CN103454413A - Bactrian camel specific antibody preparation method and immune detection method - Google Patents
Bactrian camel specific antibody preparation method and immune detection method Download PDFInfo
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Abstract
The invention relates to an optimal immune procedure and method of an immune camel. The immune method comprises the step of multipoint subcutaneous injecting at the back of a Bactrian camel, wherein the immune period is two weeks, the injected antigen dosage is 1mg/ml, and the immune is carried out three times. The invention further relates to a preparation flow and a method of a rabbit anti-camel IgG enzyme-labeled secondary antibody, a method for detecting camel anti-serum titer by applying the rabbit anti-camel IgG enzyme-labeled secondary antibody through ELISA and an optimal detection condition.
Description
(1) technical field
The invention belongs to the antibody engineering technical field, it is research object that Inner Mongol Soviet Union nit two-humped camel is take in the present invention, how anti-ly utilizes bovine serum albumin(BSA) (BSA) to prepare the camel of anti-BSA, optimizes immune condition.And the anti-camel IgG of preparation rabbit ELIAS secondary antibody, set up indirect ELISA testing kit.
(2) background technology
Immune system is divided into inherent immunity and adapts to immunity, wherein adapts to immunity and is divided into again humoral immunity and cellular immunity.
Antiserum why can and relevant antigen play immune association reaction, be because in antiserum, contain relevant antibody protein.The animal protein that will have antibody activity in early 1960s is defined as immunoglobulin (Ig) (immunoglobulin).Mainly be present in blood plasma, also see in other body fluid, tissue and some juices.The antigenicity of L chain identical (κ or λ) always on general Ig molecule.But H chain antigenicity not identical (μ chain, γ chain, α chain, δ chain and ε chain), the molecule that different H chains and L chain is formed to complete Ig according to it can be divided into five classes, be immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) and immunoglobulin E (IgE), IgG, IgA and IgM also have subclass.The basic structure of Ig molecule is comprised of the tetrapeptide chain, the light chain (L chain) of two identical molecular weights and two larger heavy chains (H chain) of identical molecular weight, consists of.
The principal ingredient of human serum immunoglobulin (Ig) is IgG, and it accounts for the 70-75% of total immunoglobulin (Ig), molecular weight approximately 150,000, sugary 2～3%.
The human serum Immunoglobulin IgG is the most lasting in primary immune response, most important antibody, and it only exists with monomeric form.That antibiotic property, mithridatism and antiviral antibody belong to IgG mostly, it plays main force's effect in anti-infective, the phagocytosis (opsonic action) of mononuclear macrophage be can promote, bacteriotoxic toxicity (neutralizing a toxin) and viral antigen neutralized in conjunction with making virus lose the ability (neutralization virus) of host cells infected.The IgG age synthetic at body will be later than IgM, and 3rd month after birth starts to synthesize, and 3-5 year approaches adult's level.It is unique can, by the Ig of placenta, playing an important role in natural passive immunity.In addition, IgG also has opsonophagocytosis and in conjunction with effects such as SPA.
1993, Hamers-Casterman etc. reported and in camellid (one-humped camel, two-humped camel, yamma etc.) body, have existed a kind of containing heavy chain, not contain the natural heavy chain antibody (Heavy chain antibody, HCAb) of light chain.Afterwards, also found the neoantigen acceptor (new antigen receptor, NAR) with camel heavy chain antibody structural similarity in some selachian bodies.
Because the camel heavy chain antibody is pure natural, can not directly act on the people, or be that the mankind are used.Therefore after cloning the variable region of heavy chain antibody in the camel body, the single domain antibody only formed by a variable region of heavy chain obtained, be referred to as heavy chain variable region gene antibody (variable domain of heavy chain of heavy-chain antibody, VHH), its diameter is 2.5nm, long 4nm, nano antibody therefore is otherwise known as.As a kind of small-sized genetic engineering antibody, the high expressed that nano antibody possesses, water-soluble, stability, strong tissue penetration and the weak advantages such as immunogenicity, make this antibody gather around in fields such as fundamental research and drug developments hold out broad prospects [10].At present, in European and American areas, the research of the VHH single domain antibody based on the camel heavy chain antibody has become a focus [11], but rarely seen report still at home.
Nano antibody is as a strict monomer, and the structure that it is special and character, make it possess the peculiar property that some other common antibody do not have.As simple as easy acquisition and expression, highly-water-soluble and stability, targeting, humanization etc.
In current domestic report, only to the serum analysis of a peak camel not synantigen produce the situation of heavy chain antibody in the immunologic process of camel, unavoidably exist biased.External research emphasis physicochemical property, application and the antigen targeting aspect of VHH antibody that be to recombinate.Top condition for preparation camel antibody mediated immunity process does not but have report at present.
Powerful advantages and application in view of nano antibody.It is research object that Inner Mongol Soviet Union nit two-humped camel is take in the present invention, the most general in line with the antigen application, as to be easy to get principle, for a certain amount of camel colony, analyzed, the anti-BSA polyclonal antibody of preparation camel, the body reaction of exploring in two-humped camel specific antibody preparation process changes, and determines the best immune time of camel.For analyzing from now on the disease-resistant protective effect of two-humped camel heavy chain antibody, prepare nano antibody and lay the groundwork, provide new direction for researching and developing from now on novel genetic engineering antibody simultaneously.
Simultaneously, because anti-camel IgG ELIAS secondary antibody on international market does not also have commercialization, for the research of nano antibody is from now on made troubles, the present invention is by the anti-camel IgG of preparation rabbit ELIAS secondary antibody, set up ELISA method diagnostic kit, detect tiring of camel Serum Antibody after immunity.
(3) summary of the invention
1. the optimum immuning program of the claimed immune camel of the present invention and method;
Immunization method of the present invention is the subcutaneous multi-point injection in two-humped camel back, and the immunity cycle is 2 weeks, and injections of antigens dosage is 1mg/mL, and immune time is 3 times;
2. related technical indicator and the result of the claimed preparation of the present invention rabbit anti-camel IgG ELIAS secondary antibody;
It is as follows that the present invention prepares the anti-camel IgG of rabbit ELIAS secondary antibody step: 1. affinity chromatography Pro A method purifying camel IgG, 2. Lowry method protein quantification 3. the SDS-PAGE purity detecting 4. prepare the anti-camel IgG of rabbit antiserum, the subcutaneous multi-point injection in back, injections of antigens dosage is 0.6mg/mL, the immunity cycle is 2 weeks, and immune time is 3 times.5. the anti-camel IgG of affinity chromatography Pro A method purified rabbit, 6. the ELISA method detect the anti-camel IgG of rabbit antiserum titre 7. simple and easy sodium periodate method labelled antibody 8. Lowry method protein quantification labelled antibody 9. the ELISA method measure labelled antibody and tire;
3. the anti-camel ELIAS secondary antibody of the claimed application of the present invention rabbit ELISA detects camel antiserum titre method and optimal detection condition.
The present invention detects camel antiserum optimal detection condition and comprises: 1. 2. 3. best confining liquid of best two anti-dilute concentrations of antigen-antibody best effort concentration.
It is research object that Inner Mongol Soviet Union nit two-humped camel is take in the present invention, utilizes bovine serum albumin(BSA) (BSA) to prepare the how anti-and anti-camel IgG of the rabbit ELIAS secondary antibody of the camel of anti-BSA, and sets up indirect ELISA testing kit.
Experiment is divided into three parts: first immunity camel prepares immune camel serum; The camel IgG immune rabbit of purifying for second portion, the anti-camel IgG of preparation rabbit ELIAS secondary antibody, prepare for ELISA detects; Third part application indirect elisa method detects immunity camel antiserum titre in each in period, finally determines best immune time, and the body reaction of setting forth in two-humped camel specific antibody preparation process changes.
(4) accompanying drawing explanation
Figure 11 is SDS-PAGE under non-reducing condition, band in 170KD be in the two-humped camel serum of purifying Inner Mongol IgG1, the IgG3 of mixing, band in the 90KD place, be in the two-humped camel serum of purifying Inner Mongol IgG2; The 2nd, SDS-PAGE under reducing condition, band is heavy chain in 55 places, 40 places are light chain.
The camel IgG Lowry method protein quantification typical curve of Fig. 2 purifying
After the anti-camel IgG of Fig. 3 rabbit enzyme mark two purifying and mark after Lowry method protein quantification typical curve
The anti-camel IgG of Fig. 4 rabbit antiserum titre
The anti-camel IgG of rabbit antiserum titre after Fig. 5 mark
The each immune P/N of Fig. 6 experimental group, under same dilutability, five road columns are respectively before immunity from left to right, just exempt from, two exempt from, three exempt to exempt from four.As seen from the figure, 0 exempts from and just exempts from P/N<2.1, without antibody, produces; The two serum greatest dilutions of exempting from P/N>2.1 are 1:400; Serum greatest dilution for the third time with four immune P/N>2.1 is 1:25600.
The each immune P/N of Fig. 7 control group, under same dilutability, five road columns are respectively before immunity from left to right, just exempt from, two exempt from, three exempt to exempt from four.As seen from the figure, just exempt from and the P/N value of booster immunization afterwards all not>2.1, and produce without antibody.
Fig. 8 experimental group increases with immune time, known in antiserum titre figure in immune serum, and immunity gets final product for three times.
(5) embodiment
1. the preparation of polyclonal antibody
Animal used as test is divided into two groups of blank group (3 peak) and experimental group (3 peak).Just exempt from front respectively Jugular vessel and extract the 20mL negative blood, separation of serum, frozen in-40 ℃ after aseptic subpackaged.The each immune physiological saline of blank group; Experimental group immunity 0.1mg/ml BSA, immunity is 4 times altogether.Reach eventually and exempt to get blood from the camel Jugular vessel, separation of serum after 15 days before each immunity.
2.ELISA the preliminary foundation of detection method
2.1 the preparation of the anti-hunchbacked IgG ELIAS secondary antibody of rabbit
1) purification of affinity chromatography camel IgG;
1. wash Protein A is fixed in to iron stand.After filler dress post, washing protective agent off with 10 bed volumes of ultrapure water is ethanol;
2. the level pad balance pillar of 10 column volumes for balance, eluate equates to get final product with initial pH of buffer;
3. loading is the serum upper prop, loading flow velocity≤60cm/h.
4. clean with the level pad of 20 column volumes and clean bed volume, until the UV line is to baseline.
5. elution buffer (glycocoll PH2.2) wash-out for wash-out, start to collect eluting peak at ultraviolet 280nm place higher than 0.1, and lower than end in 0.1 o'clock, eluent neutralized neutrality by neutralization buffer immediately, is concentrated to volume required.
2) Lowry method mensuration protein content is 2.4mg/ml;
1. according to protein concentration, the standard protein solution of accurate measuring different volumes, respectively add PBS and mend to 0.2ml respectively, makes the solubility of standard protein be respectively 0,12.5,25,50,100,200 μ g/ml;
2. the accurate measuring sample adds PBS and mends to 0.2ml;
3. by typical curve with sample respectively adds the 1ml basic cupric sulfate and 0.1ml Folin-phenol reagent mixes, survey OD at the 750nm place;
4. the concentration of standard protein of take is Y-axis, and absorbance value is X-axis, obtains a typical curve.Absorbance value by sample calculates its concentration;
3) SDS-PAGE purity detecting;
1. prepare the beaker of a clean 200ml, the separation gel that the preparation mass concentration is 10%
2. the separation gel prepared is added in 4.7ml to two glass plate, with a small amount of isopropyl alcohol fluid-tight;
3. standing solidifying, outwell isopropyl alcohol, with filter paper, blots;
4. in clean 50ml beaker, the preparation mass concentration is 5% concentrated glue (when preparation separation gel and concentrated glue, in the time of the ammonium persulfate that to add mass concentration be 10% and TEMED, limit to shake the beaker limit and add, guarantee evenly), fill a prescription in Table
5. add the concentrated glue of 1mL to separation gel top with liquid-transfering gun, and plug gently broach, guarantee that each tooth rim encloses without bubble;
6. place half an hour, after gelling to be concentrated is solid, carefully take out comb, put into electrophoresis tank.
7. albumen runs glue: will reduce Loading Buffer, non-reduced Loading Buffer, natural test sample Buffer in Table 1
Table 1 Loading Buffer
The sample made runs respectively glue, every hole loading 10ul;
8. whole electrophoresis process ice bath, first use 80V voltage, after sample ran concentrated glue, powers up and be pressed onto 120V, powered-down when bromophenol blue is gone to the bottom of glue;
9. electrophoresis complete after, unload PAGE glue, overstain in coomassie brilliant blue staining liquid, or, with slightly heating of micro-wave oven, accelerate dyeing;
10. rotary shaker, shake decolouring, sees electrophoresis result after 1h and take pictures.
4) the sero-fast preparation of the anti-camel IgG of rabbit
5) the anti-camel IgG of rabbit purifying;
With 1)
6) the anti-camel IgG of ELISA method mensuration rabbit antiserum titre is 1:64000;
1. envelope antigen
The ultimate density of antigen diluent is 1.25 μ g/ml.Every hole application of sample 100 μ l, the amount of every hole envelope antigen is 0.125 μ g.Each blood serum sample is done 12 gradient dilutions.
After antigen diluent is good, mixing. application of sample 100 μ l in every hole cover aluminium-foil paper, 4 degree refrigerator overnight.
2. washing: take out plank from refrigerator, add PBST with the volley of rifle fire and wash three times, often, inferior to after 37 ℃ of 5min, pat dry.
3. BSA sealing
Take out plank from refrigerator, add PBST with the volley of rifle fire and wash three times, often, inferior to after 37 ℃ of 5min, pat dry.Every hole adds the BSA200 μ l mixed, and covers aluminium-foil paper, and 37 ℃ of baking ovens are placed 2 hours.
4. washing: take out plank from refrigerator, add PBST with the volley of rifle fire and wash three times, often, inferior to after 37 ℃ of 5min, pat dry.
5. add primary antibodie
By the negative and positive serum of rabbit with BSA make respectively 1:1000,1:4000,1:8000,1:16000,1:32000,1:64000 doubly dilute.Dilute complete after, every hole application of sample 100ul.37 ℃ of baking ovens are placed 2 hours.
6. washing: take out plank from refrigerator, add PBST with the volley of rifle fire and wash three times, often, inferior to after 37 ℃ of 5min, pat dry.
7. adding two resists
With BSA, the two anti-1:2500 that do are diluted.Then every hole adds 100 μ l, and 37 ℃ of baking ovens are placed 1.5 hours.
8. washing: take out plank from refrigerator, add PBST with the volley of rifle fire and wash three times, often, inferior to after 37 ℃ of 5min, pat dry.
9. colour developing
Add freshly prepared tmb substrate solution 100mL/ hole, 30min is placed in 37 ℃ of dark places, shows blue.
10. stop
Every hole adds the concentrated sulphuric acid 50ul of 2M.Its colour changed into yellow, measure each hole, 450nm place OD value by microplate reader, and what the greatest dilution of positive reaction was testing sample tires.
7) antibody labeling;
1. get 5mgHRP solution in 1ml distilled water
2. the 0.1ml/L NaIO4 solution that adds 0.2ml newly to join is in upper liquid, and lucifuge stirs 20min.
3. sodium-acetate buffer (pH4.4) dialysis with 1mmol/L by above-mentioned solution, 4 ℃ are spent the night.
4. add 0.2mol/L carbonate buffer solution (pH9.5) 20 μ l, make the pH value of above hydroformylation HRP be elevated to 9.0-9.5
5. add immediately 10mg IgG in the 1ml0.01mol/L carbonate buffer solution, the room temperature lucifuge stirs 2h
6. add the 4mg/mlNaBH4 liquid that 0.1ml newly joins, mix, place 2h for 4 ℃
7. by above-mentioned liquid 0.15mol/LPBS(pH7.4) dialysis, 4 ℃ are spent the night
8. 10000g4 ℃ of centrifugal 30min
9. supernatant is moved in another pipe, by volume 1:1 adds glycerine, puts-20 ℃ of preservations.
8) labelled antibody Lowry method mensuration protein content is 6.7mg/ml;
With 2)
9) ELISA method mensuration labelled antibody is tired as 1:64000.
With 6)
2.2 determining of antigen-antibody best effort concentration
According to the dot matrix titration measuring, the best coated concentration of antigen is 0.39ug/ml, and primary antibodie best effort concentration is 1:200.
2.3 determining of best two anti-dilute concentrations
The OD450 positive serum is close to 1.000, and the P/N maximum is as two anti-best dilute concentrations.Two anti-best effort concentration are 1:2000.
2.4 determining of best confining liquid
Choose negative OD mean value minimum, the sealing combination of P/N ratio maximum, to determine confining liquid.Best confining liquid is mass concentration 5% skimmed milk power.
2.5 what each test serum was tired determines
Be ELISA with the antigen coated concentration of above-mentioned definite the best, best primary antibodie dilutability, best two anti-dilutabilitys, best confining liquid, repeat above-mentioned steps.What P/N>2.1 greatest dilutions were test serum tires.As shown in table 2, just exempt from not give birth to potent antibodies, two to exempt from antibody titer be 1:400, and three to exempt from antibody titer be 1:25600, and four to exempt from antibody titer be 1:25600.
The each immune sero-fast antibody titer of table 2 experimental group
Annotate: the different lowercase alphabet differentials of same column shoulder mark different significantly (P<0.05).
The each immune sero-fast antibody titer of table 3 control group
Annotate: the different lowercase alphabet differentials of same column shoulder mark different significantly (P<0.05).
2.6 best immune time is determined
Known according to table 2, table 3, experimental group for the third time under immune serum maximum dilution multiple under P/N and the 4th immune serum maximum dilution multiple the P/N value without significant difference, and the obvious significant difference of nothing between each group of control group, and there is no P/N>2.1, therefore, determine that the best immune time of immune camel is three times.
Claims (5)
1. a method of carrying out Dispersal risk by immune camel, is characterized in that described method comprises the steps:
(1) the subcutaneous multi-point injection antigen in two-humped camel back, the immunity cycle is 2 weeks, and injections of antigens dosage is 1mg/mL, and immune time is 3 times;
(2) exempt from eventually to get blood from the camel Jugular vessel, separation of serum after 15 days.
2. a kind of method of carrying out Dispersal risk by immune camel according to claim 1, is characterized in that immune camel is Inner Mongol Soviet Union nit two-humped camel, and the antigen adopted is bovine serum albumin(BSA) BSA.
3. a test right requires the method for antiserum titre in the immune camel body of 1 described process, it is characterized in that, described method is the ELISA detection method of the anti-camel ELIAS secondary antibody of application rabbit, wherein antigen coated concentration is 0.39ug/ml, primary antibodie best effort concentration is 1:200, two anti-best effort concentration are 1:2000, and confining liquid is 5% skimmed milk power.
4. a kind of method detected through antiserum titre in immune camel body claimed in claim 3, it is characterized in that, described two resist for the anti-camel IgG of enzyme target rabbit, the anti-camel IgG of preparation rabbit ELIAS secondary antibody step is as follows: 1. utilize and just exempt from front camel serum, affinity chromatography ProA method purifying camel IgG; 2. Lowry method protein quantification; 3. SDS-PAGE purity detecting; 4. prepare the anti-camel IgG of rabbit antiserum, the subcutaneous multi-point injection in back, injections of antigens dosage is 0.6mg/mL, and the immunity cycle is 2 weeks, and immune time is 3 times; 5. the anti-camel IgG of affinity chromatography ProA method purified rabbit, 6. the ELISA method detects the anti-camel IgG of rabbit antiserum titre; 7. simple and easy sodium periodate method labelled antibody; 8. Lowry method protein quantification labelled antibody; 9. ELISA method mensuration labelled antibody is tired.
5. a test right requires the indirect ELISA testing kit of antiserum titre in the immune camel body of 1 described process, it is characterized in that, described kit comprises: (1), as the camel IgG of primary antibodie, primary antibodie concentration is 1:200; (2) antigen, its coated concentration is 0.39ug/ml; (3) as the two anti-anti-camel IgG of enzyme mark rabbit, its concentration is 1:2000; (4) as 5% skimmed milk power of confining liquid.
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| CN105238759A (en) * | 2015-10-14 | 2016-01-13 | 吉日木图 | Monoclonal antibody of heavy-chain IgG3 in camel milk, test paper containing monoclonal antibody and application of test paper |
| CN105367655A (en) * | 2015-12-11 | 2016-03-02 | 广东工业大学 | Hand-foot-and-mouth disease resistant specific nanobody and titer determination method thereof |
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