature of 4DEG C; the present invention further relates to a reagent kit for detecting bacterium specific lipopolysaccharide in the method and a method for rapidly detecting bacterium specific LPS by the reagent kit. The reagent kit comprises a bacterium LPS monoclonal antibody, a second enzyme labelling antibody, a standard LPS, a dilute solution and an encapsulated enzyme labelling detecting strip. The present invention has the advantages of simple LPS embedding method and reagent kit, and convenient detection.

Description

Detect the test kit and the detection method of specific lipopolysaccharides

Technical field

The present invention relates to the test kit of the specific lipopolysaccharides of a kind of bacterial detection and use the method for the specific lipopolysaccharides of this test kit rapid detection bacterium.

Background technology

Lipopolysaccharides (LPS) is the main component of gram-negative bacteria cell wall, as intracellular toxin, is again one of important virulence factor of pathogenic bacteria.LPS has the effect of the pros and cons to animal body, on the one hand, LPS is subjected to the host of infectation of bacteria to bring toxic actions such as pyrogen reaction, and make pathogenic bacteria hide the attack of animal body complement, make pathogenic bacteria success infection host, on the other hand, LPS is again the immunostimulant of animal body, but the immune resistibility of enhancing body.Since earlier 1900s is found intracellular toxin, about the existing a large amount of report of endotoxic research, to endotoxic structure, active, in the research to the mechanism of body effect and pathogenesis, commonly used to immunologic research method, and highly sensitive ELISA detection method is a method of using always, but in the ELISA of LPS antigen or antibody detection method, the antigenic embedding difficulty of LPS, can not be directly embedded in the enzyme mark bar with common embedding method of protein, generally need to adopt some comparatively complicated chemically crosslinked or centrifugation method, so the antigenic embedding trouble of LPS, unstable.

The content of invention

The objective of the invention is for test kit that the specific LPS of a kind of rapid detection bacterium is provided and the method for using the specific LPS of this test kit rapid detection bacterium.

Cardinal principle of the present invention is: the LPS of bacterium LPS monoclonal antibody and bacterium has special antigen-antibody immunity association reaction, with a certain amount of LPS embedding enzyme mark reacting hole, adding will detect solution and a certain amount of LPS monoclonal antibody and the ELIAS secondary antibody of LPS, immobilized LPS competition combines the LPS monoclonal antibody in LPS in the solution and the embedding hole, the quantity minimizing that makes and be fixed in the LPS monoclonal antibody of the LPS bonded horseradish peroxidase-labeled in the enzyme mark hole, the solution colour that horseradish peroxidase and substrate reactions show shoals until colourless, thus can qualitative or half-quantitative detection LPS.

The test kit of the present invention's specific lipopolysaccharides of a kind of rapid detection bacterium (LPS), its solvent and material comprise:

(a) be stored in bacterium LPS monoclonal antibody in the disodium phosphate soln that contains 0.01-1% sodium azide, 30-80% glycerine;

(b) immunoglobulin (Ig) of lipotropism polysaccharide (LPS) monoclonal antibody of peroxidase labelling is an ELIAS secondary antibody;

(c) be dissolved in standard lipopolysaccharides (LPS) in the pH7.4 phosphate buffered saline buffer;

(d) diluent promptly contains the phosphate buffered saline buffer of the pH7.4 of 0.05-0.5% gelatin and 0.01-0.1% tween 20;

(e) wrapped by the enzyme mapping of good lipopolysaccharides (LPS) and decided bar.

Above among (a) of described test kit, sodium azide and glycerine preferred content are respectively 0.05-0.3% and 45-60%.Above among (d) of described test kit the preferred content of gelatin be 0.1-0.3%.

The method for coating of bacteria lipopolysaccharide (LPS) comprises the following steps among (e) of described test kit

(a) 0.2-2 μ g LPS is dissolved in 50-300 μ l pH9.6 and contains in the 0.1mol/L carbonate buffer solution (coating buffer) of 0.02-2% trichoroacetic acid(TCA), and is added to the enzyme mapping of shining 10-60 minute with sterilizing viltalight lamp in advance and decides in the hole;

(b) 35-39 ℃ of temperature bathed 30 minutes-3 hours;

(c) placed 2-48 hour for 4 ℃;

(d) dry solution, add skim-milk or albumin confining liquid, 35-39 ℃ of temperature bathed sealing;

(e) dry solution, washings washing (three times) places 4 ℃ of preservations.

In the method for coating of above-mentioned bacteria lipopolysaccharide (LPS), (a) preferred version is: 0.5-1.2 μ g LPS is dissolved in 80-180 μ lpH9.6 and contains in the 0.1mol/L carbonate buffer solution (coating buffer) of 0.3-1% trichoroacetic acid(TCA), and is added to the enzyme mapping of shining 20-50 minute with sterilizing viltalight lamp in advance and decides in the hole; (b) preferred version is that 35-39 ℃ of temperature bathed 1-2 hour; (c) optimum condition is 4 ℃ and placed 12-24 hour.

Need the reagent of preparation separately to comprise when using test kit:

(1) washings (pH7.4 0.02mol/L phosphate buffered saline buffer-0.05% polysorbas20), it contains

Potassium primary phosphate 0.2 gram

Sodium phosphate dibasic 2.9 grams

Sodium-chlor 8.0 grams

Polysorbas20 0.5 gram

Add distilled water to 1000 milliliter

(2) pH5.0 phosphoric acid salt-citrate buffer solution

First liquid: citric acid (anhydrous) 19.2 grams are dissolved in 1000 milliliters of distilled waters

Second liquid: Sodium phosphate dibasic (12H ₂O) 71.6 grams are dissolved in 1000 milliliters of distilled waters

Get 24.3 milliliters of first liquid, 25.7 milliliters of second liquid, 50 milliliters of distilled waters mix phosphoric acid salt-citric acid of pH5.0

Damping fluid.

(3) substrate is used liquid (time spent now joins), is formed by following three kinds of reagent mix:

40 milligrams of O-Phenylene Diamines

100 milliliters of pH5.0 phosphoric acid salt-citrate buffer solutions

30% hydrogen peroxidase 10 .15 milliliter

(4) stop buffer

30 milliliters of vitriol oils are added in 70 milliliters of distilled waters and mix.

A kind of test kit that uses the specific lipopolysaccharides of above-mentioned rapid detection bacterium and the method for joining the specific lipopolysaccharides of reagent rapid detection bacterium in addition comprise:

(a) in every mensuration hole, add bacteria lipopolysaccharide (LPS) monoclonal antibody;

(b) detected sample is carried out adding after the suitable dilution enzyme mapping with diluent and decide in the bar, the volume that makes every hole add solution is 100 μ l, and control wells adds bacteria lipopolysaccharide (LPS) monoclonal antibody and diluent, and to make its volume be 100 μ l, abundant mixing;

(c) 35-39 ℃ of temperature bathed 1-2 hour;

(d) washings washing (3-4) is inferior;

(e) add the ELIAS secondary antibody 100 μ l that suitably dilute with diluent;

(f) 35-39 ℃ of temperature bathed 0.5-2 hour;

(g) washings washing (3-4) is inferior;

(h) add substrate and use liquid 100 μ l, lucifuge colour developing 15-30 minute;

(I) when obvious color reaction appears in control wells, add stop buffer 50 μ l, the absorbance value of measuring each hole with microplate reader is the OD value immediately;

(j) result judges: antigenic content is inversely proportional in each color depth of measuring the hole colour developing and the liquid to be measured, and as if treating that gaging hole OD value is littler than the control wells, then decidable treats that the gaging hole result is positive; Otherwise it is then negative; Test sample is carried out when quantitative, and the standard bacteria lipopolysaccharide (LPS) of getting gradient concentration detects, and according to detected result, measures the ratio drawing standard curve of hole OD value and control wells OD value by each, searches the antigenic content of testing sample according to mensuration ratio.

Can adjust the concentration of test sample in the mensuration, the OD value ratio for the treatment of gaging hole OD value and control wells is advisable at 0.3-0.8.Advantage of the present invention and positively effect:

LPS is the essential composition of gram-negative bacteria cell wall, it is again important virulence factor, with immunological method LPS being launched research and detects is a kind of method that is in daily use, and uses easy method and LPS is embedded in the enzyme mapping decide in the hole, can give to study and detection bring very big facility.And the LPS detection kit both can be used for the scientific research of relevant LPS, can be used for the detection of infected animal again, can also detect in some biological products, whether polluted the LPS that specific bacteria is arranged.This test kit has certain market application foreground.

Embodiment

Following is that the invention will be further described in conjunction with the embodiments, but should not be used as limitation of the present invention.

The method for coating of bacterium LPS is

(a) 0.8 μ g LPS is dissolved in 100 μ l pH9.6 and contains in the 0.1mol/L carbonate buffer solution (coating buffer) of 1% trichoroacetic acid(TCA), and is added to the enzyme mapping of shining 30 minutes with sterilizing viltalight lamp in advance and decides in the hole;

(b) 37 ℃ of temperature were bathed 1 o'clock;

(c) placed 12 hours for 4 ℃;

(d) dry solution, add skim-milk or albumin confining liquid, 37 ℃ of temperature are bathed sealing;

(e) dry solution, washings washing three times places 4 ℃ of preservations.

Test kit comprises

(a) be stored in bacterium LPS monoclonal antibody in the disodium phosphate soln that contains 0.1% sodium azide, 50% glycerine;

(b) immunoglobulin (Ig) of the anti-LPS monoclonal antibody of peroxidase labelling is an ELIAS secondary antibody;

(c) be dissolved in standard LPS in the pH7.4 phosphate buffered saline buffer;

(d) diluent promptly contains the phosphate buffered saline buffer of the pH7.4 of 0.3% gelatin and 0.05% tween 20;

(e) wrapped by the enzyme mapping of good LPS and decided bar.

Other joins consistent in reagent and the technology contents.

Whether use this test kit research vibrio alginolyticus LPS under the certain culture condition is secreted in the microbial culture supernatant, applicable cases is as follows: vibrio alginolyticus is inoculated in the 2.5%NaCl nutritional medium, in 30 ℃ of shaking culture, in 48 hours, took a sample centrifugation microbial culture supernatant every 4 hours.Get the microbial culture supernatant 50 μ l in each period, the enzyme that adding vibrio alginolyticus LPS detection kit provides is marked in the cylindrical void, every hole adds 50 μ l diluents and 1 μ l mouse-anti vibrio alginolyticus LPS monoclonal antibody again, abundant mixing, detect by the test kit using method, each mensuration is all done a replicate(determination), and determining does not at last have the LPS that can measure in the microbial culture supernatant, shows that vibrio alginolyticus its LPS under 2.5%NaCl nutritional medium culture condition is not secreted in the microbial culture supernatant.

Claims

1. the test kit of the specific lipopolysaccharides of bacterial detection is characterized in that solvent and material comprise:

(a) be stored in bacteria lipopolysaccharide monoclonal antibody in the disodium phosphate soln that contains 0.01-1% sodium azide, 30-80% glycerine;

(b) immunoglobulin (Ig) of the antibacterium lipopolysaccharides monoclonal antibody of peroxidase labelling is an ELIAS secondary antibody;

(c) be dissolved in standard bacteria lipopolysaccharide in the pH7.4 phosphate buffered saline buffer;

(e) wrapped by the enzyme mapping of good bacteria lipopolysaccharide and decided bar.

2. according to the test kit described in the claim 1, it is characterized in that in (a), sodium azide and glycerol content are respectively 0.05-0.3% and 45-60%.

3. according to the test kit described in the claim 1, it is characterized in that the content of gelatin in (d) is 0.1-0.3%.

4. one kind is used the test kit described in the claim 1 to reach the method for joining the specific lipopolysaccharides of reagent rapid detection bacterium in addition, and its feature comprises the following steps:

(a) in every mensuration hole, add the bacteria lipopolysaccharide monoclonal antibody;

(b) detected sample is carried out adding after the suitable dilution enzyme mapping with diluent and decide in the bar, the volume that makes every hole add solution is 100 μ l, and control wells adds bacteria lipopolysaccharide monoclonal antibody and diluent, and to make its volume be 100 μ l, abundant mixing;

(c) 35-39 ℃ of temperature bathed 1-2 hour;

(d) washings washing;

(f) 35-39 ℃ of temperature bathed 1-2 hour;

(g) washings washing;

(j) result judges: antigenic content is inversely proportional in each color depth of measuring the hole colour developing and the liquid to be measured, and as if treating that gaging hole OD value is littler than the control wells, then decidable treats that the gaging hole result is positive; Otherwise it is then negative; Test sample is carried out when quantitative, and the standard bacteria lipopolysaccharide of getting gradient concentration detects, and according to detected result, measures the ratio drawing standard curve of hole OD value and control wells OD value by each, searches the antigenic content of testing sample according to mensuration ratio.