CN103728455A - Kit and method for detecting concentration of Kappa free light chains - Google Patents
Kit and method for detecting concentration of Kappa free light chains Download PDFInfo
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Abstract
The invention discloses a kit and a method for detecting the concentration of Kappa free light chains. The kit comprises a reaction buffer solution and a Kappa free light chain antibody solution, wherein the concentration of sodium chloride in the reaction buffer solution is 1250-2400 mmol/L. According to the kit, the problem of poor clinical specimen linearity in a turbidimetry assay of the Kappa free light chains is solved, reliable Kappa free light chain detection results as diagnostic references are provided for clinical doctors, the advantages of high speed and flux of a latex-enhanced turbidimetric immunoassay can be conveniently brought into full play, the detection time is shortened, and the clinical application and patient diagnosis are facilitated.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of kit and method of the Kappa of mensuration free light chain concentration.
Background technology
Immunoglobulin (Ig) (Ig) light chain is divided into κ (kappa) and 2 types of λ (lambda), only has the light chain of a type on each Ig molecule, and the ratio of mankind κ (kappa) and λ (lambda) is 6:4.Light chain can heavily be absorbed and be got back in blood circulation at renal tubule freely by the small protein of glomerular basement membrane, so only have a small amount of light chain to exist in normal person's urine.When human body generation metabolism disorder and Huppert's disease, in blood, there are a large amount of free light chains, and discharge in urine, be Bence-Jones albumen (Bence Jones protein).In normal human, serum κ type free light chain is 3～19mg/L, and λ type free light chain is 6～26mg/L, and κ/λ ratio is 0.26～1.65.The κ type free light chain of abnormal conditions and and κ/λ ratio or abnormal λ type free light chain and κ/λ ratio or the equal situation such as abnormal of three simultaneously, susceptibility to monoclonicity gamma Globulin increase disease (monoclonal gammopathies, MGP) is 88%～98%; To non-secretory myeloma (nonsecretory myeloma, NSM) susceptibility is 65%～70%, contribute to the early diagnosis of monoclonal light chain disease, AL-amyloidosis, also can be used for the monitoring of whether recurring after chemotherapy or self autologous peripheral blood stemcell transplant, if chemotherapy or transplantation effect are good, sufferer serum κ type free light chain, λ type free light chain and κ/λ ratio all have clear improvement at normal level or before comparing treatment.
Latex intensified can be measured κ type free light chain than purifying method on full automatic biochemical apparatus, and speed is fast, flux is high.For the mensuration of free light chain, the domestic main Freelite Series Measurement reagent that uses Britain Binding site company at present, can measure Kappa free light chain than purifying method by latex intensified; But in clinical practice, Freelite reagents series exists a large amount of clinical samples to dilute non-linear problem, and these sample measurement results obviously do not meet clinical symptoms, and hospital applies very limited at home at present.(note: be diluted to linearity and refer to the manual normal saline dilution clinical samples that uses according to a certain percentage, the result of measuring the rear sample of dilution should be with the notional result deviation of calculating according to original concentration and dilution ratio in positive and negative 20%.Linearity is also one of registration basic demand of external diagnosis reagent, and it is insecure conventionally diluting non-linear sample result.Cause the main cause of linear problem to have the anti-blood constituent interference problem of agent prescription, antibody specificity problem etc.）
Meanwhile, inventor uses the Kappa free light chain reagent of preparation in a conventional manner, also occurs diluting non-linear problem condition with the similar clinical samples of Freelite reagent when not using this special inventive method; No matter owing to being the Kappa free light chain reagent that Freelite series Kappa free light chain mensuration reagent or inventor prepare voluntarily, all exist clinical samples to dilute non-linear problem, therefore, can think a whether large common difficult point in the preparation of the linear Kappa of being free light chain turbidimetric assay kit of clinical samples dilution.
Summary of the invention
Technical matters to be solved by this invention is to measure non-linear defect in order to overcome existing Kappa free light chain, and a kind of kit and method of the Kappa of mensuration free light chain concentration is provided.Assay method of the present invention can obtain reliable Kappa free light chain measurement result.
The present invention solves the problems of the technologies described above by the following technical programs:
The invention provides a kind of latex intensified that adopts and than purifying method, measure the kit of Kappa free light chain concentration, it comprises reaction buffer (R1 reagent) and Kappa free light chain antibody-solutions (R2 reagent), it is characterized in that, in described reaction buffer, the concentration of sodium chloride is 1250～2400mmol/L.
Wherein, Kappa free light chain antibody in described Kappa free light chain antibody-solutions is preferably the anti-human polyclone Kappa of rabbit free light chain antibody, be more preferably the anti-human polyclone Kappa of the rabbit free light chain antibody of Dako company (be the Polyclonal Rabbit Anti-Human Kappa Free Light Chains of Dako company, article No. is A0100).
Preferably, when reaction buffer consist of 4-hydroxyethyl piperazine ethanesulfonic acid (Hepes) 100mmol/L, sodium chloride 1250～2400mmol/L, Tween-20 1ml/L and antiseptic Proclin-3000.3ml/L time, the anti-human polyclone Kappa of the rabbit that consists of Dako company free light chain antibody (article No. is A0100) 0.5mg/ml, latex microsphere 3mg/ml, Tween-20 1ml/L and the antiseptic Proclin-3000.3ml/L of Kappa free light chain antibody-solutions.In above-mentioned composition, latex microsphere can be purchased from international well-known latex supplier, as HuoJSR company of Bangslab company.
More preferably, when reaction buffer consist of 4-hydroxyethyl piperazine ethanesulfonic acid (Hepes) 100mmol/L, sodium chloride 1250mmol/L, Tween-20 1ml/L and antiseptic Proclin-3000.3ml/L time, the anti-human polyclone Kappa of the rabbit that consists of Dako company free light chain antibody (article No. is A0100) 0.5mg/ml, latex microsphere 3mg/ml, Tween-20 1ml/L and the antiseptic Proclin-3000.3ml/L of Kappa free light chain antibody-solutions; Or, when reaction buffer consist of 4-hydroxyethyl piperazine ethanesulfonic acid (Hepes) 100mmol/L, sodium chloride 2000mmol/L, Tween-20 1ml/L and antiseptic Proclin-3000.3ml/L time, the anti-human polyclone Kappa of the rabbit that consists of Dako company free light chain antibody (article No. is A0100) 0.5mg/ml, latex microsphere 3mg/ml, Tween-20 1ml/L and the antiseptic Proclin-3000.3ml/L of Kappa free light chain antibody-solutions; Or, when reaction buffer consist of 4-hydroxyethyl piperazine ethanesulfonic acid (Hepes) 100mmol/L, sodium chloride 1500mmol/L, Tween-20 1ml/L and antiseptic Proclin-3000.3ml/L time, the anti-human polyclone Kappa of the rabbit that consists of Dako company free light chain antibody (article No. is A0100) 0.5mg/ml, latex microsphere 3mg/ml, Tween-20 1ml/L and the antiseptic Proclin-3000.3ml/L of Kappa free light chain antibody-solutions; Or, when reaction buffer consist of 4-hydroxyethyl piperazine ethanesulfonic acid (Hepes) 100mmol/L, sodium chloride 2400mmol/L, Tween-20 1ml/L and antiseptic Proclin-3000.3ml/L time, the anti-human polyclone Kappa of the rabbit that consists of Dako company free light chain antibody (article No. is A0100) 0.5mg/ml, latex microsphere 3mg/ml, Tween-20 1ml/L and the antiseptic Proclin-3000.3ml/L of Kappa free light chain antibody-solutions.In above-mentioned composition, latex microsphere can be purchased from international well-known latex supplier, as HuoJSR company of Bangslab company.
The present invention also provides a kind of method of the Kappa of mensuration free light chain concentration, it comprises the steps: to adopt latex intensified to compare purifying method, after testing sample is mixed with above-mentioned reaction buffer and Kappa free light chain antibody-solutions, calculate absorbance changes delta A, relatively can obtain the Kappa free light chain concentration of testing sample with the working curve of standard items, it is characterized in that, the sodium chloride concentration of testing sample and reaction buffer and the mixed mixed system of Kappa free light chain antibody-solutions is 1000～1600mmol/L.
In the present invention, described latex intensified refers to a kind of common method of measuring Kappa free light chain concentration on full automatic biochemical apparatus than purifying method, general under identical basic parameter (wavelength, methodology, sample reagent ratio, reaction time), first use the standard items of determining concentration to use corresponding mensuration reagent to carry out calibration testing at full automatic biochemical apparatus, obtain the calibration curve (working curve) of corresponding mensuration reagent, then, use identical mensuration reagent, on full automatic biochemical apparatus, test testing sample, by full automatic biochemical apparatus contrast calibration curve, automatically calculate measured object content in testing sample, concrete operations can be referring to < < whole nation clinical examination working specification third edition > > (publishing house of Southeast China University, in November, 9787564105839,2006) and each instrument operation instructions book number:.
Wherein, described full automatic biochemical apparatus is preferably the full automatic biochemical apparatus of Olympus AU480 or Hitachi7170 for model.
Wherein, described reaction buffer can be the reaction buffer (R1 reagent) that in the Kappa free light chain method for measurement of concentration of this area, latex intensified is more conventional than purifying method; Described Kappa free light chain antibody-solutions can be the latex intensified Kappa free light chain antibody-solutions (R2 reagent) more conventional than purifying method in the Kappa free light chain method for measurement of concentration of this area.
Wherein, Kappa free light chain antibody in described Kappa free light chain antibody-solutions is preferably the anti-human polyclone Kappa of rabbit free light chain antibody, be more preferably the anti-human polyclone Kappa of the rabbit free light chain antibody of Dako company (be the Polyclonal Rabbit Anti-Human Kappa Free Light Chains of Dako company, article No. is A0100).
Preferably, when reaction buffer consist of 4-hydroxyethyl piperazine ethanesulfonic acid (Hepes) 100mmol/L, sodium chloride 1250～2400mmol/L, Tween-20 1ml/L, antiseptic Proclin-3000.3ml/L time, the anti-human polyclone Kappa of the rabbit that consists of Dako company free light chain antibody (article No. is A0100) 0.5mg/ml, the latex microsphere 3mg/ml of Kappa free light chain antibody-solutions, Tween-20 1ml/L, antiseptic Proclin-3000.3ml/L.In above-mentioned composition, latex microsphere can be purchased from international well-known latex supplier, as HuoJSR company of Bangslab company.
Wherein, described absorbance changes delta A can be obtained by following method: after testing sample is mixed with reaction buffer, and 37 ℃ of constant temperature 5 minutes, then add Kappa free light chain antibody-solutions, after 30 seconds, measuring absorbance is A
1; 37 ℃ of constant temperature 4 minutes and 30 seconds, mensuration absorbance is A
2, calculate absorbance changes delta A=A
2-A
1.
Wherein, described working curve can be obtained by following method: after the standard items of determining concentration are mixed with reaction buffer, and 37 ℃ of constant temperature 5 minutes, then add Kappa free light chain antibody-solutions, after 30 seconds, measuring absorbance is A
01; 37 ℃ of constant temperature 4 minutes and 30 seconds, mensuration absorbance is A
02, calculate absorbance changes delta A
0=A
02-A
01, with Δ A
0with definite concentration mapping of standard items.
Wherein, the sodium chloride concentration of described mixed system is preferably 1300mmol/L.
Wherein, described testing sample is preferably serum and/or marrow separation of serum.
Meeting on the basis of this area general knowledge, above-mentioned each optimum condition, can combination in any, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material be commercially available obtaining all.
Positive progressive effect of the present invention is:
(1) the invention solves the linear poor problem of clinical samples in Kappa free light chain turbidimetric assay, for clinician provides reliable Kappa free light chain measurement result as diagnosis reference, so that it is faster than purifying method speed to give full play to latex enhancing immune, the advantage that flux is high, shorten detection time, be beneficial to clinical practice and diagnosing patient.
(2) method that this invention is used is simply effective, and advantages of nontoxic raw materials and convenient purchase are beneficial to produce and amplify and apply.
Accompanying drawing explanation
Fig. 1 is the working curve of A-a kit.
Fig. 2 is the working curve of A-b kit.
Fig. 3 is the working curve of B-a kit.
Fig. 4 is the working curve of B-b kit.
Fig. 5 is the working curve of A kit.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, according to conventional method and condition, or selects according to catalogue.
Full automatic biochemical apparatus in following embodiment is that model is the full automatic biochemical apparatus of Olympus AU480.
The present invention adopts latex intensified than purifying method, on full automatic biochemical apparatus, to measure the operation of κ type free light chain concentration can be referring to < < whole nation clinical examination working specification third edition > > (publishing house of Southeast China University, book number: in November, 9787564105839,2006).
Reaction buffer (R1 reagent) in A kit (being purchased from Shanghai Ai Kete medical product company limited) and Kappa free light chain antibody-solutions (R2 reagent) composed as follows:
The composition of reaction buffer in A kit (R1 reagent)
| | Title | Concentration | |
| 1 | 4-hydroxyethyl piperazine ethanesulfonic acid (Hepes) | 100mmol/ |
|
| 2 | Sodium chloride (NaCl) | 188mmol/L | |
| 3 | Tween-20 | 1ml/L | |
| 4 | Antiseptic Proclin-300 | 0.3ml/L |
The composition of Kappa free light chain antibody-solutions (R2 reagent) in A kit
| | Title | Concentration | |
| 1 | Kappa free light chain antibody | 0.5mg/ |
|
| 2 | Latex microsphere | 3mg/ml | |
| 3 | Tween-20 | 1ml/L | |
| 4 | Antiseptic Proclin-300 | 0.3ml/L |
Reaction buffer (R1 reagent) in B kit (being purchased from Shanghai Ai Kete medical product company limited) and Kappa free light chain antibody-solutions (R2 reagent) composed as follows:
The composition of reaction buffer in B kit (R1 reagent)
| | Title | Concentration | |
| 1 | 4-hydroxyethyl piperazine ethanesulfonic acid (Hepes) | 100mmol/L |
| 2 | Sodium chloride (NaCl) | 225mmol/L |
| 3 | Tween-20 | 1ml/L |
| 4 | Antiseptic Proclin-300 | 0.3ml/L |
The composition of Kappa free light chain antibody-solutions (R2 reagent) in B kit
| | Title | Concentration | |
| 1 | Kappa free light chain antibody | 0.5mg/ |
|
| 2 | Latex microsphere | 3mg/ml | |
| 3 | Tween-20 | 1ml/L | |
| 4 | Antiseptic Proclin-300 | 0.3ml/L |
Kappa free light chain antibody in above-mentioned A kit and B kit is the Polyclonal Rabbit anti HUM Kappa free light chains of Denmark Dako company, and its article No. is A0100; Latex beads derives from Japanese JSR company, and its article No. is P0112.
Standard items in following embodiment derive from (Landau laboratory diagnosis company limited) the specific protein Quality Control SP286LPC(of Britain Randox company article No.), testing sample derives from Shuguang Hospital of Shanghai.
Freelite reagent is purchased from Britain Binding site company.
A kit is improved, and the concentration that changes the sodium chloride in reaction buffer (R1 reagent) is 1250mmol/L, and the concentration of other constituents is constant, is designated as A-a kit, specifically composed as follows:
The composition of reaction buffer in A-a kit (R1 reagent)
| | Title | Concentration | |
| 1 | 4-hydroxyethyl piperazine ethanesulfonic acid (Hepes) | 100mmol/ |
|
| 2 | Sodium chloride (NaCl) | 1250mmol/L | |
| 3 | Tween-20 | 1ml/L | |
| 4 | Antiseptic Proclin-300 | 0.3ml/L |
The composition of Kappa free light chain antibody-solutions (R2 reagent) in A-a kit
| | Title | Concentration | |
| 1 | Kappa free light chain antibody | 0.5mg/ |
|
| 2 | Latex microsphere | 3mg/ml | |
| 3 | Tween-20 | 1ml/L | |
| 4 | Antiseptic Proclin-300 | 0.3ml/L |
The drafting of working curve:
Adopt latex intensified to compare purifying method, after 3 μ l being determined to the standard items of concentration and 240 μ l reaction buffers in A-a kit mixing, 37 ℃ of constant temperature 5 minutes, then add 60 μ l Kappa free light chain antibody-solutions in A-a kit, after 30 seconds, measuring absorbance is A
01(detect wavelength: 570nm), 37 ℃ of constant temperature 4 minutes and 30 seconds, mensuration absorbance is A
02(detect wavelength: 570nm), calculate absorbance changes delta A
0=A
02-A
01, with Δ A
0with definite concentration mapping of standard items, obtain the working curve of A-a kit, as shown in Figure 1.
Adopt latex intensified than purifying method, by 3 μ l testing samples with after 240 μ l reaction buffers in A-a kit mix, 37 ℃ of constant temperature 5 minutes, then add 60 μ l Kappa free light chain antibody-solutions in A-a kit, after 30 seconds, measuring absorbance is A
1(detect wavelength: 570nm), 37 ℃ of constant temperature 4 minutes and 30 seconds, mensuration absorbance is A
2(detect wavelength: 570nm), calculate absorbance changes delta A=A
2-A
1, more obtain the Kappa free light chain concentration of testing sample with the working curve (Fig. 1) of standard items; Wherein, the sodium chloride concentration of final mixed system is 1000mmol/L.
Above-mentioned testing sample is first added to normal saline dilution to 50% rear (being that testing sample amount is identical with the amount of physiological saline), repeat above-mentioned latex intensified than the operation of purifying method, obtain the Kappa free light chain concentration of the testing sample of 50% dilution.
Before and after the dilution that the method is measured, the Kappa free light chain concentration of testing sample compares, as shown in table 1 below.
The test result when sodium chloride concentration of table 1 mixed system is 1000mmol/L (mg/L)
(note: the notional result that dilution ratio is 0 is test result when undiluted, lower with)
A kit is improved, and the concentration that changes the sodium chloride in reaction buffer (R1 reagent) is 2000mmol/L, and the concentration of other constituents is constant, is designated as A-b kit, specifically composed as follows:
The composition of reaction buffer in A-b kit (R1 reagent)
| | Title | Concentration | |
| 1 | 4-hydroxyethyl piperazine ethanesulfonic acid (Hepes) | 100mmol/ |
|
| 2 | Sodium chloride (NaCl) | 2000mmol/L | |
| 3 | Tween-20 | 1ml/L | |
| 4 | Antiseptic Proclin-300 | 0.3ml/L |
The composition of Kappa free light chain antibody-solutions (R2 reagent) in A-b kit
| | Title | Concentration | |
| 1 | Kappa free light chain antibody | 0.5mg/ |
|
| 2 | Latex microsphere | 3mg/ml | |
| 3 | Tween-20 | 1ml/L | |
| 4 | Antiseptic Proclin-300 | 0.3ml/L |
The drafting of working curve:
Adopt latex intensified to compare purifying method, after 3 μ l being determined to the standard items of concentration and 240 μ l reaction buffers in A-b kit mixing, 37 ℃ of constant temperature 5 minutes, then add 60 μ l Kappa free light chain antibody-solutions in A-b kit, after 30 seconds, measuring absorbance is A
01(detect wavelength: 570nm), 37 ℃ of constant temperature 4 minutes and 30 seconds, mensuration absorbance is A
02(detect wavelength: 570nm), calculate absorbance changes delta A
0=A
02-A
01, with Δ A
0with definite concentration mapping of standard items, obtain the working curve of A-b kit, as shown in Figure 2.
Adopt latex intensified than purifying method, by 3 μ l testing samples with after 240 μ l reaction buffers in A-b kit mix, 37 ℃ of constant temperature 5 minutes, then add 60 μ l Kappa free light chain antibody-solutions in A-b kit, after 30 seconds, measuring absorbance is A
1(detect wavelength: 570nm), 37 ℃ of constant temperature 4 minutes and 30 seconds, mensuration absorbance is A
2(detect wavelength: 570nm), calculate absorbance changes delta A=A
2-A
1, more obtain the Kappa free light chain concentration of testing sample with the working curve (Fig. 2) of standard items; Wherein, the sodium chloride concentration of final mixed system is 1600mmol/L.
Above-mentioned testing sample is first added to normal saline dilution to 50% rear (being that testing sample amount is identical with the amount of physiological saline), repeat above-mentioned latex intensified than the operation of purifying method, obtain the Kappa free light chain concentration of the testing sample of 50% dilution.
Before and after the dilution that the method is measured, the Kappa free light chain concentration of testing sample compares, as shown in table 2 below.
The test result when sodium chloride concentration of table 2 mixed system is 1600mmol/L (mg/L)
Embodiment 3
B kit is improved, and the concentration that changes the sodium chloride in reaction buffer (R1 reagent) is 1500mmol/L, and the concentration of other constituents is constant, is designated as B-a kit, specifically composed as follows:
The composition of reaction buffer in B-a kit (R1 reagent)
| | Title | Concentration | |
| 1 | 4-hydroxyethyl piperazine ethanesulfonic acid (Hepes) | 100mmol/ |
|
| 2 | Sodium chloride (NaCl) | 1500mmol/L | |
| 3 | Tween-20 | 1ml/L | |
| 4 | Antiseptic Proclin-300 | 0.3ml/L |
The composition of Kappa free light chain antibody-solutions (R2 reagent) in B-a kit
| | Title | Concentration | |
| 1 | Kappa free light chain antibody | 0.5mg/ |
|
| 2 | Latex microsphere | 3mg/ml | |
| 3 | Tween-20 | 1ml/L | |
| 4 | Antiseptic Proclin-300 | 0.3ml/L |
The drafting of working curve:
Adopt latex intensified to compare purifying method, after 3 μ l being determined to the standard items of concentration and 200 μ l reaction buffers in B-a kit mixing, 37 ℃ of constant temperature 5 minutes, then add 100 μ lKappa free light chain antibody-solutions in B-a kit, after 30 seconds, measuring absorbance is A
01(detect wavelength: 600nm), 37 ℃ of constant temperature 4 minutes and 30 seconds, mensuration absorbance is A
02(detect wavelength: 600nm), calculate absorbance changes delta A
0=A
02-A
01, with Δ A
0with definite concentration mapping of standard items, obtain the working curve of B-a kit, as shown in Figure 3.
Adopt latex intensified than purifying method, by 3 μ l testing samples with after 200 μ l reaction buffers in B-a kit mix, 37 ℃ of constant temperature 5 minutes, then add 100 μ l Kappa free light chain antibody-solutions in B-a kit, after 30 seconds, measuring absorbance is A
1(detect wavelength: 600nm), 37 ℃ of constant temperature 4 minutes and 30 seconds, mensuration absorbance is A
2(detect wavelength: 600nm), calculate absorbance changes delta A=A
2-A
1, more obtain the Kappa free light chain concentration of testing sample with the working curve (Fig. 3) of standard items; Wherein, the sodium chloride concentration of final mixed system is 1000mmol/L.
Above-mentioned testing sample is first added to normal saline dilution to 50% rear (being that testing sample amount is identical with the amount of physiological saline), repeat above-mentioned latex intensified than the operation of purifying method, obtain the Kappa free light chain concentration of the testing sample of 50% dilution.
Before and after the dilution that the method is measured, the Kappa free light chain concentration of testing sample compares, as shown in table 3 below.
The test result when sodium chloride concentration of table 3 mixed system is 1000mmol/L (mg/L)
Embodiment 4
B kit is improved, and the concentration that changes the sodium chloride in reaction buffer (R1 reagent) is 2400mmol/L, and the concentration of other constituents is constant, is designated as B-b kit, specifically composed as follows:
The composition of reaction buffer in B-b kit (R1 reagent)
| | Title | Concentration | |
| 1 | 4-hydroxyethyl piperazine ethanesulfonic acid (Hepes) | 100mmol/ |
|
| 2 | Sodium chloride (NaCl) | 2400mmol/L | |
| 3 | Tween-20 | 1ml/L | |
| 4 | Antiseptic Proclin-300 | 0.3ml/L |
The composition of Kappa free light chain antibody-solutions (R2 reagent) in B-b kit
The drafting of working curve:
Adopt latex intensified to compare purifying method, after 3 μ l being determined to the standard items of concentration and 200 μ l reaction buffers in B-b kit mixing, 37 ℃ of constant temperature 5 minutes, then add 100 μ l Kappa free light chain antibody-solutions in B-b kit, after 30 seconds, measuring absorbance is A
01(detect wavelength: 600nm), 37 ℃ of constant temperature 4 minutes and 30 seconds, mensuration absorbance is A
02(detect wavelength: 600nm), calculate absorbance changes delta A
0=A
02-A
01, with Δ A
0with definite concentration mapping of standard items, obtain the working curve of B-b kit, as shown in Figure 4.
Adopt latex intensified than purifying method, by 3 μ l testing samples with after 200 μ l reaction buffers in B-b kit mix, 37 ℃ of constant temperature 5 minutes, then add 100 μ l Kappa free light chain antibody-solutions in B-b kit, after 30 seconds, measuring absorbance is A
1(detect wavelength: 600nm), 37 ℃ of constant temperature 4 minutes and 30 seconds, mensuration absorbance is A
2(detect wavelength: 600nm), calculate absorbance changes delta A=A
2-A
1, more obtain the Kappa free light chain concentration of testing sample with the working curve (Fig. 4) of standard items; Wherein, the sodium chloride concentration of final mixed system is 1600mmol/L.
Above-mentioned testing sample is first added to normal saline dilution to 50% rear (being that testing sample amount is identical with the amount of physiological saline), repeat above-mentioned latex intensified than the operation of purifying method, obtain the Kappa free light chain concentration of the testing sample of 50% dilution.
Before and after the dilution that the method is measured, the Kappa free light chain concentration of testing sample compares, as shown in table 4 below.
The test result when sodium chloride concentration of table 4 mixed system is 1600mmol/L (mg/L)
Comparative example 1
The drafting of working curve:
Adopt latex intensified than purifying method, after 3 μ l being determined to the standard items of concentration and 240 μ l reaction buffers in A kit mixing, 37 ℃ of constant temperature 5 minutes, then add 60 μ l Kappa free light chain antibody-solutions in A kit, after 30 seconds, measuring absorbance is A
01(detect wavelength: 570nm), 37 ℃ of constant temperature 4 minutes and 30 seconds, mensuration absorbance is A
02(detect wavelength: 570nm), calculate absorbance changes delta A
0=A
02-A
01, with Δ A
0with definite concentration mapping of standard items, obtain the working curve of A kit, as shown in Figure 5.
Adopt latex intensified than purifying method, by 3 μ l testing samples with after 240 μ l reaction buffers in A kit mix, 37 ℃ of constant temperature 5 minutes, then add 60 μ l Kappa free light chain antibody-solutions in A kit, after 30 seconds, measuring absorbance is A
1(detect wavelength: 570nm), 37 ℃ of constant temperature 4 minutes and 30 seconds, mensuration absorbance is A
2(detect wavelength: 570nm), calculate absorbance changes delta A=A
2-A
1, more obtain the Kappa free light chain concentration of testing sample with the working curve (Fig. 5) of standard items; Wherein, the sodium chloride concentration of final mixed system is 150mmol/L.
Above-mentioned testing sample is first added to normal saline dilution to 50% rear (being that testing sample amount is identical with the amount of physiological saline), repeat above-mentioned latex intensified than the operation of purifying method, obtain the Kappa free light chain concentration of the testing sample of 50% dilution.
Before and after the dilution that the method is measured, the Kappa free light chain concentration of testing sample compares, as shown in table 5 below.
The test result when sodium chloride concentration of table 5 mixed system is 150mmol/L (mg/L)
Comparative example 2
Adopt latex intensified than purifying method, use the Freelite Series Measurement reagent of current commercial Britain Binding site company, measure the Kappa free light chain concentration of dilution front and back testing sample, result is as shown in table 6 below.
Test result (mg/L) when table 6 adopts Freelite Series Measurement reagent
From above-mentioned table 1～6, can see, use method of the present invention, when control testing sample is 1000mmol/L and 1600mmol/L with the sodium chloride concentration of measuring the mixed system after reagent mix, the dilution of clinical testing sample meet test result in notional result positive and negative 20% with interior linear requirement, can meet external diagnosis reagent request for utilization; And when the concentration of the sodium chloride of mixed system is 150mmol/L, there is dilution skew in various degree in clinical testing sample, surpass positive and negative 20% linear requirement, in-vitro diagnosis is had to considerable influence, and Freelite reagent has occurred that testing sample dilutes non-linear situation too, there is false positive (False Positive) and false negative result (False Negative) in various degree, clinical practice more problems.
Claims (10)
1. adopt latex intensified than purifying method, to measure a kit for Kappa free light chain concentration, it comprises reaction buffer and Kappa free light chain antibody-solutions, it is characterized in that, in described reaction buffer, the concentration of sodium chloride is 1250～2400mmol/L.
2. kit as claimed in claim 1, is characterized in that, the Kappa free light chain antibody in described Kappa free light chain antibody-solutions is the anti-human polyclone Kappa of rabbit free light chain antibody.
3. kit as claimed in claim 2, is characterized in that, the Kappa free light chain antibody in described Kappa free light chain antibody-solutions is the anti-human polyclone Kappa of the rabbit free light chain antibody of Dako company.
4. kit as claimed in claim 1, it is characterized in that, when described reaction buffer consist of 4-hydroxyethyl piperazine ethanesulfonic acid 100mmol/L, sodium chloride 1250～2400mmol/L, Tween-20 1ml/L and antiseptic Proclin-3000.3ml/L time, the anti-human polyclone Kappa of the rabbit that consists of Dako company free light chain antibody 0.5mg/ml, latex microsphere 3mg/ml, Tween-20 1ml/L and the antiseptic Proclin-3000.3ml/L of described Kappa free light chain antibody-solutions.
5. kit as claimed in claim 4, it is characterized in that, when described reaction buffer consist of 4-hydroxyethyl piperazine ethanesulfonic acid 100mmol/L, sodium chloride 1250mmol/L, Tween-20 1ml/L and antiseptic Proclin-3000.3ml/L time, the anti-human polyclone Kappa of the rabbit that consists of Dako company free light chain antibody 0.5mg/ml, latex microsphere 3mg/ml, Tween-20 1ml/L and the antiseptic Proclin-3000.3ml/L of described Kappa free light chain antibody-solutions; Or, when described reaction buffer consist of 4-hydroxyethyl piperazine ethanesulfonic acid 100mmol/L, sodium chloride 2000mmol/L, Tween-20 1ml/L and antiseptic Proclin-3000.3ml/L time, the anti-human polyclone Kappa of the rabbit that consists of Dako company free light chain antibody 0.5mg/ml, latex microsphere 3mg/ml, Tween-20 1ml/L and the antiseptic Proclin-3000.3ml/L of described Kappa free light chain antibody-solutions; Or, when described reaction buffer consist of 4-hydroxyethyl piperazine ethanesulfonic acid 100mmol/L, sodium chloride 1500mmol/L, Tween-20 1ml/L and antiseptic Proclin-3000.3ml/L time, the anti-human polyclone Kappa of the rabbit that consists of Dako company free light chain antibody 0.5mg/ml, latex microsphere 3mg/ml, Tween-20 1ml/L and the antiseptic Proclin-3000.3ml/L of described Kappa free light chain antibody-solutions; Or, when described reaction buffer consist of 4-hydroxyethyl piperazine ethanesulfonic acid 100mmol/L, sodium chloride 2400mmol/L, Tween-20 1ml/L and antiseptic Proclin-3000.3ml/L time, the anti-human polyclone Kappa of the rabbit that consists of Dako company free light chain antibody 0.5mg/ml, latex microsphere 3mg/ml, Tween-20 1ml/L and the antiseptic Proclin-3000.3ml/L of described Kappa free light chain antibody-solutions.
6. a method of measuring Kappa free light chain concentration, it comprises the steps: to adopt latex intensified to compare purifying method, after testing sample is mixed with reaction buffer as described in any one in claim 1～5 and Kappa free light chain antibody-solutions, calculate absorbance changes delta A, relatively can obtain the Kappa free light chain concentration of testing sample with the working curve of standard items, it is characterized in that, the sodium chloride concentration of testing sample and reaction buffer and the mixed mixed system of Kappa free light chain antibody-solutions is 1000～1600mmol/L.
7. the method for mensuration as claimed in claim 6 Kappa free light chain concentration, it is characterized in that, described absorbance changes delta A is obtained by following method: after testing sample is mixed with reaction buffer, 37 ℃ of constant temperature 5 minutes, add Kappa free light chain antibody-solutions, after 30 seconds, measuring absorbance is A again
1; 37 ℃ of constant temperature 4 minutes and 30 seconds, mensuration absorbance is A
2, calculate absorbance changes delta A=A
2-A
1.
8. the method for mensuration as claimed in claim 6 Kappa free light chain concentration, it is characterized in that, described working curve is obtained by following method: after the standard items of determining concentration are mixed with reaction buffer, 37 ℃ of constant temperature 5 minutes, add Kappa free light chain antibody-solutions, after 30 seconds, measuring absorbance is A again
01; 37 ℃ of constant temperature 4 minutes and 30 seconds, mensuration absorbance is A
02, calculate absorbance changes delta A
0=A
02-A
01, with Δ A
0with definite concentration mapping of standard items.
9. the method for mensuration Kappa free light chain concentration as claimed in claim 6, is characterized in that, the sodium chloride concentration of described mixed system is 1300mmol/L.
10. the method for mensuration Kappa free light chain concentration as claimed in claim 6, is characterized in that, described testing sample is serum and/or marrow separation of serum.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105548573A (en) * | 2016-02-02 | 2016-05-04 | 潍坊三维生物工程集团有限公司 | Kit and method for detecting content of KAPPA light chain and application |
| CN105738300A (en) * | 2016-02-02 | 2016-07-06 | 潍坊三维生物工程集团有限公司 | Kit and method for detecting content of KAPPA light chain and application of kit |
| CN109164263A (en) * | 2018-09-05 | 2019-01-08 | 苏州普瑞斯生物科技有限公司 | A kind of kit and preparation method measuring light chain Kappa concentration |
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| WO2005116651A2 (en) * | 2004-05-24 | 2005-12-08 | Diasys Corporation | Method and device for testing for bence-jones protein |
| CN102175871A (en) * | 2010-12-30 | 2011-09-07 | 北京九强生物技术股份有限公司 | Liquid double-reagent kit for measuring free light chains in serum or urine by double-latex method |
| CN102692507A (en) * | 2011-07-29 | 2012-09-26 | 南京诺尔曼生物技术有限公司 | Kit for measuring lipoprotein-associated phospholipase A2 (LPPLA2) (by adopting latex enhanced turbidimetric immunoassay) |
| CN102735845A (en) * | 2012-06-12 | 2012-10-17 | 上海沪晶生物科技有限公司 | Novel detection method and kit for synchronously detecting concentration of free kappa light chain and free lambda light chain |
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2013
- 2013-09-03 CN CN201310396461.0A patent/CN103728455B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005116651A2 (en) * | 2004-05-24 | 2005-12-08 | Diasys Corporation | Method and device for testing for bence-jones protein |
| CN102175871A (en) * | 2010-12-30 | 2011-09-07 | 北京九强生物技术股份有限公司 | Liquid double-reagent kit for measuring free light chains in serum or urine by double-latex method |
| CN102692507A (en) * | 2011-07-29 | 2012-09-26 | 南京诺尔曼生物技术有限公司 | Kit for measuring lipoprotein-associated phospholipase A2 (LPPLA2) (by adopting latex enhanced turbidimetric immunoassay) |
| CN102735845A (en) * | 2012-06-12 | 2012-10-17 | 上海沪晶生物科技有限公司 | Novel detection method and kit for synchronously detecting concentration of free kappa light chain and free lambda light chain |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105548573A (en) * | 2016-02-02 | 2016-05-04 | 潍坊三维生物工程集团有限公司 | Kit and method for detecting content of KAPPA light chain and application |
| CN105738300A (en) * | 2016-02-02 | 2016-07-06 | 潍坊三维生物工程集团有限公司 | Kit and method for detecting content of KAPPA light chain and application of kit |
| CN105738300B (en) * | 2016-02-02 | 2019-04-30 | 潍坊三维生物工程集团有限公司 | Detect kit, method and the purposes of KAPPA light chain content |
| CN109164263A (en) * | 2018-09-05 | 2019-01-08 | 苏州普瑞斯生物科技有限公司 | A kind of kit and preparation method measuring light chain Kappa concentration |
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| CN103728455B (en) | 2015-07-15 |
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