CN102297966A - Progesterone semi-quantitative determining colloidal gold test paper and preparation method thereof - Google Patents
Progesterone semi-quantitative determining colloidal gold test paper and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a progesterone semi-quantitative determining colloidal gold test paper and a preparation method thereof. The invention belongs to the technical field of dairy cow pregnancy diagnosis. According to the progesterone semi-quantitative determining colloidal gold test paper, a sample pad, a gold-labeled pad containing a gold-labeled progesterone composite, and a nitrocellulose filter are connected in series, and are stuck on a substrate. The nitrocellulose filter comprises a detection line and a control line. The preparation method of the test paper comprises the following steps that: colloidal gold is prepared form chlorauric acid and trisodium citrate; purified polyclonal antibodies are prepared through steps that: artificial dairy cow progesterone antigens are prepared with a diimine coupling method, the artificial progesterone antigens are used for immunizing mice, and progesterone polyclonal antibodies are prepared; gold-labeled antibodies are prepared through steps that: colloidal gold and the antibodies are absorbed, and are purified through centrifugation; the colloidal gold test paper is prepared through steps that: a PB solution containing tween is used for soaking the gold-labeled pad, the gold-labeled antibodies are filled in a glass fiber membrane, and two lings formed by artificial dairy cow progesterone antigens and goat anti-mouse IgG secondary antibodies are sprayed on the nitrocellulose filter by using a membrane spotting machine. The operation of the method is simple, the method is convenient, applicable, highly efficient, fast, and is suitable to be popularized.
Description
Technical field
The invention belongs to milk cow cyesiognosis technical field, particularly relate to a kind of half-quantitative detection progesterone colloidal gold strip and preparation method thereof.
Background technology
Progesterone (P in the dam body
4) mainly synthetic at ovary, be the accurate indicator of reflection ovarian activity, have the function of control dam reproduction activity.Detect progesterone level and content in the body fluid, not only can understand the reproductive physiology rule of dam, and can monitor genital system diseases, and be the important means of research dam reproductive physiology and disease thereof, be a kind of effective ways of prevention and treatment female livestock breed obstacle disease.P in blood or the milk
4Determination on content also can be used for diagnosis of early gestation, infertility diagnosis and the evaluation of oestrusing; Also can be used for the research of ovary function, breeding difficulty and obstetrics' disease; Also can be used to check the artificial insemination effect, hormone test is for genetic analysis provides effective physical signs etc.Diagnosis of early gestation for improve cow breeding situation, reduce the nonpregnant time, shorten the tire spacing, to improve the economic benefit that cow raises significant.
At present, measure progesterone with radio immunoassay (RIA) and have sensitivity and characteristics such as degree of accuracy height, good reproducibility, but because of its to the equipment requirements height, during check fee, the waste liquid of generation is difficult to handle, and radiological hazard is arranged and has limited its application.Abroad carry out cyesiognosis and obtained bigger progress measuring progesterone with ELISA in recent years, some complete kits appear on the market, and make to measure progesterone on the spot in the pasture, carry out diagnosis of early gestation and become a reality.Yet, find that in production practices are used the ELISA susceptibility is lower, problem such as background is darker, and the result judges relatively difficulty, and measurement result is stable inadequately.At present, the many dairy farms of China are still by examination per rectum and carry out cyesiognosis, conceived earliest time and the Best Times of this method diagnosis do not have definite standard, and its effect depends on the development condition etc. of examiner's clinical experience, domestic animal kind, parent and fetus to a great extent.Generally must carry out after 2 months, thereby be difficult in time find the unpregnancy cow, can not in time handle, increase the nonpregnant time of cow cow in artificial insemination.The external ELISA kit of producing costs an arm and a leg, and domestic dairy farm is difficult to be extensive use of.In view of the foregoing, studying new early stage, responsive, special, easy, accurate and maneuverable progesterone detection technique and just seem very necessary, also is current problem demanding prompt solution, and this is for promoting China's Developing of Animal Industry to have very important meaning.
Summary of the invention
The present invention provides a kind of half-quantitative detection progesterone colloidal gold strip and preparation method thereof for solving the technical matters that exists in the known technology.
The immuno-gold labeling technology has mainly utilized gold grain to have the characteristic of high electron density, and the place forms macroscopic redness or pink spot in gold mark protein combination, thereby become can qualitative or semiquantitative tachysynthesis detection method.
One of purpose of the present invention provide a kind of have simple to operate, convenient and practical, for improving cow breeding situation, reduce the nonpregnant time, shorten the tire spacing, control obstetrics disease, checking the artificial insemination effect, improve the half-quantitative detection progesterone colloidal gold strip of characteristics such as economic benefit that cow raises is significant.
The technical scheme that half-quantitative detection progesterone colloidal gold strip of the present invention is taked for the technical matters that exists in the solution known technology is:
A kind of half-quantitative detection progesterone colloidal gold strip is characterized in: test strips comprises gold mark pad, nitrocellulose membrane, glass fibre membrane sample pad, adsorptive pads, the PVC base plate that contains gold mark progesterone antibody complex; The glass fibre membrane sample pad, contain gold mark progesterone antibody complex gold mark pad and the end to end PVC of the sticking on base plate of nitrocellulose filter on, the opposite side of nitrocellulose filter is pasted with adsorptive pads; Cow progesterone artificial antigen detection line and sheep anti mouse two anti-control lines are arranged on the nitrocellulose filter, and cow progesterone artificial antigen detection line is near gold mark pad one side that contains progesterone antibody.
Half-quantitative detection progesterone colloidal gold strip of the present invention can also adopt following technical scheme:
Described half-quantitative detection progesterone colloidal gold strip is characterized in: control line is apart from adsorptive pads near-end 0.3-0.6cm, and detection line and adsorptive pads near-end are at a distance of 0.8-1.3cm.
Described half-quantitative detection progesterone colloidal gold strip is characterized in: control line sheep anti mouse two anti-spraying concentration are 0.2-2mg/L, and the spraying concentration of the cow progesterone artificial antigen of detection line is 0.05-0.1mg/ml.
Described half-quantitative detection progesterone colloidal gold strip is characterized in: the test strips size is 6-8cm * 0.3-0.6cm, the long 1.8-2.2cm of adsorptive pads, the long 1.8-2.2cm of sample pad, gold mark pad long 0.6-1.0cm, the long 2-3cm of nitrocellulose filter; Nitrocellulose filter is attached on the base plate, with edge, base plate suction side at a distance of 1.3-1.8cm; Adsorptive pads and nitrocellulose filter overlap and are affixed on the base plate suction side, sample pad, gold mark pad from top to bottom overlap successively with cellulose nitrate, gold mark pad is between sample pad and nitrocellulose filter, and gold mark pad is 0.8-1.3mm with the overlapping length of nitrocellulose filter.
Two of purpose of the present invention provides that a kind of to have technology simple, easy to operate, economical and practical, the preparation method of easier half-quantitative detection progesterone colloidal gold strip in characteristics such as basic unit's popularizations.
The technical scheme that the preparation method taked of half-quantitative detection progesterone colloidal gold strip of the present invention is:
A kind of preparation method of half-quantitative detection progesterone colloidal gold strip is characterized in that preparation process is as follows:
The first step, the preparation collaurum: the heating aqueous solution of chloraurate adds trisodium citrate aqueous solution to boiling, is heated to transparent redness, makes colloid gold particle size 15nm-20nm;
Second step, preparation purifying polyclonal antibody: adopt the dicyclohexyl carbodiimide coupling method with progesterone-11 α-hemisuccinic acid ester respectively with bovine serum albumin(BSA), the coupling of oralbumin carrier, preparation cow progesterone artificial antigen; Prepare immune serum with immune mouse after artificial immunity antigen for preparing and the Freund emulsification again; How anti-adopt sad-ammonium sulfate method method to obtain the purifying progesterone;
In the 3rd step, prepare golden labeling antibody: antibody-solutions is desalted, and collaurum pH value is adsorbed with antibody between 9-9.5 the time, employing centrifuge method purifying;
In the 4th step, the preparation of colloidal gold strip: with the PB solution immersion gold mark pad 20-40min of weight content 0.3-0.6% tween, drying is poured into golden labeling antibody on the glass fibre membrane of having handled well in oven dry; On nitrocellulose membrane, be sprayed into 2 lines with cow progesterone artificial antigen and sheep anti mouse two are anti-, be respectively detection line and control line, again vacuum drying with some film machine; Sample pad, gold mark pad are sticked on the base plate with nitrocellulose filter is end to end, and adsorptive pads is attached to the opposite side of nitrocellulose filter, and half-quantitative detection progesterone colloidal gold strip is made in assembling.
The preparation method of half-quantitative detection progesterone colloidal gold strip of the present invention can also take following technical scheme:
The preparation method of described half-quantitative detection progesterone colloidal gold strip is characterized in: during the preparation collaurum, with the extremely boiling of micro-wave oven heating 0.005-0.02% aqueous solution of chloraurate, the trisodium citrate aqueous solution of disposable adding 1-5mL 0.5-2%.
The preparation method of described half-quantitative detection progesterone colloidal gold strip is characterized in: how anti-adopt sad-ammonium sulfate method method to obtain the purifying progesterone, and the antibodies to "wrong" antigens in will resisting is removed more; Recording antibody concentration at last is 1-3mg/mL.
The present invention detects the preparation method of the colloidal gold strip of early stage conceived cow, and its concrete preparation process may further comprise the steps:
The first step, the preparation collaurum: heat 0.01% aqueous solution of chloraurate to boiling with micro-wave oven, the trisodium citrate aqueous solution of disposable adding 2.5mL 1% is heated to till the transparent redness.The colloid gold particle size is 15nm-20nm all, and mean grain size (n=20) is 18.9nm ± 0.5nm;
Second step, preparation antibody purification: adopt the dicyclohexyl carbodiimide coupling method with progesterone-11 α-hemisuccinic acid ester (P
4-11 α-HS) respectively with bovine serum albumin(BSA) (BSA), oralbumin (OVAA1G) fat, quick, free from environmental pollution 0) the carrier coupling, preparation cow progesterone artificial antigen P
4-BSA and P
4-OVA.Again with the artificial immunity antigen P for preparing
4-BSA(50ug～100ug/ is only) with Freund emulsification after immune Balb/c mouse prepare immune serum.Use P
4-OVA detects antigen as ELISA, and indirect elisa method is measured tiring of progesterone antibody in the immune serum: antibody titer: 1:12800～25600; Adopting sad-ammonium sulfate method method to obtain antibody purification, is 6.37mg/mL with the determined by ultraviolet spectrophotometry antibody concentration; Further purification progesterone antibody, recording antibody concentration is 1.57mg/mL;
In the 3rd step, prepare golden labeling antibody: antibody-solutions to be marked is desalted, and finally determining to stablize the suitableeest antibody amount of 1 mL collaurum is 36.72ug.Collaurum pH value is better with many anti-absorption between 9-9.5 the time; Adopt low temperature supercentrifugation purifying;
The 4th step, the preparation of colloidal gold strip: glass fibre membrane is cut into the wide slice of 8mm, soaks gold mark pad 30min with the PB solution that contains 0.5% tween, 37 ℃ of oven dry, on the glass fibre membrane that golden labeling antibody perfusion has been handled well, dry under 4 ℃ of conditions at last.On nitrocellulose membrane, will compete former P with a film machine
4-OVA and sheep anti-mouse igg are sprayed into 2 lines, be respectively detection line (T line) and control line (C line), vacuum drying again, sample pad, gold mark pad are sticked on the base plate with nitrocellulose filter is end to end, adsorptive pads is attached to the other end of nitrocellulose filter, the assembling back is cut into the wide test strips of 4mm with cutting machine, and is standby;
In the 5th step, detect and interpretation: the application of sample end is inserted liquid to be measured (take out moistening back) or testing sample is added drop-wise on the sample pad the about 3-5min of horizontal positioned, observations.As on the test strips nitrocellulose membrane only control line to be a purplish red colour band testing result then positive; As it is then negative 2 purplish red colour bands to occur; Do not develop the color as control line, it is invalid then test strips to be considered as.
Advantage and good effect that the present invention has are:
Half-quantitative detection progesterone colloidal gold strip and preparation method thereof has been owing to adopted brand-new technology scheme of the present invention, compared with prior art,
Detection progesterone colloidal gold strip of the present invention is the product of progesterone level in the complete detection serum, simple to operate, convenient and practical, for improving cow breeding situation, reduce the nonpregnant time, shorten the tire spacing, control obstetrics' disease, checking the artificial insemination effect, the economic benefit that the raising cow is raised is significant.The present invention makes the early diagnosis of milk cow become possibility, and method is simple and reliable, promotes in basic unit easilier.
Colloidal gold strip is used for the immune detection of macroscopic level, has the following advantages: 1. sample does not need special processing, and reagent and sample consumption are minimum; 2. susceptibility and specificity height both can be used for Detection of antigen, also can be used for antibody test; 3. the time shortens greatly, has improved detection speed, just can obtain the result in several minutes; 4. simple to operate, do not need expensive instruments such as fluorescent microscope, enzyme mark detector, be more suitable for on-the-spot and basic unit's application; 5. experimental result can long preservation.This makes it that very big application potential be arranged in inspection and quarantine.Can the antibody of different infectious diseases and hormone be made test strips by preparation monoclonal or polyclonal antibody, directly detect pathogen and hormone in blood, swab, ight soil, the tissue suspension.The Antibody Preparation of infectious disease cause of disease and hormone is become test strips, and technical is ripe, is feasible on the method, and colloidal gold strip is widely used in the inspection and quarantine development trend that is inevitable.
Description of drawings
Fig. 1 is a half-quantitative detection progesterone colloidal gold strip structural representation;
Fig. 2 is Fig. 1 side-looking structural representation;
Fig. 3 is a half-quantitative detection progesterone colloidal gold strip positive findings displayed map;
Fig. 4 is a half-quantitative detection progesterone colloidal gold strip negative findings displayed map.
Among the figure, 1-sample pad, 2-gold mark pad, 3-detection line, 4-control line, 5-base plate, 6-adsorptive pads, 7-nitrocellulose filter.
Embodiment
For further understanding technology contents of the present invention, characteristics and effect, exemplify following examples now, and conjunction with figs. is described in detail as follows:
A kind of colloidal gold strip that can detect early stage conceived milk cow, it is developed by the plain film gold of the glass fibre that contains progesterone antibody mark pad 2, nitrocellulose filter 7, glass fibre membrane sample pad 1, adsorptive pads 6, PVC base plate 5 etc., and test strips detects principle and adopts competition law.
The test strips size is 6cm * 0.4cm, the long 2cm of adsorptive pads, the long 2cm of sample pad, gold mark pad long 0.8cm, nitrocellulose filter 2.5cm.Nitrocellulose filter is attached on the base plate, with edge, base plate suction side at a distance of 1.5cm; Successively adsorptive pads and nitrocellulose filter overlapping are affixed on the base plate suction side then, successively sample pad, gold mark pad are from top to bottom overlapped successively with cellulose nitrate again, gold mark pad is between sample pad and nitrocellulose filter, and gold mark pad is 1mm with the overlapping length of nitrocellulose filter.The material of each material is as follows in this test strips: base plate is homemade PVC material, and outward appearance is white in color; Adsorptive pads adopts homemade thieving paper material, and model is H-8; Nitrocellulose filter is the material of import, and model is Whantman Prima; Gold mark pad is the plain film of the glass fibre of import, and model is Alstrom 8964; Sample pad is the homemade plain film of glass fibre, and model is GF-08; Control line 4(C line) and detection line 3(T line) relative position: C line-spacing adsorptive pads near-end 0.5cm, T line and adsorptive pads near-end are at a distance of 1cm.Gold mark pad is handled: with model is that the plain film of glass fibre of Alstrom 8964 immerses gold mark pad treating fluid and (contains 5% sucrose, 0.5% tween, 0.1%NaN
3, the PBS solution of pH=7.2 0.01mol/L) and middle 30min, 37 ℃ of dryings of soaking.After golden labeling antibody purification, be diluted to 2/5 of initial volume with golden labeling antibody dilution (the PB solution that contains the pH=7.0 0.01mol/L of 1%BSA, 3% trehalose), gold mark pad is soaked in wherein, carries out low temperature drying then.C line sheep anti mouse two anti-spraying concentration are greater than 0.2mg/mL, and the C wire spraying concentration among the present invention is 2mg/mL; The P of T line
4-The best spraying of-OVA concentration is 0.072mg/mL.
A kind of preparation method that can detect the colloidal gold strip of early stage conceived cow, its preparation process is as follows:
The first step, adopt the sodium citrate reducing process to prepare collaurum: at first 100mL 0.01% chlorauric acid solution to be heated to boiling with micro-wave oven, 1% sodium citrate solution of disposable then adding 2.5mL, mixing continues to boil 3～5min after solution becomes claret rapidly.The colloid gold particle diameter for preparing among the present invention should be about 15nm-20nm.
Second step, preparation purifying polyclonal antibody: adopt the dicyclohexyl carbodiimide coupling method with progesterone-11 α-hemisuccinic acid fat (P
4-11 α-HS) respectively with bovine serum albumin(BSA) (BSA), the coupling of the pure albumen of ovum gallinaceum (OVA) carrier, preparation progesterone artificial antigen P
4-BSA and P
4-OVA is respectively as immunogene and detection former (competing former).Prepared P
4-BSA and P
4The concentration of-OVA is respectively 11.1mg/mL and 4.67mg/mL.With synthetic artificial antigen P
4-BSA, lumbar injection active immunity mouse, first immunisation adopts incomplete Freund emulsification antigen later on Freund's complete adjuvant emulsification antigen, and immunity is spaced apart 1 month, the preparation immune serum.The progesterone antibody titer that records with indirect elisa method is up to 1:25600.With antibody in sad-ammonium sulfate method purified blood serum, SDS-PAGE detects antibody purity, and purity should reach more than 95%.
The 3rd step, the preparation of gold mark progesterone antibody complex:
The pH of collaurum is transferred to 9.0, the progesterone antibody concentration is transferred to 1mg/mL, the antibody-solutions of getting different amounts (0.5uL, 0.7uL, 1uL, 2uL, 3uL, 4uL, 5uL) mixes with the colloidal gold solution of 100uL respectively, the 10%NaCl that adds 10 μ L, the collaurum that adds the antibody quantity not sufficient will become blue, and institute is when adding the antibody amount and surpassing, and the collaurum color will remain unchanged, determine the antibody amount that the 1mL collaurum is required with this, add 20% antibody amount in addition on this basis again;
Get aequum antibody and under magnetic agitation, mix, as stabilizing agent, prepare golden labeling antibody with PEG-20000 with colloidal gold solution;
Golden labeling antibody solution is first with the gold grain of the centrifugal 15min of 1500rpm with the removal aggegation; 10000rpm carries out high speed centrifugation 30min again, inhales and abandons supernatant, and bolarious precipitation is in conjunction with good collaurum-antibody complex, makes suspension;
The method that adopts golden labeling antibody and antigen directly to react on the NC film is identified the bonding state of golden labeling antibody;
The 4th step, the preparation of colloidal gold test paper film:
On the NC film, rule coating antigen P with a film machine
4-OVA is used for the demonstration of detection line, the sheep anti mouse two anti-demonstrations that are used for control line; Detection line P
4The best bag of-OVA is 0.072mg/mL by concentration, and control line two anti-concentration are 2mg/mL.
The gold labeling antibody is sprayed on the glass fibre membrane of having handled well, and is dry under 4 ℃ of conditions;
The assembling of test-strips is fixed in NC film, gold mark pad, thieving paper and sample pad on the PVC base plate, cuts with the test strips cutting machine then, and the test strips size is 4mm * 6cm;
The using method of test strips of the present invention: at milk cow breed blood sampling in back about 22 days and separation of serum, serum is dripped on the sample pad of test strips, set level test strips, will demonstrate the result through about 5min.
In the 5th step, the result judges: the principle of progesterone test strips of the present invention is a competition law.When the progesterone concentration in the serum is higher than ultimate value, progesterone in the serum can be with the whole combinations of gold mark progesterone antibody on the gold mark pad, golden labeling antibody-progesterone the compound that forms can not held back by tested survey line, when this compound chromatography during to control line, holds back colour developing thereby can be resisted by two.Therefore positive findings is: detection line (T line) does not develop the color, and control line (C line) colour developing; And when the progesterone concentration in the serum was lower than ultimate value, the progesterone in the serum can not be with the whole combinations of the mark progesterone antibody of the gold on the gold mark pad, thereby a part of golden labeling antibody compound can tested survey line be held back at detection line and developed the color.Therefore, negative findings is: detection line (T line) and control line (C line) all develop the color.When control line does not develop the color, illustrate that test strips lost efficacy.
Claims (7)
1. half-quantitative detection progesterone colloidal gold strip is characterized in that: test strips comprises gold mark pad, nitrocellulose membrane, glass fibre membrane sample pad, adsorptive pads, the PVC base plate that contains gold mark progesterone antibody complex; The glass fibre membrane sample pad, contain gold mark progesterone antibody gold mark pad and the end to end PVC of the sticking on base plate of nitrocellulose filter on, the opposite side of nitrocellulose filter is pasted with adsorptive pads; Cow progesterone artificial antigen detection line and sheep anti mouse two anti-control lines are arranged on the nitrocellulose filter, and cow progesterone artificial antigen detection line is near gold mark pad one side that contains gold mark progesterone antibody.
2. according to the described half-quantitative detection progesterone of claim 1 colloidal gold strip, it is characterized in that: control line is apart from adsorptive pads near-end 0.3-0.6cm, and detection line and adsorptive pads near-end are at a distance of 0.8-1.3cm.
3. according to claim 1 or 2 described half-quantitative detection progesterone colloidal gold strips, it is characterized in that: control line sheep anti mouse two anti-spraying concentration are 0.2-2mg/mL, and the spraying concentration of the cow progesterone artificial antigen of detection line is 0.05-0.1mg/ml.
4. according to claim 1 or 2 described half-quantitative detection progesterone colloidal gold strips, it is characterized in that: the test strips size is 6-8cm * 0.3-0.6cm, the long 1.8-2.2cm of adsorptive pads, the long 1.8-2.2cm of sample pad, gold mark pad long 0.6-1.0cm, the long 2-3cm of nitrocellulose filter; Nitrocellulose filter is attached on the base plate, with edge, base plate suction side at a distance of 1.3-1.8cm; Adsorptive pads and nitrocellulose filter overlap and are affixed on the base plate suction side, sample pad, gold mark pad from top to bottom overlap successively with cellulose nitrate, gold mark pad is between sample pad and nitrocellulose filter, and gold mark pad is 0.8-1.3mm with the overlapping length of nitrocellulose filter.
5. the preparation method of a half-quantitative detection progesterone colloidal gold strip is characterized in that preparation process is as follows:
The first step, the preparation collaurum: the heating aqueous solution of chloraurate adds trisodium citrate aqueous solution to boiling, is heated to transparent redness, makes colloid gold particle size 15nm-20nm;
Second step, preparation purifying polyclonal antibody: adopt the dicyclohexyl carbodiimide coupling method with progesterone-11 α-hemisuccinic acid ester respectively with bovine serum albumin(BSA), the coupling of oralbumin carrier, preparation cow progesterone artificial antigen; Prepare immune serum with immune mouse after artificial immunity antigen for preparing and the Freund emulsification again; Adopt sad-ammonium sulfate method method to obtain how anti-, the also further purification progesterone antibody of purifying progesterone;
In the 3rd step, prepare golden labeling antibody: antibody-solutions is desalted, and collaurum pH value is adsorbed with antibody between 9-9.5 the time, employing centrifuge method purifying;
In the 4th step, the preparation of colloidal gold strip: with the PB solution immersion gold mark pad 20-40min of weight content 0.3-0.6% tween, drying is poured into golden labeling antibody on the glass fibre membrane of having handled well in oven dry; On nitrocellulose membrane, be sprayed into 2 lines with cow progesterone artificial antigen and sheep anti mouse two are anti-, be respectively detection line and control line, again vacuum drying with some film machine; Sample pad, gold mark pad are sticked on the base plate with nitrocellulose filter is end to end, and adsorptive pads is attached to the opposite side of nitrocellulose filter, and half-quantitative detection progesterone colloidal gold strip is made in assembling.
6. according to the preparation method of the described half-quantitative detection progesterone of claim 5 colloidal gold strip, it is characterized in that: during the preparation collaurum, with the extremely boiling of micro-wave oven heating 0.005-0.02% aqueous solution of chloraurate, the trisodium citrate aqueous solution of disposable adding 1-5mL 0.5-2%.
7. according to the preparation method of the described half-quantitative detection progesterone of claim 5 colloidal gold strip, it is characterized in that: adopt sad-ammonium sulfate method method to obtain the progesterone polyclonal antibody, the antibodies to "wrong" antigens in will resisting is again removed more; Antibody concentration is 1-3mg/mL behind the purifying.
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