MODERN METHODS OF OBTAINING IMMUNOCONJUGATE FOR THE QUANTITATIVE DETERMINATION OF CEPHALOSPORIN ANTIBIOTICS

L.T. PULATOVA, Sh.S. TASHMUKHAMEDOVA, Yu.B. ARZIYEVA

It is now known that the development of highly sensitive enzyme immunoassay method, the most important task is the choice of enzyme labels, as the sensitivity of enzyme immunoassay (ELISA) largely depends on the properties of the enzyme. As it is known in the active site of the enzyme formed close-and in a certain way oriented in the space of sections of the polypeptide chain, is the binding of the substrate and its chemical transformation. Therefore, with the introduction of the enzyme label by covalent binding of the enzyme molecule to molecule antigen or antibodies, must be carried out so that modification of enzyme did not cause its inactivation.

As shows the analysis of foreign and domestic literature, for the most specific and highly sensitive conjugates using single-stage and two-stage methods of synthesis. In one-step method of cross-linkable reagent is added to a mixture of enzyme and protein, and in two stages - at the first stage, cross-linking agent handle only enzyme that removes excess dialdehyde, and then already to the modified enzyme add protein. These methods are simple and received compared to meet the necessary requirements. It is necessary to note, that at synthesis of conjugates in the reaction mixture is formed not only conjugates, but polymolecular education type of antibody-antibody or enzyme-enzyme, which often distort the results of the immunoassay. It would be expedient to prevent the formation of such compounds or to find affordable ways to get rid of them. Therefore, in this paper were developed some methods of obtaining conjugates, in particular:
- conjugates on a solid substrate with a pre-modified enzymes (oriented method);
- conjugates on a solid substrate with a pre-modified antibodies (directed method).
In our work method is illustrated for the receipt of conjugates of amylase and antibodies raised to cephalosporin antibiotics using two methods, in particular, focused and directed each with different technology.

The experimental part

The choice of antibiotics cephalosporin as the object of the research is explained by the fact that in the structure of their molecules are amino group (NH₂ - group). This fact complicates the immobilization at synthesis immunosorbent assay, providing access to knitting.

Cephalosporins are related to beta lactams and represent one of the most extensive class of antimicrobial agents. There are four generations (generation) cephalosporins, based division which is not a chronological criteria, and the main features of the spectrum of action and pharmacokinetics. For all cephalosporins there is the risk of cross Allergy with other β - lactam antibiotics (penicillins, carbapenems, carbapenems). All of cephalosporins is celebrated the same pharmacodynamics, in particular, like penicillins, they violate synthesis of microbial wall at the time of mitosis. Cephalosporins on the chemical structure similar to the penicillins and are derived 7 - aminocephalosporinic acid (7-ACA) and 7 - aminodesacetoxycephalosporanic acid (7-ADCA):

The task is to improve the specificity and catalytic activity received conjugates with sufficient simplicity and availability of the way they are received. This increases the sensitivity, accuracy and reproducibility immunochemical method in the quantitative determination of cephalosporin antibiotics for customs examination.
The method of obtaining conjugates for the quantitative determination of cephalosporins in the study the sample includes the synthesis of conjugate on a solid substrate, as a solid substrate use powdery sorbent polyamide P6, then 100 mg sorbent activate solution of hydrochloric acid, then washed with distilled water, then 0.1M borate buffer and modify 2.5% glutaric aldehyde. To remove excess glutaraldehyde cooked solution is washed, then spend covalent binding antigen (cefalo-spalinowych antibiotics) in the amount of 100 mg in 1:1 ratio in the conditions of constant stirring for 2 days at pH 7.4 in the presence of 5% solution of ethylene glycol and chloride calcium in the amount from 10 to 30 mm, after incubation rinse sorbent 0.05 M buffer solution to block free aldehydic groups add monoethanolamin, the excess of which remove triple rinsing Bortnik buffer, then immunosorbent add antibodies with the formation of immune complex AG - AB, within 12 hours spend modification of antibodies in immunosorbent assay solution NaJO₄, excess NaJO₄, is removed by washing, then add enzyme label (amylase) in the amount of 1 mg and incubated for 18 hours at the temperature of 4°C after what are desorption conjugate three - HCl buffer acidified HCl to pH 2.3, neutralize 0.5 M solution of sodium bicarbonate, cialis what against 0.15 M NaCL solution.
Received conjugate has high serological activity. This trend sasana what exactly variable plots synthesized immunoconjugate remain free, which facilitates the formation of immune complex on solid substrate when conducting immunoferment of analysis used in the customs examination and forensic practice. Besides, the obtained conjugates have high specificity and catalytic activity. It is connected primarily with the fact that in the formation of enzyme conjugate binds specifically with the Fc fragment of antibodies, with variable plots Fab - fragment of antibodies remain free, which creates the opportunity for binding of the enzyme with effector part of antibodies. Active centre of antibodies remain free, which facilitates the binding of the antibody with the appropriate antigens (cephalosporin antibiotics). It is at pH 7.4 in the incubation environment in the process of sorption with immobilized antenom contact reactive antibodies.

Positive influence on the sorption of antibodies have and calcium ions and ethylene glycol. So, adding in the process of sorption in the incubation environment 5% solution of ethylene glycol and chloride calcium in the amount from 10 to 30 mm contributes to the binding of the antibody with antenom on the media. Modification of antibodies within 10 hours NaJO₄ leads to the formation of the active aldehyde groups, which then reacts with the amino groups of the enzyme, in particular with amylase, with the formation of the bases of Shiffer.
Received conjugate quickly neutralize 0.5 M a sodium bicarbonate solution to pH 7.4 in order to save enzymatic and serological activity. Cialiswhat against 0.15 M NaCl solution is to get rid of some low-molecular components.
Synthesized conjugate has high enzymatic and serological activity, especially the immune component, because the antibodies are not modified, so that the latter retain high serological activity, specificity to the relevant haptens. In this way the obtained conjugate, which is an antibody associated with the enzyme labeled. Developed a new approach obtain enzymatic and serologically active conjugates, which will be further used during the development of highly sensitive enzyme immunoassay. ELISA will be defined low-molecular haptens (cephalosporin antibiotics)
Thus, developed an optimal methods of obtaining conjugates with high specificity, accuracy of results, which are necessary for further implementation of immunochemical analysis in the area of customs examination in a hospital outside the laboratory practice.

Peculiarity of modern stage of development of ELISA is connected with the transition from a complex of devices for simple and more affordable methods. In the developed methods all the necessary components immobilized or integrated into the porous surface. However, the only thing we need to do in case of positive result is the availability of direct contact system with the analysed sample and subsequent recording of the signal.

When conjugates as cross-linkable reagent was very convenient glutaric aldehyde. At the first stage of work we were synthesized conjugates antibodies on the solid substrate oriented method. The essence of the developed method is that the synthesis of conjugate is on a solid substrate, which previously was immobilized antigen, in this case, an antibiotic, cephalosporin. Then added antibodies that are specific to the antibiotic, forming complex antigen - antibody and introduced enzyme label in the amount of 1 mg modified 0,1% glutaraldehyde.
Received conjugate quickly neutralized 0,5 M solution of sodium bicarbonate and concentrated using polyethylene glycol up to a volume of 5 ml Then were dialyzed against 0.05 M sodium chloride. However, were obtained catalytically active conjugates with high specificity.

Immune complex inhibited 18 hours at the temperature of 4°C after incubation carried out desorption conjugates three - HCl buffer acidified HCl to pH 2.3 received conjugate neutralized. The yield of the conjugate was 35.6%. Received oriented method conjugates had high specificity and catalytic activity.
Thus developed the optimal way of getting conjugates used in enzyme-linked immunosorbent assay. Thus, the output of conjugate has increased considerably and amounted to 48.6%. Thus, compared obtained aimed method, had high specificity and catalytic activity. Serological activity was determined by the method of double diffusion. In this case, line formed precipitation from the edges of the holes, which is evidence of more education major agents of antibodies to the enzyme (Fig.1.) In addition, the formation of lines precipitation indicates that antibodies do not lose their serological activity.

Fig.1. Synthesis conjugate aimed method

After conjugating enzyme with antibodies can be expected to change certain properties of the enzyme. As a result of binding expanded the profile curve. According activity from pH. Up to conjugation amylase was active in a more narrow range of pH with pronounced maximum at pH 5.0.
Temperature optimum enzyme before and after conjugation practically unchanged. After conjugation there was a slight increase incubation at 50°C native amylase inactivated after 60 minutes, in the composition of conjugate activity persists over a long time.

The results hydrolysis of starch in the concentration range from 0.5 to 2% of native and conjugated amylase. The figure shows that when linking amylase with antibodies experience a decrease hydrolytic activity conjugated to the enzyme. Maximum activity of native and conjugated the enzyme manifested in substrate concentration of 0.5%.

Thus, were obtained compared amylase with antibodies to cephalosporin and learned from some physical - chemical properties.

Synthesis conjugate oriented method

In order to synthesize conjugate oriented method, we were first synthesized immunosorbent, which make polyamide P6. As ligand used the hapten (cephalosporin antibiotics). First 100 mg sorbent activated 2,05 n HCL solution during 2 hours at the temperature 45°C. After washing borate buffer, sorbent modified 2,5% solution of glutaraldehyde, then sorbent washed 0.05 M buffer solution and added cephalosporin antibiotic in the amount of 100 mg, incubated within 48 hours, with the subsequent washing of the sorbent. This amount communicating antigens amounted to 81 mg.

Thus was received immunosorbent on the insoluble polyamide and was later used as a solid substrate for the synthesis of conjugate oriented method.
The next stage was the sorption of antibodies in immunosorbent assay. It was established that at pH 8.0 and at the temperature of 40 C for 16 hours is the maximum sorption of antibodies immobilized on the haptens. After obtaining immune complex on immunosorbent, this Wednesday introduced enzyme Meiko, which amylase was pre-modified 0.05%-s ' solution of glutaraldehyde. Incubation of the enzyme was immunosorbent assay for 18 hours at the temperature of 4°C. Desorption conjugate with sorbent carried out three HCL buffer acidified HCL to pH 2.3. Received conjugate quickly neutralized 0.5M solution of sodium bicarbonate and concentrated using poliatilenglikola to 1 ml were dialyzed 0,05 m solution of sodium chloride. The developed method output conjugate fixed cephalosporin antibiotic was 35.6% when the ratio of the concentrations of the enzyme - antibody 1:1, which significantly increases the efficiency of the quantitative content of antibiotics in the studied objects during customs examination.
Developed oriented method has allowed the use of conjugate in the development of solid-phase enzyme Immunoassay for the customs examination. The procedure for conducting quantitative analysis simple, does not require special equipment and is consistent immersion media with immobilized antibodies in a number of solutions. Before conducting the EIA, the media (membrane) with the immobilized antibody washed 0.05 M three HCL buffer, then it poured defined antigen (low-molecular hapten, i.e. an antibiotic or a drug), incubated for 15-20 minutes at constant stirring. However, the hapten specifically bound to the immobilized on the membrane antibodies. After incubation medium was washed from unbound haptens, then added synthesized by us conjugate (antibodies associated with amylase) in the amount of 20 to 22 micrograms (for protein) and leave for 10-15 minutes with continuous stirring. During incubation, antibodies in the composition of the conjugate, specifically associated with the hapten (low-molecular substance), which is on the surface of the media. After washing added to the substrate of the enzyme amylase, which ensured the hydrolysis of the substrate and the formation of reaction products. As a result, the intensity of staining product you can visually determine the number of hapten or with a spectrophotometer to establish the activity of the conjugate, which is proportional to the concentration determined antigen (hapten). The degree of planting hapten reflects epitope the structure of the antigen, which is optimal for obtaining specific antibodies.

This approach has allowed to establish the impact of the method of conjugation of low-molecular compounds on the parameters of the developed solid-phase ELISA for customs examination outside laboratory practice in stationary conditions, i.e. the sensitivity of determination haptens, specificity and affinity antibodies, used in the analysis.

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Synthesis conjugate oriented method

Summarizing the proposed material, it should be noted that the developed methods allow identification banned in food production of antibiotics, by determining the excess of the maximum allowable content, thereby, increasing the efficiency of control quality and safety of food products. At the same time, the specificity of the binding conjugate with the object will enable identification of the lack of a functioning substance in antibacterial drugs, which confirms their falsification.