Enzyme-linked immunosorbent assay, immunofluorescent assay, and recombinant immunoblotting in the serodiagnosis of early Lyme borreliosis

Serum samples from Bulgarian patients with physician-diagnosed erythema migrans (EM) (n=105) were examined using Borrelia burgdorferi ELISA (Boehring, Germany) after previous absorption with Treponema phagedenis. For IgM antibody detection sera were additionally pretreated with anti-IgG serum (RF absorbent). Serum samples of 93% of persons from healthy control group were IgM negative and all were IgG negative. Out of 105 patients with EM, 49% were IgM positive and 14 % were borderline. IgG ELISA showed positive results for 17% and borderline for 6% of the patients. Positive and borderline serum samples were examined further by immunofluorescent assay (IFA) and immunoblot test with recombinant B. burgdorferi proteins from strain PKo (B. afzelii) - p100, flagellin, OspA and OspC, and internal flagellin fragments from strains PKo and PBi (B. garinii) [B.Wilske, V.Fingerle, P. Herzer et al. 1993. Med. Microbiol. Immunol. 182:255]. IFA detected IgM antibodies against B. burgdorferi in 47 % of the positive and in none of the borderline by IgM ELISA serum samples as well as IgG antibodies in 83% of the positive and in 50% of the borderline by IgG ELISA samples. Presence of specific antibodies was confirmed by immunoblot in 71 % of the IgM ELISA postive and in 67 % of the IgG ELISA positive sera. In addition, anti-B. burgdorferi antibodies were detected in 60 % of the borderline by IgM ELISA serum samples. IgM serum reactivity was directed mainly against OspC antigen and flagellin and IgG antibodies were directed mainly against flagellin and p100. These findings clearly showed advantages of the ELISA test based on previous pretreatment of sera and capable to detect specific antibodies in more than half of patients with early Lyme borreliosis despite the well-known delayed immune response. IFA was less sensitive than ELISA in detection of anti-B. burgdorferi antibodies. An additional examination of ELISA borderline sera by immunoblot revealed more positive results. Serum reactivity to a single OspC antigen seems to be a sufficient criterion for positive IgM immunoblot.     

IgG antibodies to Loxosceles sp. spider venom in human envenoming.

The presence and specificity of IgG antibodies produced by patients with loxoscelism were studied. The loxoscelism diagnosis was supported mainly by clinical parameters. A search for IgG antibodies anti-Loxosceles gaucho venom in patients with loxoscelism submitted to serumtherapy showed antibodies in four out of 20 patients. The IgG antibodies were detected as early as 9 days and as late as 120 days after bite. The highest IgG antibody titer was 1:640 and the lowest was 1:80. Immunoblotting tests showed that human anti-L. gaucho IgG antibodies recognize preferentially the components responsible for the dermonecrotic and lethal activities of the venom. A comparison of the clinical picture, the level of serum IgG antibodies and the dose of antivenom administered suggest that there is no relationship between these parameters.     

New methodology for the obtainment of antibothropic factors from the South American opossum (Didelphis marsupialis) and jararaca snake (Bothrops jararaca).

The antibothropic factor (ABF) from D. marsupialis was collected from perforated hollow plastic golf balls which were surgically implanted subcutaneously in anesthetized opossums, a technique originally described for the production of polyclonal antibodies. Two months after the implantation of the balls, approximately 15 ml of seromatous fluid from D. marsupialis (SFDm-50 mg total protein/ml) could be recovered monthly. Opossum serum as well as SFDm showed similar SDS-PAGE profiles and antihemorrhagic potencies against Bothrops jararaca snake venom (Bjv). The presence of ABF in SFDm was confirmed by immunoblotting, using rabbit polyclonal antibodies raised against ABF isolated from opossum serum. ABF isolated from SFDm or from serum by ion-exchange chromatography showed identical chromatographic and electrophoretic profiles. ABF fromboth sources displayed very similar antihemorrhagic and anticaseinolytic activities against Bjv. In the case of B. jararaca, polyethylene perforated tubes were inserted in the abdominal cavity and two months after implantation, approximately 4 ml of seromatous fluid from B. jararaca (SFBj-23 mg total protein/ml) were recovered. B.jararaca serum and SFBj showed the same native and SDS-PAGE band pattern. Both serum and SFBj inhibited Bjv hemorrhagic activity. We conclude that this new methodology is very suitable for continuously obtaining opossum ABF and SFBj, in large scale and in an easier way, avoiding animal suffering and eventual sacrifice.     

Proteolytic action of Bothrops jararaca venom upon its own constituents.

Many of the components of Bothrops jararaca venom are proteolytic enzymes. In the present work, we investigated the proteolytic action of B. jararaca venom upon its own constituents. Crude venom was reconstituted and incubated at pH 5.0 or 8.5 for up to 48h at room temperature. Aliquots taken at 0, 24 and 48h of incubation were then tested for proteolytic activity and several biological activities, as well as electrophoretic migration pattern and antibody recognition. Rate of hydrolysis of azocasein by venom samples was not changed by the incubation, but hemagglutinating activity decreased by 93% after 24h of incubation at pH 8.5, with no detectable changes at pH 5.0. Incubation of venom samples caused a progressive increase in phospholipase A(2) and procoagulant activities that was more evident in samples incubated at pH 5.0. The electrophoretic migration pattern showed no significant change for venom samples incubated at pH 5.0, whereas in samples incubated at pH 8.5 bands in the region between 66 and 45kDa gradually disappeared. The addition of a mixture of protease inhibitors (EDTA, PMSF, PPACK and benzamidine) effectively protected against venom degradation at pH 8.5. The cocktail of inhibitors also reduced the changes in phospholipase A(2) activity found in venom samples incubated at pH 5.0. Recognition of venom samples by polyclonal antibodies raised against crude venom was progressively lost during incubation at both pH 5.0 and 8.5; again the addition of protease inhibitors protected against loss of antibody recognition. We conclude that prolonged manipulation of B. jararaca venom at an acidic or alkaline pH can produce significant changes in its biological properties.     

Bradykinin is involved in hyperalgesia induced by Bothrops jararaca venom.

Bradykinin is involved in hyperalgesia (pain hypersensitivity) induced by Bothrops jararaca venom-intraplantar injection of B. jararaca venom (5microg/paw) in rats caused hyperalgesia, which peaked 1h after venom injection. This phenomenon was not modified by promethazine (H(1) receptor antagonist), methysergide (5-HT receptor antagonist), guanethidine (sympathetic function inhibitor), anti-TNF-alpha or anti-interleukin-1 antibodies or by the chelating agent CaNa(2)EDTA. Venom-induced hyperalgesia was blocked by the bradykinin B(2) receptor antagonist HOE 140. On the other hand, des-Arg(9), [Leu(8)]-bradykinin, a bradykinin B(1) receptor antagonist, did not modify the hyperalgesic response. These results suggest that bradykinin, acting on B(2) receptor, is a mediator of hyperalgesia induced by B. jararaca venom.     

Serological detection of 2019-nCoV respond to the epidemic: A useful complement to nucleic acid testing

Corona Virus Disease 2019 (COVID-19) has spread rapidly to more than 215 countries, with over 11.91 million reported cases and more than 540,000 deaths. Rapid diagnosis remains a bottleneck for containing the epidemic. We used an automated chemiluminescent immunoassay to detect serum IgM and IgG antibodies to the 2019-nCoV in 742 subjects, so as to observe the dynamic process of antibody production in COVID-19 disease and seroepidemiology in different populations. Patients with COVID-19 were reactive (positive) for specific antibodies within 3–15 days after onset of symptoms. Specific IgM and IgG levels increased with the progression of the disease. The areas under the receiver operating characteristic curves for IgM and IgG were 0.984 and 1.000, respectively. This antibody detection assay had good sensitivity and specificity. The understanding of the dynamic serological changes of COVID-19 patients and the seroepidemiological situation of the population will be helpful to further control the epidemic of COVID-19.     

Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica.

Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica. Toxicon 000-000. This work compared the specificity, ELISA titers and IgG subclass content of the polyvalent antivenom (anti-Bothrops asper, Crotalus durissus durissus and Lachesis muta stenophrys) of Instituto Clodomiro Picado (Costa Rica) and the bothropic antivenom (anti-Bothrops jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. alternatus) of Instituto Butantan (Brazil). The role of IgG(T) and IgGa subclasses in neutralization of some venom toxic activities and the cross neutralization of the antivenoms against B. jararaca and B. asper venoms were also evaluated. Both antivenoms were able to recognize B. asper and B. jararaca venoms by immunoblotting and presented similar antibody titers when assayed by ELISA. IgG(T) was highest, followed by IgGa, IgGb and IgGc. IgGa and IgG(T) isotypes isolated from both antivenoms by affinity chromatography were tested for neutralization of lethal, hemorrhagic, coagulant and phospholipase A2 activities of the homologous venoms. In both antivenoms, IgG(T) was the major isotype responsible for neutralization of all the tested activities, followed by IgGa. These results suggest that Instituto Butantan and Instituto Clodomiro Picado antivenoms have the same IgG profile and their neutralizing ability is due mostly to the IgG(T) isotype. Also, they neutralize lethality in mice induced by homologous and heterologous venoms, the bothropic antivenom of Instituto Butantan being more effective.     

Morphological study of accessory gland of Bothrops jararaca and its secretory cycle

The venom gland apparatus of Bothrops jararaca is composed of four distinct parts: main venom gland, primary duct, accessory gland and secondary duct. Despite the numerous studies concerning morphology and venom production and secretion in the main venom gland, there are few studies about the accessory gland and its secretion. We characterized the accessory gland of B. jararaca snake and determined the secretion cycle by morphological analysis using light and transmission electron microscopy. Our data showed that the accessory gland of B. jararaca has a simple secretory epithelium with at least six types of cells in the anterior region: two types of secretory cells, mitochondria-rich cells without secretory vesicles, horizontal cells, dark cells and basal cells, and in the posterior region a simple epithelium with two types of cells: seromucous cells and horizontal cells. Furthermore, the mucous secretory cells of the accessory gland show a delayed and massive exocytosis that occurs four days after the extraction of venom. Morphological analysis at different steps after venom extraction showed that the accessory gland has a long cycle of production and secretion, which is not synchronous with the main venom gland secretory cycle.     

Purification of a proteinase inhibitor from the plasma of Bothrops jararaca (jararaca)

A proteinase inhibitor was isolated from the plasma of Bothrops jararaca by three chromatographic steps: DEAE Sephacel, Phenyl Sepharose and Bio Gel P200. It inhibited caseinolytic and hemorrhagic activity of the whole venom of B. jararaca. Proteolytic activity of bothropasin and J protease, both metalloproteinases of the venom, were neutralized by the inhibitor. The J protease-inhibitor complex was isolated by gel filtration chromatography in HPLC and the electrophoresis pattern of this complex showed that the interaction between enzyme and inhibitor is not covalent.     

抗B血型物质单克隆抗体的研制及其免疫学特性的研究

应用杂交瘤技术,分别以人B血型红细胞,B血型红细胞加纯化的B血型物质和B血型物质为抗原,采用不同免疫程序免疫BALB/C小鼠,从6次细胞融合中筛选建立起3株分泌高滴度,高特异性抗人B血型物质单克隆抗体的杂交瘤细胞株:3—3—D_9(IgG_1),3—5—D_(12)(IgG_1)和6—1—G_(11)(IgA),用Takatsy微量血凝滴定法测得它们的96孔板组织培养上清液的血凝滴度分别为4096,4096和16384。经过凝血特异性试验,凝血抑制试验以及抗体稀释试验等广泛免疫化学研究证明这些单克隆抗体仅对人B型血特异。杂交瘤细胞在液氮中贮存数月,分泌抗体能力不变。抗体经56℃保温30minA-20℃和37℃反复冻融试验凝血活性保持稳定。这些单克隆抗体为进一步研究B血型抗体结合部位结构的多样性提供了条件,并且使在临床血液分型试验时用单克隆抗体替代人多克隆抗血清成为可能。     

O型孕妇不同孕期IgG抗A/B抗体效价的动态监测

目的动态监测夫妇ABO血型不合的O型孕妇孕早期与孕晚期血清中IgG抗A/B抗体效价,探讨其在产后ABO-HDN诊断中的价值。方法采用微柱凝胶法对1158名夫妇ABO血型不合的O型孕妇孕早期与孕晚期进行IgG抗A/B抗体效价检测,分析其抗体效价的分布、变化幅度与产后ABO-HDN发生率的关系。结果 158例夫妇ABO血型不合的O型孕妇孕早期与孕晚期血清中IgG抗A/B抗体效价的分布与血型分布无相关性(P>0.05),产后ABO-HDN的发生率与产前(包括孕早期与孕晚期)IgG抗A/B效价的高低呈正相关性(P<0.05),与孕期IgG抗A/B抗体效价的升高幅度呈正相关性。结论动态监测夫妇ABO血型不合的O型孕妇不同孕期IgG抗A/B抗体效价,可更加准确的预测ABO-HDN的发生,为临床提供诊断依据。     

Vascular endothelial cell injury induced by Bothrops jararaca venom; non-significance of hemorrhagic metalloproteinase.

To examine the effect of Bothrops jararaca venom and its major hemorrhagic metalloproteinase, jararafibrase I (JF I), on vascular endothelial cells, B. jararaca crude venom and JF I were infused intravenously into rabbits. The degree of endothelial cell injury was estimated from the plasma level of soluble thrombomodulin (TM). The fibrinogen level, prothrombin time (PT), JF I antigen level and macroglobulin activity of the plasma were also measured. The TM level was not increased even by a large quantity of JF I, while the crude venom caused an increase in TM level suggesting the occurrence of endothelial cell injury. No alterations of fibrinogen level and PT were noted with a high amount of JF I, and no systemic bleeding was observed. Macroglobulin, which is the main inhibitor of metalloproteinase in rabbit plasma, was not significantly reduced despite a high dose of JF I. The elevation of TM level in the rabbit plasma after infusion of crude venom was totally suppressed by pretreatment with heparin. These findings suggest that the endothelial cell injury caused by B. jararaca venom is not due to the hemorrhagic metalloproteinase but to the coagulating factors in the venom. Plasma macroglobulin appears to be efficient enough to neutralize the circulating hemorrhagic metalloproteinases inoculated by B. jararaca.     

Pathological changes in muscle caused by haemorrhagic and proteolytic factors from Bothrops jararaca snake venom

Haemorrhagic factor HF2 and bothropasin, two metalloproteins isolated from the venom of Bothrops jararaca, caused haemorrhage followed by myonecrosis and arterial necrosis after i.m. injection in mice. The effects of HF2 were qualitatively similar to those of bothropasin and crude B. jararaca venom, but its potency was about 20 times higher. The haemorrhagic and necrotizing actions of these components are unrelated to their proteolytic activity on casein.     

SARS患者恢复期抗体动态变化及临床意义

目的：动态监测SARS患者恢复期抗SARS病毒IgG和IgM抗体变化，探讨其临床意义。方法：采用酶联免疫吸附测定法（ELISA），检测SARS患者发病后5，10，14个月末血清IgG、IgM抗体水平，并与临床诊断进行比较。同时观察感染SARS后抗体的持续维持水平及效能。结果：IgG诊断与临床诊断符合程度不一致。随访中3个时期患者恢复期IgG抗体整体水平下降，部分患者IgG抗体阴转，随访后期少数患者IgM抗体水平仍为阳性。结论：SARS患者的血清学IgG抗体检测诊断与临床诊断不一致，SARS病毒抗体变化规律与一般传染病抗体产生规律不同。     

天津地区新冠病毒肺炎患者特异性IgM和IgG动态变化研究

目的　通过动态检测重症与非重症新冠病毒肺炎（COVID-19）患者血清特异性IgM和IgG水平,评估两者在不同患者群体的变化规律。方法　收集天津地区2020年1月—2020年3月确诊的30例新冠病毒肺炎患者血清,重症20例,非重症10例,动态检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）特异性IgM和IgG抗体,进行纵向分析。结果　1.患者发病0～6天,IgM和IgG阳性率分别为42.86%、28.57%,随着病程的进展,两种抗体的阳性率逐步增高。两者从发病大约5周后阳性率开始下降。重症与非重症组患者在发病后约28 d内IgM、IgG水平逐步升高,此后开始下降,重症患者IgM、IgG水平下降明显。2.重症组患者在发病后28～33 d IgG水平较非重症组高,差异有统计学意义(P＜0.05)。3.重症患者发病后28～33 d、34～42 d自身IgG水平显著高于IgM,差异有统计学意义(P＜0.05)。4.恢复期重症组IgG水平较非重症组高(P＜0.05);重症组患者自身恢复期IgG水平较IgM高(P＜0.05)。结论　COVID-19患者特异性IgM和IgG抗体水平随病程进展先升高后下降。重症较非重症组患者特异性IgG抗体水平高。高水平的IgG可能是病情危重的预警指标。     

Characterization of Bothrops jararaca coagulation inhibitor (BjI) and presence of similar protein in plasma of other animals.

BjI, a protein isolated from Bothrops jararaca snake blood, inhibits the coagulant activity of thrombin. This protein presents two bands of 109 and 138 kDa by SDS-PAGE under reducing conditions. In order to verify the presence of BjI-like proteins in plasma of other animals (reptiles and non-reptiles), we raised a specific polyclonal antibody in mice to it, and we verified immunological cross-reaction by western blotting, considering as positive reactions the development of bands with either 109 or 138 kDa. Similar proteins were identified in Bothrops neuwiedi and Crotalus durissus terrificus snakes. In contrast, no BjI-like protein in other classes of animals was noticeable, nor in other snakes tested. Interestingly, a prolonged thrombin time was found only in snake plasmas that showed similar BjI proteins. BjI bound to two proteins of B. jararaca venom, identified by western blotting. The N-terminal of the B. jararaca venom proteins showed similarity with thrombin-like proteins isolated from other snake venoms. In conclusion, there are similar proteins to BjI in plasmas of B. neuwiedi and Crotalus durissus terrificus, and these proteins also prolong thrombin time. Moreover, these results evidence the presence of target enzymes in snake venom for plasma BjI.     

Efficacy of bothropic antivenom and its IgG(T) fraction in restoring fibrinogen levels of Bothrops jararaca envenomed mice.

Bothropic antivenom and its IgG(T) fraction, administered 4 h after experimental envenoming by Bothrops jararaca in Swiss mice, were compared for their abilities to restore fibrinogen 24 or 48 h after treatment. IgG(T) was able to normalise fibrinogen levels as efficiently as conventional antivenom. As IgG(T) also neutralises most anti-toxic activities of Bothrops venom, our results suggest that IgG(T) could be a better alternative treatment for envenoming due to the reduced amount of extraneous proteins, which may facilitate the induction of early adverse reactions.     

新型冠状病毒B.1.617.2变异株感染儿童血清IgM、IgG特点分析及思考

目的:分析新型冠状病毒B.1.617.2变异株感染儿童的血清免疫球蛋白（Ig）M、IgG特点,为大疫情时代下疫情防控提供参考。方法:研究组为2021年5月26日至2021年6月11日广州医科大学附属市八医院确诊的28例（年龄2～17岁）新型冠状病毒B.1.617.2变异株感染患儿。对照组为同期变异株感染成人患者125例...     

Systemic haemorrhage in rats induced by a haemorrhagic fraction from Bothrops jararaca venom.

The main haemorrhagic fraction of Bothrops jararaca venom, showing in vitro fibrinogenolytic activity and an inhibitory effect on platelets aggregation induced by collagan, was studied in rats. Development of coagulopathy and/or haemorrhage was studied 2 hr after s.c. injection of batroxobin, B. jararaca whole venom and its haemorrhagic fraction. Incoagulable blood, together with low fibrinogen levels, were found only in rats injected with batroxobin and whole venom; thrombocytopenia alone was detected in rats given s.c. injections of haemorrhagin. Intravenous injection of low doses of haemorrhagin (less than 15 micrograms) resulted in significant thrombocytopenia, without any alterations in the blood coagulation mechanism. Severe damage to the vascular endothelium and skeletal muscle following s.c. injection of haemorrhagin together with signs of systemic haemorrhage in the kidneys, lungs and liver occurred. Levels of factor VIII and von Willebrand factor antigen were within the normal range in all animals. Serum levels of both whole venom and haemorrhagin were significantly correlated. This study confirms that B. jararaca haemorrhagin plays a vital role in systemic bleeding.