Suzhou Vdo Biotech Co.Ltd.

Support

Position:Home - Support -

Application guideline for medical diagnosis microspheres

Application guideline for medical diagnosis microspheres

Application guideline for medical diagnosis microspheres: Polymeric microspheres can be used in diagnostic detections including lateral flow chromatography, latex agglutination, flow chip test, and turbidity measurements. Microsphere’s performance is affected by many parameters, such as surface properties, particle size and monodispersity that can ultimately affect the performance of diagnostic reagents. Therefore, it is very important to understand how to choose the microspheres.The binding between proteins with microspheres depends largely on the surface functional groups of microspheres and their concentration. Microsphere size is closely related to the detection sensitivity and linear. In general, the smaller the particle size, the more beneficial will be to the linear range; and the bigger the particle size, the more helpful will be to the sensitivity. The monodispersity of microspheres is associated with the difference between batches.Therefore, the selection of suitable microspheres is essential for the development of stable, reproducible and high-quality diagnostic reagents.

Effect of surface properties on the hydrophobic adsorption

Microsphere surface, even the microsphere surface with sulfonic acid group and carboxyl group and other hydrophilic groups, can form hydrophobic (passive) absorption with biomolecules, including proteins, oligopeptides, and other small molecules.

    

The above-described physical adsorption will be improved as the increase of the molecular weight of protein. Most of the polystyrene microsphere surfaces consist of C-C bonds, C-H bonds and other regions formed by hydrophobic molecular chains. These regions can form physical adsorption with the hydrophobic regions of biomolecules. Proteins and microspheres interact with these regions by means of multi-point adsorption. In mixed protein solution, the large molecular weight protein will preferentially adsorb to the surface of the microsphere.

so, in the coating of mixed protein solution, the large molecular weight protein will be preferentially adsorbed onto the surface of microsphere. In the preferred pH and mixing microspheres with proteins, it is easy for microspheres to specifically adsorb such substances as antibodies. And then, non-cross-linked proteins can be removed by means of centrifugation or the like.。

Surface-functionalized microspheres can covalently coupled with proteins (or other biological molecules), in order to achieve the covalent binding between microspheres and proteins (or other biological molecules).

Carboxyl-modified microspheres react readily with proteins after EDC/NHS activation. It is noteworthy that, these microspheres are more stable in electrolyte of high concentration (up to 1 M of monovalent salt).

Aldehyde group-modified microspheres are made by modifying aldehyde group onto the surface of microsphere. Aldehyde group can react with amino group of protein by forming Schiff base. Aldehyde group-modified microspheres do not require activation, but just one step can achieve covalent coupling with proteins.

Amino-modified microspheres are prepared by carboxyl microspheres through converting the carboxyl group into amino group. In some cases, such microspheres are easier to be covalently coupled with carboxyl of antibody.

How to calculate the average parking area (PA) of single functional group

The average parking area (PA) of single functional group of microsphere refers to the average parking area of each type of functional group in surface of microsphere, such as carboxyl. It’s generally indicated by Å2, and to be a measure of the density of group in the surface of microsphere, calculated as:

Among them, P represents the average parking area (PA) of the functional group with the unit of Å2; Dc represents the superficial concentration of the functional group, achieved by titration in common with the unit of μeq/g microsphere; ρs represents the concentration of microsphere, with the unit of g/cm3; and d is the average diameter, with the unit ofμm.

Protein coating

The coated protein of microsphere surface has two way of combination: passive adsorption and covalent cross-linking.

I)Passive absorption

The passive absorption can rapidly increase the protein volume at the surface of the microsphere and forms protein coating in a short time to a protein saturation situation. Based on the experience empirical formula, the correlation between particle diameter of the microsphere and maximize protein coating can be calculated:

S=5.71/D

S represents the superficial area of 1g microsphere with the unit of m2; D represents the diameter of microsphere, with the unit ofμm

Therefore, for a set-volume microsphere, the smaller the particle diameter, the larger the superficial area and the more coated protein theoretically.

It should be noted that though the passive absorption is strong however it is not permanent. Many reagents can desorb the protein at the surface of the microsphere.

II)Covalent coupling

The protein can be covalently crosslinked to the surface of microparticle by many ways. The surface of microparticle contains epoxy group, chloromethyl group, aldehyde group, carboxyl group, etc., which can react with the amino group at the Fc end of antibody to form the covalent bond. When selecting covalent cross-linking way, validity, process complexity and last charge condition of the cross-linking shall be taken into consideration.

The first three groups can react with the amino group in the protein to form the covalent bond. It is easy to be used, but its stability is lower than that of carboxylic microparticle. The carboxyl group microparticle, through EDC/NHS activation, can react with the amino group of the protein to form the covalent bond. Because the carboxyl group microparticle itself has no performance to react with protein directly, so it has relatively strong chemical stability. The two methods shall be selected according to actual situation.

The carboxyl group microparticles are widely applied. Currently popular process uses carboxyl group of EDC (+NHS) activated microparticle, and then cross-links with the amino group of the antibody. The cross-linked charge condition is very important, which greatly influences its actual stability. In the covalent cross-linking, the carboxyl group (or the amino group) can provide covalent cross-linking groups, and is very important to the charge condition of cross-linked compounds and stable sol state of the whole system. The stability of microparticle depends on surface charges, type and quantity of coated protein, buffer solution, etc. The improper treatment easily causes agglomeration and non-specific adsorption. In particular, IgG, with relatively-low charge density and relatively-high hydrophobicity, is easy to be damaged after being cross-linked to microparticles.

 

 

 

Protein coating guide

1.Covalent cross-linking principle

1EDC/NHS activated carboxyl group coupling

In the above graph, after the carboxyl group of the microparticle is activated by EDC, the intermediates can react with the amino group of antibody to form the covalent bond. This is one-step method.

It can be hydrolyzed and reduced to carboxyl group. Once the microparticles are activated, the microparticles shall be used at once.

Moreover, it can react with NHS to form intermediates, and then with the amino group of the antibody to form the covalent bond. This is two-step method.

2)Aldehyde group coupling

In the above graph, the aldehyde group of the microparticles can react with the amino group of the antibody to remove 1 molecule H2O, and then be oxidized under the action of NaCNBH3 to form stable C-N bond. Noticeably, the aldehyde group can be hydrolyzed under certain pH, so attention shall be paid to storage stability of microparticles.

1.Activated buffer solution

pH value of 50 mM pH6.1 MES fluctuates according to the actual situation, but it is not suggested to make a big change and to be diluted excessively.

The selection of pH value is connected with the isoelectric point of coated protein. Near the isoelectric point, it is good for “precipitating” the protein to the most extent onto the surface of microparticle. Far away from the isoelectric point, it is good for “expanding” conformation of protein to avoid congestion and influence the activity.

It shall be stored under 4℃.

2.Coated buffer solution:

In view of stability of intermediates and antibody after the activation of EDC (or EDC/NHS), its pH value is suggested to be 5~7. So, it may use 50 mM, pH6.1 MES.

3.Proportion of granule and protein

The coating quantity of the antibody on the granule directly relates to coating result and cost. If the coating quantity is too low, it is impossible to capture antigen to the most extent. If the coating quantity is too high, it is impossible to save cost, and even possible to make a part of antibodies lose activity because of excessive “congestion”. In general, we suggest that 100 mg microparticles coat the 5~30mg protein. The specific surface area of the big microparticle is small, so the quantity of coated protein is fewer. The specific surface area of the small microparticle is big, so the quantity of coated protein is more. The antibodies of each project are different. The sources of antibodies of each manufacturer are different. Specific coating quantity shall be determined according to experimental results.

On the biochemical platform, in general, the coating quantity for 100 mg microparticles is suggested to be:

At the chromatographic level, the number of antibodies connected with each microsphere will directly influence the sensitivity of the final test. Theoretically, the best status is that each microsphere coup with antibody to reach the best sensitivity. Therefore, we suggest significant decrease of coating at this platform.

 

1.Wash the buffer solution

Phosphoric acid buffer solution (PBS), 0.1M, pH7.2.

Different buffer solution presents different results in different items. Clients should optimize according to the real situation and select the best buffer system and pH value.

2.Storage Buffer solution

Phosphoric acid buffer solution (PBS), 0.1M, pH7.2, 0.1% glycine and 0.1% NaN3.

Different buffer solution presents different results in the different items and measuring platforms. The above buffer solution is advised to be used at the biochemical platform. However, the clients should optimize according to the real situation and find the best buffer system and pH value.

It should be noticed that the pH value of buffer solution of chromatographic platform may be higher and the clients should make adjustment according to the specific circumstance

3.Blocker

The blocker plays important role in the linearity, sensitivity, stability and prevention of nonspecific absorption of the results. BSA, skim milk powder, casein, gelatin, Tween-20 and glycine are the common blockers. The clients should optimize according to the real situation and find the best blocker.

4.Carboxyl-modified Beads Coupling Protocol  

Activation:

Advices:

1. The pH value of MES buffer solution can be adjusted to approach the isoelectric point of antibody.

2. During the coating step, try to avoid using the buffer solution with multivalent anions, like phosphoric acid or boric acid buffer fluid.

3. Glycine is used as the blocker to avoid nonspecific absorption. However it is not omnipotent, the best blocker should be selected.

Application guideline of magnetic nano-microsphere for nucleic acid purification

Frequent asked questions

1.Low recovery rate

 

Causes

Countermeasures

The ethyl alcohol is not removed completely

After the last washing, please remove the washing buffer solution carefully. Place it under 55~60℃ for 5 min to volatilize the ethyl alcohol.

The elution is not sufficient

If holding the temperature under 55~60℃ for 2 min., it can enhance the elution efficiency effectively.

The elution time is not proper

The properest time for DNA solution is 1 min.

The stir is not sufficient upon eluting.

Upon eluting, if the mixture of magnetic beads is not sufficient, the recovery quantity will be decreased. After separating the magnetic beads, the magnetic beads will be agglomerated on the wall of the centrifugal tube, please scatter the beads carefully.

2.The absorbancy of recycled nucleic acid is not correct

 

Causes

Countermeasures

Washing is not sufficient

The adsorption solution contains the substances adsorbing the UV. To quantitate the recovered nucleic acid, please wash it with the washing liquid and 70% ethyl alcohol solution.

The adsorption solution is mixed

The adsorption solution contains the substances adsorbing the UV. Please remove the adsorption solution carefully. Wipe the adsorption solution on the cover of the test tube with the absorbent paper, etc., which can improve conditions effectively.

3.The recovered nucleic acid can’t react well

 

Causes

Countermeasures

The salt hinders the reaction

The adsorption solution contains high concentration salt, please add the working procedure of washing with 70% ethyl alcohol solution twice.

The ethyl alcohol hinders the reaction

Please follow the operating procedure to dry it fully to remove the ethyl alcohol.

发表留言
 
Name:
*
小于等于20个字符(包括A-Z、a-z、0-9、汉字、不含特殊字符)
Tel:
*
Content:
* 已输入字符:0
小于等于500字符
Verification:
   
留言列表

 

  • 示例:example@mail.com您的邮箱:
    • Commenting time: 2017年10月10日
  •  
    Content: This message&nb...
  • 示例:example@mail.com您的邮箱:
    • Commenting time: 2017年7月25日
  •  
    Content: И что бы&n...
  • 示例:example@mail.com您的邮箱:
    • Commenting time: 2017年7月3日
  •  
    Content: Крем от пс...
  • 示例:example@mail.com您的邮箱:
    • Commenting time: 2017年7月3日
  •  
    Content: Обустраивая гос...
  • 示例:example@mail.com您的邮箱:
    • Commenting time: 2017年7月2日
  •  
    Content: Тинедол – ...
  • 示例:example@mail.com您的邮箱:
    • Commenting time: 2017年6月16日
  •  
    Content: http://www.gagprince...
  • 示例:example@mail.com您的邮箱:
    • Commenting time: 2017年6月13日
  •  
    Content: Revolutional up...
  • 示例:example@mail.com您的邮箱:
    • Commenting time: 2017年5月19日
  •  
    Content: Absolutely NEW&...
Suzhou Vdo Biotech Co.Ltd.