CN1529148A - Bacteriotoxin quantitive detecting method-dynamic end-point method - Google Patents
Bacteriotoxin quantitive detecting method-dynamic end-point method Download PDFInfo
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Abstract
The invented testing method solves blindfold determination of important parameter-terminal hour in prior art, making selection of terminal hour possess rationality. Adopting photoelectric detecting instrument, the invention records, plots dynamic curve of reaction, and searches out optimal terminal hour by using computer. Then, end-point method is carried out for testing content of endotoxin in sample quantificationally by utilizing the said terminal hour. The method is applicable to horseshoe crab reagent in end-point method as well as reagents in dynamic turbidimetry or dynamic colorimetry.
Description
Technical field
The present invention relates to the bacterial endotoxin quantitative detecting method.
Background technology
Bacterial endotoxin is the lipopolysaccharides composition in the gram-negative bacterial cell wall structure, has multiple biologically active, is a kind of main pyrogen material.
The employed reagent of detection of bacterial endotoxin is tachypleus amebocyte lysate (TAL or LAL).Contain factor C, factor B, proclotting enzyme and the coagulagen that can be activated by the denier bacterial endotoxin among the TAL.(temperature, pH value and noiseless material etc.) under optimum conditions, bacterial endotoxin can make TAL produce agglutinating reaction.
The methodology of detection of bacterial endotoxin is classified as follows:
Bacterial endotoxin detection by quantitative ratio juris is to use photoelectric detector, turbidity/colourity change transitions that endotoxin and TAL agglutinating reaction are produced is the variation of shading value, set up the mathematical model of endotoxin concns and shading value and correlation parameter relation, thus the endotoxin content of quantitative measurement sample.
It is to set up mathematical model (typical curve) according to shading value and bacterial endotoxin concentration that limulus test is reflected at when arriving the default reaction time (terminal point) that end-point method detects principle, carries out detection by quantitative then.
It is that luminosity according to the limulus test reaction changes when arriving preset value needed reaction time and bacterial endotoxin concentration and sets up mathematical model (typical curve) that dynamic method detects principle, carries out detection by quantitative then.
The endotoxin sensing range of end-point method is generally 1 order of magnitude, is characterized in that the reaction time short.The dynamic method reaction time is generally longer, and its characteristics are that the endotoxin sensing range is big, can reach 3 to 4 orders of magnitude.End-point method has nearly 10 times raising with the detection sensitivity of dynamic method than gel method.
The endotoxin detection technique of existing end-point method is described below:
Get (bottle) standard endotoxin, tire by sign and check that with bacterial endotoxin water is diluted at least three geometric ratio concentration C to it
1, C
2And C
3, under 37 ℃ ± 1 ℃ constant temperature, react certain time interval T with TAL respectively, this time is terminal time.Read shading value OD again after reading the shading value of each reactant or cessation reaction by the photoelectricity instrument
1, OD
2And OD
3With parameters C
1-OD
1, C
2-OD
2And C
3-OD
3Do regretional analysis, obtain a typical curve, and try to achieve its related coefficient (r).If related coefficient | r| 〉=0.98, then can use this typical curve to come endotoxin content unknown in the detection by quantitative sample.
The defective of existing end-point method technology is that the selection of important parameter terminal time is had blindness, and it is selected mainly according to related coefficient.According to existing end-point method, when definite terminal time T, if at terminal time T
1The related coefficient that obtains | r|＜0.98, must change another terminal time T2 test of reforming, up to related coefficient | r| 〉=0.98.
Summary of the invention
The problem to be solved in the present invention is that existing end-point method technology exists the defective that the selection of important parameter terminal time is had blindness, and a kind of new method---dynamic end-point method of bacterial endotoxin detection by quantitative is provided.
The new method of bacterial endotoxin detection by quantitative of the present invention---dynamic end-point method, its method step is as follows:
1. use gel method, end-point method, kinetic turbidimetric assay or dynamic colourimetry tachypleus amebocyte lysate to do dynamic end-point method bacterial detection endotoxin;
2. determine to set up the required standard endotoxin concns of typical curve, the standard endotoxin concns of at least three concentration of preparation, react on photoelectric detector with tachypleus amebocyte lysate respectively, photoelectric detector is gathered response data, by computer recording and depict the performance graph of each reaction;
3. computing machine is determined the rational reaction end time period automatically according to the reaction performance graph, and searches out best terminal time T automatically in the section at this moment
0
4. utilize best terminal time T
0Set up typical curve, on photoelectric detector, carry out the endotoxin content of end-point method detection by quantitative sample.
Every lot number tachypleus amebocyte lysate and specific series concentration standard endotoxin reaction, its best terminal time T
0It is a fixing value.
The endotoxin content of negative control is less than the minimum point concentration of typical curve.
The computer program of described bacterial endotoxin quantitative detecting method comprises:
Five parameters of sequence number, title, sample lot number, extension rate, standard endotoxin of record test tube and the penetrating light intensity that photoelectric detector reads;
With document form data the data of record are preserved;
Open data file, sequence number, title, sample lot number, extension rate and five parameters of standard endotoxin of each test tube in the test are write the first five row of form; The penetrating light intensity of each test tube is at the two-dimensional array datak[i of correspondence, j] and test tube sequence number i=1,2 ..., 96, sampled data j=1,2 ..., in, carry out data analysis;
The method of data analysis is selected;
Search for best terminal time;
The end-point method that utilizes best terminal time to carry out the sample bacteria endotoxin content detects test.
Searching for best terminal time comprises:
1. the end-point method analytical approach is selected:
A, optical densitometric method (OD)---Is/Ie, along with the increase of test period, the OD value increases gradually, and variation range is between 1～1.7.
The regression equation of OD method is Lg (Is/Ie)=a+bC, and C is an endotoxin concns, and a, b are the regression equation coefficient.
B, penetrability method (RT)---Ie/Is, along with the increase of test period, the RT value reduces gradually, and variation range is between 0.3～1.
The regression equation of RT method is Ie/Is=a+bC, and C is an endotoxin concns, and a, b are the regression equation coefficient.
2. after from form, taking out the sequence number of standard pipe, set up the standard pipe array;
3. the endotoxin concns value leaves array X[I in the standard pipe] in, calculate:
Lxx=∑ Xi
2-(∑ Xi)
2/ n; N is the standard pipe number, I=1, and 2 ..., n:
4. since 5 minutes, the penetrating light intensity data of one group of standard pipe were got in every increase in 1 minute, calculated OD value or RT value to array Y[i] { OD method Y=Lg (Is/Ie) is among the RT method Y=Ie/Is}
Wherein: Is---initial light intensity, get wdq stationary phase (being generally 12 sampling periods) three light intensity mean values afterwards as initial light intensity;
Ie---attitude light intensity, the penetrating light intensity under the sampling time arbitrarily;
Dt---sampling interval, 5 seconds or 10 seconds; From the data file head, obtain;
5. calculate:
Lyy＝∑Yi
2-(∑Yi)
2/n；
Lxy=∑ Xi*Yi-(∑ Xi) * (∑ Yi)/n; N is the standard pipe number, I=1, and 2 ..., n; Related coefficient: R=Lxy/ (sqrt (Lxx) * sqrt (Lyy))
6. when coefficient R＞0.9999, jump out circulation, obtain the reference value that this time is best terminal time; Otherwise the time when getting related coefficient absolute value maximum is as the reference value of best terminal time.
Determine terminal time:
1. the endotoxin concns of standard pipe is at an order of magnitude, the concentration number three and more than;
2. the performance graph of Cmin pipe separates with the performance graph of negative control pipe;
3. the performance graph of Cmax pipe does not enter the smooth phase as yet.
The advantage of dynamic end-point method:
1. compare with existing end-point method; The selection of important parameter terminal time is more reasonable, and the linearity of typical curve is better.
The dynamic end-point method is determined rational terminal time section with the reaction performance graph as a reference, utilizes computer search to go out best terminal time.Among Fig. 1, E
a, E
b, E
cAnd E
dBe the reaction performance graph of standard endotoxin and TAL, NC is the reaction performance graph of checking water and TAL, the i.e. performance graph of negative control.If the selection terminal time is 24 minutes, at this moment E
a, E
b, E
cAnd E
dShading value (OD) be respectively: 1.09195,1.03826,1.02041 and 1.01485, pass through E
a-OD
a, E
b-OD
b, E
c-OD
cAnd E
d-OD
dDo regretional analysis, obtain a typical curve, the related coefficient of this typical curve | r|=0.9895＞0.98, but as can see from Figure 1, least concentration E in the time of 24 minutes
dAnd E
cPerformance graph almost overlap with the performance graph of negative control NC, illustrate this moment E
d, E
cAnd the shading value of NC is very approaching, and is obviously unreasonable with the typical curve that this terminal time is set up.The dynamic end-point method is utilized response curve, searches for and select rational, best terminal time (T
0) set up typical curve.
2. compare with dynamic method: shorten test period, improve detection sensitivity.
The dynamic end-point method has the short characteristics of test period, improves the efficient of detection of bacterial endotoxin.The length of its test period is relevant with reagent and endotoxin concns.Usually the test period of dynamic end-point method is all obviously short than dynamic method.
The dynamic end-point method can improve detection sensitivity.Endotoxin concns E with Fig. 1
dBe example, the reaction performance graph did not intersect with default 92% OD value (thick horizontal line) in 60 minutes, showed that the detection sensitivity that this dynamic method is tested does not reach E
dIf, but change the dynamic end-point method into, then can make detection sensitivity reach E
d
3. can use dynamic method reagent to do end-point method detects.
Description of drawings
Fig. 1 is the endotoxic reaction dynamic curve diagram of standard of the best terminal time of search;
Fig. 2 is the endotoxic reaction dynamic curve diagram of sample detection standard;
Fig. 3 is the reaction dynamic curve diagram of test sample and test sample positive control;
Fig. 4 is a computer program process flow diagram of the present invention.
Embodiment
Bacterial endotoxin quantitative detecting method of the present invention---dynamic end-point method comprises dynamic end-point turbidimetry and dynamic end-point colourimetry.The dynamic end-point method is to utilize photoelectric detector to gather response data, utilize computer recording and describe to react performance graph, determine rational terminal time section, search out best terminal time, set up typical curve with this terminal time then, thus the endotoxin content of quantitative working sample.
The selection of best terminal time: with the dynamic end-point nephelometry is the selection problem that example illustrates best terminal time.The selection of best terminal time must rely on the dynamic response curve, by analyzing the dynamic response curve, selects rational terminal time section, goes out best terminal time according to computer search then.As shown in Figure 1, should select the performance graph of endotoxin concns minimum point obviously to descend, the performance graph of concentration peak does not also reach the zone of minimum point, promptly 34 minutes to 50 minute time period, utilizes computing machine to search out the time T of best terminal point in the section at this moment
0Be 47 minutes, the corresponding standard curve related coefficient | r|=0.9977.
The endotoxin content that it is exactly negative control that endotoxin detection by quantitative method has a requirement is less than the minimum point concentration of typical curve, if concentration minimum point performance graph does not obviously descend, but overlap with the performance graph of negative control, as Fig. 1, test period was less than 20 minutes, the endotoxin content that obtains negative control this moment equals the minimum point concentration of typical curve, does not just meet the requirement of detection by quantitative method.If the performance graph of concentration peak touches the bottom, as Fig. 1, test period was greater than 54 minutes, and at this moment, the shading value of the peak concentration of typical curve has reached mxm., no longer changed, and the typical curve of Jian Liing is obviously also unreasonable like this.
Best terminal time (T
0) meaning: the endotoxic a series of concentration of the standard of the TAL of a lot number and a lot number are (as E
a, E
b, E
c) T that obtained of reaction
0Be a fixed value, this means if when reusing the TAL of this lot number, again dependent reaction performance graph removal search T
0, utilize the T of test for the first time
0Just can directly do the end-point method inspection.This makes that test is very convenient, also can do end-point method with dynamic method reagent and detect.
Embodiment:
1. test material
Standard endotoxin (CSE), tachypleus amebocyte lysate (TAL), bacterial endotoxin is checked water (W);
Testing sample is test sample (S);
Photoelectric detector.
2. test method and step
2.1 determine to set up the needed standard endotoxin concns of typical curve.As: 0.3,0.15,0.075,0.03Eu/ml, be designated as E0.3, E0.15, E0.075, E0.03 respectively;
2.2 get 1 (bottle) CSE, tiring by sign is diluted to desired concn with W, carries out reaction in 60 minutes with TAL on photoelectric detector respectively; Photoelectric detector is gathered response data (light intensity value) dynamically in course of reaction.
2.3 analyze by photoelectric detector by computer recording and the reaction performance graph depicted, determine rational terminal time section, utilize computer search to go out the terminal time T of the best in the terminal time section
0
2.4 utilize best terminal time T
0, on photoelectric detector, carry out the endotoxin content of end-point method detection by quantitative sample:
Prop up 2.4.1 get CSE1, by specification dissolves and is diluted to desired concn, as E
0.3, E
0.15, E
0.075And E
0.03
2.4.2 S is to detectable concentration in dilution.As: the S detectable concentration is that dilution in 1: 4 (is designated as S
4);
2.4.3 preparation S
4Positive control (PPC), make S
4Contain 0.075Eu/ml standard endotoxin, be designated as S
4E
0.075
2.4.4 get TAL respectively with the CSE dilution series solution, the S that prepare
4And S
4E
0.075On photoelectric detector, carry out with T
0End-point method detection for the reaction end time.
3. test findings
3.1 the endotoxic reaction performance graph of standard is seen Fig. 2.
3.2 test sample (S
4) and test sample positive control (S
4E
0.075) the reaction performance graph see Fig. 3.
3.3 examining report sees Table 1.
4. interpretation of result
4.1 endotoxin detection by quantitative method requires: the related coefficient of typical curve | r|＞0.98; The positive control recovery is in 50%-200%; The endotoxin content of NC (negative control) is less than the minimum point concentration of typical curve.
4.2 the related coefficient of this test standard curve | r|=0.9976, can be found out positive control S by examining report table 1
4E
0.075The recovery be 114.93%, the endotoxin content of NC is 0.0216Eu/ml, less than the minimum point concentration 0.03Eu/ml of typical curve, this test is effective.
The present invention utilizes photoelectric detector and computing machine to carry out the bacterial endotoxin detection by quantitative.
The penetrating light intensity that five parameters such as the sequence number of this computer program recorded test tube, title, sample lot number, extension rate, standard endotoxin and photoelectric detector read is preserved with document form data.
After program is opened data file, five parameters such as sequence number, title, sample lot number, extension rate, standard endotoxin of each test tube in the test are write the first five row of form.The penetrating light intensity of each test tube is at the two-dimensional array datak[i of correspondence, j] and test tube sequence number i=1,2 ..., 96, sampled data j=1,2 ..., in, carry out data analysis.Search for best terminal time, the end-point method that utilizes best terminal time to carry out the sample endotoxin content detects test.Its main process flow diagram 4.
Search for best terminal time
1. the end-point method analytical approach is selected:
1. optical densitometric method (OD)---Is/Ie, along with the increase of test period, the OD value increases gradually, and variation range is between 1～1.7.
The regression equation of OD method is Lg (Is/Ie)=a+bC, and C is an endotoxin concns, and a, b are the regression equation coefficient.
2. penetrability method (RT)---Ie/Is, along with the increase of test period, the RT value reduces gradually, and variation range is between 0.3～1.
The regression equation of RT method is Ie/Is=a+bC, and C is an endotoxin concns, and a, b are the regression equation coefficient.
2. after from form, taking out the sequence number of standard pipe, set up the standard pipe array;
3. the endotoxin concns value leaves array X[I in the standard pipe] in, calculate:
Lxx=∑ Xi
2-(∑ Xi)
2/ n; N is the standard pipe number, I=1, and 2 ..., n;
4. since 5 minutes, the penetrating light intensity data of one group of standard pipe were got in every increase in 1 minute, calculated OD value or RT value to array Y[i] and OD method Y=Lg (Is/Ie), among the RT method Y=Ie/Is},
Wherein: Is---initial light intensity, get wdq stationary phase (being generally 12 sampling periods) three light intensity mean values afterwards as initial light intensity;
Ie---dynamic light intensity, the penetrating light intensity under the sampling time arbitrarily;
Dt---sampling interval, 5 seconds or 10 seconds; From the data file head, obtain;
5. calculate:
Lyy＝∑Yi
2-(∑Yi)
2/n；
Lxy=∑ Xi*Yi-(∑ Xi) * (∑ Yi)/n; N is the standard pipe number, I=1, and 2 ..., n; Related coefficient: R=Lxy/ (sqrt (Lxx) * sqrt (Lyy))
6. when coefficient R＞0.9999, jump out circulation, obtain the reference value that this time is best terminal time; Otherwise the time when getting related coefficient absolute value maximum is as the reference value of best terminal time.
Annotate: the terminal time that above-mentioned algorithm obtains is only for reference, in fact should select best terminal time by the concentration range and the performance graph form of standard pipe; The specific requirement of determining terminal time is:
1. the endotoxin concns of standard pipe is at an order of magnitude, the concentration number should three and more than;
2. the performance graph of Cmin pipe separates with the performance graph of negative control pipe;
3. the performance graph of Cmax pipe does not enter the smooth phase as yet;
If do not satisfy above-mentioned requirements, end-point method is analyzed inapplicable.
The core source program of best terminal time search: (Delphi language)
Procedure TForm3.BitBtn23Click (Sender:TObject); ∥ searches for terminal time " button " var ∥ automatically ... types of variables definition begin ∥ ... for i:=1 to such as initialization of variable 96 do ∥ Eustd=' endotoxin standards ' begin if (pos (EuStd, TubeHead[i, 2])=0) or (pos (' invalid ', TubeHead[i, 2] then continue)＞0) or (Length (trim (TubeHead[i, 5]))=0); // non-standard pipe, invalid pipe, there is not the Guan Jun of concentration to skip tubm:=tubm+1; ∥ tubm standard pipe number z[tubm] :=strTofloat (TubeHead[i, 5]); ∥ endotoxin concns num[tubm, 1] :=i; 1 of ∥ num classifies pipe end as; K:=0; For i:=1 to 96 do x[i] :=0; Fori:=1 to tubm do begin if num[i, 2]＞0 then continue; K:=k+1; Num[i, 2] :=k; X[k] :=z[i]; The ∥ endotoxin concns is at X[] for j:=1 to tubm do if abs in the array (z[j]-x[k])＜1e-4 then num[j, 2]: 2 of=k ∥ num classifies group number end as; Tubm1:=k; ∥ concentration number if tubm1＞1 then begin xm:=0; For j:=1 to tubm1 do xm:=xm+x[j]; Lxx:=0; Forj:=1 to tubm1 do lxx:=lxx+x[j] * x[j]; Lxx:=lxx-xm*xm/tubm1; Rdmax:=0; Kmax:=5; Tk:=CTime (StrToInt (DataHead[9])); ∥ full test time _ minute for k:=5 to tk do begin for j:=1 to 96 do begin z[j] :=0; Y[j] :=0; End; M:=(k*60) div StrToInt (DataHead[9]); Flag:=1; For i:=1 to tubm do begin ave:=0;＜!--SIPO＜DP n=" 8 "〉--〉＜dp n=" d8 "/for j:=wdq+1 to wdq+3 do ave:=ave+datak[num[i, 1], j]; Ave:=Round (ave/3); If RadioButtonl.Checked then z[i] :=ave-datak[num[i, 1], m] and eise if ave＞0 then z[i] :=datak[num[i, 1], m]/ave else z[i] :=0; End; For i:=1 to tubm1 do begin ym:=0; N:=0; For j:=1 to tubm do if (num[j, 2]=i) and (z[i]＞0) then begin n:=n+1; Ym:=ym+z[j]; End; If n＞1 then y[i] :=ym/n; ∥ y[i] in put average OD or RT value end under the same concentration; For j:=1 to tubm1 do begin if y[j]＜=0 then begin flag:=0; Break; End; If RadioButtonl.Checked then y[j] :=log10 (y[j]); ∥ is to the OD end that takes the logarithm; If flag=0 then continue; Ym:=0; For j:=1 to tubm1 do ym:=ym+y[j]; Lxy:=0; For j:=1 to tubm1 do lxy:=lxy+x[j] * y[j]; Lxy:=lxy-xm*ym/tubm1; Lyy:=0; For j:=1 to tubm1 do lyy=lyy+y[j] * y[j]; Lyy:=lyy-ym*ym/tubm1; Rd:=lxy/ (sqrt (lxx) * sqrt (lyy)); If abs (rd)＞abs (rdmax) then begin rdmax:=rd; Kmax:=k; End else if abs (rd)＞0.9999 then break; End; ∥ ... write terminal time and related coefficient in the corresponding assembly messageDlg (' automatically search time only for reference! ', mtInformation, [mbOK], 0); (' endotoxin concns is less than 2 to end else messageDlg, can not search for automatically! ', mtInformation, [mbOK], 0); End;
The OD value (or RT value) of calculating each test tube by terminal time with reference to the best terminal time of search automatically and the performance graph of standard pipe, is specified terminal time 1. by aforementioned requirement; 2. by behind " determining " button, the OD value (or RT value) of calculating each pipe writes form the 7th row.Calculate the core source program of OD value (or RT value): (Delphi language)
Procedure TForm3.BitBtn20Click (Sender:TObject); The ∥ end-point method time " is determined " var ∥ ... types of variables definition begin t0:=StrToInt (DataHead[9]); ∥ acquisition interval---second t2:=StrToInt (SpinEdit5.Text); ∥ terminal time---minute k:=(t2*60) div t0;＜!--SIPO＜DP n=" 9 "〉--〉＜dp n=" d9 "/Edit22.Text:="; Edit23.Text:=SpinEdit5.Text+ ' minute '; If RadioButtonl.Checked then StringGrid2.Cells[6,0] :=' optical density O.D. ' else StringGrid2.Cells[6,0] :=' penetrability '; For i:=1 to StringGrid2.RowCount do begin ave:=0; For j:=wdq+1 to wdq+3 do ave:=ave+datak[i, j]; T0:=Round (ave/3); ∥ Is t1:=datak[i, k]; ∥ Ie if RadioButtonl.Checked then if t1＞0 then diff:=t0/t1 else diff:=0 else if t0＞0 then diff:=t1/t0 else diff:=0; If diff＞0 then stringGrid2.Cells[6, i] :=FloatToStrF (diff, ffFixed, 8,6); End; ∥ ... push-botton operation logic control end;
The regretional analysis of typical curve
1. obtain standard pipe concentration from form the 5th row and leave in the Xi array, the OD value (or RT value) that obtains standard pipe from form the 7th row is placed on the Yi group;
2. if average the recurrence, with the OD value (or RT value) of the standard pipe of same concentration on average, having what concentration then to think has several standard pipes.
3. calculate:
Xm＝∑Xi
2/n，??Ym＝∑Yi
2/n，
Lxx＝∑Xi
2-(∑Xi)
2/n：
Lyy＝∑Yi
2-(∑Yi)
2/n；
Lxy＝∑Xi*Yi-(∑Xi)*(∑Yi)/n；
N is the standard pipe number, i=1, and 2 ..., n;
The regression equation parameter:
Aod＝Lxy/Lxx，
Bod＝Ym-b*Xm；
Rod=Lxy/ (sqrt (Lxx) * sqrt (Lyy)) is a related coefficient
4. according to endotoxin measured value and other parameter of each pipe of regression equation calculation,, write respectively in the respective column of form as the recovery, standard variance, the coefficient of variation.The core source program of end-point method regretional analysis: (Delphi language)
Procedure Regress (ff:Integer); The ∥ end-point method is asked recurrence: ff=0 directly returns, and ff＞0 on average returns " process " var ∥ ... types of variables definition begin tubm:=0; For i:=1 to 96 do begin Eutmp[i] :=0; If (pos (EuStd, TubeHead[i, 2])=0) or (pos (' invalid ', TubeHead[i, 2])＞0) or (Length (trim (TubeHead[i, 5]))=0) then continue; The non-standard pipe of ∥, invalid pipe, there is not the standard pipe of concentration all to skip tubm:=tubm+1; Eutmp[tubm] :=strToffoat (TubeHead[i, 5]); ∥ endotoxin concns end; ∥ tubm standard pipe number j:=0; Eu1:=0; For i:=1 to tubm do begin eu2:=Eutmp[i]; ∥ endotoxin concns if abs (eu2-eu1)＞1e-5 then begin j:=j+1; SEu[j] :=floatTostr (Eutmp[i]); Eu1:=eu2; End; End; ∥ sEu[j] deposit endotoxin concns value word string, j is an endotoxin standard concentration numbers 2,3,4 ..., 8 k:=j; If k＞=2 then begin for i:=1 to, 96 do begin x[i] :=0; Y[i] :=0; End; J:=0; For i:=1 to 96 do begin fi (pos (EuStd, TubeHead[i, 2])=0) or (pos (' invalid ', TubeHead[i, 2] then continue)＞0) or (Length (trim (TubeHead[i, 5]))=0); S1:=Trim (form3.StringGrid2.Cells[6, i]); If length (s1)＞0 then if StrToFloat (s1)＞0 then begin j:=j+1; X[j] :=StrToFloat (TubeHead[i, 5]); ∥ standard pipe concentration value y[j] :=StrToFloat (s1); ∥ obtains the OD or the RT value end of effective standard pipe; End; ∥ obtains standard endotoxin coordinate points x[j], y[j], take the logarithm, j be the standard pipe number＜!--SIPO＜DP n=" 11 "〉--〉＜dp n=" d11 "/tubm:=j; If ff＞0 then ∥ is to the test tube of same concentration, and OD under the terminal time or RT value are averaged k concentration begin of begin for k1:=1 tok do ∥ yav[k1] :=0; M1:=0; Xav [k1] :=strTofloat (sEu[k1]); For i:=1 to tubm do if abs (strtoFloat (sEu[k1])-x[i])＜1e-5 then begin m1:=m1+1; Yav[k1] :=yav[k1]+y[i]; End; If m1＞0 then yav[k1] :=yav[k1]/m1; End; For i:=1 to k do begin x[i] :=xav[i]; Y[i] :=yav[i]; End; Tubm:=k; End; The data that ∥ on average returns are ready to complete for j:=1 to tubm do if Form3.RadioButtonl.Checked then y[j] :=log10 (y[j]); Xm:=0; For i:=1 to tubm do xm:=xm+x[i]; Ym:=0; For i:=1 to tubm do ym:=ym+y[i]; Lxx:=0; For i:=1 to tubm do lxx:=lxx+x[i] * x[i]; Lxx:=lxx-xm*xm/tubm; Lxy:=0; For i:=1 to tubm do lxy:=lxy+x[i] * y[i]; Lxy:=lxy-xm*ym/tubm; Lyy:=0; For i:=1 to tubm do lyy:=lyy+y[i] * y[i]; Lyy:=lyy-ym*ym/tubm; Bod:=lxy/lxx; Aod:=ym/tubm-Bod*xm/tubm; Rod:=ky/ (sqrt (kx) * sqrt (lyy)); ∥ ... write and return equation, related coefficient CalEu03 () in the corresponding assembly; ∥ calculates the endotoxin measured value of each pipe, and as the recovery, standard variance, the coefficient of variation, ∥ writes respectively in the respective column of form.∥ ... push-botton operation logic control end; End;
Sample bacteria endotoxin content determination test is specified " terminal time "
To the reagent of a certain lot number, test if be applicable to end-point method, when data acquisition, experimental period can be set to the terminal time of this lot number reagent, the experimental period that general terminal time is all tested less than nephelometry; When data acquisition, specify " end-point method " image data time, when test arrives terminal time, test automatically and finish.Table one:
Bacterial endotoxin is measured [Raw070703-14] examining report
---end-point method
The date of inspection: 2003-7-7_ checker: Done tachypleus amebocyte lysate lot number: H450L
Sensitivity of the limulus reagent: 0.06 temperature of reactor: 37.0 BET water lot numbers: 0301220
Standard endotoxin lot number: 2002-12 room temperature: 26
Regression equation: Lg (Is/Ie)=-the 0.0148+1.0544C related coefficient: 0.9976 terminal time: 47 fens work wavelength: 405nm
| The pipe number | The test tube title | The sample lot number | Extension rate | Normaltoxin E | Recovery % | Optical density O.D | Actual measurement EU/ml | Variance SD | Variation CV% |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 | Negative control negative control negative control endotoxin standard endotoxin standard endotoxin standard endotoxin standard endotoxin standard endotoxin standard endotoxin standard endotoxin standard sample sample | - - - - - - - - - - - - - - - | - - - - - - - - - - - - - - - | 0.3 0.3 0.15 0.15 0.075 0.075 0.03 0.03 0.075 0.075 | 114.93 114.93 | 1.019404 1.017322 1.018658 1.987850 2.021794 1.390736 1.412708 1.129792 1.125221 1.060685 1.058534 1.264415 1.284261 1.578882 1.563428 | 0.0216 0.0216 0.0216 0.3005 0.3005 0.1531 0.1531 0.0635 0.0635 0.0379 0.0379 0.1139 0.1139 0.2001 0.2001 | 0.001 0.024 0.016 0.003 0.002 0.014 0.011 | 0.098 1.197 1.141 0.266 0.189 1.099 0.700 |
Claims (7)
1. a bacterial endotoxin quantitative detection method---dynamic end-point method, its method step is as follows:
1. use gel method, end-point method, kinetic turbidimetric assay or dynamic colourimetry tachypleus amebocyte lysate to do dynamic end-point method bacterial detection endotoxin;
2. determine to set up the required standard endotoxin concns of typical curve, the standard endotoxin concns of at least three concentration of preparation, react on photoelectric detector with tachypleus amebocyte lysate respectively, photoelectric detector is gathered response data, by computer recording and depict the performance graph of each reaction;
3. computing machine is determined the rational reaction end time period automatically according to the reaction performance graph, and searches out best terminal time T automatically in the section at this moment
0
4. utilize best terminal time T
0Set up typical curve, on photoelectric detector, carry out the endotoxin content of end-point method detection by quantitative sample.
2. according to the described bacterial endotoxin quantitative detecting method of claim 1, it is characterized in that: every lot number tachypleus amebocyte lysate and specific series concentration standard endotoxin reaction, its best terminal time T
0It is a fixing value.
3. according to the described bacterial endotoxin quantitative detecting method of claim 1, it is characterized in that: the endotoxin content of negative control is less than the minimum point concentration of typical curve.
4. according to the computer program of the described bacterial endotoxin quantitative detecting method of claim 1, comprising:
The penetrating light intensity that five parameters such as the sequence number of record test tube, title, sample lot number, extension rate, standard endotoxin and photoelectric detector read;
With document form data the data of record are preserved;
Open data file, sequence number, title, sample lot number, extension rate and five parameters of standard endotoxin of each test tube in the test are write the first five row of form; The penetrating light intensity of each test tube is at the two-dimensional array datak[i of correspondence, j] and test tube sequence number i=1,2 ..., 96, sampled data j=1,2 ..., in, carry out data analysis;
The method of data analysis is selected;
Search for best terminal time T
0
Utilize best terminal time T
0Carry out the end-point method test, the test sample endotoxin content.
5, according to the computer program of the described bacterial endotoxin quantitative detecting method of claim 4, it is characterized in that: the method for data analysis is selected OD method and RT method.
6, according to the computer program of the described bacterial endotoxin quantitative detecting method of claim 4, it is characterized in that: search for best terminal time and comprise:
1. the end-point method analytical approach is selected:
A, optical densitometric method (OD)---Is/Ie, along with the increase of test period, the OD value increases gradually, and variation range is between 1～1.7;
The regression equation of OD method is Lg (Is/Ie)=a+bC, and C is an endotoxin concns, and a, b are the regression equation coefficient.
B, penetrability method (RT)---Ie/Is, along with the increase of test period, the RT value reduces gradually, and variation range is between 0.3～1;
The regression equation of RT method is Ie/Is=a+bC, and C is an endotoxin concns, and a, b are the regression equation coefficient;
2. after from form, taking out the sequence number of standard pipe, set up the standard pipe array;
3. the endotoxin concns value leaves array X[I in the standard pipe] in, calculate:
Lxx=∑ Xi
2-(∑ Xi)
2/ n; N is the standard pipe number, I=1, and 2 ..., n;
4. since 5 minutes, the penetrating light intensity data of one group of standard pipe were got in every increase in 1 minute, calculated OD value or RT value to array Y[i] { OD method Y=Ig (Is/Ie) is among the RT method Y=Ie/Is}
Wherein: Is---initial light intensity, get wdq stationary phase (being generally 12 sampling periods) three light intensity mean values afterwards as first light intensity;
Ie-dynamic light intensity, the penetrating light intensity under the sampling time arbitrarily;
Dt---sampling interval, 5 seconds or 10 seconds; From the data file head, obtain;
5. calculate:
Lyy＝∑Yi
2-(∑Yi)
2/n；
Lxy=∑ Xi*Yi-(∑ Xi) * (∑ Yi)/n; N is the standard pipe number, I=1, and 2 ..., n;
Related coefficient: R=Lxy/ (sqrt (Lxx) * sqrt (Lyy))
6. when coefficient R＞0.9999, jump out circulation, obtain the reference value that this time is best terminal time;
Otherwise the time when getting related coefficient absolute value maximum is as the reference value of best terminal time.
7. according to the computer program of the described bacterial endotoxin quantitative detecting method of claim 6, it is characterized in that: determine terminal time:
1. the endotoxin concns of standard pipe is at an order of magnitude, as 0.03～0.3, the concentration number should three and more than;
2. the performance graph of Cmin pipe separates with the performance graph of negative control pipe;
3. the performance graph of Cmax pipe does not enter the smooth phase as yet.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102348984A (en) * | 2009-03-13 | 2012-02-08 | 兴和株式会社 | Method for measuring biogenous biologically active substances, a program for implementing the same, and apparatus for measuring biogenous biologically active substances |
| CN101960294B (en) * | 2008-03-19 | 2013-01-23 | 小幡彻 | Gel particle measuring apparatus |
| CN103901083A (en) * | 2014-01-10 | 2014-07-02 | 中国科学院苏州生物医学工程技术研究所 | Micro-fluidic chip for detecting endotoxin by adopting electrochemical method |
| CN111007253A (en) * | 2019-12-26 | 2020-04-14 | 常州千红生化制药股份有限公司 | Method for detecting bacterial endotoxin in heparin |
-
2003
- 2003-09-26 CN CN 03146974 patent/CN1276248C/en not_active Expired - Lifetime
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101960294B (en) * | 2008-03-19 | 2013-01-23 | 小幡彻 | Gel particle measuring apparatus |
| CN102348984A (en) * | 2009-03-13 | 2012-02-08 | 兴和株式会社 | Method for measuring biogenous biologically active substances, a program for implementing the same, and apparatus for measuring biogenous biologically active substances |
| CN102348984B (en) * | 2009-03-13 | 2014-04-02 | 兴和株式会社 | Method for measuring biogenous biologically active substances, a program for implementing the same, and apparatus for measuring biogenous biologically active substances |
| CN103901083A (en) * | 2014-01-10 | 2014-07-02 | 中国科学院苏州生物医学工程技术研究所 | Micro-fluidic chip for detecting endotoxin by adopting electrochemical method |
| CN103901083B (en) * | 2014-01-10 | 2016-04-20 | 中国科学院苏州生物医学工程技术研究所 | A kind of electrochemical process detects endotoxic micro-fluidic chip |
| CN111007253A (en) * | 2019-12-26 | 2020-04-14 | 常州千红生化制药股份有限公司 | Method for detecting bacterial endotoxin in heparin |
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