CN102735845A - Novel detection method and kit for synchronously detecting concentration of free kappa light chain and free lambda light chain - Google Patents
Novel detection method and kit for synchronously detecting concentration of free kappa light chain and free lambda light chain Download PDFInfo
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Abstract
The invention discloses a time-resolved fluoroimmunoassay (TRFIA) kit and a detection method thereof for synchronously detecting the concentration of a free kappa light chain and a free lambda light chain. The kit comprises: 1) an antibody synchronously aiming at a heavy chain and a light chain of a human antibody, 2) an anti-free kappa light chain antibody labelled by lanthanide, 3) an anti-free lambda light chain antibody labelled by another lanthanide, 4) a standard substance of the free kappa light chain and a standard substance of the free lambda light chain, 5) a buffer, 6) a washing liquid, and 7) an enhancement solution. According to the invention, the antibody synchronously aiming at the heavy chain and the light chain of the human antibody is used as a capture antibody, other two antibodies respectively aiming at the free kappa light chain and the free lambda light chain are used as the labelled antibodies, double labeling TRFIA detection principles are used, thus the novel detection method for synchronously detecting the concentration of the free kappa light chain and the free lambda light chain is established. The method provided by the invention has the advantages of high sensitivity, strong specificity and good stability, and can realize high degrees of automation.
Description
Technical field
The invention belongs to field of immunodetection, the time-resolved fluorescence immunoassay method and the kit thereof of free kappa light chain of particularly a kind of synchronous detection and free lambda light chain.
Background technology
Immunoglobulin (Ig) (Ig) light chain is divided into κ (kappa) and 2 types of λ (lambda), has only the light chain of a type on each Ig molecule, and the ratio of human κ (kappa) and λ (lambda) is 6: 4.Light chain is the free small protein through GBM of ability, is heavily absorbed at renal tubule and gets back in the blood circulation, so have only a small amount of light chain existence in normal person's urine.When metabolism disorder and Huppert's disease take place, a large amount of free light chains can occur in the blood, and, be Bence Jones protein (Bence-Jones albumen) by discharging in the urine.
Existing significant basis and clinical research show that the free serum light chain detects, and can be used for the MG primary dcreening operation, and its susceptibility is 88%～98%; Can be used for AL amyloidosis disease early diagnosis; Can be used for conventional electrophoresis method can't detected not secreted Huppert's disease (NSMM) patient detect, and its susceptibility reaches 65%～70%; In addition myeloma state of an illness progress is had important prognosis effect, can carry out the rapid evaluation of risk stratification and result of treatment, also can be used for the monitoring of whether recurring behind chemotherapy or self autologous peripheral blood stemcell transplant not qualitative MG patient.
Normal human serum free light chain concentration is 3～19mg/L, and wherein λ type free light chain is 6～26mg/L, and κ/λ ratio is 0.26～1.65.Up to the present, still do not have the international referene preparation of free light chain on the market, detection method is ununified yet, so the testing result of different manufacturers kit is not had a comparability yet.Detection method commonly used in the market is an immunoturbidimetry, exists sensitivity and precision all not ideal enough and can produce shortcoming such as hook effect because of the antigen amount is excessive.
Time resolved fluoro-immunoassay method (TRFIA) is the new immunoassay that last century, early eighties grew up.It is a kind of special fluorescence analysis that on the basis of fluoroimmunoassay, grows up.Fluorescence analysis has utilized the greatest differences of wavelength of fluorescence and its excitation wavelength, but when carrying out ultramicro-analysis, the parasitic light of exciting light can have a strong impact on the detection to fluorescence.Solve the best method of stray light yes the existence that does not have exciting light when measuring.But the fluorescence lifetime of common fluorescent marker is very short, and exciting light disappears, and fluorescence also disappears.But there is the fluorescence lifetime of considerably less rare earth metal (Eu, Tb, Sm, Dy) longer; Can reach 1/2ms, can satisfy measurement requirement, therefore produce time-resolved fluoroimmunoassay; Promptly use long-acting fluorescent marker, after closing exciting light, measure the analytical approach of fluorescence intensity again.The used fluorescent marker of TRFIA is a lanthanide chelate, utilizes the long and big characteristics of Stokes (Stokes) displacement of this type fluorescent material fluorescence lifetime, through wavelength and time two kinds of resolution techniques; Effectively got rid of the interference of non-special background fluorescence, had highly sensitively, label preparation is simple; Good stability; Advantages such as the typical curve range of linearity is wide, and is easy to operate become the most rising ultramicro-analysis technology and current labelled immune analysis and develop into the representative of new stage.In practical application, also in fluorescent marker, add a kind of enhancing liquid (Enhancement solution) usually, can strengthen up to a million times of fluorescent effects.
At present, also do not adopt TRFIA to detect the technology of free lambda light chain, more lack of synchronization detects the technology of free kappa light chain and free lambda light chain.
Summary of the invention
Therefore, the technical matters that the present invention will solve is exactly in the existing method, and free kappa light chain and free lambda light chain are detected respectively; Testing process is complicated and loaded down with trivial details; Sensitivity and precision are all not ideal enough and can produce shortcoming such as hook effect because of the antigen amount is excessive, and the method and the kit thereof of free kappa light chain of a kind of synchronous detection and free lambda light chain is provided, and this detection method and kit thereof are highly sensitive; High specificity; Good stability, simple to operate, fast, can greatly improve diagnosis efficiency.
The inventor is through lot of test and research, and pleasantly surprised discovery utilizes TRFIA can realize immune double-tagging analysis with comparalive ease, measures free kappa light chain and free lambda light chain simultaneously, thereby accomplishes the present invention.
Therefore, the present invention solves the problems of the technologies described above one of technical scheme of being adopted and is, a kind of kit that is used for the time resolved fluoro-immunoassay of free kappa light chain of synchronous detection and free lambda light chain concentration comprises following reagent:
1) simultaneously to the antibody of human antibody heavy chain and light chain,
2) with free kappa light chain antibody of resisting of a kind of lanthanide series mark,
3) with free lambda light chain antibody of resisting of another kind of lanthanide series mark,
4) standard items of free kappa light chain and free lambda light chain,
5) damping fluid,
6) cleansing solution,
7) strengthen liquid.
According to the present invention, reagent 1) be the antibody that is directed against human antibody heavy chain and light chain simultaneously.The described antibody that is directed against human antibody heavy chain and light chain simultaneously is the polyclonal antibody from the inhuman mammiferous anti-human IgG antibody in source.Preferably, the described antibody that is directed against human antibody heavy chain and light chain simultaneously is the anti-people's antibody of rabbit, more preferably is rabbit anti-human igg H&L polyclonal antibody.
Preferably, the described antibody that is directed against human antibody heavy chain and light chain simultaneously is insolubilized antibody.The solid phase carrier of said insolubilized antibody is preferably selected the low high adsorption reaction plate of fluorescence background, preferred 96 orifice plates or 48 orifice plates for use.Described insolubilized antibody can adopt the method preparation of the conventional antibody sandwich solid phase carrier in this area; Preferably comprise the steps: to adopt said reaction plate as solid phase carrier; With the said antibody sandwich reaction plate that is directed against human antibody heavy chain and light chain simultaneously; With the confining liquid sealing, vacuum is drained then, promptly gets insolubilized antibody again.Preferably, when encapsulating, the said antibody that is directed against human antibody heavy chain and light chain simultaneously is diluted to 1～10mg/L as coating buffer with damping fluid; Said damping fluid is that pH is 9.0～9.8 Na
2CO
3-NaHCO
3Damping fluid; Said confining liquid is 7.0～7.4 Tris-HCl damping fluid for 50mmol/L, the pH that contains 0.2 (w/v) % gelatin and 2 (w/v) % sucrose.After vacuum was drained, the insolubilized antibody of gained (like reaction plate) preferably can be placed on-20 ℃ of freezing preservations in sealing.
According to the present invention, reagent 2) be anti-free kappa light chain antibody with a kind of lanthanide series mark.Described anti-free kappa light chain antibody is from mammiferous monoclonal antibody in inhuman source or polyclonal antibody; Preferably, described anti-free kappa light chain antibody is anti-free kappa light chain polyclonal antibody, and it only combines with the kappa light chain of free form, does not all combine with complete antibody and heavy chain district thereof and light chain district.More preferably, described anti-free kappa light chain antibody is the free kappa light chain polyclonal antibody of the anti-people of rabbit.Described anti-free kappa light chain antibody can adopt the conventional protein labeling method preparation in this area, preferably comprises the steps: to use R
3+-N
2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (R
3+-DTTA) with anti-free kappa light chain antibody response, wherein R represent europium or samarium, reactant liquor passes through column chromatographic isolation and purification, promptly gets.Wherein, described anti-free kappa light chain antibody and R
3+The mass ratio of-DTTA preferably is 1: 0.2～0.5.
According to the present invention, reagent 3) be anti-free lambda light chain antibody with another kind of lanthanide series mark.Equally, said anti-free lambda light chain antibody is from mammiferous monoclonal antibody in inhuman source or polyclonal antibody; Preferably, described anti-lambda light chain antibody is polyclonal antibody, and it only combines with the lambda light chain of free form, does not combine with complete antibody and heavy chain district thereof and light chain district.More preferably, described anti-free lambda light chain antibody is the free lambda light chain polyclonal antibody of the anti-people of rabbit.Described anti-free lambda light chain antibody can adopt the conventional labeling method preparation in this area, preferably comprises the steps: to use R
3+-DTTA and anti-free lambda light chain polyclonal antibody reaction, wherein R represents samarium or europium, and reactant liquor promptly gets through column chromatographic isolation and purification.Wherein, described anti-free lambda light chain polyclonal antibody and R
3+The mass ratio of-DTTA preferably is 1: 0.2～0.5.
According to the present invention; Reagent 2) described anti-free kappa light chain antibody; Having a kind of lanthanide series mark, reagent 3) described anti-free lambda light chain antibody has another kind of lanthanide series mark, and described lanthanide series preferably is selected from any in europium and the samarium; In same kit, the lanthanide series of the said anti-free kappa light chain antibody of mark and the lanthanide series of said anti-free lambda light chain antibody are inequality.
According to the present invention, reagent 4) be the standard items of free kappa light chain and free lambda light chain.Preferably, described standard items are the hybrid standard article of free kappa light chain and free lambda light chain.More preferably, described standard items are the hybrid standard article of said free lambda light chain that comprise said free kappa light chain and the same concentrations of 0～600mg/L.Best, described hybrid standard article comprise the said free kappa light chain of following concentration and the said free lambda light chain of same concentrations: 0mg/L, 5mg/L, 20mg/L, 100mg/L, 200mg/L, 500mg/L.
According to the present invention, reagent 5) be damping fluid.Preferably, described damping fluid is the Tris-HCl damping fluid; More preferably, described damping fluid is for containing 8mmol/L NaCl, 0.1wt% gelatin, 0.2wt%IgG, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s, 0.1ml/L Tween-80 and 0.1wt%NaN
350mmol/L, the Tris-HCl damping fluid of pH7.2.
According to the present invention, reagent 6) be cleansing solution.Preferably, described cleansing solution is the Tris-HCl damping fluid; More preferably, described cleansing solution is for containing 14.5mmol/L NaCl, 0.2ml/L Tween-80 and 0.2wt%NaN
350mmol/L, the Tris-HCl damping fluid of pH7.2.
According to the present invention, reagent 7) for strengthening liquid.Preferably, described enhancing liquid is the damping fluid that contains beta-diketon; More preferably, described enhancing liquid is the Potassium Hydrogen Phthalate damping fluid that contains the pH3.2 of 15 μ mol/L β-naphthoyltrifluoroacetones, 50 μ mol/L TOPOs and 0.1 (v/v) %TritonX-100.
The present invention solves the problems of the technologies described above two of the technical scheme that adopted, and a kind of time-resolved fluorescence immunoassay method (TRFIA) according to free kappa light chain of above-mentioned kit synchronous detection and free lambda light chain concentration may further comprise the steps successively:
1) in the solid phase carrier that is coated with the antibody that is directed against human antibody heavy chain and light chain simultaneously, add free kappa light chain and free lambda light chain standard items or testing sample and damping fluid, hatch;
2) with after the cleansing solution washing, add with a kind of anti-free kappa light chain antibody of lanthanide series mark with free lambda light chain antibody of resisting of another kind of lanthanide series mark, hatch;
3) with the cleansing solution washing, add enhancing liquid then and react, carry out time-resolved fluorescence afterwards and detect.
The present invention uses the antibody that is directed against human antibody heavy chain and light chain simultaneously as capture antibody; Only use respectively other two kinds of antibody to free kappa light chain and free lambda light chain antibody that serves as a mark; Utilize the double-tagging time resolved fluoro-immunoassay to detect principle, foundation can detect the novel detection method of free kappa light chain and free lambda light chain concentration simultaneously.Preferably, it is capture antibody that the present invention adopts rabbit anti-human igg H&L polyclonal antibody, adopts Sm
3+Free lambda light chain antibody of the anti-people of mark and Eu
3+The free kappa light chain antibody of the anti-people of mark is labelled antibody, and the principle of its detection is as shown in Figure 1.
According to the present invention, step 1) is: in the solid phase carrier that is coated with the antibody that is directed against human antibody heavy chain and light chain simultaneously, add free kappa light chain and free lambda light chain standard items or testing sample and damping fluid, hatch.Wherein, said solid phase carrier preferably is the low high adsorption reaction plate of fluorescence background, is about to the described antibody sandwich that is directed against human antibody heavy chain and light chain simultaneously on reaction plate; Said condition of hatching preferably is 25～37 ℃, 1～2 hour with conventional.After hatching, the antibody to human antibody heavy chain and light chain in the time of the free kappa light chain in said free kappa light chain and free lambda light chain standard items or the testing sample and free lambda light chain and solid phase forms immune complex.
According to the present invention, step 2) be: after the cleansing solution washing, add, hatch with a kind of anti-free kappa light chain antibody of lanthanide series mark with free lambda light chain antibody of resisting of another kind of lanthanide series mark.Wherein, described anti-free kappa light chain antibody has a kind of lanthanide series mark, and described anti-free lambda light chain antibody has another kind of lanthanide series mark, and described lanthanide series preferably is selected from europium and samarium; With in detecting operation, the lanthanide series of the described anti-free kappa light chain antibody of mark is inequality with the lanthanide series of the described anti-lambda light chain antibody that dissociates of mark.
Because lanthanide series is relatively large in be stimulated back emission wavelength and fluorescence lifetime, the time resolved fluoro-immunoassay Guttae Phacosylini is different with these lanthanide series wavelength and fluorescence lifetime, and the label difference is come.See from emission wavelength, if select Eu for use
3+(615nm) and Tb
3+(544nm) do double-tagging pairing, distinguishes more obvious, but to measure them simultaneously, just need are with fluoride fat beta-diketon sequestrant, and it is to Eu
3+The mensuration effect relatively poor, thereby, Eu
3+With Tb
3+When doing the double-tagging pairing, measure result and bad.Eu
3+With Sm
3+Though maximum emission wavelength only differ about 30nm, because of being mostly narrow emission peak, can be by abundant resolution, under the effect of 340nm exciting light, both can both form highly fluorescigenic chelate with β-NTA (beta-diketon), and their fluorescence lifetime differs significantly (Sm
3+Be 50s, Eu
3+Be 730s), can fully differentiate through time delay.Therefore, preferred Eu
3+And Sm
3+Double-tagging as pairing is used for the time resolved fluoro-immunoassay method.
According to the present invention; Step 2) be for washing step 1 with cleansing solution in) hatch after solid phase carrier; Described solid phase carrier is uploaded the described antibody that is directed against human antibody heavy chain and light chain simultaneously; It is capture antibody; The free kappa light chain of described capture antibody and adding and the free kappa light chain in free lambda light chain hybrid standard article or the testing sample and free lambda light chain (being antigen) combine because of the effect of Ag-Ab, and free not with other compositions of capture antibody reaction, remove with the cleansing solution washing.
Among the present invention; As antigen; Free kappa light chain in free kappa light chain and the free lambda light chain compound and free lambda light chain all with solid phase carrier on the time to the antibodies of human antibody heavy chain and light chain; But these free light chains can also combine other anti-free light chain antibody, i.e. steps 2 at other epitope) described anti-free kappa light chain antibody and anti-free lambda light chain antibody.Through after hatching, formed sandwich complex respectively.The method of hatching preferably is 25～37 ℃, 1～2 hour with conventional.
According to the present invention, step 3) is: with the cleansing solution washing, add enhancing liquid then and react, carry out time-resolved fluorescence afterwards and detect.Wherein, described cleansing solution is in order to remove free anti-free kappa light chain antibody and anti-free lambda light chain antibody with the cleansing solution washing as stated.
Among the present invention, strengthen beta-diketon in the liquid fluorescence signal that can double.After adding enhancing liquid carries out oscillating reactions, form highly fluorescigenic chelate.The temperature and time of reaction, preferable is 25～37 ℃, 5 minutes.Under the effect of 340nm exciting light, the very strong fluorescence of formed chelate emission, Sm
3+Fluorescence lifetime be 50s, Eu
3+Fluorescence lifetime be 730s, differentiate fluorescent instrument with the time and measure its fluorescence intensity.Fluorescence intensity respectively with sample in free kappa light chain and free lambda light chain concentration be directly proportional, the reference standard curve can be confirmed the amount of antigen in the sample.
Raw material that the present invention is used or reagent except that specifying, all commercially available getting.
Than prior art, beneficial effect of the present invention is following: the present invention adopts high-sensitive time resolved fluoro-immunoassay technology, has set up the method that detects free kappa light chain and free lambda light chain simultaneously.This method is highly sensitive, high specificity, and good stability is specially adapted to serum, the detection of free kappa light chain and free lambda light chain content in urine or other body fluid (like pericardium hydrops, ascites, urine etc.).Agents useful for same cost of the present invention is low; Method of operating is simple, and analytic system can realize increasingly automated, can improve the speed of clinical examination; Can reduce personal error significantly again; Increase the reliability that detects the result, in addition, detect the diagnosis efficiency that free kappa light chain and free lambda light chain also can greatly improve relevant disease simultaneously.
Description of drawings
Below in conjunction with description of drawings characteristic of the present invention and beneficial effect.
Fig. 1 time resolved fluoro-immunoassay detects the principle schematic of free kappa light chain and free lambda light chain simultaneously.
Fig. 2 detects the double-tagging TRFIA typical curve and the precision figure of free kappa light chain and free lambda light chain for the present invention.A figure: free kappa light chain, B figure: free lambda light chain.
Embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer." room temperature " described in the following embodiment is the laboratory temperature of test operation, is 18～25 ℃.
Wherein, in the time of in the following example to the antibody of human antibody heavy chain and light chain available from the rabbit anti-human igg H&L of Abcam company polyclonal antibody (Rabbit anti-Human IgG H&L secondary antibody, ab6715); Anti-free kappa light chain antibody is free kappa light chain polyclonal antibody (the Polyclonal Rabbit Anti-Human kappa Free Light Chains of the anti-people of rabbit; Code No./Code/Code-Nr.A0101); Available from dako company; Anti-free lambda light chain antibody is that the free lambda light chain polyclonal antibody (Polyclonal Rabbit Anti-Human Lambda Free Light Chains, Code No./Code/Code-Nr.A0101) of the anti-people of rabbit is available from dako company; 96 hole microwell plates (8 * 12) are the labsystem Company products; Eu
3+And Sm
3+Marker cassette is the PerkerElmer Company products; Sephadex G-50 is the Amersham product; It is pure that other reagent are homemade analysis; Full-automatic TRFIA detector (Auto DELFIA 1235) is the Wallac product.
The preparation of embodiment 1 kit
Reagent in each box enough carries out 96 measurements, and the material in the box is following:
(1) 1 * 96 orifice plate (8 * 12 hole can be split as single hole) is coated with rabbit anti-human igg H&L polyclonal antibody.
(2) 6 * free kappa light chain/free lambda light chain hybrid standard article, dried frozen aquatic products comprises 6 concentration: 0mg/L, 5mg/L, 20mg/L, 100mg/L, 200mg/L, 500mg/L.Respectively use the 1ml dissolved in distilled water for every bottle before the use.
(3) 1 * europium labelled antibodies 1 dried frozen aquatic products is used the 0.5ml dissolved in distilled water during use.
(4) 1 * samarium labelled antibodies 2 dried frozen aquatic productses are used the 0.5ml dissolved in distilled water during use.
(5) 1 * damping fluids, 30ml.
(6) 1 * cleansing solutions, 30ml dilutes by 1: 25 with distilled water during use.
(7) 1 * enhancing liquid, 15ml.
Be contained concrete composition of each material in the kit and preparation method thereof below.
1, micro reaction plate preparation
With 50mmol/L pH 9.6Na
2CO
3-NaHCO
3Damping fluid rabbit anti-human igg H&L polyclonal antibody is diluted to 1 μ g/mL as coating buffer; The every hole of 96 hole microwell plates adds 100 μ l; 4 ℃ of placements are spent the night, and discard coating buffer, with cleansing solution (14.5mmol/L NaCl, 0.2ml/L Tween-80 and 0.2wt%NaN
350mmol/L Tris-HCl pH7.2) flushing four times; Add the Tris-HCl damping fluid sealing that 200 μ l contain the 50mmol/L pH7.2 of 0.2wt% gelatin and the multitudinous sugar of 2wt%, 4 ℃ of placements are spent the night, and discard confining liquid; Vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing.
2, free kappa light chain/free lambda light chain hybrid standard article preparation
With commercially available free kappa light chain antibody and free lambda light chain pure antibody article, with containing 0.2wt%BSA, the 50mmol/L pH7.8Tris-Hcl damping fluid of 0.1wt%NaN3 is made into above-mentioned definite value serum the potpourri of the free kappa light chain of dissociate comprising of following 6 concentration kappa light chain and same concentrations: 0mg/L; 5mg/L; 20mg/L, 100mg/L, 200mg/L; 500mg/L; Thereby constituted the hybrid standard article of series concentration, then with the hybrid standard article of every kind of concentration with every bottle of 1ml packing freeze-drying ,-20 ℃ of kept dry.
The hybrid standard article that this each bottle freeze-drying is preserved can each be used the 1ml dissolved in distilled water during use, and free kappa light chain is identical with the concentration of free lambda light chain in the standard solution that obtains; Be respectively: 0mg/L, 5mg/L, 20mg/L; 100mg/L, 200mg/L, 500mg/L.
3, Eu
3+The free kappa light chain polyclonal antibody preparation of the anti-people of the rabbit of mark
With reference to Eu
3+The operation of labelling kit (PerkerElmer company of production firm) instructions.Be specially: get the free kappa light chain polyclonal antibody 1mg of the anti-people of rabbit and be dissolved in 0.25mol/L NaHCO
3Among the liquid 200 μ l, add 0.2mg Eu
3+-N
2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (Eu
3+-DTTA) abundant mixing, 4 ℃ of reactions 24 hours.(1 * 20cm) chromatography, protein peak is collected in the A280 monitoring to reactant liquor, merges the peak pipe, makes protein content with Sephadex G-50 post.Use the Eu of PerkinElmer company simultaneously
3+Titer is measured the Eu that merges the peak
3+Concentration.Mark rate (the Eu of gained
3+Mol/IgG mol) be 8.91, protein recovery is 87%.The solution dilution of collecting is become Eu
3+The free kappa light chain polyclonal antibody concentration of the anti-people of the rabbit of mark is 0.75mmol/L, every bottle of 0.5ml packing ,-20 ℃ of kept dry.
4, Sm
3+The free lambda light chain polyclonal antibody preparation of the anti-people of mark rabbit
With reference to Sm
3+The operation of labelling kit (PerkerElmer company of production firm) instructions.Be specially: get the free lambda light chain polyclonal antibody 1mg of the anti-people of rabbit and be dissolved in 0.25mol/L NaHCO
3Among the liquid 200 μ l, add 0.2mg Sm
3+-N
2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (Sm
3+-DTTA) abundant mixing, 4 ℃ of reactions 24 hours.(1 * 20cm) chromatography, protein peak is collected in the A280 monitoring to reactant liquor, merges the peak pipe, makes protein content with Sephadex G-50 post.Use the Sm of PerkinElmer company simultaneously
3+Titer is measured the Sm that merges the peak
3+Concentration.The solution dilution of collecting is become Sm
3+The free lambda light chain polyclonal antibody concentration of the anti-people of mark rabbit is 0.73mmol/L, every bottle of 0.5ml packing, and-20 ℃ of kept dry are the Sm in the kit
3+The anti-people of the mark rabbit lambda light chain polyclonal antibody dried frozen aquatic products that dissociates.
5, damping fluid: the 50mmol/L Tris-HClpH7.2 of 8mmol/L NaCl, 0.1wt% gelatin, 0.2wt%IgG, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s, 0.1ml/L Tween-80 and 0.1wt%NaN3.
6, cleansing solution: 14.5mmol/L NaCl, 0.2ml/L Tween-80 and 0.2wt%NaN
350mmol/L Tris-HCl pH7.2.
7, strengthen liquid: 1 liter of pH3.2 Potassium Hydrogen Phthalate damping fluid contains 15 μ mol β-naphthoyltrifluoroacetones, 50 μ mol TOPO and 1mlTritonX-100.
Points for attention are following before using this kit measurement:
(1) before the use all reagent is taken out to room temperature (18～30 ℃)
(2) immediately all reagent are put back to 2～8 ℃ of preservations after the use.
(3) if the big suggestion of sample size moves device with hyperchannel, to reduce the difference of each sample on the reaction time.
(4) hatch in the process at all constant temperature, avoid irradiate light.
(5) taking-up needs the microwell plate with quantity, no microwell plate is put in the former Fresco Bag and with the drying agent that provides reseal, and is stored in 2～8 ℃.
The preparation of embodiment 2 kits
Material in the kit: except that not containing 96 orifice plates that are coated with rabbit anti-human igg H&L polyclonal antibody, other same embodiment 1.
Each preparation methods in the kit is following:
(1) Eu
3+The free kappa light chain polyclonal antibody preparation of the anti-people of the rabbit of mark
With reference to Eu
3+The operation of labelling kit (PerkerElmer company of production firm) instructions.Be specially: get the free kappa light chain polyclonal antibody 1mg of the anti-people of rabbit and be dissolved in 0.25mol/L NaHCO
3Among the liquid 200 μ l, add 0.2mg Eu
3+-N
2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (Eu
3+-DTTA) abundant mixing, 4 ℃ of reactions 24 hours.(1 * 20cm) chromatography, protein peak is collected in the A280 monitoring to reactant liquor, merges the peak pipe, makes protein content with Sephadex G-50 post.Use the Eu of PerkinElmer company simultaneously
3+Titer is measured the Eu that merges the peak
3+Concentration.Mark rate (the Eu of gained
3+Mol/IgG mol) be 6.91, protein recovery is 77%.The solution dilution of collecting is become Eu
3+The free kappa light chain polyclonal antibody concentration of the anti-people of the rabbit of mark is 0.95mmol/L, every bottle of 0.5ml packing ,-20 ℃ of kept dry.
(2) Sm
3+The free lambda light chain polyclonal antibody preparation of the anti-people of mark rabbit
With reference to Sm
3+The operation of labelling kit (PerkerElmer company of production firm) instructions.Be specially: get the free lambda light chain polyclonal antibody 1mg of the anti-people of rabbit and be dissolved in 0.25mol/L NaHCO
3Among the liquid 200 μ l, add 0.2mg Sm
3+-N
2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (Sm
3+-DTTA) abundant mixing, 4 ℃ of reactions 24 hours.(1 * 20cm) chromatography, protein peak is collected in the A280 monitoring to reactant liquor, merges the peak pipe, makes protein content with Sephadex G-50 post.Use the Sm of PerkinElmer company simultaneously
3+Titer is measured the Sm that merges the peak
3+Concentration.The solution dilution of collecting is become Sm
3+The free lambda light chain polyclonal antibody concentration of the anti-people of mark rabbit is 0.88mmol/L, every bottle of 0.5ml packing, and-20 ℃ of kept dry are the Sm in the kit
3+The anti-people of the mark rabbit lambda light chain polyclonal antibody dried frozen aquatic products that dissociates.
(3) preparation method of the other materials in the kit (cleansing solution strengthens liquid for free kappa light chain/free lambda light chain hybrid standard article, damping fluid) is all with embodiment 1.
The preparation of embodiment 3 kits
Reagent in each box enough carries out 48 measurements, and the material in the box is following:
(1) 1 * 48 orifice plate (4 * 12 hole can be split as single hole) is coated with the free kappa light chain monoclonal antibody of mouse-anti people.
(2) 6 * free kappa light chain and free lambda light chain hybrid standard article, dried frozen aquatic products comprises 6 concentration: 0mg/L, 5mg/L, 20mg/L, 100mg/L, 200mg/L, 500mg/L.Respectively use the 1ml dissolved in distilled water for every bottle before the use.
(3) 1 * europium labelled antibody dried frozen aquatic productses are used the 0.5ml dissolved in distilled water during use.
(4) 1 * samarium labelled antibody dried frozen aquatic productses are used the 0.5ml dissolved in distilled water during use.
(5) 1 * damping fluids, 30ml.
(6) 1 * cleansing solutions, 30ml dilutes by 1: 25 with distilled water during use.
(7) 1 * enhancing liquid, 15ml.
Each preparation methods in the kit is following:
1, micro reaction plate preparation
Damping fluid with 50mmol/L pH 9.6Na2CO3-NaHCO3 is diluted to 10 μ g/mL as coating buffer with rabbit anti-human igg H&L polyclonal antibody, and 48 hole microwell plates respectively add 100 μ l, and 4 ℃ of placements are spent the night; Discard coating buffer, wash four times, add the Tris-HCl damping fluid sealing that 200 μ l contain the 50mmol/L pH7.2 of 0.2wt% gelatin and the multitudinous sugar of 2wt%; 4 ℃ of placements are spent the night; Discard confining liquid, vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing.
2, (free kappa light chain/free lambda light chain hybrid standard article, the Eu of the other materials in the kit
3+The mouse-anti people of the mark kappa light chain polyclonal antibody that dissociates, Sm
3+The anti-people of the mark rabbit lambda light chain polyclonal antibody that dissociates, damping fluid, cleansing solution strengthens liquid) the preparation method all with embodiment 1.
Embodiment 4 adopts the concentration of free kappa light chain of TRFIA synchronous detection and free lambda light chain
The kit that adopts embodiment 1 to provide detects.
One, the dilution of each material in the kit of embodiment 1
6 * free kappa light chain/free lambda light chain hybrid standard article, dried frozen aquatic products comprises 6 concentration: 0mg/L, 5mg/L, 20mg/L, 100mg/L, 200mg/L, 500mg/L.Respectively use the 1ml dissolved in distilled water for every bottle before the use.
Europium labelled antibody dried frozen aquatic products is used the 0.5ml dissolved in distilled water.
Samarium labelled antibody dried frozen aquatic products is used the 0.5ml dissolved in distilled water.
Cleansing solution dilutes by 1: 25 with distilled water.
Testing sample 200 examples are taken from the health examination personnel of Renji Hospital Attached to Medical College of Shanghai Jiaotong Univ., all selected personnel, and its hematology and imaging examination are all no abnormal.The 2ml that phlebotomizes on an empty stomach, isolate serum after, be used for the present invention and detect.
Two, detect
Get the micro reaction plate that is coated with rabbit anti-human igg H&L polyclonal antibody of above-mentioned preparation, add in the free kappa light chain of 50 μ l/free lambda light chain hybrid standard article or testing sample to the micropore separately, add 100 μ l damping fluids, room temperature oscillating reactions 1 hour.Add 200 μ l after the washing and make thinning agent, the free kappa light chain polyclonal antibody of the anti-people of rabbit of 1: 100 dilution europium mark and the free lambda light chain polyclonal antibody of the anti-people of rabbit of samarium mark, room temperature oscillating reactions 1 hour with damping fluid.With cleansing solution washing 6 times, add to strengthen and measure fluorescence intensity with full-automatic TRFIA detector after liquid 200 μ l vibrated 5 minutes, the free kappa light chain from typical curve difference calculation sample and the content of free lambda light chain.
The content of free kappa light chain of table 1 normal human serum and free lambda light chain
Three, the TRFIA performance index examination of free kappa light chain/free lambda light chain
1. the sensitivity and the range of linearity
The free kappa light chain/TRFIA representative standard curve of free lambda light chain is seen Fig. 2 with the coefficient of variation of each concentration value.Each concentration point repeats to survey 10 times, calculates to become variable coefficient.Fig. 2 shows that the Variation Lines number average of each standard items concentration value is less than 10%.
Be used as sample measurement 20 times with zero normative reference article; Calculate its fluorescence average and standard deviation; Deducting the concentration value that the fluorescent value substitution typical curve Equation for Calculating of 2 times of standard deviation gained draws with this fluorescent determining value and be its sensitivity, is 0.81mg/L (free kappa light chain) and 0.35mg/L (the lambda light chain dissociates) through measuring this assay sensitivity.
2. accuracy (recovery test) is free kappa light chain concentration with the series standard article dilution of the freeze-drying among the embodiment 1: 11.89; 51.06 110.33mg/L is added to that (its free kappa light chain concentration is followed successively by 28.65 in the serum sample of 5 known free kappa light chain concentration; 56.74; 90.92,154.56,299.68mg/L).Free lambda light chain concentration: 10.67,58.81,226.03mg/L, be added in the blood serum sample of 5 known free lambda light chain concentration (10.59,23.13,58.63,116.43,277.45mg/L.Through calculating the ratio of measured value and theoretical value, draw this experiment and can survey its recovery in the scope.Free kappa light chain is 95.32%-101.6%; Free lambda light chain is 96.78%-101.24%.
3. precision is got 7 parts of serum samples that contain free kappa light chain of variable concentrations and free lambda light chain; Sample source and processing are with embodiment 4; By identical experiment condition; Duplicate detection is fixed 10 times in 10 days, and every part of sample repeats to survey 10 times when testing at every turn, calculates the coefficient of variation between batch interior criticizing.Variation within batch coefficient of this method and interassay coefficient of variation meet the requirement of kit regulation all less than 10%.
Claims (10)
1. a kit that is used for the time resolved fluoro-immunoassay of free kappa light chain of synchronous detection and free lambda light chain concentration is characterized in that, comprises following reagent:
1) simultaneously to the antibody of human antibody heavy chain and light chain,
2) with free kappa light chain antibody of resisting of a kind of lanthanide series mark,
3) with free lambda light chain antibody of resisting of another kind of lanthanide series mark,
4) standard items of free kappa light chain and free lambda light chain,
5) damping fluid,
6) cleansing solution,
7) strengthen liquid.
2. kit as claimed in claim 1 is characterized in that, the described antibody that is directed against human antibody heavy chain and light chain simultaneously is the polyclonal antibody from the inhuman mammiferous anti-human IgG antibody in source.
3. kit as claimed in claim 1 is characterized in that, the described antibody that is directed against human antibody heavy chain and light chain simultaneously is insolubilized antibody; The solid phase carrier of said insolubilized antibody is the low high adsorption reaction plate of fluorescence background.
4. kit as claimed in claim 1 is characterized in that, reagent 2) the anti-free kappa light chain antibody of a kind of lanthanide series mark of described usefulness is monoclonal antibody or polyclonal antibody from the free kappa light chain of the mammiferous anti-people in inhuman source; Reagent 3) the anti-free lambda light chain antibody of the another kind of lanthanide series mark of described usefulness is monoclonal antibody or polyclonal antibody from the free lambda light chain of the mammiferous anti-people in inhuman source.
5. kit as claimed in claim 1 is characterized in that, reagent 4) described standard items are the hybrid standard article of free kappa light chain and free lambda light chain.
6. kit as claimed in claim 5 is characterized in that, described hybrid standard article comprise the said free kappa light chain of 0～600mg/L and the said free lambda light chain of same concentrations.
7. kit as claimed in claim 1 is characterized in that, described lanthanide series is selected from any in europium and the samarium.
8. kit as claimed in claim 1 is characterized in that, reagent 5) described damping fluid is the Tris-HCl damping fluid; Reagent 6) described cleansing solution is the Tris-HCl damping fluid; Reagent 7) described enhancing liquid contains beta-diketon.
9. kit as claimed in claim 1 is characterized in that, reagent 5) described damping fluid is for containing 8mmol/L NaCl, 0.1wt% gelatin, 0.2wt%IgG, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s, 0.1ml/L Tween-80 and 0.1wt%NaN
350mmol/L, the Tris-HCl damping fluid of pH7.2; Reagent 6) described cleansing solution is for containing 14.5mmol/L NaCl, 0.2ml/L Tween-80 and 0.2wt%NaN
350mmol/L, the Tris-HCl damping fluid of pH7.2; Reagent 7) described enhancing liquid is 3.2 Potassium Hydrogen Phthalate damping fluid for the pH that contains 15 μ mol/L β-naphthoyltrifluoroacetones, 50 μ mol/L TOPOs and 0.1 (v/v) %TritonX-100.
10. the time-resolved fluorescence immunoassay method of free kappa light chain of a basis such as each said kit synchronous detection of claim 1～9 and free lambda light chain concentration may further comprise the steps successively:
1) in the solid phase carrier that is coated with the antibody that is directed against human antibody heavy chain and light chain simultaneously, add free kappa light chain and free lambda light chain standard items or testing sample and damping fluid, hatch;
2) with after the cleansing solution washing, add with a kind of anti-free kappa light chain antibody of lanthanide series mark with free lambda light chain antibody of resisting of another kind of lanthanide series mark, hatch;
3) with the cleansing solution washing, add enhancing liquid then and react, carry out time-resolved fluorescence afterwards and detect.
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