CN104407159A

CN104407159A - IgM (Immune Globulin M) immune turbidimetry test kit

Info

Publication number: CN104407159A
Application number: CN201410765259.5A
Authority: CN
Inventors: 甘宜梧; 董雯; 谭柏清; 谢清华; 王绮; 李静; 包兴艳
Original assignee: Biobase Biodustry Shandong Co Ltd
Current assignee: Biobase Biodustry Shandong Co Ltd
Priority date: 2014-12-15
Filing date: 2014-12-15
Publication date: 2015-03-11
Anticipated expiration: 2034-12-15
Also published as: CN104407159B

Abstract

The invention discloses an IgM (Immune Globulin M) immune turbidimetry test kit, and belongs to the technical field of clinical in-vitro test reagents. The kit disclosed by the invention comprises a reagent R1, a reagent R2 and a calibration material. The stability and the analysis sensitivity of the kit are effectively improved by adding a certain amount of silicon dioxide-cladded magnetic nano-particles into the reagent R1 and adding a certain amount of bovine serum albumin and Kathon-CG into the reagent R2; the linear range of the kit is also larger; the accuracy of each reagent is high; popularization and application of the kit in markets are facilitated.

Description

A kind of Immunoglobulin IgM immunoturbidimetry detection kit

Technical field

The present invention relates to clinical vitro detection reagent technique field, particularly a kind of Immunoglobulin IgM immunoturbidimetry detection kit.

Background technology

Immunoglobulin (Ig) is made up of heavy chain and light chain, according to heavy chain (Heavy chain, H chain) difference of constant region chemical constitution, corresponding immunoglobulin (Ig) is divided into polytype, is respectively immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin A (IgA), immunoglobulin E (IgE) and immunoglobulin G (IgG).

Human immunoglobulin M (IgM) is present in the serum of people, account for 5 ~ 10% of serum immune globulin total amount, serum-concentration is about 1mg/ml, synthesize primarily of spleen mesoplasmatocyte, an IgM molecule connects into pentamer by 5 monomers by J chain, has higher antigenic valence, is also that five immunoglobulin like protein (IgA, IgD, IgE, IgG, IgM) middle-molecular-weihydroxyethyl is maximum, its molecular weight is 950k dalton, therefore is also called macroglobulin.

IgM is a kind of immunoglobulin (Ig) of very early time, is the immunoglobulin (Ig) first synthesized after first contacts antigen.In the serum of adult, account for 5% of total immunoglobulin (Ig).IgM concentration declines and occurs in primary and secondary immunodeficiency syndrome.The minimizing of IgM value is common in protein-loss intestines problem and burn.Severe infections and autoimmune disease can cause IgM concentration to rise.Multiple macroglobulinemia, bacterium and parasitic infection, liver diseases, rheumatoid arthritis and cystic disease can make IgM concentration increase.

At present, the IgM assay method that laboratory is conventional has HPLC and ELISA method.HPLC has high specificity, the advantage such as highly sensitive, reproducible, generally acknowledge now that it is the prefered method measuring blood plasma IgM concentration, but the equipment needed because of HPLC is complicated and expensive, be difficult to adapt to the application of routine clinical chemical laboratory, and ELISA method operating process is a bit loaded down with trivial details, the reaction needed time, can not accomplish in one move.Immunoglobulin IgM immunoturbidimetry detection kit, based on the reaction between IgM antibody and IgM antigen, forms immune complex, and detect the change of its turbidity at 340nm wavelength place, its intensity of variation is directly proportional to the IgM content in sample.The method is a kind of without the need to pre-service sample, and technology and equipment is less demanding, and precision and the higher analytical approach of specificity.Because the method does not need expensive equipment, can robotization be realized, and a large amount of sample can be measured, therefore be subject to clinical extensive popularization.But common Immunoglobulin IgM immunoturbidimetry detects that reagent stability is bad, accuracy and sensitivity are not high yet, thus limit its applying clinically.

Summary of the invention

Be directed to prior art Problems existing, the invention provides a kind of Immunoglobulin IgM immunoturbidimetry detection kit, this kit is compared with the kit of routine, stability and the range of linearity better than the detection kit of routine, sensitivity for analysis is high, is conducive to reagent applying clinically.

The present invention is achieved by the following measures:

A kind of Immunoglobulin IgM immunoturbidimetry detection kit, it is characterized in that, it comprises reagent R1, reagent R2 and calibration object, and wherein reagent R1 consists of: the magnetic nanoparticle of 18.16mmol/L pH 7.6 Tris damping fluid, 123.20mmol/L sodium chloride, 6% PEG400,0.1%-2% coated with silica; Reagent R2 consists of: 18.16mmol/L pH 7.6Tris damping fluid, 30ml/L goat-anti human IgM antibody, 20-40g/L bovine serum albumin(BSA), 0.05% Kathon-CG.

The magnetic nanoparticle of described coated with silica is Fe ₃o ₄/ SiO ₂composite nanoparticle, particle diameter is 20-50nm.

Described reagent R1 and reagent R2 ratio are in use R1:R2=250:50.

The magnetic nanoparticle of coated with silica used in the present invention adopts following methods to prepare:

Polyol process is adopted to prepare Fe ₃o ₄nano particle.Get certain ferric acetyl acetonade and triethylene glycol joins in reflux heating reaction unit, under the condition of magnetic agitation and Ar gas shielded, device is slowly heated to boiling, and keeps backflow a period of time.Cool in backward reaction solution and add the ferriferrous oxide nano-particle generation flocculation that ethyl acetate makes generation, magnetic resolution black product, cleaning for several times, is distributed in ethanol, namely obtains stable Fe ₃o ₄nano particle alcohol colloidal solution.Afterwards by gained Fe ₃o ₄nano particle adopts Stober Hydrolyze method to obtain SiO ₂coated Fe ₃o ₄nano particle.Gained Fe ₃o ₄/ SiO ₂the particle diameter of composite nanoparticle is 20-50nm.

Calibration object used in the present invention is the IgM calibration object that Jiu Fengrunda Bioisystech Co., Ltd produces.

Kit of the present invention carries out on the automatic biochemistry analyzer with double reagent function, and its concrete using method is as follows:

Add physiological saline, sample or calibration object 2 μ l, after adding R1 reagent 250 μ l preincubate 5min afterwards again, read absorbance A 1, after the reagent R2 adding 50 μ l afterwards again reacts 5min, read absorbance A 2, and calculate Δ A.

Beneficial effect of the present invention:

Immunoglobulin IgM immunoturbidimetry detection kit provided by the invention, by adding the magnetic nanoparticle of coated with silica in reagent R1, this magnetic nanoparticle is combined with goat-anti human IgM antibody under the effect of Electrostatic Absorption, thus make antibody form aggegation on magnetic nanoparticle, improve the sensitivity for analysis of reagent greatly.In reagent R2, add bovine serum albumin(BSA) simultaneously, solve antibody this difficult problem unstable in lean solution, it can make antibody stabilization in test, and it is relatively neutral, but the character of antibody can not be affected, and Kathon-CG is a kind of novel high-efficiency environment friendly type wide-spectrum bactericide, antiseptic, in this kit, use Kathon-CG to efficiently solve BSA preserve easily mouldy shortcoming for a long time, therefore BSA and Kathon-CG acting in conjunction effectively enhances the stability of kit, but can not have an impact to the accuracy of reagent, be conducive to this reagent further to promote in the market.

Accompanying drawing explanation

Fig. 1 embodiment 2 accuracy validation laboratory test results and control group testing result correlativity;

Fig. 2 embodiment 3 accuracy validation laboratory test results and control group testing result correlativity;

Fig. 3 embodiment 4 accuracy validation laboratory test results and control group testing result correlativity.

Embodiment

For a better understanding of the present invention, further describe below in conjunction with specific embodiment.

Embodiment 1

An Immunoglobulin IgM immunoturbidimetry detection kit for routine, it comprises reagent R1, reagent R2, calibration object.

Wherein reagent R1 consists of:

PH 7.6 Tris damping fluid 18.16 mmol/L

Sodium chloride 123.20 mmol/L

PEG400 6%

Reagent R2 consists of:

Tris pH of buffer 7.6 18.16 mmol/L

Goat-anti human IgM antibody 30ml/L

The kit that the present embodiment describes, in use, its assay method adopts Toshiba 120 automatic analyzer with double reagent function, operates as follows:

The IgM calibration object that the calibration object that the present embodiment uses is produced for Jiu Fengrunda Bioisystech Co., Ltd.

Embodiment 2

A kind of Immunoglobulin IgM immunoturbidimetry detection kit, it comprises reagent R1, reagent R2, calibration object.

Wherein reagent R1 consists of:

PH 7.6 Tris damping fluid 18.16 mmol/L

Sodium chloride 123.20 mmol/L

PEG400 6%

The magnetic nanoparticle 0.1% of coated with silica

Reagent R2 consists of:

Tris pH of buffer 7.6 18.16 mmol/L

Goat-anti human IgM antibody 30ml/L

Bovine serum albumin(BSA) 20g/L

Kathon-CG 0.05%

Concrete assay method is with embodiment 1.

Embodiment 3

Wherein reagent R1 consists of:

PH 7.6 Tris damping fluid 18.16 mmol/L

Sodium chloride 123.20 mmol/L

PEG400 6%

The magnetic nanoparticle 1% of coated with silica

Reagent R2 consists of:

Tris pH of buffer 7.6 18.16 mmol/L

Goat-anti human IgM antibody 30ml/L

Bovine serum albumin(BSA) 30g/L

Kathon-CG 0.05%

Concrete assay method is with embodiment 1.

Embodiment 4

Wherein reagent R1 consists of:

PH 7.6 Tris damping fluid 18.16 mmol/L

Sodium chloride 123.20 mmol/L

PEG400 6%

The magnetic nanoparticle 2% of coated with silica

Reagent R2 consists of:

Tris pH of buffer 7.6 18.16 mmol/L

Goat-anti human IgM antibody 30ml/L

Bovine serum albumin(BSA) 40g/L

Kathon-CG 0.05%

Concrete assay method is with embodiment 1.

Experimental verification is carried out to kit assay performance obtained in above-described embodiment 1-4.

accuracy validation is tested:

Using the kit of embodiment 2,3,4 as experimental group, the Immunoglobulin IgM kit (production of Changchun company) market obtaining a kind of accuracy excellence of accreditation carries out contrast experiment as a control group, detect 40 samples, the result of detection is as Fig. 1-Fig. 3.

Known by the detection data of Fig. 1-Fig. 3, the testing result correlativity of embodiment 2,3,4 detection kit and control test kit is respectively 0.998,0.9991,0.9981, correlativity is relatively good, show that kit of the present invention and market obtaining the Immunoglobulin IgM detection kit with excellent accuracy approved has high consistency, prove that other various compositions that kit of the present invention adds can not impact its accuracy, kit still keeps good accuracy.

linear dependence confirmatory experiment:

Immunoglobulin IgM high level sample is found to be 500 mg/dL, serial dilution is carried out with physiological saline, the sample of preparation 6 variable concentrations, be followed successively by the sample of 37.00 mg/dL, 27.75 mg/dL, 18.50 mg/dL, 9.25 mg/dL, 0 mg/dL concentration, each concentration level various kinds originally measures three times respectively, gets its mean value respectively.The reagent of embodiment 1,2,3,4 is utilized to detect respectively.Testing result is as shown in table 1.

Table 1 embodiment 1-4 linear correlation confirmatory experiment testing result

Theoretical concentration (mg/dL)	Embodiment 1 testing result (mg/dL)	Embodiment 2 testing result (mg/dL)	Embodiment 3 testing result (mg/dL)	Embodiment 4 testing result (mg/dL)
					500	494.94	503.29	505.44	496.72
400	401.21	406.77	398.42	405.71
					300	288.30	297.48	300.28	298.87
200	204.22	200.03	197.67	201.59
					100	99.23	100.95	99.69	101.29
0.00	0.17	0.23	0.21	0.20
					Correlation coefficient r	0.9990	0.9997	0.9998	0.9997

Above-mentioned testing result display, embodiment 1-4 testing result correlativity is all greater than 0.990, but the testing result of embodiment 2,3,4 is greater than 0.997, has better linear dependence compared with embodiment 1, and this illustrates that reagent of the present invention has better linear dependence.

stability confirmatory experiment:

2 DEG C ~ 8 DEG C, store reagents in the light protected environment of non-corrosiveness gas, detect the stability of four kinds of embodiment reagent.Four kinds of reagent are monthly chosen same sample and are measured its absorbance three times, average, and contrast, thus determine the stabilization time of reagent with fresh embodiment 1 reagent testing result.Detect data as table 2.

Table 2 stability confirmatory experiment testing result

Time	Embodiment 1 reagent testing result	Embodiment 2 reagent testing result	Embodiment 3 reagent testing result	Embodiment 4 reagent testing result	Fresh embodiment 1 reagent testing result
						12 months	0.02457	0.02506	0.02487	0.02498	0.02479
13 months	0.03528	0.03587	0.03616	0.03632	0.03622
						14 months	0.04346	0.04188	0.04214	0.04204	0.04174
15 months	0.03328	0.03398	0.03382	0.03358	0.03407
						16 months	0.01967	0.02943	0.02967	0.03001	0.02957
17 months	0.00684	0.02734	0.02686	0.02691	0.02729
						18 months	0.00032	0.04826	0.04757	0.04811	0.04843
19 months	0.00002	0.03725	0.03733	0.03688	0.03698
						20 months	0.00001	0.04685	0.04596	0.04623	0.04703
21 months	0.00000	0.05374	0.05421	0.05417	0.05461
						22 months	0.00000	0.03284	0.03314	0.03321	0.03358
23 months	0.00000	0.03263	0.03198	0.03167	0.03265
						24 months	0.00000	0.02178	0.02213	0.02121	0.02235
25 months	0.00000	0.03357	0.03443	0.03500	0.04232
						26 months	0.00000	0.01365	0.01932	0.02397	0.05328

Experimental result shows, embodiment 1 reagent 2 DEG C ~ 8 DEG C, in the light protected environment of non-corrosiveness gas storage within 15 months, stablize, and embodiment 2,3,4 reagent 2 DEG C ~ 8 DEG C, in the light protected environment of non-corrosiveness gas storage within 24 months, stablize, illustrate and add bovine serum albumin(BSA) and Kathon-CG in reagent, the two acting in conjunction effectively raises the stability of Immunoglobulin IgM detection kit.

sensitivity for analysis confirmatory experiment:

With the sample of Immunoglobulin IgM kit test concentration known of the present invention at 170mg/dL, record absorbance difference.The reagent of embodiment 1,2,3,4 is utilized to detect respectively.Testing result is as shown in table 3.

Table 3 sensitivity for analysis experimental result

Theoretical concentration (mg/dL)	Embodiment 1 testing result Δ A	Embodiment 2 testing result Δ A	Embodiment 3 testing result Δ A	Embodiment 4 testing result Δ A
					170	0.1968	0.2561	0.2498	0.2514

Known by detecting data, the absorbance difference of embodiment 2,3,4 detection kit is all than the height of embodiment 1, illustrate that the magnetic nanoparticle adding coated with silica in reagent is combined with goat-anti human IgM antibody under the effect of Electrostatic Absorption, thus make antibody form aggegation on magnetic nanoparticle, expand response signal, improve the sensitivity for analysis of reagent greatly.

Comprehensive above analysis, Immunoglobulin IgM immunoturbidimetry detection kit provided by the invention, by adding the magnetic nanoparticle of a certain amount of coated with silica and add stability and the sensitivity for analysis that a certain amount of bovine serum albumin(BSA) and Kathon-CG effectively can improve kit in reagent R2 in reagent R1, the range of linearity is better, and the accuracy of reagent is also better.Therefore, Immunoglobulin IgM immunoturbidimetry detection kit provided by the invention is conducive to further promoting the use of in the market.

Claims

1. an Immunoglobulin IgM immunoturbidimetry detection kit, it is characterized in that, it comprises reagent R1, reagent R2 and calibration object, and wherein reagent R1 consists of: the magnetic nanoparticle of 18.16mmol/L pH 7.6 Tris damping fluid, 123.20mmol/L sodium chloride, 6% PEG400,0.1%-2% coated with silica; Reagent R2 consists of: 18.16mmol/L pH 7.6Tris damping fluid, 30ml/L goat-anti human IgM antibody, 20-40g/L bovine serum albumin(BSA), 0.05% Kathon-CG.

2. kit according to claim 1, is characterized in that, the magnetic nanoparticle of described coated with silica is Fe ₃o ₄/ SiO ₂composite nanoparticle, particle diameter is 20-50nm.

3. kit according to claim 1, is characterized in that, described reagent R1 and reagent R2 ratio are in use R1:R2=250:50.