CN103454413B - Bactrian camel specific antibody preparation method and immune detection method - Google Patents

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

The preparation of two-humped camel specific antibody and immunologic detection method

(1) technical field

The invention belongs to antibody engineering technical field, the present invention is with Inner Mongol Soviet Union nit two-humped camel for research object, and the camel utilizing bovine serum albumin(BSA) (BSA) to prepare anti-BSA resists more, optimizes immune condition.And prepare rabbit anti-camel IgG 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.

Why antiserum can play immune association reaction with relevant antigen, is because in antiserum, containing relevant antibody protein.In early 1960s, the animal protein with antibody activity is defined as immunoglobulin (Ig) (immunoglobulin).Mainly be present in blood plasma, also see in other body fluid, tissue and some juices.(κ or λ) that on general Ig molecule, the antigenicity of L chain is always identical.But H chain antigenicity not identical (μ chain, γ chain, α chain, δ chain and ε chain), according to it, molecule that different H chain and L chain form complete Ig can be divided into five classes, i.e. 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 made up of four peptide chains, is namely made up of the heavy chain (H chain) that the molecular weight that the light chain (L chain) of two identical molecular weights is identical with two is larger.

The principal ingredient of human serum immunoglobulin (Ig) is IgG, and it accounts for the 70-75% of total immunoglobulin (Ig), molecular weight about 150,000, sugary 2 ~ 3%.

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 can be promoted, neutralize bacteriotoxic toxicity (neutralizing a toxin) and viral antigen and combine the ability (neutralization virus) making virus lose host cells infected.IgG will be later than IgM at the age that body synthesizes, within 3rd month, start after birth synthesis, 3-5 year close to adult's level.It is uniquely by the Ig of placenta, plays 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. report existed a kind of containing heavy chain not containing the native heavy antibody (Heavy chain antibody, HCAb) of light chain in camellid (one-humped camel, two-humped camel, yamma etc.) body.Afterwards, in some selachian bodies, have also discovered the neoantigen acceptor (new antigen receptor, NAR) similar to camel heavy chain antibody structure.

Because camel heavy chain antibody is pure natural, can not people be directly acted on, or be that the mankind are used.Therefore after cloning the variable region of heavy chain antibody in camel body, the single domain antibody be only made up of a variable region of heavy chain obtained, be referred to as heavy chain variable region gene antibody (variable domain of heavy chain of heavy-chainantibody, VHH), its diameter is 2.5nm, long 4nm, be therefore otherwise known as nano antibody.As a kind of small-sized genetic engineering antibody, the high expressed that nano antibody possesses, water-soluble, stability, the strong advantage such as penetration into tissue and more weak immunogenicity, make this antibody gather around hold out broad prospects [10] in the field such as fundamental research and drug development.At present, in European and American areas, the research based on the VHH single domain antibody of camel heavy chain antibody has become a focus [11], but still rarely seen report at home.

Nano antibody as a strict monomer, its special structure and character, the peculiar property making it possess some other common antibody not have.As easily obtain and express, highly-water-soluble and stability, targeting, humanization are simple etc.

In current domestic report, only to the serum analysis of a peak camel not synantigen in the immunologic process of camel, produce the situation of heavy chain antibody, unavoidably exist biased.Then external research emphasis is to recombinate the physicochemical property of VHH antibody, application and antigen targeting aspect.But report is not had at present for the top condition preparing camel antibodies immunologic process.

In view of powerful advantages and the application of nano antibody.The present invention revives nit two-humped camel for research object with the Inner Mongol, the most general in line with antigen application, to be easy to get principle, analyze for a certain amount of camel colony, the anti-BSA polyclonal antibody of preparation camel, explore the body reaction change in two-humped camel specific antibody preparation process, determine the best immune time of camel.For analyzing the disease-resistant protective effect of two-humped camel heavy chain antibody from now on, preparing nano antibody and laying the groundwork, simultaneously for researching and developing the direction that novel genetic engineering antibody provides new from now on.

Simultaneously, because camel IgG ELIAS secondary antibody anti-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 preparation rabbit anti-camel IgG ELIAS secondary antibody, set up ELISA method diagnostic kit, detect tiring of the camel Serum Antibody after immunity.

(3) summary of the invention

1. application claims protects optimum immuning program and the method for immune camel;

Immunization method of the present invention is two-humped camel dorsal sc multi-point injection, and the immunity cycle is 2 weeks, and injections of antigens dosage is 1mg/mL, and immune time is 3 times;

2. the technical indicator involved by application claims protection preparation rabbit anti-camel IgG ELIAS secondary antibody and result;

It is as follows that the present invention prepares rabbit anti-camel IgG ELIAS secondary antibody step: 1. affinity chromatography Pro A method purifying camel IgG, 2. Lowry method protein quantification 3. SDS-PAGE purity detecting 4. prepare rabbit anti-camel IgG antiserum, dorsal sc multi-point injection, 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. ELISA method detect rabbit anti-camel IgG antiserum titre 7. simple and easy Over-voltage protection labelled antibody 8. Lowry method protein quantification labelled antibody 9. ELISA method measure labelled antibody and tire;

3. the anti-camel ELIAS secondary antibody ELISA of application claims protection application rabbit detects camel antiserum titre method and optimal detection condition.

The present invention detects camel antiserum optimal detection condition and comprises: 1. antigen-antibody best effort concentration 2. best two anti-dilute concentrations 3. best confining liquid.

The present invention for research object with Inner Mongol Soviet Union nit two-humped camel, utilizes bovine serum albumin(BSA) (BSA) to prepare the many anti-and rabbit anti-camel IgG ELIAS secondary antibody of camel of anti-BSA, and sets up indirect ELISA testing kit.

Experiment is divided into three parts: Part I immunity camel, prepares immune camel serum; The camel IgG immune rabbit of Part II purifying, preparation rabbit anti-camel IgG ELIAS secondary antibody, prepares for ELISA detects; Part III application indirect elisa method detects immunity camel antiserum titre in each in period, finally determines best immune time, sets forth the body reaction change in two-humped camel specific antibody preparation process.

(4) accompanying drawing explanation

Figure 11 is SDS-PAGE under non reducing conditions, band in 170KD be in the two-humped camel serum of purifying Inner Mongol IgG1, IgG3 of mixing, band in 90KD place be in the two-humped camel serum of purifying Inner Mongol IgG2; 2 is SDS-PAGE under reducing condition, and band is heavy chain in 55 places, and 40 places are light chain.

The camel IgG Lowry method protein quantification typical curve of Fig. 2 purifying

With mark Lowry method protein quantification typical curve afterwards after Fig. 3 rabbit anti-camel IgG enzyme mark two purifying

Fig. 4 rabbit anti-camel IgG antiserum titre

Fig. 5 marks rear rabbit anti-camel IgG antiserum titre

The each immune P/N of Fig. 6 experimental group, under same dilutability, five road columns are before immunity from left to right respectively, just exempt from, two to exempt from, three exempt to exempt from four.As seen from the figure, 0 exempts from and just exempts from P/N < 2.1, produces without antibody; The two serum greatest dilutions of exempting from P/N > 2.1 are 1:400; Be 1:25600. in third time and the serum greatest dilution of the 4th immune P/N > 2.1

The each immune P/N of Fig. 7 control group, under same dilutability, five road columns are before immunity from left to right respectively, just exempt from, two to exempt from, three exempt to exempt from four.As seen from the figure, just exempt from and all non-> 2.1 of P/N value of booster immunization afterwards, and produce without antibody.

Fig. 8 experimental group increases with immune time, known in antiserum titre figure in immune serum, immunity three times.

(5) embodiment

1. the preparation of polyclonal antibody

Animal used as test is divided into blank group (3 peak) and experimental group (3 peak) two groups.Before just exempting from, Jugular vessel extracts 20mL negative blood respectively, and separation of serum is frozen in-40 DEG C after aseptic subpackaged.The each immune physiological saline of blank group; Experimental group immunity 0.1mg/ml BSA, immunity 4 times altogether.Blood is got from camel Jugular vessel, separation of serum before each immunity and after exempting from 15 days eventually.

The preliminary foundation of 2.ELISA detection method

The preparation of the anti-hunchbacked IgG ELIAS secondary antibody of 2.1 rabbit

1) purification of affinity chromatography camel IgG;

1. wash and Protein A is fixed on iron stand.After filler dress post, wash protective agent and ethanol off with ultrapure water 10 bed volumes;

2. the balance equilibration buffer pillar of 10 column volumes, eluate is equal with start buffer pH;

3. loading is by serum upper prop, loading flow velocity≤60cm/h.

4. the cleaning level pad cleaning bed volume of 20 column volumes, until UV line is to baseline.

5. wash-out elution buffer (glycocoll PH2.2) wash-out, starts to collect eluting peak higher than 0.1 at ultraviolet 280nm place, lower than 0.1 time terminate, eluent neutralizes neutrality by neutralization buffer immediately, is concentrated to volume required.

2) Lowry method measures protein content is 2.4mg/ml;

1. according to protein concentration, the standard protein solution of accurate measuring different volumes respectively, respectively adds PBS and mends to 0.2ml, make the solubility of standard protein be respectively 0,12.5,25,50,100,200 μ g/ml;

2. accurate measuring sample adds PBS benefit to 0.2ml;

3. typical curve and sample are respectively added 1ml basic cupric sulfate and the mixing of 0.1ml Folin-phenol reagent, survey OD at 750nm place;

4. with the concentration of standard protein for Y-axis, absorbance value is X-axis, obtains a typical curve.Its concentration is calculated by the absorbance value of sample;

3) SDS-PAGE purity detecting;

1. the beaker of the 200ml that preparation one is clean, preparation mass concentration is the separation gel of 10%

2. the separation gel prepared is added in 4.7ml to two piece of glass plate, with a small amount of isopropyl alcohol fluid-tight;

3. leave standstill and solidify, outwell isopropyl alcohol, blot with filter paper;

4. in clean 50ml beaker, prepare mass concentration is 5% concentrated glue (when preparation separation gel and concentrated glue, time to add mass concentration be ammonium persulfate and the TEMED of 10%, limit to shake beaker limit and add, ensure evenly), fills a prescription in Table

5. add 1mL with liquid-transfering gun and concentrate glue to separation gel top, and plug comb gently, ensure that each tooth rim encloses bubble-free;

6. place half an hour, after gelling to be concentrated is solid, carefully take out comb, put into electrophoresis tank.

7. albumen runs glue: by reduction Loading Buffer, non-reduced Loading Buffer, natural test sample Buffer in table 1

Table 1 Loading Buffer

Obtained sample runs glue, every hole loading 10ul respectively;

8. whole electrophoresis process ice bath, first uses 80V voltage, ran after concentrated glue, and powered up and be pressed onto 120V, the powered-down when bromophenol blue goes to the bottom of glue until sample;

9. electrophoresis complete after, unload PAGE glue, overstain in coomassie brilliant blue staining liquid, or hot slightly with micro-wave oven, accelerate dyeing;

10. rotary shaker, shake decolouring, sees electrophoresis result after 1h and takes pictures.

4) the sero-fast preparation of rabbit anti-camel IgG

Parallel 2 of animal used as test.Before just exempting from, Jugular vessel extracts 20mL negative blood respectively, and separation of serum is frozen in-40 DEG C after aseptic subpackaged.Dosage is in 0.5mL PBS/ only the anti-camel IgG of 0.6mg/mL rabbit, adds 0.5mL adjuvant.Immunity 3 times altogether.Blood is got from Jugular vessel, separation of serum before each immunity and after exempting from 15 days eventually;

5) rabbit anti-camel IgG purifying;

With 1)

6) ELISA method measures rabbit anti-camel IgG 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, namely the amount of every hole envelope antigen is 0.125 μ g.Each blood serum sample does 12 gradient dilutions.

After antigen diluent is good, mixing. every hole application of sample 100 μ l, covers aluminium-foil paper, 4 degree of refrigerator overnight.

2. wash: take out plank from refrigerator, add PBST with the volley of rifle fire and wash three times, often inferior to after 37 DEG C of 5min, pat dry.

3. BSA closes

Take out plank from refrigerator, add PBST with the volley of rifle fire and wash three times, often inferior to after 37 DEG C of 5min, pat dry.Every hole adds the BSA200 μ l mixed, and covers aluminium-foil paper, and 37 DEG C of baking ovens place 2 hours.

4. wash: take out plank from refrigerator, add PBST with the volley of rifle fire and wash three times, often inferior to after 37 DEG C of 5min, pat dry.

5. primary antibodie is added

The negative and positive serum BSA of rabbit is made 1:1000,1:4000,1:8000,1:16000,1:32000,1:64000 respectively doubly dilute.Dilute complete after, every hole application of sample 100ul.37 DEG C of baking ovens place 2 hours.

6. wash: take out plank from refrigerator, add PBST with the volley of rifle fire and wash three times, often inferior to after 37 DEG C of 5min, pat dry.

7. add two to resist

With BSA, the two anti-1:2500 that do are diluted.Then every hole adds 100 μ l, and 37 DEG C of baking ovens place 1.5 hours.

8. wash: take out plank from refrigerator, add PBST with the volley of rifle fire and wash three times, often inferior to after 37 DEG C of 5min, pat dry.

9. develop the color

Add freshly prepared tmb substrate solution 100mL/ hole, 30min is placed in 37 DEG C of dark places, and display is 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, the greatest dilution of positive reaction is tiring of testing sample.

7) antibody labeling;

1. 5mgHRP solution is got in 1ml distilled water

2. add 0.1ml/L NaIO4 solution that 0.2ml newly joins in upper liquid, lucifuge stirs 20min.

3. dialysed by the sodium-acetate buffer (pH4.4) of above-mentioned solution 1mmol/L, 4 DEG C 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 in 10mg IgG to 1ml0.01mol/L carbonate buffer solution immediately, room temperature lucifuge stirs 2h

6. add the 4mg/mlNaBH4 liquid that 0.1ml newly joins, mixing, place 2h for 4 DEG C

7. by above-mentioned liquid 0.15mol/LPBS(pH7.4) dialysis, 4 DEG C are spent the night

8. 10000g4 DEG C of centrifugal 30min

9. moved in another pipe by supernatant, by volume 1:1 adds glycerine, puts-20 DEG C of preservations.

8) labelled antibody Lowry method measures protein content is 6.7mg/ml;

With 2)

9) ELISA method mensuration labelled antibody is tired as 1:64000.

With 6)

The determination of 2.2 antigen-antibody best effort concentration

According to dot matrix titration measuring, antigen the best bag is 0.39ug/ml by concentration, and primary antibodie best effort concentration is 1:200.

The determination of 2.3 best two anti-dilute concentrations

OD450 positive serum is close to 1.000, and P/N is maximum as two anti-best dilute concentrations.Two anti-best effort concentration are 1:2000.

The determination of 2.4 best confining liquids

Choose negative OD mean value minimum, the closed combination that P/N ratio is maximum, to determine confining liquid.Best confining liquid is mass concentration 5% skimmed milk power.

The determination that 2.5 each test serums are tired

Be ELISA with the antigen coated concentration of above-mentioned the best determined, best primary antibodie dilutability, best two anti-dilutabilitys, best confining liquid, repeat above-mentioned steps.P/N > 2.1 greatest dilution is tiring of test serum.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

Note: the different lowercase alphabet of same column shoulder mark shows significant difference (P<0.05).

The each immune sero-fast antibody titer of table 3 control group

Note: the different lowercase alphabet of same column shoulder mark shows significant difference (P<0.05).

2.6 best immune time is determined

Known according to table 2, table 3, under experimental group third time immune serum maximum dilution multiple under P/N and the 4th immune serum maximum dilution multiple P/N value without significant difference, and without obvious significant difference 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 (2)

1. carried out a method for Dispersal risk by immune camel, it is characterized in that described method comprises the steps:

(1) two-humped camel dorsal sc multi-point injection antigen, the immunity cycle is 2 weeks, and injections of antigens dosage is 1mg/mL, and immune time is 3 times;

(2) blood is got from camel Jugular vessel after exempting from 15 days eventually, separation of serum;

Immune camel be Inner Mongol Soviet Union nit two-humped camel, the antigen adopted is bovine serum albumin(BSA) BSA.

2. the method for antiserum titre in the camel body passing through immunity described in a test right requirement 1, 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, and described camel is Inner Mongol Soviet Union nit two-humped camel.

3. a kind of method detecting antiserum titre in the camel body of immunity according to claim 2, it is characterized in that, described two resist for the anti-camel IgG of enzyme target rabbit, preparation rabbit anti-camel IgG ELIAS secondary antibody step is as follows: 1. utilize and just exempt from front camel serum, affinity chromatography Pro A method purifying camel IgG; 2. Lowry method protein quantification; 3. SDS-PAGE purity detecting; 4. prepare rabbit anti-camel IgG antiserum, dorsal sc multi-point injection, 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 Pro A method purified rabbit, 6. ELISA method detects rabbit anti-camel IgG antiserum titre; 7. simple and easy Over-voltage protection labelled antibody; 8. Lowry method protein quantification labelled antibody; 9. ELISA method mensuration labelled antibody is tired.

4. test right requires the indirect ELISA testing kit passing through antiserum titre in immune camel body described in 1, and 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 bag is 0.39ug/ml by concentration; (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, described camel is Inner Mongol Soviet Union nit two-humped camel.