The serological evaluation of the Cytomegalovirus and Chlamydia pneumoniaeinfections in patients with cardiovascular diseases Jia Xiaohui Science & Technologies, 2013,3(1): 26-31
Contents 1. Key words 2. Introduction 3. Materials and Methods 4. Results and Discussion 5. Conclusions
1. Key words Cytomegalovirus[,saɪtə(ʊ)‘meg(ə)lə(ʊ),vaɪrəs] --巨细胞病毒(CMV) Chlamydia[klə‘mɪdɪə] pneumoniae--肺炎衣原体(Cpn) Cardiovascular[,kɑ:dɪəʊ‘væskjʊlə] disease--心脑血管疾病(CVDs) Atherosclerosis[,æθərosklɪ‘rosɪs]--动脉粥样硬化(AS) coronary[‘kɔrənɛri] heart disease--冠心病(CHD)
2. Introduction CVDs remained the most significant threat to the health of human population all over the world. At present, known risk factors for the developing of AS do not clarify all cases of the CHD. The ability of various infectious agents to developed chronic infections and their secreted products to altered vascular cells may play a crucial role in the pathogenesis of CVDs.
In recent years, various studies have indicated that both CMV and Cpn were associated with AS. It was not clear whether the infectious burden could explain the role of inflammation in cardiac instability and AS or not.
Increasing evidence supported the thesis that AS was based on a chronic inflammatory process, which elevated IL-6 levels and cytokine production. Reports of such associations had raised the possibility that anti-infective treatments might be able to prevent CVDs. Mediated by this systemic inflammatory process, CMV and Cpn infections might contribute to the AS and atherothrombotic processes.
This article investigated the serum levels of IgG antibodies to CMV and Cpn in patients with different CVDs and the risks related to persistent infection for late cardiac events.
3. Materials and Methods 1) Patient characteristics and Groups 93 patients: 55 men, 38 women 41-85years chronically myocardial ischemic disease(IHD) acute coronary syndrome(ACS) Groups stable angina pectoris(AP) healthy controls
Blood samples Centrifuge, 4000g 10min 2) CMV and Cpn serological studies separate serum and frozen at -80℃ ELISA Kits Test IgG to CMV and to Cpn A semi-quantitative tests, in which results are expressed as a ratio of extinction value of the control or patient sample over extinction value of the calibrator (ratio=extinction of the control or patient sample/extinction of the calibrator). Quantitative values of antibodies for both tests were obtained from the standard curve by point-to-point plotting of extinction values against the corresponding units.
3) Statistical analysis Tests were done in triplicate and in two separate experiments. Categorical data were analyzed by the χ2-test. Variables with control distribution are presented χ±S. A value of p<0.05 was considered statistically significant.
4. Results and Discussion Patients were divided generally into 4 groups: （1）22 (23,7%) patients with IHD; （2）30(32,6%) with ACS; （3）19(20,4%) patients with AP； （4）22(23,7%) patients in the control group(Fig. 1).
??? Of all patients with CVDs studied 52 cases(94.5%) and 23 cases(60.5%) had anti-CMV IgG. Optical density, an indicator of CMV antibody titre, was higher in the patients with ACS than in other groups: median 36 NTU/mlvs25-26 NTU/ml in IHD, stable AP and in controls. The ratio relating CMV seropositivity to incidents with ACS was 5.4 (32.6% from all cases with CVDs). ???
However, the proportion of cases with CMV positive antibody titres was not statistically different between the 4 groups (94.5% in ACS, 90.2% in chronically IHD, 90.5% in stable AP and 92.4% in the controls). Among controls, seropositivity was not associated with age and gender. Fig.2 The proportion of cases with CMV positive antibody titer
93 patients 15(62.5%) man Anti-Cpn IgG positive: 24(25.8) 9(37.5%) women This coincided in published data according which Cpn antibody seroprevalence rates tend to be higher in men than in women, suggesting that men were more susceptible to Cpn infection than women.
However, only serum concentrations of IgG antibodies to Cpn were significantly associated with acute coronary events, smoking behaviour and hypertension. Several limitations must be considered in interpretation of these findings. We used the presence of IgG antibodies(for Cpn, IgG titer) measured on a single occasion late in life to characterize prior infection. This result may have had inadequate statistical power to detect very high risk of cardiac death associated with the presence of prior infection with Cpn. In addition, the data from this study may not be generalizable to young and middle-aged adults.
5. Conclusions This study founded that CMV and Cpn were unlikely to be strong predictors of risk of CVDs and future cardiac death. Probably, antibody titer-dependent connection of CMV and Cpn may be implicated in ACS, but is unlikely to be a strong risk factor for development of myocardial infarction, chronically IHD and stable AP.
Our data were in a contrast with a various studies that these agents may be implicated in the pathogenesis of atherosclerotic disease and being associated with a bad prognostic clinical outcome. The study does not however completely exclude that CMV and Cpn infection or reactivation/reinfection plays any role in AS disease of the coronary arteries.
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Antibody Production 1 Introduction 2 Materials 3 Method 4 Notes
1 Introduction Antibodies are proteins that are produced by the immune systems of animals in response to foreign substances. Antisera are derived from individual animal bleeds and because of this are subject to batch variation.
As individual clones produce the antibody molecules and there are many of them, the resulting antibody mix in the blood is known as polyclonal and the fluid derived from the clotted blood is known as Polyclonal antibody. Monoclonal antibodies are produced from highly cloned cell lines that are stable and reliably produce a defined antibody product.
Polyclonal antibodies are generally less specific than monoclonals, as cross-reactivity may occur with nontarget proteins. Monoclonal antibodies can be too specific, as they will recognize only a single epitope that may vary on the protein of interest. Both antibody types have their place in the research laboratory, and a careful evaluation of the required use should be undertaken before deciding which would be most applicable.
Small quantities: Rabbits,Rats and mice(5–10mL of polyclonal ascitic fluid). unless very large quantities: Sheep, goats, donkeys, and horses. Chickens(antibodies are produced in the eggs), 1.1 Donor Animals
1.2 Adjuvants Increase the immune response to antigen by an animal. Ideally an adjuvant should not induce an antibody response to itself. Chemicals: alum(adsorbs and aggregates proteins).
Freund’s adjuvant: complete(primary immunizations ), incomplete(subsequent immunizations). Freund’s incomplete is a mixture of 85% paraffin oil and 15% mannide monooleate. Freund’s complete is a mixture of 85% paraffin oil,15% mannide monooleate and 1 mg/mL of BCG.
1) As close in structure and chemical identity to the target protein as possible. 2) Soluble, stable at dilutions of approx 1 mg/mL 3) physiological pH (6.5–7.5). 4) As pure a form as practically possible, to avoid the generation of antibodies to contaminating materials. 5) Proteins are derived from a different species. 1.3 Antigens
Affinity: plate-trapped double-antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA). Cross-reactivity: Ouchterlony double-immunodiffusion. Radioimmunoassay and other related techniques can also be used to test the avidity of the antiserum to the antigen and will also give a measure of antibody titer. In all the above tests preimmune antisera should be included to ensure that results obtained are a true reflection of antibodies produced by immunization and not due to nonspecific interactions. 1.4 Test Protocol
2 Materials 1. At least two rabbits(New Zealand whites rabbit) should be used for each polyclonal antibody production project, as they may have different immune responses to the antigen. 2. The antigen should be in a buffer of pH 6.5–7.5 and be free of toxic additives. A concentration of 1 mg/mL is desirable but anything above 100 μg/mL is acceptable. 3. Complete and incomplete Freund’s adjuvant
3 Method 1. Prior to a course of immunizations a test bleed should be taken from the rabbits to provide a source of preimmune antiserum for each animal. The blood should be allowed to clot at 4°C for 12 h and the serum gently aspirated from the tube. 2. The antigen should be mixed with the appropriate adjuvant according to the manufacturer’s instructions to achieve a final volume of 0.5 mL/injection containing 50–500 μg of antigen. The first injection only should contain the complete formulation and the incomplete one should be used for all subsequent immunizations.
3. The rabbit should be restrained and the antigen–adjuvant mixture injected into the thigh muscle. 4. The immunization should be repeated 14 d after the primary one and a test bleed taken 30 d after that. 5. If the antiserum shows that the desired immune status and antibody quality has been achieved then donor bleeds can be taken. Each bleed should be assayed individually, and once the antibody titer has started to fall a further immunization can be given.
6. Antiserum collected from rabbits can be stored for extended periods of time at 4℃ but the addition of 0.02% sodium azide is recommended to prevent adventitious bacterial growth. Antiserum quite commonly has functional antibodies even after years of refrigerated storage, but storage at –20℃ is recommended for long-term preservation.
4 Notes 1. Some antigens will consistently fail to induce an antibody response in certain animals, and other species should be investigated as potential donors. In very rare occasions antigens will not elicit an immune response in a range of species and the nature of the antigen will then have to be investigated with a view to modifying it to increase its immunogenicity. 2. Female rabbits are less aggressive, and although smaller and yielding smaller quantities of antiserum are preferable to male rabbits for antiserum production.
3. The use of excessive amounts of antigen in immunizations should be avoided, as this can lead to a poor immunological response. High doses of antigen in the secondary and subsequent immunizations can cause anaphylactic shock and death of the donor animal. 4. Increased stress levels in animals can depress the immune response, and appropriate measures should be taken to ensure that immunizations and bleeds are performed with the minimum of stress to the animals. Make sure that animals for polyclonal production are maintained under suitable conditions.